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I’ I 3, t 1.! , ‘1“: 11‘" (1.11?“ 31““ 1111351 331.11%.“ '11‘. 1‘4 '1'1' ‘ THESIS LIBRARY Michigan State University dissertation entitled I | This is to certify that the 1 l Explorations in Design and Performance of an Instrument Utilizing the Method of Cell Rotation in Molecular Spectrometric Chemical Analysis presented by Karlis Adamsons has been accepted towards fulfillment of the requirements for Ph . D . degree in Chemistry Major professor Date ‘ May 20, 1985 ucn.‘.....ur .~ A- .- -n - , ~ ~ 0427." EXPLORATIONS IN DESIGN AND PERFORMANCE OF AN INSTRUMENT UTILIZING THE METHOD OF CELL ROTATION IN MOLECULAR SPECTROMETRIC CHEMICAL ANALYSIS BY Karlis Adamsons A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Chemistry 1985 ABSTRACT EXPLORATIONS IN DESIGN AND PERFORMANCE OF AN INSTRUMENT UTILIZING THE METHOD OF CELL ROTATION IN MOLECULAR SPECTROMETRIC CHEMICAL ANALYSIS BY Karlis Adamsons A computerized instrumental system has been develOped integrating a single beam spectrOphotometer and a right- angle spectrofluorometer to allow routine fluorescence measurements corrected for the effects of primary and/or secondary absorptions by the sample. In addition, the instrumental system permits determination of total quantum efficiencies by the comparative method, evaluation of relative fluorescence efficiencies, output of primary absor- bance calculated spectrOphotometrically or spectrofluoro— metrically (and the corresponding transmittances), output of secondary absorbance calculated spectrofluorometrically (and the corresponding transmittance), evaluation of single or multiple derivatives of any spectral functions, applica— tion of fluorophore absorbance curve stripping procedures, and determination of both turbidimetric and nephelometric quantities. The system incorporates a unique off-center sample cell rotation to obtain differences in the thickness of the solution through which the excitation and emission beams penetrate. These differing pathlengths permit determination Adamsons, Karlis of the primary and secondary absorptions occurring by evalu- ation of the fluorescence intensity attenuations along the excitation and emission optical axes. Knowledge of these attenuations enabled absorption factors to be develOped and applied for on-line correction of fluores- cence measurements. The equations written for this purpose required accurate definition of the viewing window geome- try. The resulting corrected fluorescence is linear with the fluorophore concentration to absorbances as high as 2.5 A (primary and/or secondary absorbance units). The corrections are independent of the chemical system studied. This fluorescence correction capability offers the clinical laboratory the opportunity to extend the working curves of many fluorometric assays (including those where fluores— cence measurements could be obtained directly and those where the fluorescence had to be generated indirectly by chemical pre-treatment. Useful applications are in the analyses of various drugs, hormones, vitamins, amino acids and porphyrins. In one case, the direct fluorometric analysis of quinidine sulfate, the linear working range was extended from approximately 1-15 ug/ml realized by routine spectrofluorometric determinations to 1.5-200 ug/ml with this method. ACKNOWLEDGEMENT AND THANKS ... TO my SAGE, MAJOR PROFESSOR and TRUE BELIEVER - Dr. ANDREW TIMNICK To UNCLE JACK, the COMMANDER OF THE UNIVERSE - Dr. JOHN HOLLAND To the KEEPER OF THE ELECTRONIC GATES - Dr. JOHN SELL TO the NOTORIOUSLY TALENTED UNDERGRADUATE PROGRAMMERS - MS. KELLY ADAMSKI Mr. PATRICK SIMON Mr. MICHAEL REID To THOSE WHO GAVE FREELY of their chemical and wisdom - Dr. CLARENCE SUELTER Dr. WILLIAM FRANTZ Dr. STANLEY CROUCH Dr. ARNOLD REVZIN Dr. JOHN SPECK To the LATVIAN SPIRIT, and my own proud parents - Mrs. Austra Adamsons Mr. Arvids Adamsons To the various sources for my CONTINUAL FINANCIAL SUPPORT — MSU CHEMISTRY DEPARTMENT for TA's and RA's MSU BIOCHEMISTRY DEPARTMENT for an RA FELLOWSHIP/TRAINEESHIP from COLLEGE OF NATURAL SCIENCE YATES MEMORIAL SCHOLARSHIP from MSU To my BUBBLY, BOUNCY and BEAUTIFUL BUNCH who always stood ready with BICYCLE and BACKPACK and DANCE SHOES - MS. HOLLY FORTNUM —ii- 55.2% 125:0 uEemZomSmmm mjpomaoz 7: 22:93 .55 no. cores: are REESE: aZmzpmemE z< mo moznquoom oaochpnmfin mo .muowononu .Emon cofiumufloxo coumEfiHHoo on» no numm on» on noH50flpcomumm we :ofiuoouop cocoommnosam .coaflmuop we pcwfiunmmfioo Haoo onu mo ucwEomcmuum .COMumusmHmcoo HOOfimmu wee ca HmuofiouosHmonuoomm oamchanmflH m Emergence :8 SEES . so I— saemm Em I... LessoEuocoE 8745 U [1 JOIeuIOJuaouow uoIIEIpxg 8528 8582 so: .v musmflm BDJHOS -14- Figure 5. Optical track arrangements in instruments integrating the capabilities of spectrophotometers and Spectrofluorometers. A. System employing the Moving (Vibrating) Mirror Method; sample cell remains stationary; R =reference cell, S== sample cell. B. System employing the Cell Shift Method; sample cell moves along perpendicular tracks. C. System employing the Cell (Elliptical) Rotation Method; sample cell undergoes a three stop rotation pattern. -15- absorption and fluorescence measurements. The usual photomultiplier tube as detector was replaced by a photon counter so that the detector output could be converted to a number which was proportional to the relative number of photons. An inter-calibration of the photon counter output with the photomultiplier tube output of the emission detector for the same beam intensity resulted in an emission detector output also in relative numbers of quanta. The mathematical model developed for correction of fluorescence measurements for absorption attenuations was based on knowledge of the excitation beam attenuation as it penetrates the sample solution as shown in Figure 1B. The corrections could be applied in real-time, thus making these corrections on a routine and practical basis. The primary absorption corrected fluorescence was linear with the fluorophore concentration in solutions with Absorbances up to 2.0. The primary absorption correction was found to be independent of the nature of the absorbing Species and the excitation and emission wavelengths. The Cell Shift Method as illustrated in Figure 5B. This was the first computer based system to employ sample cell positioning along both the excitation and emission axes. The mathematical model developed for correction of fluorescence measurements for absorption attenuations was based on knowledge of fluorescence attenuation along both of these axes as shown in Figure 1B,D. Unfortunately, the slowness of data acquisition while in a scanning mode and —16- the potential for misalignment in cell positioning made this system impractical for routine analyses. The primary and secondary absorption corrected fluorescence was reported linear with the fluorophore concentration in solutions with Absorbances as high as 2.7. The primary and secondary absorption corrections were found to be independent of the absorbing Species and the excitation and emission wavelengths. The Elliptical Cell Rotation Method is illustrated in Figures 5C, 6 and 8. This system was initially developed as an engineering solution to the slowness of data acquisi— tion and misalignment problems that were characteristic of the Cell Shift Method. This instrument was able to make accurate corrections of the fluorescence measurements for the effects of primary and secondary absorption on a practical, rapid and routine basis. Additional features include ease of operation, reliability, precision, accuracy, and real-time data acquisition and processing. CHAPTER III THEORETICAL BASES FOR THE VARIOUS OUTPUTS OF AN INSTRUMENT EMPLOYING THE CELL ROTATION METHOD (CRM) A. Introduction to the Cell Rotation Method The Cell Rotation Method employs three cycle positions obtained by unit ellipse off-center viewing of the rotating sample cell as illustrated in Figure 6. Patterns other than that of a unit ellipse are also possible during cell rotation. Figure 7 Shows a number of cell centered ellip- tical rotation patterns with major axes along either the excitation beam or the emission detection beam. For all subsequent work documented in this dissertation, the unit ellipse is used to allow maximizing the effects of beam attenuation along either axis. The distance between the viewing window centers, including either positions 1 and 2 or positions 1 and 4, must be known prior to very accurate determination of the primary and secondary absorption of the sample. The adjustment of the cell position on the cell holder platform is accurate to about 0.01 inch along either the excitation or emission detection axis. Therefore the distance between the viewing window centers is known. These distances are the bases of the geometric factors defined in Figure 8, -17- -13- F EM EX T E] . R (”I ,9.) S Position in, 1 E ._ ...___I r. V) b U; 2 z E’ I: E Z E . . < POSItIon 3 Lu 2 I... m: In EX 8 r}“*’“°9 E Z o. m R S E E. < m V) fr EX R (”1,69L—J S Position 4 Figure 6. Cell orientations employed in the Cell Rotation Method. Cell positions 1 and 4 provide information on the extent of primary absorption. Cell positions 1 and 2 provide information on the extent of secondary absorption. Key: R =reference beam; S =samp1e beam; F =f1uorescence detec— tion beam; EX =excitation beam axis; EM =emission detection beam axis; b =cell pathlength along the excita— tion axis; b' =cell pathlength along the emission detection axis; ml and M2 =known centers of the fluorescence measure— ment observation windows while in cell positions 1 and 4, respectively; 81 and 92 =known centers of the excitation beam cross-section while in cell positions 1 and 2, respectively. Figure 7. -19- 1 . —-— ———>EX 2 ‘EJ ........ III" ELI-j PD: Ed D ‘3., EU The elliptical cell rotation patterns. All reported fluorescence measurements were conducted with centered and circular (unit ellipse) rotation patterns. The cell holder platform permits setting a variety of rotation patterns as illustrated. Figure 8. -20- Definition of the geometric factors (G#'s), equations for their evaluation, and identification of quantities that must be measured for their evaluation. A. The 61 factor multiplies the primary absorbance measured between cell positions 1 and 4 to give the primary absorbance taking place between the excitation port cell wall and position 1. B. The G2 factor multiplies the secondary absorbance measured between cell positions 1 and 2 to give the secondary absorbance taking place between cell positions 1 and the emission port cell wall. C. The G3 factor multiplies the primary absorbance measured between cell positions 1 and 4 to give the primary absorbance taking place along the entire excitation pathlength. D. The G4 multiplies the secondary absorbance measured between cell positions 1 and 2 to give the secondary absorbance taking place along the entire secondary pathlength of the cell. F1, F2 and F4 are the fluorescence intensities measured in cell positions 1, 2 and 4, respectively. ml and w2== planes (centers of the observation windows) along the excitation axis, values known. 01 and 62==planes (centers of the observation windows) along the emission axis, values known. b =ce11 pathlength along excitation axis. b'==cell pathlength along emission detection axis. All geometric factors are wavelength independent constants. Optimum settings with a 2 cm by 2 cm (b==b' from top View) sample cell: G1 =62 =0.75 and G3 =G4 =2.50. A2 -21- Aw,,CALCULATED = (..fi’ ...) AMOEHHQ can ouos3 oan .m .haamOHuuoeouonmouuoomm haco COCHEHODOC on coo oamemw may mo monmnnomnm mHmEflHm OED CROSS ommo .d .ononmouosam AwaamOHEOEO- mcfiuomuoudHIcoc .SOEOHOHMMO Esucmnw smfln m we mcofipmuucmucoo 30H m0 coauflppm an COHDSHOO Ca monoanEonso mo OOEMQHOme Tau mo coflumcflsuouoo .m madman -26- IIIIII 5 5 5 5 5 5 5 5 _5 5 515 615 5 . S 5 S 5 5 5 515 51 5 5 5 5 5 5 5E5 5L_5 5 5 5 5 5 5 5 5“. 5 515 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 22:95 5325. 255325555 5 2555505QO 2555505QO ..m 2m .50 :4. -27- the attenuation of the excitation radiation can be monitored. 2. Secondary Absorption Measurements The extent of the fluoresced radiation absorption can be readily evaluated by the following function: A3 = (G4)1og(Fl/F2) (III.3) where the G4 factor multiplies the secondary absorption measured between cell positions (1) and (2) to give the secondary absorption taking place along the entire possible cell pathlength as shown in Figure 8D. Spectral distor— tions caused by the effects of secondary absorption are illustrated in the section on results and discussion in Figures 39 and 42. 3. Absorption Corrected Fluorescence The detailed derivation of the expressions applied to correct fluorescence for primary and/or secondary absorptions can be found in a previously reported study (Reference [2]). Fluorescence readings taken with the cell in position (1) are corrected as follows: A. Fluorescence corrected for primary absorption. (G1)1n(F1/F4) FP = (Fl)e (III.4) -28- B. Fluorescence corrected for secondary absorption. (G2)1n(F1/F2) FS = (Fl)e (111.5) C. Total absorption corrected fluorescence. FO = (Fl)e(Gl)1n(F1/F4)e(G2)ln(F1/F2) (III.6) D. Source and absorption corrected fluorescence. FR = (Fl/Rl)e(Gl)1n(F1/F4)e(G2)ln(F1/F2) (III 7) where G1 and G2 are the geometric factors (defined in Figure 8A,B), and F1, F2 and F4 are the quantized relative fluorescence intensities monitored in cell positions (1), (2) and (4), respectively. The corrections were applied to the position (1) fluorescence readings because the largest signal is available and it is subject to the least amount of attenuation by either primary or secondary absorption (note Figures 6 and 39). The utility of absorption corrected fluorescence in the quantitative and qualitative aSpects Of fluorescence intensity measure- ments is demonstrated in the section on results and discussion (Shown in Figures 38 and 39). 4. Absorbance Curve Stripping This technique allows primary absorption due to the fluorophore components of a sample to be subtracted from -29- the total primary absorbance profile to generate a primary absorbance curve of the non-fluorescing Species. In principle, the fluorescence excitation curve of a pure fluorophore mimics exactly the primary absorbance curve. Note that the excitation spectrum will be the same as the absorbance spectrum assuming that the absorbance spectrum is due to a Single electronic transition. Only a linear, wavelength independent scale factor difference exists between the two curves, Al and FR. If some point along the excitation scan axis can be identified where all of the absorption is produced by the fluorophore, a scale factor can be Obtained that will convert the excita- tion curve into a primary absorbance curve for the fluoro- phore. Direct subtraction at each wavelength removes this contribution from the total primary absorbance curve. Figure 10 illustrates the process of absorbance curve stripping. Studies utilizing absorbance curve stripping are provided in the experimental section. 5. Turbidimetric and Nephelometric Measurements Scattering of the excitation radiation by particles in solution can be monitored along either the excitation or emission Optical axes permitting turbidimetric or nephelometric analyses, respectively [36-46]. A treatise by F.W. Billmeyer Jr. is available summarizing the theory and documenting the equations describing scattered light intensity appearing at any angle as a phenomenon with a complex dependence on a number Of factors including the Figure 10. -30- W U z . ( A1 m .- m D b V] d] .. < .. ...-.',..':...'.',-,....-.... AVELEN TH vv ii Aex (FRXSF) o - ... .0. A1 ABEDRBANEE WAVELENQTH A“ Lu 9 ID = A1-(FR)(SF) 4: [fl .- d a i- UT 0 I < WAVELENGTH A” Process Of fluorophore absorbance curve stripping. A. Curve A1==total primary absorbance. Curve FR== fluorescence intensity observed in cell position 1 corrected for total absorption attenuations and reference beam intensity fluctuations. B. Curve [(FR)(SF)] =resu1t Of multiplying the excitation curve by a scale factor, SF, which is equal to [(A1)(FR)] determined at a wavelength selected to have minimal absorbance contributions from the sample solution chromophores. C. Curve 1D== difference spectrum between the Al curve and the [(FR)(SF)] curve and represents the primary absorbance of the chromophores in the sample solution. _31_ number of particles, size of particles, shape of particles, refractive index of the particles, refractive index of the cell walls and medium, and the wavelength of the radiation [49]. However, the theory is too complex to utilize directly. Simplifications are possible if the areas of application are restricted. In cases where the wave— length of the scattered light is not shifted from that of the incident light, it is possible to distinguish Rayleigh scattering from Tyndall scattering. Rayleigh scattering is based on particles which are small compared to the incident wavelength and is proportional to the inverse fourth power of the wavelength according to the following equation: 5 = log(Po/P) = kbN (111.8) where S is the turbidance, k is a proportionality factor called the turbidity coefficient, b is the pathlength, and N is the number of scattering particles per milliliter. The proportionality factor is theoretically represented as: k = 0.4343{(2/3)TI5d6>(—4[(m2—l)/(m2+2)]2} (111.9) where d is the particle diameter, A is the wavelength, and m is the ratio of the refractive index of the particles to that of the solvent. Tyndall scattering is based on particles which are large compared to the incident -32- wavelength and is proportional to the inverse second power of the wavelength according to a similar equation: 6A‘2[(m2-1)/(m2+2)]2} (111.10) S = 0.4343{(2/3)H5d These equations require absolute measurements for their evaluation, such as the values of d and m. Obtaining these values can be time consuming and requires access to other types Of spectroscopic instrumentation for their determina- tion. Fortunately, most Of the applications of the measure- ment of particle light scattering do not require such a rigorous approach to generate analytically useful results. A simplified and highly empirical approach to quantita- tively using particle light scattering measurements has proven successful in many analyses and can be readily implemented with the CRM instrument. Turbidimetric measurements are based on comparison Of an unknown sample with a working curve that has been developed from a series Of standard solutions containing the same chemical system. These working curves are determined in a manner analogous to those of absorption Spectrometry and are given as follows: log(Reference Beam Intensity/Sample Beam Intensity) vs Concentration of the Light Scattering Particles (III.11) -33- The Optical configurations used in Obtaining turbidimetric measurements in commercially available turbidimeters (or spectrophotometers) and the CRM instrument are compared in Figure 11. Nephelometry is also based on comparison Of an unknown sample with a working curve that has been developed from a series of standard solutions of the same chemical system. The working curve is determined from a plot of particle scattered light detected orthagonal (at 90°) to the excitation beam axis relative to the light scatterer concentration and is given as follows: log Scattered Light Intensity at 90° Reference Beam Intensity vs Concentration of the Light Scattering Particles (111.12) The Optical configurations used in Obtaining nephelo— metric measurements in commercially available nephelo- meters (or Spectrofluorometers) and the CRM instrument are compared in Figure 12. A third form Of particle light scattering measurement makes available a new method of Obtaining turbidimetric- like quantities by taking a ratio of nephelometric measure— ments from cell positions 1 and 4. This output is unique to the CRM instrument. The working curve is develOped from a series of standard solutions with known particle concentrations and can be represented by a plot Of: -34- BS VB Vs Figure 11. A comparison between typical commercially available turbidimeter versus the CRM instrument opical configura- tions used to obtain turbidimetric measurements. A. Commercial instrument with excitation beam passing through center Of sample cell; BS==beam Splitter, R==reference beam intensity, 5 =sample beam intensity. B. The CRM instrument with excitation beam passing through near edge of sample cell; BS, R and S defined as above, M==front surface mirror. -35- Figure 12. R A comparison between a typical commercially available nephelometer versus the CRM instrument Optical configura— tions used to Obtain nephelometric measurements. A. Commercial instrument with excitation beam passing through center of sample cell and particle scattered radiation detected from an Observation window at the center of the cell; BS =beam Splitter, R==reference beam intensity, Si =90° light scatter intensity. B. The CRM instrument with excitation beam passing through near edge of cell and particle scattered radiation detected from cell position 1; BS and R are defined as above, 811 =90° light scatter intensity from cell position 1. -36- (G3)log(Sil/Sl4) vs C (111.13) where 811 and 814 are the right-angle measurements of the scattered light intensity at cell positions 1 and 4, and CT is the concentration of light scattering particles. The Optical configuration of the CRM instrument used to Obtain this quantity is shown in Figure 13. Note that the two measured quantities, $11 and 514, are nephelometric measurements. Potential advantages of this turbidimetric- like measurement would be two-fold: l. in the case where light scattering components are being adsorbed onto cell surfaces, right-angle measurement of light scattering intensity from two independent positions along the excita- tion axis permits compensation for these adsorbed Species; and 2. incorporation Of Off-center sample cell rotation capability into a right-angle spectrofluorometer would allow turbidimetric—like measurements to be made without the need to monitor the reference and sample beam intensities. Fluorescence intensity measurements can be corrected for the effects of particle light scattering in a manner Similar to that employed in the calculation of absorption corrected fluorescence. Light scattering corrections can be made by application of the following expressions: A. Fluorescence can be corrected for primary axis light scattering by an expression analogous to that employed to Obtain fluorescence corrected for primary absorbance. -37- S11 541 BS / Figure 13. R Measurement Of turbidimetric~analog quantities employing an instrument Operating under the CRM. BS =beam splitter, R==reference beam intensity, 81 and SD =90° light scatter intensity from cell positions 1 and 4, respectively. -38- F .= (Fl)e(GS)1n(Fl/F4) llP (III.14) B. Fluorescence can be corrected for secondary axis light scattering by an expression analogous to that employed to Obtain fluorescence corrected for secondary absorbance. (G6)1n(Fl/F2) F = (Fl)e 11S (III.15) C. Fluorescence can be corrected for total light scattering by an expression analogous to that employed to obtain fluorescence corrected for primary and secondary absorbance. (G5)ln(F1/F4)e(G6)ln(F1/F2) F = (Fl)e 11T (111.16) The constants, G5 and G6, are empirically determined geo— metric factors for use in light scattering corrections along the excitation and emission detection beam axes, respectively. These geometric factors are selected to provide linear fluorescence intensity versus fluorophore concentration working curves based on standard solutions containing known fluorophore and light scattering particle concentrations. When precipitates are difficult to filter, due to small particle Size, gelatinous nature, etc., they usually make ideal suspensions to be measured by these light -39- scattering techniques. In many cases, the light scattering techniques are chosen because Of their Speed and simplicity compared to the (alternative) gravimetric procedure. When properly controlled, light scattering measurements can Often eliminate the time consuming analytical Operations of filtration, washing, drying, igniting and weighing. Light scattering methods are nondestructive and respond rapidly to changes in concen— tration Of the scattering system. The rotation Of the sample cell in the CRM instrument provides continual periodic sample mixing which prevents concentration gradient formation by the particles settling to the bottom Of the cell. This tends to be a serious limitation Of instruments measuring turbidimetric or nephelometric quantities that do not have a means Of ensuring a uniform size distribution of the light scatterers and a uniform solution density. 6. Total Quantum Efficiency, QE, by the Comparative Method The quantum efficiency of a fluorophore can be defined as the following ratio: Number of Photons Emitted per Unit Time (III 17) QE = Number of Photons Absorbed per Unit Time The comparative method provides the following exact expression: 2 QEz/QEl = (FZ/Fl)(Al/A2)(nD2/nDl) (III.18) -40- where QE is the quantum efficiency, F is the fluorescence emission, A is the absorbance, and nD is the refractive index. The subscripts 1 and 2 refer to the chemical species of unknown quantum efficiency and the standard of known quantum efficiency, respectively. To Obtain quantum efficiencies by the comparative method, first F2, A2 and n 2 are evaluated for the reference standard, D for which the quantum efficiency is known. Subsequent measurements of the same quantities for the unknown, leads to evaluation of the quantum efficiency of the unknown. Note that normally the refractive indices are not evaluated because the solutions are so dilute. An alternate and approximate approach to the compara- tive method is available to determine quantum efficiencies using the CRM instrument. The quantum efficiency approxi- mation is given as follows: , _ Area Under the (FS/Rl) Emission Curve QE — (Rl _ Sl)(fw) (III.19) where FS is the measured fluorescence intensity from cell position 1 corrected for secondary absorption, R1 is the measured reference beam intensity from cell position 1, $1 is the measured sample beam intensity from cell position 1, and fw is the window factor that accounts for all of the radiation absorbed by the solution not absorbed in the window of Observation. All of these measured quantities have been converted to relative numbers of -41- quanta. To Obtain quantum efficiencies by this approxima- tion method, first the above ratio is calculated for a solution containing the standard fluorophore. This is then equated with a manually input value of the documented quantum efficiency for that substance determined under (ideally) equivalent instrumental and environmental conditions. Completion of this calibration procedure allows subsequent evaluations of QE' for other fluorophores. 7. Relative Fluorescence Efficiency (RFE) A new mathematical function, RFE, arises from the instrumentation required to make the necessary measurements to calculate fluorescence quantum efficiencies. This function is an analog of fluorescence quantum efficiency made possible by the ability to determine the quanta of excitation radiation absorbed and the quanta of fluo- rescence emitted at any given wavelength. Just as in the determination of fluorescence quantum efficiencies, the following quantities must be available: quantized reference beam intensity, quantized sample beam intensity, and quantized fluorescence detection beam intensities. The RFE of a fluorophore can be defined as the following ratio: Number of Photons Emitted per Unit Time RFE = Number of Photons Absorbed per Unit Time (111.20) The RFE differs from the total quantum efficiency in that the efficiency is evaluated for a fixed fluorescence -42- wavelength at various excitation wavelengths. Effectively, RFE'S are evaluated for a narrow fixed emission band for the selected excitation wavelength range. Originally [27] the RFE function was approximated according to the following equation for the Moving (Vibrating) Mirror Method: F . . RFE = across the(;bs:§vation w1ndow (III.21) where F is the integral Of the quanta fluoresced (calcu- lated by the method Of summation of trapazoids), and the term (R -S) carries the Significance of the number of quanta in the reference beam less the number Of quanta in the sample beam, or in essence, the number of quanta absorbed by the fluorophore. Since the integral under the fluorescence curve produces a number related to the number of quanta fluoresced, this ratio will satisfy the classical definition of total quantum efficiency [Eqn. (III.17)]. Figure 14 illustrates the relative Size of the Observation window in the Moving Mirror Method compared to that employed by the CRM. The Moving Mirror Method viewed about 0.8 cm of a 1.0 cm cell surface orthogonal to the excitation beam, while the CRM views about 0.3 cm of a 2.0 cm cell surface orthogonal to the excitation beam in each cell position. The limitation encountered in the previous approximation was that the -43- .v COHDHmOm HHOO DO sopcflz coDDO>DOon OED mo DODCOO s3ocx O£Du :mox 1v COHDHmom HHOO mo mOmpo BOUQDS CODDO>HOOQO CBOCRCS OLD" :x can mx 1H CODDDmom HHOO DO 30pca3 CODDM>HOOQO OSD mo MODQOO csocx ODD" NHox “H coflDflmom HHOO Mo mOmpO BOOCDB CODDm>hOon csocxcs OSDN Nx can fix in can A mCODDDmoa HHOO DO msopcfls CODDO>HOODO OOCOOmOHODHwn N> can D> "COSDOE EOHDODom HHOU .m .EOCCDS coDDm>DOon OnD mo DODCOO csocx OED" ox “wOmpO BOUGDS ODD mo mcoDDmooH csocx ODD” Nx can Hx 13OCCD3 CODDO>DOOQO OOGOOOOMODHMN Qo> “OOEDOZ Donna: mcD>OE .m .EODDOEDM >OCODOHMMO OOCOOOODOSHD O>HDOHOH OED mo CODDODHO>O ODD ca OODDOz coflDmDom HHOO ODD msmuo> COEDOS Donna: mafi>oz OLD mcfluonEO ODCOEDHDmcD COOzDOn EBOOCHB COHDO>DOOQO OLD mo mONDm O>DDOHOD OED mo EOOHHOQEOU v * m .._‘:)>4 2! O N O M --->< ----_------_---_x ----------_----Ix I m> HP so.» ZoEZeom mommHE .Smo OZH>OE m AV .vH Ousmflm -44- fluorescence measurements were not corrected for secondary absorption. The new version (or approximation) Of the RFE function for the CRM is given as follows: (Fl)e(G4)ln(F1/F2) RFE = (R1 -Sl> (f2) (111.22) where the numerator is the fluorescence intensity observed across the observation window Of cell position 1 corrected for secondary absorption, and the denominator includes the difference between the quantized reference and sample beam intensities, (R1 —Sl), which is equivalent to the number Of quanta absorbed by the fluorophore at each excitation wavelength and fw is the window factor that accounts for all of the radiation absorbed by the solution not absorbed in the window of Observation. This expression for RFE overcomes the limitation encountered in the previous version of RFE (Moving Mirror Method) to provide a routine RFE output. The correction Of fluorescence measurements for secondary absorption is an essential factor in many chemical and biochemical systems where secondary absorption is significant. 8. Derivative Spectrometry Provisions for Single or multiple derivative calcula- tions are available for Spectral functions. The manner in which derivatized spectra are output is Shown in Figure 15. The data to be treated are plotted at the -45- P _ -:' i { "5% is ............. r ...... ‘ .. X T b - Figure 15. Derivative spectrometery. Curve X =wavelength dependent spectral data plotted along the baseline. Curve X/== first derivative calculated from the X curve data file plotted along the midscreen. Second and higher order derivative calculations are plotted at midscreen in a similar manner. -45- baseline Of the terminal screen and the n-th derivative of that data set is plotted midway up the screen. An eleven point Savitsky-Golay type of smoothing function can be applied to either the original data or after any level derivative. It is known and it can be readily observed in the above figure that derivative plots Often reveal spectral data that is less apparent in ordinary Spectral plots. CHAPTER IV EXPERIMENTAL A. Instrumentation 1. System Configuration An exploded view Of the Cell (Elliptical) Rotation System is shown in Figure 16. A block diagram of the entire system configuration is Shown in Figure 17. The system is constructed around a Perkin-Elmer Model 512 Double-Beam Spectrofluorometer. Of the original Model 512 only the source, excitation and emission monochroma- tors, and the photomultiplier tube were left intact, the remaining electronics (controls) have been either substituted or dismantled. Specifications for all of the intact Model 512 components are listed in the Appendix in Table XVI. Details concerning the three detectors, the Signal amplifier circuits, the A/D converter cichitry, the 4 channel multiplexer circuitry, the stepper motor pulsing circuits, and the omnibus interface circuitry, are all available in the Appendix in Figures 91 through 95. The stepper motor and associated power supply specifica- tions are listed in the Appendix in Table XVII. The entire system is run and all Operations co-ordinated by -47- .AOQSD HODHQDDHDEODOEQV HODOODOC EOOD OOGOOOODOSHMN.H “-HHOO OHODHO>ODODQV DODOODOC EOOQ Oamfimmn.m a-HHOO ODODHOPODODQV HODOODOE EOOD OOQOHOMOHN w “DODOEOHEOOEOE BODOODEO OGDDmHmn m “Houses OOOMHSO DSODMH.M «HHOO OHmEmmnua “HODDDHQO EOOD w\mn.0 “HODOEODEOOGOE comeDfloxO OGHDOMOHIm “:oDDODpOD COHDODDOxO mo OODDOOH.< “>OM .DGOEDDDOGD Emu ODD CD pOonmEO HODOSOHODHMODDOOQO paw DODOEODOEQOHDOOQO pODOMOODdfi OED mo Emummflp xooam -43- . OH 853m O... 0.. 0.0.0.... COMPUTER A/D CONVERTE , I -49- R}-——+fWULTIPLEXER I m H In Is Ir DISK STEPPER CELL P-E MODEL 512 DRIVES MOTOR ROTATION SPECTROFLUOROMETER DRIVER [z DEVICE IR F 1r VIDEO TERMINAL STEPPER DETECTOR ‘ MOTOR EXCITATION VOLTAGE ‘ DRIVER MONOCHROMATOR SUPPLY X-Y W a 1 PLOTTERI * STEPPER _J XENON MOTOR EMISSION LAMP THETYPE DRIVER MONOCHROMATOR POWER PRINTER ‘ SUPPLY c Figure 17. Component configuration of the entire Cell Rotation System. The component manufacturers and model numbers are indicated: 1 =Digital Equipment Corporation model pdp 8/e computer; 2 =Data Systems Single Density Dual (floppy) Drive; 3 =Tektronix model 4002A Graphic Computer Video Terminal; 4 =Houston Instruments Omnigraphic X—Y Plotter; 5 =Perkin—Elmer Corporation model 512 Double— Beam Spectrofluorometer; 6 =Perkin-Elmer Corporation model 150 W Xenon Lamp Power Supply; 7 =Kepco model OPS-2000 High Voltage Power Supplies (one for each system detector); 8 =Teletype Corporation Standard Printer; 9 =Stepper Motors (for Cell Rotation Device = Superior Electric Company Rapid-Syn 23D—6102, for monochromator wavelength controls =Superior Electric Company Slo—Syn H825); 10 =Analogic A/D model MP—2112 12—Bit Converter; 11 =Datel Systems model MXD—409 Four Channel Analog Multiplexer; and 12 =Superior Electric Company model STM—1800 Stepper Motor Controllers. -50.... the computer, a standard pdp 8/e from the Digital Equipment Corporation which has been dedicated to the system. The wavelength functionality of the source, mono- chromators, sample cell composition and detectors of the above instrument relative to that of other spectrosc0pic instrumentation on the commercial market is shown in Figure 99 of the Appendix. 2. The Cell Rotation Device This device is the very heart of the instrument based on the Cell Rotation Method and is illustrated in Figure 18. Components included are (from the bottom upward) as follows: base platform; cell holder Z-axis adjustable post; the universal cell holder; and the quartz sample cell. The various views of the universal cell holder are shown by mechanical drawings in Figure 19. The universal cell holder is capable of supporting quartz sample cells ranging in base dimensions from 1 X1 cm2 to 4 X4 cmz, therefore the adjective "universal" was deemed apropo. The stepper motor and the adjustable cell holder support platform are shown in Figure 20. Due to the requirements for very fine adjustment of the sample cell position, it was necessary to equip the platform with two perpendicular vernier caliper tracks (along the X and Y axes) that are each adjustable to +/- 0.001 inch. When the desired cell position is achieved, the entire platform is clamped onto the bench tOp. Once the cell holder Z-axis adjuStable post is in place it is locked in position along the -51.. CELL ROTATION DEVICE Figure 18. The cell rotation device. Key: A =base platform to support the stepper motor; B =stepper motor (Rapid— Syn model 23D-6102); C =cell holder Z—axis adjustable post; D =universal cell holder. -52- “0---.-. Coco-0-0.4 1:! F/‘ UNIVERSAL CELL HOLDER ==D ==D q \ .0 F” TOP BOTTOM Figttre 19. The universal cell holder. Key: A==geometric View of the support post and the cell seating platform; B==side View of the platform; C=;front View of the platform; D==top View of the bottom portion of the platform; E =bottom view of the bottom portion of the platform. The universal cell holder used in the CRM instrument can support sample cells ranging from 1 X1 cm2 (base) to 4 x4 cm2 (base). -53- EuowumHm mcflCOfluHmom mnu cu pmusoom me Monoe memem mmwaommHMMoMMamwww . >n nocwn wuoumuonma mnu cu pmuonocm ma Ehomumam mcacowuamom $59 mucmfimuoce node Hoo.o CM ucprmsnpm Hmflcum> meMIw paw wx ..Euomumam mcHGOHufimom moa>mp coaumoou Hawo pom HouoE memmum may mo ucmemcmnud .om mnomam mw_zmm> mm_zmu> V A. mmmmmmmwwaaazzv s§§§sasmww§mxmwm PE: mmAQMHwh wnu msocm pwumuumsaafi wwmo one .cofluflmom Hamo some CH popuoomu mum mamcmflm hufiwcwuofl wocoomwuooaw pom OHQEMm roocouomwn 3mm .pmum0flpcfi mum pcmEmMSmme pwuoum mfimcflm m mm pmmmuw>m paw pwpnoomu mum mu:flom dump ow Scans mcfludp Hm>umDCH mpmfiflxoummm one .v pom N .H mQOAOHmom ca Hawo wnu How c30£m mum mamcmflm .mCOADHmom HHoo mo macho wamcflm m Mo>o qcflHmEMm Hmcmflm .HN onzoflh mozoomw 2. m2: 5 B m._ 2 mo mo mo no 8 _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ -59- ‘IVNOIS AllSNEiNI mozEumpcooom pom FNMEHHQ m an conflmop pawflm cofluowuwo .mm wusmflm ._. -63— r \\\\\\\\\\\\\\\\\\\\\\\\\\\\\x. \\\\\\\\\\\\\\\\ \\ \\\\\\\\\\\\\\\\\\\\. \\ H ‘ W\\\\\\\\\\\\\\\\\\\\ g -64- size of the masks relative to the size of the sample cell is drawn to scale. 2. Mapping of the Emission Port Window A study was completed to determine the transmission profile and sensitivity in detection of white light across the emission port window. A collimated source of white light, with a cross-sectional area of 0.05 by 0.25 inz, was positioned orthagonal to the window surface at each of 25 0.05 divisions along both the X (horizontal) and Y (vertical) axes. At each position intensity readings were recorded from the PMT detector. The source of collimated white light consisted of a brass tube, 12 inches long with an ID of 0.5 inches, having a slot with area 0.05 by 0.25 in2 at one end and a small 1.5 volt (DC) rod flashlight attached to the other end. The tube was spray painted flat-black on the inside to minimize internal reflections. 3. Focusing onto the Emission "Hot Spot" In an attempt to increase the detection sensitivity a study was conducted wherein a series of double convex lenses with differing focal lengths were used to focus wider beams of the emitted radiation onto the region of maximal sensitivity, to be subsequently referred to as the "hot spot". The lenses used were obtained from an optician's ocular lens set. Each lens had an identical holder permitting a single bracket to be built to house any of them. The focal lengths were slightly shorter, -65- equal, or slightly longer than the distance between the lens and the "hot spot" on the mirror in the emission window port. The typical arrangement within the cell compartment is shown in Figure 24. 4. Cell Positioning of the Excitation and Emission Detection Beams To avoid beam attenuations due to reflections from the quartz cell walls it was necessary to know how far from any particular cell wall the centers of the colli- mated excitation beam or emission detection beam should be located. Since the beam positions are always fixed, it was necessary to use the base platform vernier calipers to re-position the sample cell along either the excitation or emission ports. The incremental movements were 0.5 mm along each axis with intensity readings recorded after each shift. The study was completed using a sample cell with a 2.0 X2.0 cm2 base and a 4.5 cm height. 5. Error due to Temperature Changes in Instrument Fluctuations in temperature of the instrumental components and sample solution can cause variations in the fluorescence intensity. The instrument attains a constant running temperature in 15 to 20 minutes. After— wards, the source lamp temperature is maintained constant by a fan and by a specially designed ozone venting system. The cell rotation device is well shielded thermally from the lamp chamber. The stepper motor maintains a constant temperature whether in stepping-mode -66- w _ _ 1 / \ FOCUSING / LENS VV22-- V/flé WVZ CELL POSITION 1 Figure 24. Focusing of a larger emission observation window onto the emission port "hot spot". Double convex lenses, with centered 1/1+inch2 windows, W1 and wz, in front and behind the lens, and of various focal lengths were used to focus various larger areas of the emitted radiation. -67- or holding mode. To ascertain the significance of the error observed if the instrument is operated during the warm-up phase, sample analyses were made in both the visible region (Quinine Sulfate in a matrix of 0.1 N H2804) and the ultraviolet region (Tryptophan in a matrix of 0.02 M Tris). These analyses were then compared to results obtained after the warm-up period. 6. Error due to Internal Reflections Internal reflections of excitation and/or emission radiation from sample cuvette surfaces can result in elevation of the fluorescence intensity near these surfaces. A short discussion concerning the significance of such an error is provided in the next chapter. The following steps were taken to both minimize and account for such errors. First, the sample cell walls were ordered from the manufacturer (NSG Precision Cells, Inc.) to be constructed from highly matched quartz plates to minimize reflection and the absorption of radiation. Second, the design of the detector calibration routine attempts to compensate for the reflection phenomena. This is done by matching both reference and sample beam detector outputs while the sample cell is filled with distilled water (the typical solvent matrix in chemical systems studied herein). In this manner a wavelength dependent look—up table of f factors (described earlier 1 in chapter) is created and subsequently used to -68- mathematically correct for reflection and absorption artifacts not caused by the presence of sample solutes. 7. Source Flicker as a Source of Random Error Instabilities in the spectral output of the 150 W Xenon lamp results in fluctuations in the fluorescence intensity readings. The significance of this source of error was determined in the following manner. The steadiness of each of the system detected outputs, including that of the R—PVC, S-PVC and F-PMT, was monitored after instrumental warm-up (about 1/2 hr.) for periods of up to several hours. This type of study was completed approximately once every two months to monitor changes over the lifespan of the lamp. Whenever this source of error is considered to be significant the source-corrected output of the fluorescence intensity can be selected. This is made possible by the system software since the raw 1R, 2R, 4R, ls, ZS, 48, 1F, 2F and 4F measured signals are digitized and stored for each selected wavelength setting. 8. 60 Hz Line Noise Line noise can be readily detected by monitoring photovoltaic cell or photomultiplier tube outputs during selected intervals. An oscillosc0pe is a convenient means of ascertaining the presence and significance of this source of noise. Since this source of error follows a regular pattern (sinusoidal), the software invOlved in signal sampling was designed to average readings obtained -69- over several cycles of the line noise. Each detector reading that is stored is currently an average of 40 readings taken at regular intervals over a period of 200 milliseconds. The number of readings collected and averaged can be set from the operating program, however this influences and is proportional to the collection period. 9. Stepper Motor Uncertainty in Cell Positioning Cell positioning uncertainty can fall into several catagories. One is skewing of the sample cell base that could result if the stepper motor in the cell rotation device mis-steps in cycling through the various positions during a typical scan. A study was conducted to determine the frequency of mis-stepping by the stepper motor wherein fluorescence output was monitored over 10,000 cycles. A lateral position misalignment caused by improper setting of the cell in the universal cell holder can create a measurement error. Fortunately, such misaligned positioning can be detected when unit-ellipse rotation patterns are selected. Also the support post for the cell holder permits vertical alignments to be made which avoids another source of position mis-alignment due to rotation wobble. 10. Back-Reflection of Sample Beam Intensity from Sample Detector Another source of error exists when back-reflection of the sample beam from the face of the S-PVC occurs. A study was conducted to determine the effect of the back- reflection of the excitation beam from the S-PVC. -70- Figure 25 illustrates the problem and the solution to the problem. In A the S-PVC surface is at the proper angle so that the beam is completely back—reflected to the mirror and, in turn, to the sample cell. In B the S-PVC is rotated 30° from the perpendicular to the mirror so that the beam to the detector surface is not back-reflected to the mirror. 11. Raman Scattering The significance of Raman scattering was determined in the following manner. During irradiation by the excitation beam, the spectrum of the scattered radiation was monitored at right-angles to the excitation beam. The sample cell contained distilled water, a common solvent matrix used in the studies of chemical systems herein. This study allowed determination of the relative magnitudes of the Rayleigh and Raman peaks, and the location dependence-of the Raman peak maxima with regard to the excitation wavelength. 12. Effect of Solution Absorption on the Observation Medians The current instrument employs a detector with a non-linear detection sensitivity across the fluorescence observation window. Although the sensitivity profile is not known exactly, an approximation is shown in Figure 33. Since only the sample cell itself moves (rotation pattern), identical collection Optics are used in all three cell positions. This figure also -71- .AL, PROBLEM OF BACK IHWLECTION CELL S-PVC B. SOLUTION FOR BACK REFLECTION C E l. L 15.-3:1.- S-PVC Figure 25. Back—reflection of sample beam radiation from the S-PVC detector. In case A the beam is partially back reflected from the S—PVC surface. In case B the S-PVC surface is rotated so that the beam cannot be back-reflected. _72_ illustrates how an increase in solute concentration, and thus the solution absorbance, causes the detection median, or center of intensity, to shift toward the excitation source. This effect has been characterized and is shown in Figure 58 using quinine sulfate in 0.1 N H2804 as the chemical system. In the CRM it is assumed that the difference between the medians in positions 1 and 4 remains unchanged with increases in solute concen— tration and is equal to the difference between the known window centers as illustrated in Figure 8. l3. Stray Light Analysis There are many sources of stray light in instruments such as spectrophotometers and Spectrofluorometers. A cross-section of the sources is as follows: a. leakage of room light into the cell compartment of instrument; b. reflection and scattering of radiation from walls, optics, slits and baffles; c. scattering of light within prisms and lenses; d. reflection and scattering on optical surfaces; e. scattering of light by dust particles; f. unused orders in grating spectra; g. fluorescence of optical materials; h. imperfections in the grating rulings which yield satellites, Rowland and Lyman ghosts; and i. diffraction at the slit edges. Fortunately, in well designed instruments stray light is mainly confined to any light outside the spectral region isolated by the excitation monochromator that reaches any one of the detectors. Generally, it is produced by scatter from -'73- the optics and walls of the monochromator and is present in varying amounts in all spectrophotometers and spectro— fluorometers. The amount of stray light passing through the excitation monochromator exit port can be conveniently monitored by a comparison of primary absorbance determined spectrophotometrically, obtained by output A1, with primary absorbance determined spectrofluorometrically, obtained by output A2. The magnitude and wavelength dependence of this stray light effect is illustrated in Figure 35 of the results from the next chapter. 14. Wavelength Calibration with Standard Reference Materials The high-accuracy spectrophotometer housed in the Center for Analytical Chemistry at the National Bureau of Standards, described in the NBS book "Accuracy in Analytical Spectrophotometry" by Burke and Mavrodineanu [57,58] provides transmittance standards to compare to transmittances measured by other instruments. The accurate transmittance values determined with the NBS instrument of well characterized materials, "Standard Reference Materials" or SRM's, in the Spectra range from 200 to 800 nm are available to users of spectrophoto- meters and Spectrofluorometers. A number of SRM's have been prepared at NBS for verification of the accuracy of the wavelength and transmittance scales of conventional spectrOphotometers and Spectrofluorometers. To check the accuracy of the -74- calibration of the wavelength settings of the excitation and emission monochromators, absorption curves of a solution of a recommended SRM, H0203, were recorded. The solution consisted of 0.2 9 H0203 dissolved in 10 ml of l M HClO4. The expected absorption wavelength maxima for this solution are at 279, 288, 338, 361, 386, 418, 453, 536 and 637 nm. This solution in the same cuvette was used to check the wavelength calibration of the emission monochromator. A diffusing screen set at 45° was used to direct the beam through the cell into the emission monochromator. C. Procedures to Operate System 1. Overview of System Operation All analyses were conducted using the same instrument that employs the Cell Rotation Method. The three system programs, FLCAL, CALEDT and FLUOR, controlling the functions of the instrument allow the operator the necessary detector calibrations, setting of instrumental parameters, control of the excitation, emission and linked monochromator scans, storing of the reference, sample and fluorescence measurements, application of on-line calcula— tions and corrections on stored spectral data, and displaying a variety of convenient means of presenting a selected spectral output. For each study described in the chapter on results and discussion, the wavelength _75_ and scan parameters are indicated on the figure legends of the spectral data outputs. Also indicated are the component concentration and, where appropriate, the pH of the sample medium. 2. Calibration of System Detectors All of the following steps are to be completed in the sequence indicated. This procedure permits calibration of the three system detectors and subsequent normalization of outputs to relative numbers of quanta. a. R CALEDT / Enters CALTAB from data floppy disk. ON: 1 / Sets all PAC—2's to 1'5. ON: 2 / Sets all FAC—l's to 1'5. b. R FLUOR / Place distilled water in sample cell. EX: Scan / Complete scan over desired wavelengths. BU: Ol / The FAC—l's are entered into buffer DU: 1 / The FAC—l's are dumped into CALTAB file. R CALEDT / Water can be removed from sample cell. MU: 2 / FAC-l table is fully calibrated. c. R FLUOR / Use sample system that has ideal P2 curve. EX: Scan / Complete scan over same wave- lengths. BU: CF / The PAC-2's are entered into buffer DU: 2 / The FAC—2's are dumped into CALTAB file. R CALEDT / Sample can be removed from sample cell. MU: l / FAC—2 table is fully calibrated. d. R FLUOR / Place barium sulfate suspension in cell. SF: # / Set scale factor to wavelength for which you desire to have (FO)/(Rl) =l.00. LI: Scan / Link monochromators and scan over the same wavelengths. -76- BU: 03 / The PAC-3's are entered into buffer. DU: 3 / The FAG—3's are dumped into CALTAB file. Additional notes for obtaining full calibration are as follows: 1. Scans must be at 1 nm intervals; 2. Floppy disks should not be changed during calibration; 3. DO not forget to buffer functions with the BU command; 4. Use a disk which you can afford to lose (a back-up copy); and 5. Record all steps you take in the calibration sequence and keep this information with the CALTAB you make (create file CALTAB.TX via TECO on same disk). 3. Setting_of Parameters Prior to a Scan The parameter settings are independent Of each other, therefore the order of setting them is optional. Some Of the parameters are routinely set while others are dependent on the type Of analysis being performed. If a parameter is not currently set, it will automatically assume a "normal" default value. The following list includes the parameters which can be set by the Operator. a. All Of the geometric factors (commands G1, G2, G3, G4 and GF). b. The excitation and emission wavelength scan pointers (commands EX, EM and LI). c. Wavelength interval between consecutive readings in multiples Of Vunm (command IN). d. Number Of readings to be taken at one cell position and averaged for a single recorded measurement Of either 1R, 2R, 4R, lS, ZS, 48, 1F, 2F or 4F (command PA; default is 40 readings over 200 ms). e. Delay period in ms after the sample cell completes rotation to a new position (command DE; default 50 ms). f. Current position Of the sample cell (command CU). -77- 9. Threshold value for the minimum allowable DF value to be applied to the RFE function (command TH). h. Nephelometric pathlengths in cms (commands BX and BY). 4. Primary and Secondary Command String for Obtaining an Output The primary command designates the activity to be performed with a data set. The secondary command desig— nates the specific data set. The following examples illustrate the use Of the command string. a. The desire is tO display the raw (unmodified) fluorescence values from position 1 on the video terminal. Commands - DI: lF: Carriage return b. The desire is to plot the absorption corrected fluorescence values from an excitation scan on the X-Y recorder. Commands - PL: FO: Carriage return c. The desire is to print the secondary absorption values from an excitation scan on the printer. Commands - PR: A3: Carriage return d. The desire is tO Obtain a real-time output Of the primary transmittance determined spectrofluoro- metrically On either the video terminal or printer. Commands - RT: T2: Carriage return 5. Total Quantum Efficiencies by the Comparative Method An emission scan is completed on a solution containing a standard Of known (documented) quantum efficiency. Thereafter the QC command is employed to calculate the quantized area below either the FR or F0 emission curves (as requested by the Operator). Either Of these values are subsequently equated with a manually input reference -78- quantum efficiency value and stored for future comparisons. Without altering the non-wavelength dependent parameters or amplifier settings, an emission scan is completed on a solution containing a dissolved substance Of unknown quantum efficiency. The QR command calculates the quantized area below the FR emission curve Of the unknown and outputs the value relative to the reference quantum efficiency value (based on the reference FR emission curve). The QO command calculates the quantized area below the F0 emission curve Of the unknown and outputs the value relative to the reference quantum efficiency value (based on the reference FO emission curve). The total quantum efficiency values Obtained by this comparative method can be printed out on either the video terminal or the printer. 6. Smoothing Of Spectral Functions Any wavelength dependent data can be smoothed by the application Of an eleven point weighted smoothing function (based on the Method Of Golay). The applied factors are listed below: -36 +9 +44 +69 +84 +89 +84 +69 +44 +9 -36 429 429 429 429 429 429 429 429 429 429 429 (l) (2) (3) (4) (5) (6) (7) (8) (9) (10) (ll) These factors are centered around and applied sequentially to each consecutive data point throughout a scan. The software routine has been designed to leave the first five and the last five data points Of a spectral function _79- unchanged (unsmoothed) to avoid the distortion that would result otherwise. The smoothing function is used as a primary command, which is followed by a secondary command designating the specific data set. The command string below illustrates its application: SM: BU: Carriage return This command string will smooth and store whatever was located in the buffer. 7. Fluorophore Absorption Curve Stripping The process of curve stripping permits a convenient means of outputting the difference spectrum between two spectral functions determined from the same data set (file). Fluorophore absorption curve stripping is just one example or type of difference spectrum that can be output following an excitation scan. The process is accomplished in the following manner: CH: CMD—l =? / Here CH is used as a primary CMD-2 =¢ command. It will require the ' operator to input the requested nm SF =? information. CMD-l is a spectral function (Al in this case). CMD—2 is a spectral function (FR in this case). And SF is a ratio (Al/FR) constant calculated from the input wavelength. DI: CH: CR \ Here CH is used as a secondary command. This command string will display the difference spectrum, CH =(Al) —(SF)(FR), on the video terminal. CR is a carriage return. PR: CH: CR / This command string will print the values from the difference spectrum on either the video terminal or the printer. PL: CH: _30_ CR / This command string will plot the difference spectrum on the X-Y plotter. 8. Multiple Derivative Capability The programs permit calculation Of derivative spectra from any buffered spectral data (file). The process of Obtaining multiple derivatives is shown below: BU: SM: DV: DI: PR: PL: XX: BU: DV: DV: DV: CR CR CR CR CR / / The desired spectral function, XX, is buffered. The buffered spectral function is processed by the smoothing function and stored over the unsmoothed spectral function in the buffer. The derivative command, DV, is followed by an integer input (1, 2, 3 ... N) which informs the routine how many successive derivatives are to be taken. Note that for each derivative taken a Vzinterval is lost on either end Of a spectral scan. This command string will display the derivative spectrum on the video terminal. This command string will print the values from the derivative spectrum on either the video terminal or the printer. This command string will plot the derivative spectrum on the X-Y plotter. 9. Integration Capability The program FLUOR permits integration Of the area below any spectral function. The process Of Obtaining an integral is detailed as follows: D. _81_ IT: XX: CR / Here IT is used as a primary func- tion. XX is the desired spectral function to be integrated. Prior to this step the OU command is useful in changing the wavelength pointers so that the integration is only across a portion of the entire spectrum (usually a single peak). The integration is performed in the following manner: fn(l) fn(A) S-l fn(>x)d)\ = —————°—+——S+ 2 mm. _ 2 2 i=1 1 where lo is the starting wavelength and As is the ending wavelength. DI: IT: CR / This command string will display the value for the integration on the video terminal. PR: IT: CR / This command string will print the value for the integration on the printer. Reagents All chemicals and biochemicals were used without further purification. Solute concentrations and matrix composition are detailed in the data figure legends in the chapter on results and discussion. The following list includes all substances used in this work. 1. Bovine Serum Albumin — Sigma Chemical Company; fraction V powder, Lot A—7906; 98 — 99% Albumin, remainder globulins. Bovine Serum Albumin — Sigma Chemical Company; essentially fatty acid free, Lot A-6003; prepared from fraction V powder. Human Serum Albumin — Sigma Chemical Company; crystallized and lyophilized, Lot A—8763; essentially globulin free. Human Serum Albumin - Sigma Chemical Company; essentially fatty acid free, Lot A-1887; prepared from fraction V powder. 10. ll. 12. l3. 14. 15. l6. l7. l8. 19. 20. 21. 22. -32- Human Serum Albumin — Sigma Chemical Company; fraction V powder, Lot A-l653; 96 — 99% Albumin, remainder globulins. Chicken Egg White Lysozyme - Sigma Chemical Company; crystallized 3X, Lot L-6876; dialyzed and lyophilized. beta—Prolactin — Provided by National Institutes of Health; further purified by Dr. W. Frantz (Physiology Department of Michigan State University; electrophoretically pure. L-Phenylalanine - Sigma Chemical Company; Sigma reagent grade, Lot P—2126. L-Tyrosine — Sigma Chemical Company; Sigma reagent grade, Lot T—2006. L-Tryptophan — Sigma Chemical Company; Sigma reagent grade, Lot T-0254. Soluble Starch - Merck and Company; Merck reagent grade, Lot 74881. Sodium Dodecyl Sulfate — Pierce Chemical Company; Pierce sequanal grade, Lot 2—8364. L—Ascorbic Acid - Fisher Scientific Company; Fisher reagent grade, Lot A—6l. Rhodamine B — Sigma Chemical Company; Sigma laboratory grade, Lot R-6626. Anthracene - Eastman Organic Chemicals; Eastman laboratory grade, Lot X—480. Quinine Sulfate Dihydrate — Aldrich Chemical Company; Aldrich laboratory grade, Lot 14591—2. Quinidine Sulfate - Sigma Chemical Company; Sigma laboratory grade, Lot Q-0875. Uranine (Sodium Fluorescein) — Fisher Scientific Company; Fisher laboratory grade, Lot A—833. Eosin — Allied Chemical Company; Allied laboratory grade, Lot 45380. Trizma Base (TRIS BUffer) - Sigma Chemical Company; primary standard and buffer, Lot T—1503; purity at 99.9%. Guanidine Hydrochloride — Pierce Chemical Company; Pierce sequanal grade, Lot 24115; 8.0 Molar in distilled water, free of UV absorbing material in 225 — 300 nm region. Ethylene Glycol - Mallinckrodt Incorporated; Mallinckrodt reagent grade, Lot 5001. 23. 24. 25. 26. 27. 28. 29. 30. ~83— Ethanol (Anhydrous) - Aldrich Chemical Company; Aldrich Spectrophotometric grade, Lot E—lOOO. Sulfuric Acid - Mallinckrodt Incorporated; Mallinckrodt reagent grade, Lot 2468. Perchloric Acid - Aldrich Chemical Company; Aldrich ACS reagent grade, Lot P—4550; acid 69.0 — 72.0% in distilled water. Acetic Acid (Glacial) — Aldrich Chemical Company; Aldrich reagent grade, Lot A—1090; acid 99.8% in distilled water. Sodium Hydroxide - MCB Manufacturing Chemists Incorporated; MCB reagent grade, Lot SC—0590-l. Holmium Oxide — G. Frederick Smith Chemical Company; Smith reagent grade, Lot M—l. Glycerol — MCB Manufacturing Chemists Incorporated; MCB reagent grade, Lot D—7P08A. Water — House distilled in the Biochemistry Department at Michigan State University. CHAPTER V RESULTS AND DISCUSSION A. Instrumental Performance 1. Quantization Of Detector Output The calibration procedures allow all CRM instrument detector outputs to be normalized to relative numbers Of quanta. The calibration is available over the 220 tO 550 nm range if a quantum counter solution Of Rhodamine B in ethylene glycol is used to generate the energy-to- quanta (look-up) table. Since this table is stored electronically the quality Of the factors will remain constant. The above quantum counter solution has remained stable over a period of several years as demonstrated by the reproducibility Of the energy-to-quanta table over time. Note that electronic storage Of this table benefits the user Of such instruments by avoiding the possibility Of Rhodamine B concentration through ethylene glycol evaporation (fortunately ethylene glycol has a low vapor pressure), by eliminating the Opportunity for accidental contamination or photo-decomposition Of the quantum counter solution, and by minimizing the Size requirements for the sample cell compartment. -84- -85- 2. Signal Sampling A complete set Of raw signal intensity readings at a single wavelength position, including 1R, ZR, 4R, lS, ZS, 48, 1F, 2F and 4F, each an average Of 40 individual measurements, is taken and electronically stored in approximately 1.5 seconds. Such a rate Of data handling permits the instrument to output both uncorrected and corrected spectral scans on the order Of several minutes routinely, depending on the range and interval Of wavelengths scanned. Commercially available spectro- fluorometers Obtain uncorrected, with the exception of source (intensity fluctuation) corrected, spectra in a similar time period. Primary and/or secondary absorption corrected fluorescence, for example, can now be Obtained and output on a rapid and, thus, practical basis with the CRM instrument. Limitations on the improvement Of cycling speed are as follows: a. waiting 50 milliseconds prior to data sampling to avoid the spike (due to reflection Of the excitation) beam from the cell surface into the emission port during each rotation) from the F-PMT; b. taking a sufficient number Of individual measurements over a period Of time to be able to average out the 60 Hz line noise (with a minimum Of 40 readings required); c. taking the diagnostic readings Of the raw reference and sample beam intensities in cell positions 2 and 4 (divergence indicating a FLUOR program malfunction); and d. taking 2.5 milliseconds —86- per single step for the stepper motor at its maximum advance rate (which is normally employed). In a typical wavelength scan covering a 200 nm range and taking sets of readings at each wavelength, the following summarizes the types and number of readings taken: REFERENCE (R-PVC) = (200 nms)(40 readings/mm) (3 cell positions) = 24,000 individual readings. SAMPLE (S—PVC) = (200 nms)(40 readings/mm) (3 cell positions) = 24,000 individual readings. FLUORESCENCE (F-PMT) = (200 nms)(40 readings/mm) (3 cell positions) = 24,000 individual readings. The total number of measurements taken over a 200 nm scan sampling at each nm is equal to 72,000. This rate of data uptake and handling can only be accomplished with the application of a computer. 3. Detection Field Characteristics The studies conducted employing both primary and secondary masks to determine the narrowness Of the field of fluorescence detection verify the initial assumption in the CRM instrument that the emission is viewed as a narrow slice relative to the overall cell pathlength. The width of the slice is somewhat less than Vginch. The height of the slice is somewhat greater than Phinch. These dimensions make the typical 1 XI cm2 (base) sample cell of borderline utility, however a 2 X2 cm2 (base) cell -87- presents no problem with regard to detection field overlap between cell positions 1 and 4. The use Of a larger sample cell presents a disadvantage in the requirement for a larger sample volume (4-fold in the above case). 4. Mapping Of the Emission Port Window The emission port window is 1 inch in diameter. Figure 26 illustrates the Optical transmission profile of an orthogonally incident 0.04 by 0.25 in2 (cross section) beam Of collimated white light positioned in 0.04 inch increments along the X and Y axis diameter lines. The longer axis (0.25 inch) of the beam cross section was positioned perpendicular and centered on the respective diameter line under study. In all measurements the inci- dent beam intensity and spectral composition was constant. The results indicate that the relative intensities are highest in a region centered in the emission port window. The maximum effective transmission Of white light is Obtained over only a fraction of the window diameters considered, 2 and 3 increments along the X and Y axes, respectively. Figure 27 shows the topographic intensity map Of the emission port window based on the above findings. In this figure the emission port window is partitioned into 25 0.04 inch wide segments along both the X and Y axes. Within the grid it can be readily seen that a "hot spot" exists for Obtaining maximum transmission Of white light. The effective region viewed is X-AXIS IIIIIIW. IIIIIIIIII IIIIIIII IIIIIIIIIII IIIIIIIII IIIIIIIIIIIIIIIII IIIIIJ .IIIIIIIIIIIIIIIIIIIIIIIIII 1’15 IJ -88— ¢é I' :IJ - — _ h - >e-mzmez- m>HP<4mm 25 20 15 10 Y-AXIS IIIIIIIIIIIIIIIIIIIIII IIIIIIIIIIIIIIIIIIIIIIJ. rIIIIIIIIIIIIIIIIIIIIIIIIIIIJ IIIIIIIIIIIIIIIIIIIIIII .IIIIIIIIIIIIIIIIIIIIIIIII VIIIIIIIIIIIIIIIIIIIIIJ IIIIIIIIIIIIIIIIIIIIIII IIIIIIIIIJ 2 III; 4.I 4" 10 >H-mzmez- m>HH<4mm 25 20 15 EMISSION PORT WINDOW Optical transmission profile Of the emission port window. Figure 26. Window Source =collimated white light. The X and Y axis divisions are 0.04 inch apart. diameter =1 inch. -89— INCH Y-AXIS ‘1...1.---.:J-..n.nnnn moo 020 Q90 use use 190 X-AXIS INCH Figure 27. Topographic intensity map of the emission port window. The black squares locate regions of maximal response and the shaded squares locate regions where response is 50 to 99% of maximal with respect to a collimated beam of white light. The divisions along each axis are 0.04 inch apart. _90_ sufficiently narrow to provide the necessary independence in measuring fluorescence intensities from cell positions 1 and 4. Determining accurate absorbance correction factors would be difficult if overlap existed between these viewing windows. 5. Shrinking of the Cross-Section of the Detected Emission Beam onto the Emission Port "Hot Spot" Attempts to use double convex lenses to help concen— trate or focus more of the fluorescence radiation onto the parabolic mirror immediately behind the emission port window in the vicinity of the "hot spot" proved unsatisfactory. The purpose of this study was to more efficiently measure the fluorescence radiation from the observation windows. The higher signal levels (increase in detected beam intensity) could feasibly lead to greater accuracy in the absorption correction factors Vused in the generation of absorption corrected fluorescence in highly absorbing solutions. The use of these lenses caused distortions in the absorption correction factors and in no case were improvements obtained in these factors. A variety Of focal lengths were employed, some longer (up to 2 cm) and some shorter (up to 2 cm) than the distance to the surface of the mirror. The Observed distortions were dependent on the focal length Of the lenses. In all cases an increase in fluorescence signal levels was Observed. The magnitude of the increase was dependent on both the focal length of the lense and the size of the collimated opening of the lense support _91- cage. Since the study was conducted with quinine sulfate in 0.1 N H2804 with the excitation wavelength set to 350 nm and the emission wavelength set to 460 nm, a wavelength dependence of the distortions could not be ascertained. Also, although the lenses were made of glass, the absorption of fluorescence at 460 nm was found to be negligible. The conclusion was reached that no apparent improvements were obtained with any such arrangement involving double conves lenses. 6. Cell Positioning of the Excitation and Emission Detection Beams The available positioning of the emission detection beam along the perpendicular sample cell surface it must penetrate is shown in Figure 28B. The available positioning of the excitation beam along the perpendicular sample cell surfaces it must penetrate is shown in Figure 28A. The problem of attenuation of beam intensity arises when these beams are located closer than 2 mm to a parallel cell surface as illustrated in both figures. The reasons for the attenuation of beam intensity are as follows: a. scattering and/or reflection of part of the excitation beam away from the emission window of observation; and b. elimination of part of the emission window of observation. TO provide an adequate distance between observation window centers (to allow determination of accurate absorption corrected factors) and to prevent attenuation of beam intensity (for reasons other than _92- WALL CENTER WALL l i I 1 T j fl l l 7 1100 >_ 3: _. U? l\ I—Z ’ (Lu EJJI— TIE 0 2 4 6 BIO 8 6 4 2 O DISTANCE FROM CELL WALLS, mm. >_ . DJ 2 L: ._ g B. < Lu J] I— o: .2. O 2 4 6 8 IO 8 6 4 DISTANCE FROM CELL WALLS, mm. Figure 28. Available positioning of the excitation and emission detection beams along the perpendicular sample cell surfaces they must penetrate. The plots show the relative signal intensities across the cell along both beam axes (unshaded areas). Key: A. Positioning Of the excitation beam. B. Positioning Of the emission detection beam. -93_ the presence of the sample solution) it was necessary to use a larger sample cell. The sample cell is 2 X2 cm2 (base) which allows for a distance Of 1.6 cm between both the far edges of the emission observation windows when in cell positions 1 (or 2) and 4, and the far edges of the excitation beam when in cell positions 1 (or 4) and 2. 7. Error Due to Temperature Changes in the Instrument Fluctuations in temperature of the various instru— mental components and sample solution can cause varia- tions in the fluorescence intensity. The CRM instrument warms up to a constant running temperature in 15 to 20 minutes after Xenon lamp ignition. The source lamp temperature is maintained almost constant by a fan and by a specially designed ozone venting system. The inside walls of the sample cell compartment closest to the lamp chamber reach an operating temperature of 26 to 28°C as measured by an Anspec liquid crystal (flat) thermometer. The cell rotation device is well shielded thermally from the lamp chamber. The stepper motor maintains a constant temperature Of 28 to 30°C as measured by an Anspec liquid crystal (flat) thermometer whether in stepping-mode or holding-mode. The time elapsed between introduction of a sample solution into the cell and completion of a typical scan is insufficient for equilibrating the solution temperature to that of the stepper motor. Solution temperature changes of less than 1°C higher are common. -94- The room temperature Of the laboratory where the CRM instrument is housed is found in the range Of 21 to 24°C. Both solution and room temperature measurements were made with a standard mercury thermometer. It has been concluded that this source Of error is negligible provided that measurements are taken only after the instrumental warm-up period. 8. Error Due to Internal Reflections On passage Of light through a cuvette containing a solvent or sample solution, some Of the incident radia- tion is reflected at each Of the two quartz-air and the two quartz-liquid interfaces. For the case Of perpendicu- lar incidence, the fraction, f, reflected on passing from a medium Of refractive index nl tO a second having a refractive index of n2 is given by the familiar Fresnel expression: f = [(nl -n2)/(nl +n2)]2 (v.1) Note that for an air-glass interface, f =0.04 (approxi- mately). The values reported [4] for air-quartz interfaces are slightly lower. Also note that for-a water-glass interface, f =0.0035 (approximately). The values reported [51] for water-quartz are, likewise, slightly lower. When more than one interface is involved, the effects Of multiple reflections must be considered. The following equations are excerpted from characterization studies _95_ done on a high precision double—beam spectrophotometer built at the National Bureau Of Standards [58]. The case where the reflections from all interfaces perpendi- cular to the light beam on the two sides of the sample solution are grouped together to form two effective reflection coefficients, r and r2, and is shown in 1 Figure 30. Considering only first and second order reflections, the observed absorbance is: AObserved 109(P1/P2) (v.2) = log(Po/POT)(1 —rl +rlr2) Z X (l —r +rlr2T ) (v.3) l where P0 is the incident excitation radiation, P1 is the total detected excitation radiation passing to the sample beam detector when only solvent is in the cell, P2 is the total detected excitation radiation passing to the sample beam detector when the sample solution is in the cell, and T is the transmittance. After transformation and series expansion, the above equation can be reduced to the following expression: 2 Aobserved A + (O.4343)(l -T ) >< [(rlr2)/(l —rl)] (v.4) where T is evaluated from T =10_A. -96- O 90 Figure 29. IA Al Non-parallel incident radiation and the sample cell. Shown are three rays Of excitation radiation incident on and pentrating through the sample solution. Key: Q==angle Of incidence; R==angle Of refraction; b==perpendicular distance between parallel cuvette walls. .OOCMHUHEmcmHuu B -DCOHOHMMOOO :Ofiuomawmu unaccocom O>Hpowmmwu NM -ucwfl0fimmwoo coflpooamwu xumeflum O>fipoowmou HM -HHOO ecu CH mH COHDDHOm wamfimm Onp cogs uouomuop U>mIm Ono Ou daemmmm coeumflpmu COAOODAOxO pouoouop Hmwoun No “HHOO OLD Ce we uco>Hom >Hco owns Houomuwp U>mIm OLD Op mafimmmm coaumfipwu coflumpfloxw pmuowuwp Hmuoun Hm “coeumflpmu :oflumpfloxw ucwpflocfln om “Swm .coflusaom Oamfimm .m .Ocon uco>aow .< .HHOO OHmEmm OS» canvas mcofluowamou wamfiuHSE HmcuOUCH _97_ .0m ousmfim m4m2AO® -v.-. TIP at m.- rkéfhon- M .9; M mum -Q _ o I o I I S.I-.£u.h ...-LIE. .H. 1m 0&1 A V a.» _93_ Considering only the reflections from the two Opposite cuvette faces (the magnitude Of the contributions Of subsequent reflections from these interfaces rapidly becomes negligible) and assuming, for example, that rl =IQ =0.05, the variation of AObserved With A can be put into perspective: A 0.1000 0.2000 0.5000 1.0000 2.0000 Aobserved 0.1004 0.2007 0.5010 1.0011 2.0011 Therefore, because Of the contributions from internal multiple reflections, the measured absorbance should always be larger than the true absorbance with the percentage difference being greatest at low absorbances. Although it was realized that the deviation from true absorbance, and for that matter, true fluorescence, was quite small due to multiple internal reflections, the detector calibration procedures used in the CRM instrument were in part designed to compensate for the effects Of these reflections. 9. Sources Of Noise There are various sources Of noise in instruments such as the commercially available spectrophotometers and Spectrofluorometers. Detailed listings and descrip- tions Of these sources Of noise are available in a number Of current texts on analytical instrumentation [157,158]. The following sub—sections report the results on studies -99- of the major sources of noise and their significance to the normal operation of the CRM instrument. a. Source Flicker as a Source of Random Noise. If measurements are conducted prior to instrumental warm—up, source flicker becomes significant as a source of random error. Reference, sample and fluorescence beam intensity signals have been Observed to fluctuate i5% immediately following ignition of the Xenon lamp. As the lamp and lamp housing reaches it's equilibrium operating tempera- ture, the signal rapidly stabilizes. At the operating temperature, reached in 15 to 20 minutes, the error caused by source flicker is negligible. However, if desired, source corrected fluorescence outputs are available by output options from the FLUOR system operating program. b. Baseline Noise as a Cause of Error. Baseline noise, referred to as zero percent noise in absorption measurements, includes several types of noise associated with transducers and amplifiers. These are all involved with an uncertainty in making the baseline adjustment. They include the following: dark current shot noise, dark current excess noise and amplifier excess noise. An observation of the Operating instrument, the baseline setting for each Of the detectors, R-PVC, S—PVC and F—PMT, remains quite constant if the instrument has been previously warmed—up for at least 20 minutes. Virtually no baseline drift is found to occur. The typical random -100— variation in baseline signal is $0.003 volts after amplification. Prior to Operation of the instrument in a chemical study the excitation beam shutter is closed and the baseline signal for the sample, reference and fluorescence detectors is set to 0.000 volts. c. Johnson Noise as a Cause of Error. Johnson noise is due to Brownian motion of electrons in resistive components of instruments such as spectrophotometers and Spectrofluorometers. This noise, found in an instrument's photomultiplier tubes and amplifiers, was found low in magnitude and essentially negligible in comparison to sources of noise such as instability in the radiant output of the Xenon lamp and 60 Hz line noise. Also, this noise is not dependent on the magnitude of the current and may occur in the absence of a current. Due to its low magnitude no efforts were made with the instrument to reduce or compensate for this noise. d. Signal Shot Noise as a Cause of Error. Signal shot noise is due to two factors: the random rate of arrival Of photons at the photocathode, and the random emission of photoelectrons down the dynode chain. The linearity of the signal output in the range from 0.0 to 10.0 volts, and the stability of the signal (after compensating for 60 Hz line noise by 40 point averaging), indicates that signal shot noise is a negligible source of error in the instrument. No efforts were made with the instrument to compensate for this noise. -101- e. 60 Hz Line Noise as a Cause Of Error. Line noise Of this frequency appears to be a significant and prominent source Of error in individual readings. The oscillOSCOpe (Tektronix Type 502A Dual-Beam) revealed a very regular sinusoidal oscillation at 60 Hz which remained constant with time. The magnitude Of the line noise oscillation was found constant at all tested Output levels, from 0.0 to 10.0 volts. In order to average out line noise, 40 readings were taken over a period of 200 milliseconds and subsequently averaged. The system software Offers the Option Of taking more readings over a proportionately longer period Of time, however the above setting was sufficient to eliminate 60 Hz line noise as a source of error. The only real sacrifice in eliminating the noise in this manner is in the longer sampling time required for each stored measurement. 10. Stepper Motor Uncertainty in Cell Positioning The problem Of cell skewing caused by potential mis- stepping Of the stepper motor in the cell rotation device during rotation was studied. A single step away from cell orthagonality to the excitation beam axis, equivalent to 11.8 degrees Of rotation, caused an attenuation of fluorescence intensity Of about 3%, while three steps away from cell orthagonality, equivalent to i5.4 degrees of rotation, caused an attenuation of slightly more than 10%. These attenuations were found to be independent of the fluorophore containing solution studied. Note that —102- these positions were set manually and not caused by any mis—stepping Of the stepper motor. An experiment was conducted using a single cycle sequence involving 50 steps (cell position 1 to 2), 100 steps (cell position 2 to 4) and 50 steps (cell position 4 to l). The observed frequency of mis—stepping in 10,000 full cycles was zero. Repetition Of this experiment gave the same result each time. It was concluded that under normal operation of the instrument the skewing Of the cell was of very low probability. However, caution must be exercised in always confirming the cell orthagonality immediately after turning on the stepper motor driver circuits and running of the FLUOR system operating program. If the cell orthagonality is off, the cell can be readily re— positioned by turning off the driver circuits and manually returning the cell to the correct position. The driver circuits are then turned back on. A lateral misalignment of the cell during rotation can be caused by either of the following: the excitation and emission detection beams do not intersect along the diagonal (top view) of the sample cell, or their exists improper Z-axis alignment of the post and platform sup— porting the sample cell. Fortunately, the design of the cell rotation device is such that it allows a high degree Of precision in the manual installation of the cell, thus minimizing the opportunity Of a lateral misalignment. The use of set screws in most of the adjustments prevents —lO3— screw loosening during continued Operation of the instrument. 11. Back-Reflection of Sample Beam Intensity from the Surface Of the Sample Beam Detector When the sample photovoltaic cell surface was aligned orthagonal to the incoming sample beam, the fluorescence readings from cell position 4 were between 10 to 15% higher than expected based on the known absorbances of prepared standard solutions. The elevation in the readings was found to possess a slight dependence on the excitation wavelength and on the chemical system under observation, therefore the reported range. When the sample photovoltaic cell surface was shifted 30° from orthagonal to the sample beam, the primary absorbance determined spectrofluorometrically matched the expected values. It was concluded that slightly more than 10 to 15% of the radiation impinging on the surface of the sample photovoltaic cell is reflected back in the ortha— gonal position. The shift in the angle of the detector surface appears to eliminate any problems due to back— reflection. The beam is reflected away from the mirror onto a flat-back surface and, thus, cannot return to the sample cell to induce higher cell position 4 readings. 12. Raman Scattering as a Cause of Error The results of a study comparing the Rayleigh scattering wavelength maxima with the Raman scattering -104- (Stokes) wavelength maxima of distilled water are shown in Figure 31. The difference between the Rayleigh and Raman scatter band maxima appear constant in terms of frequency (cm—1), but the wavelength difference increases with increasing wavelength. This is expected and verifies that it is indeed Raman scatter that is being Observed. Since Raman scatter is thought to be the interaction of photons (excitation radiation) with the Vibrational and/or vibrational-rotational energy levels Of the solvent and solutes (if present) producing scattering Of photons with both higher energy (anti-Stokes) and lower energy (Stokes), it will be dependent on the composition of the chemical system. Water is the solvent (and by far the major component in the solutions) used in most of the chemical analyses. The observed frequency difference between the Rayleigh scatter band maxima and the major Raman scatter band maxima was in the range 3200 to 3400 cm_l. This range correlates with the reported [159] -OH functional group frequency. A comparison of Rayleigh and Raman peak areas indicates that the Raman peak is smaller by two orders of magnitude (approximately). In most cases the contribution from this Raman peak was found to be negligible. Occasionally, the Raman bands of solvents such as water can prove troublesome. This occurs mainly if the emission wavelength of a dilute fluorophore falls close to the excitation wavelength. It is always wise to run a -105- 7oo — <2 2 ° soo - X E , <5 :1 0"” :EE ;{ A ¢ 500 — dd X I .o' 5.. ~ 0 p" ._1 PF Z LL] . 099 < <>C dd 2 3 300 — pp" 3: 200 I 350 I #50 l 560 l GOO l 700 WAVELENGTH , nm. RAYLEIGH PEAK MAXIMA Figure 31. Rayleigh scattering versus Raman (Stokes) scattering. The plot compares the Rayleigh peak (wavelength) maxima with the Raman peak (wavelength) maxima. The cell contained distilled water at room temperature (24°C). —106— blank of the solvent used in the analysis. It is Often possible to eliminate Raman interference by exciting the sample, not at the peak of the excitation band, but at a shorter wavelength on the shoulder of the excitation band. This will reduce sensitivity somewhat, but will move the Raman band toward shorter wavelengths, thereby reducing its interference with the emission band to be measured. 13. Effect of Absorption on the Intensity Median Detected Within the Fluorescence Observation Window A slight absorption dependence has been Observed on the magnitude (but not the shape) of RFE curves in highly absorbing solutions. These are solutions that absorb 1.5 Absorbance units per a 2 cm cell pathlength and above. About a 5% decrease in RFE magnitude is Observed when a comparison is made between solutions of a particular chemical system absorbing at 2.5 Absorbance units with those absorbing at 1.0 Absorbance units and below. At the lower absorbances the RFE value remains constant. This effect is independent of the chemical species in the absorbing solution, but rather on the solution's overall absorbance. It is for the purpose of compensating for this absorption effect that the fw factor was incorporated into the determinations of quantum efficiency and RFE. For all studies reported in the dissertation concerning these quantities the value of fw was set equal to unity. In future modifications of the FLUOR system Operating program there are tentative plans to develOp and -107- incorporate a look—up table of absorption dependent fw factors to be automatically implemented in the quantum efficiency and RFE evaluation equations. However, this is not a serious limitation to the present appliations of either of these quantities. In the reported studies the overall solution absorbance is changed only slightly, if at all, and is typically below the absorbance threshold wherein the effect Of absorption is apparent. The above statements note the apparent correlation between absorbance levels and the relative magnitudes of RFE values for a particular chemical system. The following is an explanation of the phenomenon. Regardless Of the solution absorbance, the position (or path) of the collimated excitation beam is fixed and the location of the emission observation window is fixed. Also, in strongly absorbing solutions containing fluorophores it can be visually seen (readily if the laboratory lights are extinguished) that the fluorescence intensity rapidly tapers off along the path of the excitation beam away from the source. This effect is illustrated in Figure 32. Case A for a solution of a dilute fluorophore shows that negligible absorption attenuation of the excitation beam is Observed across the observation window. Here the fluorescence emission is proportional to the concentration of the fluorophore. Case B for a solution of a concentra— ted fluorophore shows that excessive absorption occurs between the A and B planes prior to the window of -108- .SOOGHB COMDO>HOOQO Ono mo mmmpmn.u pom m «HHOO wamfimm MO coco mcflpmoan.¢ u-Houowump- Onsu HOAHQHDHSEOHOLQ OOGOOmOHOOHMn.EzmIm ->uflmcmucfl anon OOcOOmOHosamnum “muflmcmpse Econ mocmummmunum uxox .Ommo pmumuucmocoo .m .Ommo OUDHHQ .4 .Ononmouosam m mo GOAODHOO pmumuucmocoo m can madaflp m mcflcfimusoo HHOO OHmEmm n no mBOH> mos Ea..- 08.: .Nm OHDmHm ---0---.-. --------.. ------u--. -------..' _-o------. ---—-----. -----.o--. ---------. ----.---.. a ... n L 1 ......- Li ‘I m 0 II ...-...... ......... .. ......... ..... ......... .. ......a-. < 0‘00.....m < ......... .. ......... 6 O¢Q44 <¢Gd<10'5 M tryptophan (TRP), 1 x10"2 M Tris, pH 7.0. lex scan =Z30-350 nm, len(=370 mm. Al =primary absorbance determined spectrophotometrically, A2 =primary absorbance determined spectrofluorometrically. . . _ -121- A theoretically based comparison is provided contrast— ing the ideal case where stray light does not exist to the more realistic case where a small stray light contribu- tion does exist, presented in Table VII. In practice, only a maximum stray light contribution of 2 to 3% has been observed in the 240 to 270 nm range. 4. As a Monitor of Cell Wall Adsorption by Light Absorbing Species Another one of the added benefits Of measuring primary absorbance both spectrophotometrically and spectrofluoro— metrically is the ability to monitor the quantity of an absorbing Species that has adsorbed onto the quartz walls of the sample cell. The process of determining the Al function involves measurement of both the reference and sample beams. To Obtain these measurements the excitation radiation must (in the case of the sample beam) pass through a quartz/sample interface, the sample solution, and then through another quartz/sample interface. This is typical for spectrophotometry. On the other hand, the process of determining the A2 function involves measurement of the emitted fluorescence from two precisely known positions, F1 and F4, along the excitation axis. Since the emission readings are obtained at the fluorescence emission wavelengths of a solution fluorophore (and hope- fully removed from the excitation or absorbing wavelengths), these values are much less likely to be attenuated by species adsorbed onto the quartz cell walls. The —122- TABLE VII. The Effect of Stray Light on Absorbance Evaluations. In all cases: A2 =0.301; R1 =2.00'and Sl =1.00. A. Ideal case where Al =A2. A1 = log(Rl/Sl) A2 = (G3)log(Fl/F4) and therefore, log(Rl/Sl) = (G3)log(Fl/F4) B. In practice, where Al #A2. A1 = log[(Rl +R15L)/(51 +SlSL)] A2 = (G3)log(Fl/F4) and therefore, log[(R1 +RlSL)/(Sl +31SLH :5 (G3)log(F1/F4) where R1SL and SlSL are the stray light components in the reference and sample beams, respectively. C. Significance of the stray light contributions. 1. % stray light contribution (ideal case). = + + Al log[(Rl RlSL)/(Sl SISL)] = 10g[(2.00 +0.00)/(1.00 +0.00)] = 0.301 2. 1% stray light contribution. A1 = iog[(2.oo+0.02)/(1.00+0.02)] = 0.297 3. 2% stray light contribution A1 = log[(2.00-+0.04)/(1.00-+0.04)] = 0.293 4. 3% stray light contribution A1 = logI(Z.00 +0.06)/(l.00 +0.06)] = 0.289 -123- difference between the Al and A2 readings provides a good estimate of material adsorbed on the cell walls and, thus, no longer free in the sample solution. A considera- tion not to be neglected, however, is that the adsorbing species may undergo a change in the molar absorptivity relative to the same species free in the solution. Figure 36 illustrates a case where a species that adsorbs provides an absorbing film. The biochemical species is beta—Prolactin, a growth hormone in mammals, in a matrix . . of Tris buffer at pH 8.0. After an incubation period of Vzhour, the Al and A2 curves have attained maximum divergence. The curves change only in magnitude. A post incubation substitution of the matrix alone for the sample solution reveals the presence of the adsorbed beta— Prolactin on the cell walls from the Al spectra. The unchanged spectra (shape) indicate that a new species is not formed in the process of adsorption. An A2 spectrum could not be obtained in this latter case indicating that the beta—Prolactin remains bound to the cell walls. With lipophilic compounds such as beta-Prolactin, cell wall adsorption may be a common phenomenon. The CRM instrument can readily determine the extent to which such adsorption takes place. Potentially, it may prove feasible to monitor the kinetics of the adsorption process using repetitive scans at constant intervals. ” EXCITATION SCAN ' 0f BETA-PROLACTIN Aem = 350 nm _ SPECTRO—PHOT0/FLUOR0-METRICALLY I DETERMINED ABSORBANCE l I l l l J l l l 240 260 280 300 320 340 ‘EXCITATION WAVELENGTH, nm Figure 36. Evidence for cell wall adsorption by absorbing chemical or biochemical species based on Al and A2 values. Sample - 1000 mg beta—Prolactin in 10 ml 1 XlO'2 M Tris, pH 8.0. >‘ex scan =240—340 nm, )‘em =350 nm. A1 =primary absorbance determined spectrophotometrically, A2 =primary absorbance determined spectrofluorometrically. —125- C. Secondary Absorbance Measurements l. Determined Spectrofluorometrically This output is the result Of a unique method of calculating the secondary absorbance of a chemical solu— tion. Here the absorbance is determined spectrofluoro— metrically, more precisely, from two fluorescence readings taken at independent (regarding the excitation beam position within the cell) positions along the emission detection axis. The approach requires the presence of I; . a fluorophore in the chemical system and may have other fluorophores and/or chromophores present. This version of secondary absorbance, A3 (= (G4)log(Fl/FZ)), is accurate (approximately) to absorbances as high as 2.5 absorbance units (compared to Spectrophotometric determinations of the solution absorbance at the emission wavelength). Figure 37 illustrates the results of applying secondary absorption corrections, and thus the accuracy of the secondary absorbance determinations, to mixtures of quinine sulfate and sodium fluorescein. At a quinine sulfate emission wavelength of 435 nm there is negligible sodium fluorescein fluorescence. However, sodium fluorescein in acidic media has an absorption maximum at 435 nm and permits analysis of secondary absorption on the emission of quinine sulfate. The limitation in the calculation of A3 at higher absorbances is not in theory, but in the difficulty in obtaining accurate F2 measurements. The use of smaller distances between the .cofiumhomnm unopcoowm now pouoouuoo Hm" mm .H cofluflmom HHOO EOHM hunGOpofi oocwommuosam pONHpcmsqn Hm .Ec mmvu flax .EC mmvNUEOI .8: com” XOI .Awmcmuv camomwuooaw Edepom z mloax ml 2 mloax S van Aucmumcoov wuowasm OCACADO S mloax m I mOHmEmm .Jommm z H.o SH cemommuosam SoapOm pom oumeDm wcflcfioq mo wousuxfie MOM mcofluowuuoo coflumuomom xumpcoowm .nm moz<10—5 M was determined by extra- polation from data points obtained at lower concentrations where the primary and secondary absorption effects are negligible as illustrated in Figure 38. As expected for this compound, the primary absorption effect is considerab— ly more significant than the secondary absorption effect. The difference between curves F1 and FP is due to primary absorption. The difference between curves FF and F0 is due to secondary absorption. Curve FP is generated by application of the primary absorption correction only. Curve F0 is generated by application of both the primary and secondary absorption corrections. The latter curve is virtually identical with the theoretical curve extrapolation up to a 2.5 XlO—4 M quinine sulfate concen- tration which corresponds to a primary absorbance of approximately 2.5 absorbance units per cell pathlength. The negative deviation from linearity at 2.5 absor— bance units is within 1.6% compared to the theoretically calculated value. TO achieve this degree Of accuracy, .coHumMOQO >HOpcoomm pom wumEHHm How pouoouuoo Hm" om .coHumuomom >MOEHHQ How pwuownuoo Hm” mm .H :oHunom CH HHOO zuH3 qumcouoH oocmommuoon pONHucmoqu Hm .E: oovu Ew< .Ec ommn XOI .10mum z H.o CH oumeDm OCHCHDq How o>u=o mcquoz oozoomwuosHm pwuoounoo cOHumuOmnfl .mm musmHm EH o-x zo_eH5mOOmHH0 \ mm HMHSEOHSB \ mm pHos OHmHucww \ Hp onsmHHcNOHsm \ we mchoasoouosHmIm \ mm CHozfioumouum \ mm OchHnomnum \ mm OcouOMHocouHmm \ Nb cwmoupwm \ em Hum Honuom \ Hp mcHummm \ mm osmeHooumm \ on mwumuouHQHmnwxo pmuopHumnsmHnnm.m \ mm .mm cHom OHHSOHHMm \ mo Hum wcHamueerscwsmHo \ mm OCHEOCCHOmwm \ ww .wo OCHEOHQHEHHwnuoEmOQ \ mm OGHQHDO \ mm OQOHOHusmo \ mmHINeH wcHwacHso \ we mcHocou \ mm OCHHOOCHUO \ mm manonooHo \ mm OQHOOOum \ mm OCHUHCOQOCHU \ mm GHxOHOHH>£m0pom \ mm Ochmu0m5m \ mm cmxouOmHm \ mm prEOthumHQ pHom OHmHmmaHOEOHm \ mm HmuHQumnoucom \ NO GHQQHOEOQ¢ \ ms cHsamemm \ HO a afloseHuca \ me :HSUOHOHfiouxO \ om HOHHQHMQOE¢ \ me ocHusmwc>mooz \ mm pHod OHHonHMmosHeaIm \ me :OSQOSOCHOOOZ \ mm EHuouQOCHE< \ Hm pHOd OHHOCOSQOO%2 \ mm pH0< UHONQOnOCHE¢Im \ ow .46 mcHsmuoz \ mm mcHrmuosHsHHa \ mmozmmmmmm DZDOQEOU mmozmmmmmm QZDOmEOU .mponumz OHHuoEOHODHm HOOHHQ wn mHHOOCHHU pmuhHmc< coon m>mn umcu mpcsomaoo HMOHUDOOOEHMSO Mo umHA m .HHH> mqmde -133~ HHH mcHuwsm \ mMH .OOH .NoH wcHNmeHuoHre \ SHH cHouqmoerscwsmHo \ me wcHHosomuuoe \ mHH wcHscuwanscmsmHo \ mm OHOmeOwHMHDm \ vHH OsHHOCquomHOHO>£HQ \ mm OOHEOHHSOMHDO \ NOH ocmtzponmnoHno \ mm mHonxorposmmHSm \ OHH .mOH mcHansuocwnmuoHso \ mm mchonumesm \ wOHuooH .NOH quumeoumuoHrO \ mm OGHNOHOOMHDO \ OOH conuouoHSU \ mm CHO>EoumOHum \ NOH OCHNOOHHOHSU \ OMH mwOHHoanws wcHsmHHOHHmm \ HOH mcHchozmumo \ mMH cHOHOENMHm \ MOH mmooHnumo \ vMH .Om ochHomwm \ NOH OCHNOOHHEOHm \ MMH OcHEMHSQOCHDO \ HOH OCHOHcmnuOm \ NMH .mm OCHEmwcothmm \ OOH OGHQOHUO \ mm Hum OSHmOOHm \ mm OGHHnmud \ HMH .OMH OOHEMCHOOOHO \ mm OHo< OHcOOHOumHnd \ mm OCHEMHOGOQOHmHhsonm \ mm CHHHHOHQEH \ OOH OOHNchuocwzm \ mm wpmesw ocHEmuonmE< \ OOH OCHEMHHCOSQ \ om OCHEmumnmfifl \ mm oHHHHOHcom \ mm pHOd OHH>OHHmwocHEx0HO>mImIocHEHIh \ me .FNH OCHHowomHuwumxo \ mm EMQONMCOHOOGHE \ omH .Hm mcHrmnoz \ mm uHoa onoucwnocHESIO \ mNH ocououmoumouH>£HOz \ hm OchmHOEHOCH>HHOHHmmH>uood \ mmozmmmmmm OZOOAZOU mmuzmmmmmm ozoomzoo .moorums OHHuofionoon DOOHHOCH >9 >HHmOHcHHO OONSHmsd coon O>m£ umru mOcoomEoo HOOHOOOOOEHMLA mo uwHH m .xH OHQOB >pHHHQHOOOOHmOH Room .OGHO woowomeOSHm OHMHOOmw Opp OGHOHOOOH poo .OCHHDHO .msHmmuom wn OOHMHHCMDO Ono mwuHHOQmuwS >£mmumoumeouco mvH IHGHOO mmuswmoz whom Scum ooumummom paw pouomuuxw ODHO HO>MHICHLB .m mccsomfioo Hocoo coHumuucwocoo OQHOHCHSO SUH3 >9H>Huomou ou HmcoHuHomoum AE: OOMHme- ODOHCLOOB ImmOHo oEom .OGHO :OEEOO mononuomnm CH ommwuosH “xOHmEOO womwOOGSEEH va IHCHSO mousmmoz pwoz Os>NCOImDHO ou mccHn >ponHuc¢ OOHHQHUHDZIOahucm .m mOHHOp IOHODMH -E: cow wHHMOHMHoomm m0 :OHu M 2m .Ec OOMN xm- OOCOOOOHODHM mouHHonmuoE Iuomonm Ho -E¢ Omm HO Ea Ommn xmv mnmmuooumfiouco muH pom OCHO HHmEm COHumHOme >9 uwcun Owusmmofi pom OHSOHH L. mvH IHGHDO monommmz :H pomp wwuHHonmuOE EOHM OoumummOm mono oucmeuomuwmlanm .o 1- d mHHmOHMHUOmm mmuHHOQmuOE :oHuOOUOO cooouuHc Ho :OHumuHcoH muH pom msHO 0EMHw lmwuHHonmqu EOHM omH .th .OvH IHCHSU mousmmmz mumm Umumummwm Ugo OONHuo>HHOO Oouo SSQMMOOOMEOHSU wow .0 coHumuucousoo ou HMCOHuHomoum ocHOHcHDO cocoomouosHm -ESHOOe OHOO chou mo muHomOH msowsqm oucH pouomuuxmlxumn con» AOMMOcmum- MVH oumusoom mo>HO COEEOU .wmmcm OHcmmuo oucH pwuomuuxo mono mHuoEOHOSHMOHUOOmm .m 5338 :H E: omen 2m .5: mom" xm- chEmu mpcsomeoo EOHOOE OHOHOO :H ucoomouous ucoomouous Hmo OCHOHGHSO “pHum OHHocmwonmmuOE Acumwcmum- NvH Hwnuo >cm2 IHHoumHm >2 wouMUHmHomHm mH :Hououm wuuofiOHODHmouuoomm .d mmozmmmmmm mezmzzoo momma mHmHOszm oomemz .mOHHOumuonmq HOOHSHHU :H poms memHmcd OOMMHSm OchHcHDO mom mpocuwz .x WHmdfi -l35- HHOO mHaamm HE\O1 NED Nx N OONI m.O :uHs HE 0.0 Oocuoz .HHOO OHmEmw coHumuom HHOU HE\m1 NED Hx H oxu OCHonmEm OOH oom- m.H BOHB HE m.H JOmNm z H.O O.H ucmHna< suuwsoHoSHuouuommm .m memvm <0 .ouH< onm wstcnuws HE\O1 acmmfiou m>>m >3 OHom hmmwOOGDEEH vVH ml m.O HE m.O :oHumHmmoum anumz How “HM ml O UoOm OOHHmHuHozIOEANGm .O Hocmcumz nuHs Aqu- o.m mo mm m on now HHm .mox z H0.0 cam .Hom mcHsm ssmmnmoumeouso HE\O1 IHwnuofiHHB 2 H0.0 .Ocomcom OHSOHH mvH OHI m.O HE O.H .momz 2 mm.O mo wusutz O.vH ucOHnfid oucmfiuomummlcmHm .O Hocmnuoz CH Oponupzm EDHCHHHcmenumE OmH HE\m1 IHHB z N.O pom .mcmncom .PVH .OVH NH: N.O HE O.H .mooz z mm.O Mo OHprHz O.VH OHQOHHO> wgmmumoumEouco wow .m HE\O1 -pHmpcmum- mvH mHI O.H HE m.O :owmm z H.O O.H ucwHQE< >HUOEOHOSHMOHDOOmw .d mmUZmHmm-hmm EHMJHm-ZHH mZDHO> yam-HRH.- mm gag mnHEm-E QOEBWE mHmEmmm< mo :owHHmmEou .Hx mqmme -136- RELATIVE FLUORESCENCE INTENSITY Figure 39. A 00000 _ em 460 nm 0 o — o 00 0 0 F0 0° 00000 o 0 O _. ° 0 0 O O .- O o 4 o o O 0 ° _. . ° F1 0 80°°°o 0 ° °°o 000 o A AAA Oooooo 0000000000000000000000 _ O .23. Agog op A AB F2 A 8 00°00 28 93 0° 0 a. — A ”0 CIO‘L'LIDU F4 Duonu Donooggg [,0 ODDUDUDDUDDDUUDDUDDDOUOO Ouggé I 1 LI I I I I I 285 335 385 EXCITATION WAVELENGTH , nm Excitation scans for total absorption corrected and uncorrected cell position fluorescence for l ><10"l+ M quinine sulfate in 0.1 N HZSOO. Aex scan =270—400 nm, Aenl=460 nm. F1, F2 and F4 =quantized fluorescence intensity measurements with cell in positions 1, 2 and 4, respectively, F0 =F1 corrected for primary and secondary absorption. RELATIVE FLUORESCENCE INTENSITY —137— I I I I l I l l I ._ 000000 F0 ° °. EMISSION SCAN O o A — o 0 ex 350 nm 0 0 - o ._ O O 0 o b _ 0 - O O O 0 0° ._ o 00 .. O 0OO 0 F1 0°00 - o0 00000000000 00° .— 0° OOZAAAAAAQUAAO 00000000 33A!) F2 AAAAlljimooo _ 3333 W .. 33 Ifibqfi%§I,IyuuUuUTnDUDD‘iuuuoUUIUUUUDHT” IIII 'l I . 400 480 560 EMISSION WAVELENGTH , nm Figure 40. Emission scans for total absorption corrected and uncorrected cell position fluorescence for l XlO'“ M quinine sulfate in 0.1 N H2804. Aem scan =380-580 nm, Aex =350 nm. F1, F2 and F4 =quantized fluorescence intensity measurements with the cell in positions 1, 2 and 4, respectively, F0 =Fl corrected for primary and secondary absorption. -l38- Fluorescence, a.u. Fluorescence, a.u. lllll-l-_ ILIIII. Fluorophore Absorbance, a.u. Conc.,a.u. ' ATyfi/EFJ .' Fluorescence, a.u. Fluorescence, a.u. - O I— .00 00 0 III J- 0°I°IITII_ Fluorophore Fluorophore Conc.,a.u. Conc.,a.u. 1 Figure 41. Effects of primary and secondary absorbance on fluorescence working curves. A. Total absorbance attenuations as fluorophore concentration increases; dotted line extrapo- lates working curve (linear region). B. Fluorescence intensity decrease with increase in the overall absorbance of fluorophores and chromophores. C. Effect on fluorescence intensity of increasing the fluorophore concentration in a matrix that possesses a constant amount of an absorbing Chromophore. D. Effect on fluorescence intensity of increasing the fluorophore concentration in a matrix that could have a variable amount of a Chromophore present (most common case in a clinical laboratory where no working curve can readily be determined). —l39— :heoretically calculated. Note that a rather extreme :ase of high absorption attenuation has been selected :0 emphasize the utility of applying these corrections. It these high absorbances the spectral distortions are so severe as to make compound identification highly Improbable using a commercial spectrofluorometer. Certain chemical systems have substantial overlap >etween their absorption and emission spectra. Figure 42 .llustrates such a system in a mixture of l X10.4 M Inthracene in anhydrous ethanol. The comparison between :orrected and uncorrected spectra from cell position 1 .ndicate a large secondary absorption component. The :imilarity of this corrected curve is, within experimental :rror, identical with the curve Obtained with prior .bsorption correcting instrumentation [27]. As expected 'rom such a chemical system, secondary absorption (i.e., 'e-absorption) is pronounced in the region of overlap. ‘his absorption phenomenon poses no problem when the Lathematical correction is applied and re-inforces the Totential of this technique for obtaining true fluorescence pectra for identification purposes. A common type of analysis encountered in many linical laboratories involves taking fluorescence ntensity measurements of samples that possess widely arying background absorbances. Figure 43 shows the esults of a study where a constant amount of a fluoro- hore (L—tryptophan) was present in solutions containing .coHumHomnm mumocooom ocm >HmEHHm Mow OOHOOHHOU Hm" Oh .H coHunom HHOO Eouw uGOEOHSmmofi huHmcoucH oocoomouosHm OONHUGMDOH Hm .Es ovmn xOI .Ec OmVIOmmN cmom 80‘ .Hocmnuo moonpmzcm :H Ocmomunuco S :IOHx H I OHmEow .COHDQHOQO >Hmpc000m >3 pwmomo muuommm conmHEo mo mcoHuuouwHQ .mv OHDOHh . E: IHOZwI-m><>> ZO_ww__>_w owe cow com H _ u — - - - _ - l—J ..l I l n” nU n_. U 11- M S . 1 Ga I 3 MN Gd 3 r I I MN I- E: 8m E 3N. l l S :mom scam-Em- M — - — — p _ r - — .52-EB z_ m2mo<1o‘2 M Tris, pH 7.0. Aex scan= 250-300 nm, Aem =370 nm. SF curve matching at 295 nm. Figure 47. -150— - _ c : 2E _ ’,,-//’//////’£L\\\\\\\\\‘ — h I I 1 I “’1///TTT\\\T‘~—1, I 240 260 280 300 320 340 EXCHATKNV WAVELENGTH , nun Study employing the fluorophore absorbance curve stripping procedure with mixtures of a Chromophore and a fluorophore. A. Plots of RFE. xex scan =240-340 nm, xem.=37o nm. Sample solutions = a. 5 ><10'5 M Bovine Serum Albumin (BSA), 1 ><1O'2 M Tris, pH 7.0; b. solution a. and 2 x10"“ M NaNOg; c. solution a. and 4 ><10'5 M BSA, 8 X104I M NaN03, ‘ 1 x10"2 M Tris, pH 7.0. D. Plots of Al. Aex scan =24o- 340 nm. Samples solutions - a. Z XlO'” M NaNOg, 1 no2 M Tris, pH 7.0; b. 4 x10“+ M NaNO3, 1 x10'2 M Tris, pH 7.0. -151- applied to a chemical system consisting of two fluorophores. In the latter case one of the fluorophores is effectively converted into a chromOphore by locating an emission wavelength to monitor that which is characteristic only of the other fluorophore. The ability to apply absorbance curve stripping to mixtures of fluorphores or mixtures of chromophores and fluorophores is a powerful tool for both quantitative and qualitative purposes. Quantitation and identification of the Chromophore composition of a sample solution is simplified by removal of the primary absorbance contribu— tions of the fluorophores. 2. Limitations of the Absorbance Curve Stripping Process The most significant limiting factors on the application of the absorbance curve stripping procedure are as follows: a. selection of the most appropriate scale factor, SF, to multiply the fluorophore excitation curve, FR, by prior to evaluating the difference spectrum, (Al) —(SF)(FR); b. need for a sufficiently high quantum efficiency for the fluorophore to permit determination of an accurate FR curve; c. solutions with absorbances higher than 2.5 absorbance units lead to errors in determination of the Al and FR spectra; and d. overlap of Rayleigh or Raman (Stokes) scattering bands with the fluorescence emission wavelengths being detected decrease accuracy of the FR curve. —152— F. Spectrofluorometric Determination of Chromophore Absorption 1. Addition of Fluorophores The primary absorbance curve of a solution containing a Chromophore can be determined successfully using only spectrofluorometric intensity measurements. This, of course, implies the presence of an additional solution component, the fluorophore. With the present CRM instru— ment a l XlO—5 M concentration of a moderate or 5 X10_6 M concentration of a high quantum efficiency fluorophore I are required to provide the fluorescence signals necessary for accurate F1 and F4 determinations and, subsequently, AZ evaluations. A fluorophore of moderate quantum efficiency is L—tryptophan and one of high quantum efficiency is quinine sulfate. Figure 48 illustrates the results of this procedure with a chemical system composed of a constant amount of a moderate quantum efficiency fluorophore (L-tryptophan) and varying amounts of a Chromophore (L—ascorbic acid). The determination of primary absorbance in this manner is in agreement with that determined spectrophotometrically in Al. 2. Conversion of Spectrofluorometers to Primary Absorbance Measuring Instruments Providing the sample cell compartment of a right-angle spectrofluorometer is sufficiently large, the instrument cxauld be adapted to handle Off—center cell rotation to Ioperate under the CRM. The minimum size requirements for ~153— 100 1 1 l 1 .4 u— v E Z )‘em 370nm O E m... .— O G- : LLJ ° 5 |__.. .— < Lu 0 E U V)“ _- 3? 4 O 3 LL- l -_ _ k 2 O E S 3 _ o_—~ : '63 2 p— < - n.“ 1 U E U m a _ CURVES: _ O 3 1/ 1.0E—5M TRP alone LL. 2; 2 I (1)+ 2.0E-6M ViI.C O ..J - 3 / (1)+ 4.0E-6M VILC \ t 4 I (1)+ 1.0E-5M VILC \\ 5/ (1)+1.5E-5M Vlt.C I I I I 250 260 270 280 290 300 EXCITATION WAVELENGTH, nm O Figure 48. Spectrofluorometric determination of primary absorbance of mixtures containing both fluorophores and chromophores. Samples — mixtures Of L—ascorbic acid (Vit. C, the chromophOre) and L—tryptophan (TRP, the fluorophore), solute concentrations listed on graph in computer nota— tion (lE—5M =1 ><10-5 M), matrix =1 XlO'2 M Tris, pH 7.0. Aex scan =250—300 nm, Aem =37O nm. —154- the sample cell compartment, assuming continued use of the same 2 X2 cm2 by 4.5 cm (height) sample cell and the Slo— Syn stepper motor, are approximately 3 X3 in2 by 6 in (height). Most commercially available right-angle spectro— fluorometers have at least this amount of space available. The optical configuration Of a right—angle spectrofluoro— meter equipped to measure primary absorbance by the CRM is shown in Figure 5. 3. Limitations of Spectrofluorometric Determination of Chromophore Primary Absorption The fluorophore used must not chemically react with the Chromophore for this method to be valid. Generally a chemical reaction will alter the quantum efficiency of the original fluorophore. The excitation spectrum of the fluorophore must be in the same wavelength range as the Chromophore absorption. The upper total absorbance limit is dependent on the ratio of fluorophore to chromo— phore and quantum efficiency of the fluorophore. G. Light Scattering Measurements l. Turbidimetry and Nephelometry with the CRM Instrument Applications of the CRM instrument in this section are associated with light scattering measurements of solutions that contain either colloidal suspensions or evenly distributed precipitate particles. The light scattering in the visible wavelengths can be observed visually as a cloudiness in an otherwise transparent -155- 100 l I I l CURVES: 1 I 1.0E-5M TRP alone 2/ (1)+ 2 0E-6M Vit C — 3 I (1)+ 4.0E-6M VII.C 4 I (1)+ 1 OE-SM ViI.C 5 I (1)+ 1 SE-SM VII.C 6 I (1)+ 2.0E-5M ViI.C INTENSITY) SAMPLE / INTENSITY LOG (REFERENCE l T I I 250 260 270 280 290 300 EXCITATION WAVELENGTH, nm Figure 49. Spectrophotometric determination Of primary absorbance of mixtures containing both fluorophores and chromophores. Samples — mixtures of L-ascorbic acid (Vit. C, the Chromophore) and L-tryptophan (TRP, the fluorophore), solute concentrations listed on graph in computer notation (lE—SM =l ><10‘2 M Tris, pH 7.0. Aab scan =250—3OO nm. -156— medium, or as a muddy dullness of a colored medium. The instrument has the capability of evaluating the effect of the light scatterers in the following ways: a. determining turbidimetric quantities spectrophotometrically (using R1 and 81 measurements); b. determining nephelo— metric quantities based on right-angle light scattering and reference beam intensity (using R1 and S measure- ll ments); and c. determining analog-turbidimetric quantities spectrofluorometrically (using S and 814 measurements). ll Also, both fluorescence and light scattering measurements can be corrected for beam attenuations along the primary and/or secondary optical axes. 2. Turbidimetric Quantities Based on R1 and 81 Figure 50 shows the results of a study employing the Al function in spectral analysis of a solution containing varying amounts of light scattering particles. Figure 51 shows the results of this function in spectral analysis of a solution containing a Chromophore and varying amounts of light scattering particles. In both cases, it was found that the measured light scattering is directly proportional to the amount of light scatterer added. An important benefit obtained from using the CRM instrument is that the rotation of the sample cell during scanning mode ensures that the light scatterer density remains uniform, a feature which is a significant advantage over commercially available spectrophotometers or turbidimeters. -157— 100 ‘ J l I ’; - CURVES: _ ._.. E 1 I 14 uglml starch Z t: 2/ 28 uglml starch Z "' .— — 3 I 42 uglml starch ‘3 4/ 56 uglml starch & E _ SI 70 uglml starch __ "‘ 6 I 84 uglml starch ‘ 7 I 98 uglml starch I. >— Z ‘ 8 / 112 uglml starch - (I) Z LIJ .— Z — _ 7 __ LIJ L) Z LLJ CZ E: - 5 _ Lu 5 4 0 <3 - _ 3 Af/éx fl 0 I I l I 250 260 270 280 290 300 EXCITATION WAVELENGTH, nm Figure 50. Turbidimetry of soluble starch solutions at neutral pH. Samples — concentrations of soluble starch in ug/ml are listed on graph, matrix =1 ><10"5 M in all cases, concentrations of soluble starch in ug/ml are listed on graph, matrix =1 X10"2 M Tris, pH 7.0. ...-div- —l64— is observed which appears to follow a predictable pattern directly related to the concentration of the light scatterers. A comparison of Figures 43 and 54 illustrates the magnitude of the under—correction. It appears possible to compensate for the attenuation due to the excitation beam light scattering by the following expression: (GLS) (Gl)ln(Fl/F4) (G2)1n(Fl/F2) FO = (Fl)e e e where FOL is total absorption, source fluctuation and S light scattering corrected fluorescence, and GLS is an empirically determined light scatterer concentration and species dependent factor. The use of such a factor assumes prior knowledge of the light scattering Species and its concentration. H. Total Quantum Efficiency by the Comparative Method 1. Theoretical Approximation of the QE Function The fluorescent quantum yield for a compound is determined by the relative rates for the processes by which the lowest excited singlet state is deactivated (including fluorescence, intersystem crossing, external and internal conversion, predissociation, and dissocia— tion). The quantum energy function, QE, is generally described as the ratio between the number of photons emitted per unit time (numerator) and the number of photons absorbed per unit time (denominator). However, —l65— obtaining absolute values directly for these quantities can be difficult. Thus an approximation was develOped for this function. The model was develOped to utilize quantities that are made accessible by the CRM instrument. Since all of the system detectors, R—PVC, S—PVC and F—PMT, are calibrated and normalized to provide output relative to numbers of quanta, both the numerator and denominator values necessary in the QE approximation function can be determined and the ratio evaluated. The procedure followed for the determination of total quantum efficiency by the comparative method is as follows. k First a standard solution of a compound with a known (documented) QE value is run and the ratio evaluated. In this work the standard solution was 2.3 ug/ml rhodamine B in ethanol. The value of QE determined for rhodamine B is set equal to the known value obtained under similar instrumental conditions. Rhodamine B QE Rhodamine B QE determined by (X) = value known from CRM instrument the literature Here X is the normalization value. All subsequent analyses of compounds of unknown QE employ the following expression to allow determination of relative QE values: -l66— Unknown Species QE Unknown Species QE determined by (X) relative to CRM instrument rhodamine B QE Table XII presents a study comparing the results in using the CRM instrument with those reported in the work of Parker and Rees [55] in determining total quantum efficiency by the comparative method. All of the fluo- phore solutions considered were characteristic of relatively high (compared to most fluorophores) fluo— rescent quantum yields. This selection was based on the desire to minimize errors due to low fluorescence intensity signals. 2. Use of QE Values and Comparison to Standards Normalization of the CRM instrument determined quantum efficiencies to standards referred to in the literature permits (facilitates) comparisons with values obtained in other laboratories. Unknowns are thus evaluated on the same scale as the reference compounds. Unfortunately, there are many types of instruments and mathematical models used in determining quantum efficiencies, causing the wide range of values reported in the literature for identical chemical systems. All of this, of course, assumes a high degree of purity for the fluorophores analyzed (perhaps a great deal of liberty is taken by some research groups in making this assumption). Many of the earlier values are in error because the emission ~167- TABLE XII. Total Quantum Efficiencies by the Comparative Method for Fluorescein, Eosin and Quinine with Rhodamine B as the Reference Standard. Fcompound/Fstandard 85595 EEEER Aex ’ nm FF / FR 1.35 1.38 366 FE / FR 0.40 0.34 366 FQ / FR 0.86 0.80 366 52y; FF = QE for 1.5 ug/ml fluorescein in 0.1 N NaOH FB = QE for 2.5 ug/ml eosin in 0.1 N NaOH FQ = QE for 1.0 ug/ml quinine sulfate in 0.1 N H2504 FR = QE for 2.3 ug/ml rhodamine B in ethanol MCR = Method of Cell Rotation P&R = Results from work of Parker and Rees *The reference standard was assigned a QB value of 0.69. -l68— spectrum was not corrected for primary and secondary absorption when needed. The experimentalists that were aware of the absorption problems merely made the measurements for low concentrations to minimize these sources of error. I. Relative Fluorescence Efficiency 1. A Pure Fluorophore The first goal in the study of relative fluorescence efficiency (RFE) was to develop a theoretically sound I model to permit evaluation of this quantity. Such an I expression has been provided in Equation (III.19). When all CRM instrument detectors, R—PVC, S—PVC and F-PMT, are calibrated and their outputs normalized to relative numbers of quanta, the profile of the RFE curve for a chemical system (solution) consisting of a pure fluorophore in a non-absorbing background matrix is expected to be box—like and to be centered on the peak maximum of the excitation spectrum. Theoretically predicted RFE curves are illustrated in Figure 55. Figures 56 and 57 provide examples of two pure fluorophores in solution and the RFE curves of each. The RFE curves are horizontal across the wavelength range covered by the absorption band as expected. Thus, the terms of the first goal have been satisfied. RFE ~169— RELATIVE FLUORESCENCE EFFICIENCY abs ideal 7 ex'citation wavelengths ---—-.---—.q .............—o, ——-——--—-.J Figure 55. Model relative fluorescence spectra. A. Case representative of samples possessing a single (pure) fluorophore in a non—absorbing matrix; solid line = shape of the ideal RFE curve centered on the absorption peak maximum; dotted line =absorbance curve. B. Case typical of samples that possess a single fluorophore in a strongly absorbing matrix containing one or more broad (absorption) band chromophores; solid line = RFE curve shape mimicing the absorption profile of the fluorophore; dotted line =ideal RFE curve for same fluorophore expected if not in the presence of the absorbing chromophores. C. Case illustrative of an RFE curve for a mixture of two fluorophores of differing quantum efficiencies absorbing in the same wavelength range; solid line =RFE curve characteristic of such a mixture; dotted lines =ideal RFE curve for each fluorophore. D. Case typical (in protein solutions) of RFE curve of narrow (absorption) band Chromophore in presence of a broader (absorption) band fluorophore; solid line =RFE curve characteristic of such a mixture; dotted line =ideal RFE curve for the fluorophore not in the presence of the Chromophore. Figure 56. ~170— The constancy of the RFE curve with respect to the excitation spectrum of L—tryptophan. Sample — 5 x10'5 M L-tryptophan, 1 x10“7- M Tris, pH 7.0. Aem =37O nm. A. Plot of total absorbance and source corrected fluorescence vs Aex- B. Plot of RFE vs Aex- TOTAL ABSOR BANCE AND -171- I 1 I I I I 1 I I a; _ - O D _ - _I LI... 8 '_ - a U LIJ " .I a m I- a— O U LIJ F q o __ . _ CI 3 O - -. U) 240 260 280 300 I 320 340 EXCITATION WAVELENGTH , m. B LIJ u. r a a 240 260 280 300 320 340 EXCITATION WAVELENGTH , hm. —172— Figure 57. The constancy of the RFE curve with respect to the excitation spectrum of rhodamine B. Sample - 8 ug/ml‘rhodamine B in ethylene glycol. Aen‘= 610 nm. A. Plot of total absorbance and source corrected fluorescence vs xex‘ B. Plot of RFE vs Aex' TOTAL ABSORBANCE AND -l73- I I I I I l I I l 05.. _ O 3— 4 LI. C) lJJ- .. *— U L”— "l as o:_ a O U I— .— m U_ _ cc 3 O— .. U) I I I I I l I J I . 220 4IO GOO EXCITATION WAVELENGTH , mm. B I I I I I I I I I RFE ‘I AA‘A. Vvv I - I I I 220 4IO GOO EXCITATION WAVELENGTH , hm. —l74- The second goal of the RFE work was to document the utility of the RFE output. The remaining sections listed under the RFE heading will explore the function at various levels of application. Among these are included the following: a. fundamental effects due to sample solution condition and composition; b. elucidating information on the three dimensional structure of a particular molecule or molecular complex; and c. routine quality control analyses to determine the purity of a fluorophore. Table XIII provides an overview of the applicability of RFE " at the aforementioned levels. 2. Effects of Large Changes in Solution Absorbance The RFE values are independent of the compound concen— tration in solution with total absorbance below 1.0 absorbance units. Above this absorbance level a slight absorbance dependent decrease in the RFE value is observed. Figure 58 illustrates the correlation between RFE value and absorbance for quinine sulfate in 0.1 N H2504. At absorbances below 0.02 absorbance units the fluorescence of quinine sulfate is low and results in decreased accuracy in the FS and (R1 —Sl) quantities used in evaluating RFE. The probable cause of the lower RFE values in highly absorbing solutions is illustrated in Figure 33. It should be pointed out that this effect does not usually pose a serious limitation to the usefulness of the RFE curve since in most studies the solution absorbance would be altered slightly, if at all. TABLE XIII. -l75- Utility of Relative Fluorescence Efficiency. A. Factors affecting RFE: O mflmmwaI-J PIA H‘OIO 12. l3. 14. 15. 16. Viscosity of the solution Temperature Solvent polarity Ionic strength State of chemical ionization (pH effects) Presence of matrix fluorophores Presence of matrix chromophores Emission spectrum position Concentration effects (aggregation/dissociation) Quantum efficiency Light scattering Changes in refractive indices Secondary absorption effects Presence of metal vs non—metal ions Presence of large vs small ions Presence of molecular oxygen in matrix B. Biochemical application of RFE: 1. 2. Fingerprinting 3D structure of proteins (micro-environments) Selective perturbation of proteins (quenching of TRP, TYR and/or PHE; effects on internal vs external location of these amino acids; 3D mapping of macromolecules) Correlation of energy transfer and the relative locations of TRP, TYR and PHE C. Quality control application of RFE: l. 2. 3. Chemical synthesis Biomolecular isolation Clinical laboratory compound purity monitor Figure 58. —l76— Correlation between RFE values and concentration for quinine sulfate in 0.1 N H2504. Samples - quinine sulfate (08) in 0.1 N H2504, 1. 1.00 x10'” M QS, 2. 0.75 X10“+ M 85' 3. 0.50 x10'” M gs, 4. 0.25 ><10‘l+ M QS, 5. 1.25 X10” M QS, 6. 1.50 X10" M QS. A. Plot of total absorbance and source corrected fluorescence vs Aex' B. Plots of RFE vs Aex- Note — 1.00 X10“+ M QS in acidic media is approximately equal to 1 absorbance unit when measured with the CRM instrument. TOTAL ABSORBANCE AND SOURCE CORRECTED FLUOR. -177- I I I I l I I I 270 300 33G 360 390 420 EXCITATION WAVELENGTH , mm. RFE I I I L I I I 270 BOO 330 360 390 420 EXCITATION WAVELENGTH , nm. —l78— 3. Chromophore Contamination in Fluorophore Solutions When the presence of the contaminating Chromophore has a negligible effect on the quantum efficiency of the fluorescence process, only the denominator of the RFE equation will be influenced. The denominator, R1 -Sl, is a measure of the number of quanta of excitation radia— tion absorbed by the sample at a given wavelength. If portions of the Chromophore and fluorophore absorbance spectra overlap, then it is these regions of overlap that will show a decrease in the RFE value. This effect is illustrated in Figure 55D. Typically, if the amount of the Chromophore contaminant is small, then the decrease in that region of the RFE value will be accordingly small. Such localized attenuations in the RFE curve can be used to monitor the purity of the chemical system with regard to absorbing contaminants. 4. Effect of a Second Solution Fluorophore Solutions containing two fluorophores fall into either of two categories with reSpect to RFE values. The first category involves mixtures of fluorophores where many of the excitation wavelengths overlap, but none of the emission wavelengths. Here the RFE equation numerator, FS, is not altered, but the denominator, R1 -Sl, is. In this case the second fluorophore behaves as a chromo- phore. This situation can be routinely observed in protein solutions. Since most proteins are found to contain both L—tryptophan and L—tyrosine amino acid —l79- residues, which are both fluorophores absorbing in approxi— mately the same wavelength range, but with emission bands in different ranges, it is possible to selectively observe the emission of the L—tryptophan amino acid residues, Aem =370 nm, without observing the emission of the L- tyrosine amino acid residues. An example of this case is illustrated in Figure 55D. The second category involves mixtures of fluorophores where many of the excitation and emission wavelengths overlap. Here both the RFE equation numerator and denominator will reflect the presence of the two fluorophores. In this case the RFE curve will be a composite (average) of the individual RFE curves of the two fluorophores. This type of composite RFE curve is generally difficult to use for interpreting effects involving the individual fluorophores. In these cases it is useful to separate numerator and denominator value changes. An extreme example of this case would be a mixture of quinine sulfate and quinidine sulfate in 0.1 N H2804. These two fluorophores are stereoisomers and possess identical excitation and emission spectra, and also exhibit identical fluorescence quantum efficiencies. Another, less extreme, example is a mixture of L-tryptophan and L—tyrosine with the emission monitored in the 310—350 nm range where there is substan- tial overlap between the emission bands. -180- 5. Effect of a Change in Solvent Polarity Change in the solvent polarity (including mixtures of solvents) surrounding fluorophore molecules occasionally has a notable influence on the fluorescence process. The effect of polarity changes on RFE values can be quite dramatic. Figure 59 illustrates the effect of polarity on L-tryptophan. In this study the matrix polarity was altered by preparing various ratios of water and ethanol. Initially, a small percentage of ethanol (20%) would have a large effect on RFE (20% decrease). Above a certain percentage of ethanol (40%) the effect on RFE remains virtually unchanged (remains at a 30% decrease). In this case the emission process is influenced more than the excitation process. The range of operation of the CRM instrument is between 220 and 550 nm. Here two types of electronic transitions are normally observed, n to n* and n to n*. Typically, the energy for n to n* transitions is often increased in going to more polar solvents, while that for a n to n* transition suffers the opposite effect. The L-tryptophan molecule contains a conjugated chromOphore which often involves the n to n* transition. Thus, the observed decrease in the RFE value is appropriate and expected. The other two common electronic transitions, 0 to 0* and n to 0*, are normally observed at around 120 nm and 180 nm, respectively. Such transitions are not monitored by the instrument. -181- Em .2: Ohm". K .Hocmnum wom .uwums wom I 0am>aom .v .Hocmsum woe .Hmum3 wow I ucm>Hom .m .Hocmsum wow .prmz wow I ucm>Hom .N .Hmumz wOOH I ucw>H0m .H .mowmo HHm :H ccnmoummuul IQ z :IOHx H I mchEmm .Hocmnuw cam umHMB mo mmusuxHE cH cosmoum>HuI IA mo w>uso mum can so >UHHmHom Hcm>HOm mo uommmm one Ihozm4m><>> ZO_._.<._._oxm .5: .. 05 00m 00m Omm Ohm me 0mm Gum .mm musmHm 4 _ / L H L K N\\ K, TI I I I I I I I I I I I 338 -182- 6. Effect of Changes in the pH of Aqueous Solutions The fluorescence emitted by many aqueous fluorophores is effected by changes in the state of protonation of some functional groups. Many published procedures for preparing aqueous fluorophores are careful to specify the optimum pH range, with frequent recommendations for the use of buffer controls. Variations in pH usually change the ionic/molecular structure of the species under study. The H+ typically bonds with the n-electrons of either oxygen or nitrogen. Since only the neutral or dipole molecules, negative or positive ions, in the univalent or polyvalent state may be the fluorescing species, the pH must be controlled to produce and maintain this species. In some cases it was possible to identify and dif— ferentiate solution components at two different pH values. One example of such a chemical system studied includes o-hydroxybenzoic acid and m-hydroxybenzoic acid. The 0- and m-hydroxybenzoic acids both fluoresce at pH 12 where they are assumed to be in the doubly associated form. Only the ortho form fluoresces at a pH of 5.5 where they are assumed to be in the singly dissociated form. In other cases the changes in pH can induce conformational changes in macromolecules such as proteins and enzymes. Generally this is undesirable and solutions containing these macromolecules must be buffered against pH changes. —183- All of the solutions involving macromolecules in this dissertation work have been appropriately buffered (with Tris in most instances). All of the changes in fluorescence that are documented above can be monitored by corresponding changes in the magnitude of the RFE values. Proteins and enzymes are known in some cases to also change the shape of the RFE curve. This is possibly a function of the extent of intramolecular energy transfer between phenylalanine, tyrosine and tryptophan (amino acid) residues and the conformation of the macromolecule. 7. Protein Studies The ultraviolet absorbance spectra of proteins and enzymes can be (roughly) divided into three regions: a. above 250 nm; b. between 250 and 210 nm; and c. below 210 nm. These divisions can be paraphrased by calling the first region simple since there are only a few absorbers and that they are easily sorted out, calling the second region complex due to the multiplicity of the contributions to absorptivity, and calling the third region very complex because of the peptide bond absorption and its conformation dependence. As a consequence of the first region being simple and most readily accessible to experimentation, the majority of the reported protein and enzyme Spectral studies concern it. The literature documents the most extensive informa- tion on the main contributors to this region. For proteins -184— and enzymes there are only three intrinsic fluorophores (unless fluorescing prosthetic groups are present), the amino acid residues tryptophan (TRP), tyrosine (TYR) and phenylalanine (PHE). At wavelengths greater than 200 nms all three amino acid residues show spectra composed of two major absorption bands, a strong band at 210 to 220 nm, and a weaker band at 260 to 280 nm. The separa- tion of these two bands is approximately the same for all three of these aromatic chromophores. It is fortunate, however, that there are large differences in the quantum efficiencies of these species. This is particularly notable in the weaker (longer wavelength) bands. The absorption maxima are sufficiently separated to permit excitation of the tryptophan residues without exciting (significantly) either the tyrosine or phenylalanine residues. This can be accomplished with excitation at 295 nm. These absorption bands represent similar transitions in all cases. In practice, tryptophan fluorescence is most commonly studied, because phenyl- alanine has a very low quantum efficiency (relatively) and tyrosine fluorescence is frequently very weak due to quenching. The fluoroescence of the tyrosine is almost totally quenched if it is dissociated, or near an amino group, a carboxyl group, or a tryptophan. The present studies on protein fluorescence and the RFE evaluations can be grouped according to the following general rules: -l85— a. All of the fluorescence emitted by a protein molecule is due to residues of TRP, TYR and/or PHE unless the protein contains another fluorescent component. This, of course, assumes a high level of protein purity to avoid contamination by other fluorescing compounds. Besides electrophoretic determination of purity, different lot numbers (company bookkeeping for purified batches) can be readily compared using the RFE curve height and shape. In this manner it was ascertained that the commercially available purified proteins were sufficiently pure for use in these studies. b. The maximum wavelength of the TRP fluorescence spectrum shifts to shorter wavelengths and the intensity of the emission increases as the polarity of the solvent decreases. If the maximum wavelength is shifted to shorter wavelengths when the protein is in a polar solvent, the TRP must be internal and in a nonpolar environment. Note that these shifts are relative to the maximum intensity wavelength of the free amino acid in water or in 0.02 M Tris buffer. If the maximum intensity wave— length is shifted to shorter wavelengths when the protein is in a nonpolar medium, either the TRP is on the surface of the protein or the solvent induces a conformational change that brings it to the surface. The RFE curve magnitude is observed to change as the solvent polarity changes indicating the accessibility of the TRP residues to the solvent in most cases. The change in magnitude —186— corresponds directly to the emission peak shift. The shape of the RFE curve is generally observed to remain the same. In all of the reported RFE evaluations the TRP emission is monitored at 370 nm. At this wavelength the TYR and PHE emissions are negligible. The selection of the excitation wavelength was based on the desired or acceptable optical transparency. PHE is effectively optically transparent above 270 nm, TYR above 295 nm, and TRP above 315 mm. c. If an ion or molecule is known to be a quencher of the fluorescence of the free TYR or TRP, such as the iodide, nitrate or cesium ions, then any protein affected by these species probably has TYR or TRP residues in accessible positions, probably on its surface. When these species fail to quench, or quench partially, there are the following possibilities: a. the TYR or TRP residues may be internal and (effectively) hidden; or b. the TYR or TRP may be in a highly charged region and the charge might repel the quencher. Since many of these quenchers absorb in the same wavelength range as the protein, the RFE curve will show shape changes in the region where the quencher absorbs. Due to the ability of the CRM instrument to separately output the numerator and denominator of the RFE function, true quenchers (exhibiting numerator effects only), absorbing quenchers (exhibiting both numerator and denominator effects) and non—quenching species that effect only the conformational —187- structure of the protein (generally exhibiting both numerator and denominator effects) can be distinguished. d. When a substance does not affect the quantum yield (fluorescence) of either the free TYR or TRP, but does affect the quantum yield of a protein, it may do so by inducing a conformational change in the protein. A chemical substance that causes such a change is sodium dodecyl sulfate, a denaturant of proteins. It does not absorb in the same wavelength range as either the TYR or TRP, and it does not quench either of these amino acids in the free state. However, its effect on the observed fluorescence intensity of proteins can sometimes be quite dramatic. It has been reported [161] that sodium dodecyl sulfate denatures proteins by forming rod—like complexes with each polypeptide chain as illustrated in Figure 60. A study showing the effect of incremental additions of sodium dodecyl sulfate to a native protein, bovine serum albumin, is presented in Figure 61. The RFE curve is altered in both magnitude and shape. The ramifications of such RFE curve changes with regard to intramolecular energy transfer will be discussed later in this section. The RFE curves or "fingerprints" were obtained for several proteins under the following conditions: a. native protein buffered in a matrix of 0.02 M Tris at pH 7.0; b. protein denatured by two hour incubation in a matrix of 6.0 M guanidine hydrochloride; and c. stoichiometric -188— 6 E G G G G 6 THE SDS—POLYPEPTIDE COMPLEXES Figure 60. The process whereby sodium dodecyl sulfate causes protein denaturation. Upper diagram — polypeptide (protein) in a typical native configuration. Lower diagram - rod—like complex that is thought to form between sodium dodecyl sulfate ions, represented as a chain with a polar (negative) head group, and the polypeptide, represented as a rod. N =NH2 terminus of the polypeptide, C =COOH terminus of the polypeptide. .2 To: 6 .s .95 z mIon m .o .26 2 To? v .m .2 To: m .v 690. 2 To: m .m .mom 2 To? H .m .mom 02 .H .oK mm 639 2 To: H 3 En z muoax m u 835mm .Emm: 553$ Ednwm mcH>on mo COHumucumcmp Amomv quMHsm Hwowpop EDHGOm .Hw mHDmHm .Es Ihozm4m><3 ZO_._.<._._oxm m3” mam _ _ _ _ _ (H, _ _ _ _ ,///, % I h l 1. .. .m I 0., l HEW .n . Smog" A a I A ZOHHN0m>H wUHsz moo cwaHno .0 .QHEDQHM Eduwm amass .m .CHESQHM Esuwm mcH>on .< to.h mm um mHMB z NIOHx H CH chuoum z mIOHx m I memEmm .CHuomHOHmImuwn paw cE>NOm>H wuH£3 mmw :monso tCHESQHM Edhwm omen: .CHEDQHM Eduow 0:H>on How =mucHHmemcHw= Ammmv xocwHonmm mocoomwuosHm w>HumHom £11523m><3 zoionm mmm mmm 9% 3m mmm mmm T E -.llllfll\ - a - . m a": m a”: I m .m: - - m a: I - m .mxa - w - n .wxa m. 3 3 £x.Ihozw4w><3 mmm mmm mmw mmm _ .dmc. I ww.m>h H U - N¢.w1d .3 A _J a. .mo musmam ‘192- 100 I I I I I a ..I >. c) 5% 5 E ._ Aem = 370 nm _ /\ J EI- -I g C (I) LIJ or. ._ . c> D s B :I f5 :2- I 0. I I 1 I I 250 270 290 310 EXCITATION WAVELENGTH, nm Figure 63. Relative fluorescence efficiency (RFE) of bovine serum albumin (BSA, both in native and denatured conformations) versus the stoichiometric mixture of free PHE, TYR and TRP molecules equivalent to the protein composition. Samples —. A. 5 x10'5 M BSA, 1 x10"2 M Tris, pH 7.0; B. 5 ><10"2 M Tris, pH 7.0. 5 .0 -l93— 4’447 I I I T T I I I LI.) L) 2: LLJ u _ _ m LIJ m C) I) ._I LI. _ ._ Q —- —I LLI I— (.3 E! g _ nm Excitation — L) nm Excitation I— —I LIJ U cc 2) o _ _ m 2: C) : _ _ O. M c: U? CD < e _ Q I I L a, I I I J l 300 320 340 360 380 400 EMISSION WAVELENGTH, nm Figure 64. Total absorption and source corrected fluorescence of bovine serum albumin (BSA) in the native state. Sample — 5 ><10"5 M BSA, 1 ><10'2 M Tris, pH 7.0; A. Aex=275 mm, B. Aex =295 nm. Curve matching at 340 nm. o .0 I -l94- FLUORESCENCE CORRECTED ABSORPHON ISOURCE 9 275 nm Excitation ' 295 nm Excitation i I I I I L I I I I r 300 Figure 65. 320 340 360 , 380 400 EMISSION WAVELENGTH, nm Total absorption and source corrected fluorescence of bovine serum albumin (BSA) in a denatured state. Sample a 5 X10“5 M BSA, 6 M guanidine hydrochloride, 2 hour incubation period at room temperature; A. Xex==275 nm, E. Aex==295 nm. Curve matching at 350 nm. 8 9 —l95— FLUORESCENCE CORRECTED ABSORPTIONI SOURCE O nm Excitation I I I J I I l I I 300 Figure 66. 320 340 360 380 400 EMISSION WAVELENGTH, nm Total absorption and source corrected fluorescence of the stoichiometric mixture of free phenylalanine (PHE), tryo- sine (TYR) and tryptophan (TRP) molecules equivalent to the bovine serum albumin composition. Sample — 1.45 ><10~3 M PHE, 5 XlO'L‘ M TYR, 1 x10‘“ M TRP, 1 x10'2 M Tris, pH 7.0; A. Aex=275 nm, B. xex=295 nm. Curve matching at 350 nm. H ~196— FLUORESCENCE EFFICIENCYS RELATIVE I l I I I Figure 67. 270 290 310 EXCITATION WAVELENGTH, nm Relative fluorescence efficiency (RFE) of human serum albumin (HSA, both in native and denatured conformations) versus the stoichiometric mixture of free PHE, TYR and TRP molecules equivalent to the protein composition. Samples - A. 5 x10'5 M HSA, 1 x10'2 M Tris, pH 7.0; B. 5 x10‘5 M HSA, 6 M guanidine hydrochloride; c. 2.35 ><10-2 M Tris, pH 7.0; A. Aex=275 mm, B. xex =295 nm. Curve matching at 350 nm. Figure 69. ... o ,0 -198- FLUORESCENCE CORRECTED 275 nm Excitation LIJ L) C! D — —t O 295 nm Excitation V) g A : _ s D. o: O W to < t' _ O. I l l I I I I l l I 293 310 330 350 370 390 EMISSION WAVELENGTH, nm Total absorption and source corrected fluorescence of human serum albumin (HSA) in a denatured state. Sample — 5 ><10‘5 M HSA, 6 M guanidine hydrochloride, 2 hour incubation period at room temperature; A. Aex =275 nm, B. Dex =295 nm. Curve matching at 350 nm. Figure 70. ~199— 100' I I I I I I I T I I E 2 LI.) U h —I m LLJ D: O D E _ £3 E s \ z _ 275 nm Excitation ‘ O Q 295 nm Excitation [- _ E m D *- .. O m 2 O : - _ G. o: O V) m < — .. o. I I I I I I I I I 290 310 330 350 370 390 EMISSION WAVELENGTH, nm Total absorption and source corrected fluorescence of the stoichiometric mixture of free phenylalanine (PHE), tryosine (TYR) and tryptophan (TRP) molecules equivalent to the human serum albumin composition. Sample - 2.35 x10'3 M PHE, 1.3 x10‘3 M TYR, 5 x10"5 M TRP, 1 xlo‘2 M Tris, pH 7.0; A. Aex =275 nm, E. Aex =295 nm. Curve matching at 350 nm. —200- ._. O O EFFICIENCY _ FLUORESCENCE RELATIVE 0. 1 | l I | 250 270 290 310 EXCITATION WAVELENGTH, nm Figure 71. Relative fluorescence efficiency (RFE) of chicken egg white lysozyme (LYS, both in native and denatured conformations) versus the stoichiometric mixture of free PHE, TYR and TRP molecules equivalent to the protein composition. Samples - A. 5 x10'5 M LYS, 1 x10“2 M Tris, pH 7.0; B. 5 x10'5 M LYS, 6 M guanidine hydrochloride; c. 1.5 x10"+ M PHE, 1.5 ><10°l+ M TYR, 3 x10'“ M TRP, 1 x10"2 M Tris, pH 7.0. 6 9 —201- FLUORESCENCE CORRECTED ABSORPTION I SOURCE p nm Excitation nm Excitation I I I I I I I I I 290 Figure 72. 310 330 350 370 390 EMISSION WAVELENGTH, nm Total absorption and source corrected fluorescence of chicken egg white lysozyme (LYS) in the native state. Sample - 5 x10'5 M LYS, 1 x10“2 M Tris, pH 7.0; A. )‘ex =275 mm, B. Aex =295 nm. Curve matching at 350 nm. —201— mo , , T 1 1 Her I I I I Z Z 0 _ .. LI) LU (r O D D _ _ E L) E m )- .—I O U 3 D: D .. .. O m 2 3 EC 295 nm Excitation D! O m m “—275 nm Excitation < _ _ O I J IZ I I I I I I I 290 310 330 350 370 390 EMISSION WAVELENGTH, nm Figure 72. Total absorption and source corrected fluorescence of chicken egg white lysozyme (LYS) in the native state. Sample — 5 Xl0’5 M LYS, 1 ><10'2 M Tris, pH 7.0; A. )‘ex =275 nm, B. )‘ex =295 nm. Curve matching at 350 nm. -202— 5 9 I I I I I f I l l | LLJ U Z LLI L) _. _ m LIJ 0’: O D _I u. _ _ D _ LIJ )— U LL] 0: Q: .— .. O (J Lu L) DC 3 O _ J U) 2 275 nm Excitation O ._ _ _ CL . . a, 295 nm EchtatIon O m as <1: ~ _. o. I I J I I I I I l I 290 310 330 350 370 390 EMISSION WAVELENGTH, nm Figure 73. Total absorption and source corrected fluorescence of chicken egg white lysozyme (LYS) in a denatured state. Sample — 5 XlO' M LYS, 6 M guanidine hydro- chloride, 2 hour incubation period at room temperature; A. )‘ex =275 nm, B. )‘ex =295 nm. Curve matching at 350 nm. —203— .. o .0 FLUORESCENCE CORRECTED 275 nm Excitation “295 nm Excitation ABSORPTION I SOURCE I I I 330 350 370 390 EMISSION WAVELENGTH, nm I 20 M0 Figure 74. Total absorption and source corrected fluorescence of the stoichiometric mixture of free phenylalanine (PHE), tyrosine (TYR) and tryptophan (TRP) molecules equivalent to the chicken egg white lysozyme composition. Sample - 1.5 x10'” M PHE, 1.5 x10"+ TYR, 3 x10-“ M TRP, l xlo‘2 M Tris, pH 7.0; A. Aex =275 nm, B. Aex =295 nm. Curve matching at 350 nm. ‘_—__—_‘—‘ —204— simplified form in Figure 76. The numerator of the RFE function is evaluated by monitoring the TRP emission at 370 nm. Contributions to emission by the PHE and TYR residues are negligible at this wavelength. The denominator of the RFE function is evaluated by scanning the excitation wavelengths. With protein solutions the excitation wavelengths scanned are from 250 to 310 nm. The kinds of RFE curve shapes that are typically observed for solutions containing the native or denatured conformations of a protein and their stoichiometrically equivalent mixtures of the PHE, TYR and TRP amino acids are illustrated in Figure 75. The RFE curves are normalized at wavelength "a as shown in the figure. At this wavelength, usually 295 nm, the absorbance of PHE and TYR is very low. (b/a) — (b/a) . Energy Transfer Index = sample StOlCh (v.6) (b/a)pure TRP-(b/a) stoich At wavelength "b", usually 275 nm, the major contributors to the primary absorbance are TYR and TRP. A protein with TRP residues, but no TYR and PHE residues, will produce a box-like RFE curve centered on the TRP absorp— tion peak. An exception to this would be a case where the protein possesses a prosthetic group that absorbs in this region. However, the proteins selected for these studies do not possess such prosthetic groups. A .m numcon>m3 um prHHmEuoc mw>Mso mum .AE: mum wHHmonwuv mmB pom mwe mum cocmnMOMQM 0p mHOHDQHHucoo HommE muons numcwHo>m3N Q .AE: mom >HHmon>uV 30H MH mwB Ucm mmm mo cosmnHOQO ohm£3 cpmcmHm>m3n 0 Emma pom mwa .mmm mo wHDHxHE ucon>H5vo wHHMOHHquOHSOHoum .Q .ckuoum mo coHu Imshomcoo consumcwp .0 .cproum m0 SOHHMEHOMGOU o>Humc .m .mmh comm .¢ I monEom .coHpsHOM msoosgm CH mcHouOHm How xwch wamcmuu amumco can mcHumsHm>w Mom MHmmn >ocoHUHmmw cocoowwHODHm w>Humeu wfiu m0 coHumucommhmwu OHQQMHO .mn wusmHm Ieozmnm><>> ZOC.Nomwn muHQB 4 no.0 i\+ 00.0 no.0 -\+ 00.0 no.0 I\+ 00.0 000 soxonso .o ansdnna no.0 I\+ mm.0 no.0 I\+ em.0 no.0 -\+ 0m.o esnmm eases .m sneenna no.0 I\+ mn.o no.0 I\+ 0m.0 no.0 I\+ om.0 esnwm msn>om .0 poncumcoo onsutz onsutz :Hwnonm m> w>Hucz UHHuoEOH£UHoum OHHMmEoHcoHoum m> consumcmo m> o>Humz .mcHouonm cosfioo oonSB now mcoHumsHm>m Moch nommcmnfi >mnocm wocwHonmm oocmumonosHm m>HumHom .>Hx mamfiB —210- transfer; 0. inability to correct the fluorescence measurements for attenuations due to primary and secondary absorptions; and d. scanty or non—existent verification of protein and solvent purity. These problems could be greatly minimized by a concerted effort to standardize the instrumentation, theoretical models, spectral corrections, and protein and solvent purification techniques. CHAPTER VI CONCLUSION The title of this dissertation, "Explorations in Design and Performance of an Instrument Utilizing the Method of Cell Rotation in Molecular Spectrometric Chemical Analysis", appears appropriate considering the instrument versatility and range of the overall project. The primary goal of this work was to design and construct an improved instrument that could make accurate correc— tions on molecular fluorescence measurements for the effects of primary and secondary absorption on a practical, rapid and routine basis. Work completed and described in an M.S. Thesis [2] initiated the way toward reaching this goal. That work resulted in an instrument that was a crude prototype of the present system that revealed the potential of integrating both a spectrophotometer and a spectrofluorometer to operate under the Cell Rotation Method. This instrument showed that accurate corrections could be made of primary and secondary absorption attenuation of molecular fluorescence measurements. The subsequent goal of this work was to explore and charac- terize a vareity of other capabilities afforded by an instrument operating under the Cell Rotation Method. A -211- -212- summary of the instrumental outputs that are currently available on a practical, rapid and routine basis is as follows: a. reference, sample or fluorescence signals normalized to numbers of quanta; b. source corrected fluorescence; c. primary and/or secondary absorption corrected fluorescence; d. total quantum efficiencies by the comparative method; e. relative fluorescence efficiencies; f. primary absorbance and the corresponding primary transmittance determined spectrophotometrically; g. primary absorbance and the corresponding primary transmittance determined spectrofluorometrically; h. secondary absorbance and the corresponding secondary transmittance determined spectrofluorometrically; difference spectroscopy, subtraction capability of any two spectral functions generated from the same data file; j. turbidimetric and nephelometric quantities; k. fluorophore absorption curve stripping; and 1. multiple derivative capability for all spectral data. The instrument based on the Cell Rotation Method has a number of desirable features and advantages over commercially available spectrofluorometers and/or spectrophotometers. They can be considered among the following. 1. The system is very "User Friendly" in its operation. 2. Automatic correction of fluorescence measurements for source fluctuations, primary and/or secondary absorption attenuations is possible. 3. Absorp- tion corrected fluorescence is linear with the fluorophore —213— concentration in solutions with primary and/or secondary absorption as high as 2.5 Absorbance units. 4. All system detectors (R—PVC, S—PVC and F—PMT) are calibrated to provide output in relative numbers of quanta. 5. The "Cell Rotation Device" provides a very high degree of precision in sample cell positioning. 6. Two indepen— dent methods of absorbance calculation provide an internal diagnostic of stray light and enable accurate measurements on compounds of low solubility. 7. The relatively small size of the cell positioner assembly allow its incorpora— tion into the sample cell compartments of other spectro- fluorometers of right-angle geometry for conversion to a "Cell Rotation" system. 8. Computer control of the operation of the system is of sufficient speed to permit routine sample analysis, corrective calculations, and long term data storage and retrieval. 9. Turbidi- metric and nephelometric measurements are proportional to the amount of light scatterer present. 10. The rotation of the sample cell results in continual and thorough solution mixing and, therefore, preventing solute gradient formation and inconsistensies in the density of light scattering particles. 11. Real—time acquisition and processing capability permits use of the non—scanning mode for studies of photophysical effects and monitoring of the instrumental performance. 12. The fluorophore absorption curve stripping function results in solution Chromophore spectra, extracted from mixtures —214— of chromophores and fluorophores, matching closely the primary absorbance spectra of the chromophores alone. This list is by no means considered to be exhaustive, but rather to emphasize the currently realized potential of this type of instrumentation. Spectral data are very conveniently outputted. The "Display" command plots a graph of any raw or modified wavelength dependent data on the video terminal. The "Print" command tabulates and prints out any raw or modified wavelength dependent data either on the video terminal or the printer. The "Real-Time" command allows selection of any raw or modified wavelength dependent data to be repeatedly output to the video terminal or printer once per second. The "Pointer Reset" command allows the resetting of the beginning and end data file pointers to permit exploded viewing of a fraction of a spectral scan and subsequent ouputting by the "Display" or "Print" commands. The "Average" command calculates the average value of the raw or modified wavelength dependent data between pointers pre— set with the "Pointer Reset" command. The "Plot" command will cause a hardcopy to be made of any plots of spectral data that can be displayed on the Video terminal. Both the "Display" and "Plot" commands permit overlapping of an unlimited number of spectral scans provided the scanned wavelengths are matching. The plots of buffered spectral data can be displaced along the Y-axis, usually set at —215— mid—field, and output through the "Display" or "Plot" commands whenever negative values are expected (as in multiple derivative outputs). Selective application of a broad range of Y-axis scaling factors (multiplier constants), from 0.000001 to 1000000, is possible for any spectral data output by the "Display" or "Plot" commands. A variety of chemical and biochemical systems were used to investigate the applicability of the CRM instru- ment outputs. Table XV is a summary of the systems reported in this dissertation, correlating them with the solute concentrations applied, background matrices, specific CRM instrument output functions used, and the chemical and/or physical limitations encountered during their use. The relative fluorescence efficiency (RFE) function has been applied in the study of a variety of chemical and biochemical systems. Both chemical and physical factors have been found to influence the shape and magnitude of the RFE curves. A summary of these factors can be found in Table XIII. The shape of the RFE curve is not dependent on factors affecting the fluorescence quantum efficiency of the fluorophore, but is directly related to the composite of solution component absorbances in the excitation region of the fluorophore and on any associated energy transfers between these components and the fluorophore. 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E OHx oo.H Cmnmoumwualn l a s fil . .2 H m e z 0nx om 0 one wsnmouseun _ .m swnwsm suns mm conumun5nn 0200 I .m .N .n z Mionx mv.n .mcHsmnmnmconmun .e meuoHCoonphm wCHpHCmso z o 0 .m .4 an anesnnm .n swpmsm nuns mm 00n000n5nn 050m r .m .m .n z m onx m Edumm 00n>om .m 0.0 mm pm : Ionx o mnue z no.0 an m on mnmnnsm z 0nx n nsoeooo senoom 0 .m .0 mI UCm CHECQHC .H Eoumwm CHHB mm CoHumuHEHH wEmm « .m .N .H E mIOHx m Edhom oCH>om .m MCOHHMHHEHH HMUHm>£m\HmoHEwCU usmuso memm COHHmnuCooCOU anmhm quEDHHMCH Ho COHHCHHCTUCOU HCOHEoCoon zmo mnemonnmmm \nmonsmno .UmSCHHCOU >x OHQMB “CUCNUWQHODHM COHUQHHOU QOHDOUIH m .HHHHHQCQMU o>Hum>HHcp meHuHCEN MH UCm nmCHmmHHum OUCMQHoQO THOCQOHOCHMH NH nmeuHHCCCw UHHuoEoHoCQoC pCM OHHquHpHQHCHN HH “MHmcme Nm pCm Hm Co comma ooCmuuHEmCmnu mumpCoowm mCHUCOQ IonHoo can pCm OUCMQHomnc humpCoowmn OH “MHMCmHm gm pCm Hm Co woman wOCMHHHEmCmHu mHmEHHm uCHpCommoHHoo 05H pCm moCm9H0mnm wnmEHHmn m “MHmcmHm m CCM C CO comma ooCCHuHEmCmnu NHmEHHm mCHpCommmHHoo map pcm TOCMQ Inomnm HHMEHHQN w “moHUCoHOHHmo woCoommHosHH o>HUMHoHN 0 uponuoa o>HumnmmEoo may HQ moHonHOHmwo Edqusg Hmuoun w “woCoomeosHm popoonuoo CoHumHomnm whmpCoowmu m “oonommHous Uwuownnoo COHumHomnm mumEHHmu v “mucmsw mo mHoQECC o>HumHoH Op prHHmEHOC MHMCmHm MUCoumeOCHw no meEMm .wocwnwmmnn N «Enom 30H CH MHmCmHm 00CcomoHOCHm Ho meEom .ooCcnowmnu H "mudmuso HCwECHuwCH Emu o» >0M a 0.0 mm on mnue z no.0 z Ionx 00.m an cosmoumsneun _ 0 .m .0 E vIOHx om.H pCm oCHmonhfiIH M” .C Ecummm CHHB mm COHumHHEHH ofimm * .m .N .H E WIOHX om.H .oCHCMHMHwaCmIH .N J opHHOHCOOthm oCHpHCmso z o 0 .m .v CH oshuoqu .n seemmm suns mm sonumHHEHn 0200 I m .N .n 2 m 0nx m eons: mom coroneo .H 0.» mm or mnne 0 .m .0 z no.0 CH 05>M0mwn .n smumsm gene we :0nuMannn warm I m .m .n z m onx m mung: 00m coronno .x 0.0 mm um mHHB z H0.0 z 0nx oo.m an cmnmonmsheun s .m .0 z mionx om.n osm esnmonsein .C Eouwxm CHHB mm CoHumuHEHH meow * m .N .H z MIOHX mm.N .oCHCMHmeCoCmIH .3 MCOHHMUHEHH HCUHm>Cm\HmoHEoCU HCQHCO omCmm COHuwnquoCoo Eoummm HCoECHumCH Ho CoHumHuCooCoo HCOHEoCUOHm EMU wHQCOHHmmC \HCOHEoCU . CODE ..nnvCOU >X CHAN”? —221— on factors affecting the fluorescence quantum efficiency of the fluorophore. The purity of a chemical or biochemical fluorophore can be ascertained through use of the RFE curves. Simple fluorophores that do not possess chromophoric regions in the same molecule or do not have chromophores present in the surrounding matrix result in a box—like RFE curve that is centered on the absorption peak of the fluorophore. The use of the term Chromophore in this context implies the ability to absorb a portion of the same wavelengths of radiation that the fluorophore absorbs. Two pure chemical systems demonstrating such box—like RFE curves are L—tryptophan (in a matrix of 1 ><10_2 M Tris at pH 7.0) and rhodamine B (in ethylene glycol) and are shown in Figures 56 and 57, respectively. Complex fluorophores that do possess chromophoric regions in the same molecule but that do not have chromophores present in the surrounding matrix result in very characteristic RFE curve shapes, or "fingerprints". Examples demonstrating such species specific RFE curves are found in solutions of proteins such as Bovine Serum Albumin, Human Serum Albumin, Chicken Egg White Lysozyme and beta—Prolactin in the native and/or denatured conformations and are shown in Figures 62, 63, 67 and 71, respectively. Due to the convenience of long term electronic storage of data files with the CRM instrument it is easy to compare RFE spectra from previously prepared solutions of the ~222- fluorophores with those that have been currently prepared. Any changes in the RFE spectra may indicate degradation of the stored fluorophore, accidental introduction of chromophoric contaminants, or less than adequate purifica— tion of these substances. The latter case is especially significant when obtaining different lots of proteins from biochemical supply houses where the required purification schemes are often complex and where quality control may be difficult to maintain. The RFE curve shape permits study of the extent of I energy transfer between donor and acceptor groups that are present in macromolecules such as proteins. The tyrosine (TYR) amino acid residues would be considered potential energy donors and the trypt0phan (TRP) amino acid residues would be the potential energy acceptors in proteins. Equation (v.6) and Figure 75 detail the basis and process for evaluating an energy transfer index for proteins in both native and denatured conformations. Table XIV provides a summary of these energy transfer index evaluations. Supportive evidence for the results of these evaluations is provided by the emission spectra taken at the excitation wavelengths of 275 nm (where both TYR and TRP residues absorb) and 295 nm (where only TRP residues absorb). The protein emission spectra are shown in Figures 64, 65, 68, 69, 72 and 73. Limitations in the evaluation of the RFE function are as follows. The concentration and/or fluorescence quantum -223- efficiency of the fluorophore must be sufficiently high to provide adequate signals, specifically for 81, F1 and F2, to allow accurate determinations of RFE values at all desired wavelengths. The minimum concentration and/or fluorescence quantum efficiency that will provide the necessary signal levels is, of course, dependent on the chemical or biochemical system. There is difficulty in obtaining accurate evaluations on the edges of an absorption (excitation) band due to low fluorescence signal levels and, thus, poor S/N ratios. In general the fluorescence signals that occur after amplification give reproducible RFE evaluations that are between 2 to 3 orders of magnitude above the background noise. This noise is usually low, at +/— 0.003 V, but is found to occasionally go higher, to +/— 0.010 V. CHAPTER VII LOOKING TO THE FUTURE The excitement in looking to the future is that it is open ended. Present day technology is so bountiful that those exploring the realm of new design in analytical instrumentation have but to exercise their imagination. No longer are we locked into a technological straight- jacket. Students of the newly developing analytical fields should find the near future as rewarding as their minds are full of ideas. Modern microelectronics have removed much of the drudgery from obtaining desired instrument control, information collection and processing, final data display and output options, so that attention may now be focused on the information and need not be focused on the tools that gather and make the information accessible. The work undertaken and described in this dissertation has led to a number of successes in solving problems in both instrumental design and analysis of chemical and biochemical systems. As this type of computer operated instrumentation evolves, more of the problems encountered in making accurate photophysical measurements will have become history. It appears inevitable that instruments -224— —225— of the future will have the capability for elaborate multi-dimensional analysis. The road leading to the future is long and the steps are many. What follows is a prediction of where the next few steps may lead. A. Two excitation monochromators can be used in a tandem arrangement to reduce the amount of stray light transmitted. This arrangement is shown in Figure 77. The use of two dispersing elements, either two prism monochromators, two grating monochromators, or a prism monochromator and a grating monochromator, will markedly reduce the amount of stray light and will provide greater dispersion and spectral resolution. Note that if one of the elements is a grating monochromator, higher order wavelengths are removed by the second element or by filters. B. A tunable dye laser could be used as a source in an absorbance correcting spectrofluorometer. The arrange- ment could be as shown in Figure 78. This arrangement would provide a considerably more intense (and probably much purer) source allowing the study of highly absorbing solutions. Employing a pulsed laser would permit the resolution of fluorophore mixtures according to their fluorescent lifetimes. The ability to identify chemical species according to emission lifetimes is well known. If the lifetime resolution capability were to be added to the absorption correction capabilities of the present -226- .uCoEmmCmHHm HoumEOHCUOCoE CoHu ImuHoxo EopCmu m OCHmonEo HouofiouosHmonuommm OCHuooHHoo mOCMQHomnm CC .00 onsmHm nCoCSCdQnCoo Zoo CC 08 CC 08 GOH 50m a. ....... IL/ ....... I .flm CPHu OH 08 80 -227- . ooHDom 0 mm HomoH ohm oHQCCCu w CHH3 kuofionosHuonuoomm @CHuocHHoo TOCCQHOMQC .m0 musmHm 4 0‘3 ll QOHHHOW. L / ......... . .803 who onnannn IE fie..- O. 0.... 0.. OHHOHHH am HCHQ 0H —228- instrument, much more accurate resolutions of complex mixtures would become possible. C. A high—speed absorbance correcting spectrofluoro— meter could be constructed using a tunable dye laser as a source and a photo diode array for spectral detection. The arrangement could be as shown in Figure 79. Besides the advantages of the instrument described in the previous paragraph, this system would have the added benefit of simultaneously observing each wavelength of an entire spectrum using photo diode array detection. This type of detection could potentially greatly increase the speed of sample analysis in obtaining emission spectra relative to that feasible with the current instrument. The actual increase would, of course, depend on the wavelength range that was being scanned and the absorption corrections desired. D. Absorbance correcting spectrofluorometers can be constructed without the necessity for moving parts within the sample cell compartment. Note the contributions in this area by Eugene Ratzlaff at Michigan State University [162]. The required information to perform the absorption corrections is made accessible by the use of fiber optics (or light pipes). The arrangement could be as shown in Figure 80. In this arrangement the sample cell does not move as it would in the Cell Shift Method or in the Cell Rotation Method, nor are moving mirrors involved in relocating the excitation beam or emission .CoHHowuwp Hmupuwmm Hem wound TCOHC ovosm m UCC condom 0 mm HoMMH who mHQMCCH m CHHB kuoEOHOCHMOHuoomm mCHuooHHoo ooCan0mnm comQMICmHC C .00 wHCmHm I I 00%50m L Home: 030 03.93:. 3 —229- _ _ QQOOOIOOIOQOOO 000.... henna oUoHHo onoCQ —230- .choEonosHmonuoomm mCHuownHoo ooCMQHownC .00 mesons 000E Em: Coaaono i Hnoo 03500 0... 00000000000“ ‘ 0 oCowé ooCCom H E .. j J 0000000000 0000000000 00000 000- CCmH onCOHH —23l— detection axis as it would in the Moving (Vibrating) Mirror Method. The sample cell always remains stationary. Light pipes are secured to the outside surface of the cell. They are positioned in such a way as to permit fluorescence intensity measurements to be made as follows: a. from two independent slices along the excitation axis; and b. from two positions along the emission detection axis. Since there is no movement of the sample cell, there is no time consumed in repositioning. A rotating chopper would be used in selecting fluorescence intensity from cell positions 1, 2, or 4. Also, some light reflection radiation losses would be reduced. E. Automated monitoring of sample solution tempera— ture and pH is feasible with an arrangement as that shown in Figure 81. The diagram shows the position of both the excitation beam and the emission detection beam passing through the lower half of the sample cell. The temperature could be monitored by use of a thermocouple (information transduced and forwarded to the multiplexer onboard the system computer) incorporated in a probe jacket. The pH could be monitored by a pH electrode (information transduced and forwarded to the multiplexer onboard the system computer) incorporated in a probe jacket. Both probes would be located above where the excitation beam and emission detection beam is located. Also, the probes would be located in such a position within the cell to avoid any contact with the cell walls during rotation. -232- Figure 81. Automated monitoring of sample solution temperature and pH. ~233— F. A jacketed sample cell for heating or cooling the sample solution could be built as shown in Figure 82. The diagram shows the position of both the excitation beam and the emission detection beams passing through the lower half of the sample cell. Also shown is the placement of the temperature controlling jacket. Not shown is the thermostated heating or cooling bath which cycles liquid through the IN/OUT port tubes on the upper portion of the jacket. This type of jacket is not intended for use with the current cell rotation system, but rather with an I instrument based on light pipes to obtain fluorescence I measurements from the internal cell positions (1), (2) and (4) (note Cell Rotation Method). The jacket would be made from stainless steel to facilitate temperature equilibration. Adjusting the sample contents temperature should be considerably quicker than use of a jacket surrounding the entire cell compartment. G. A jacketed sample cell compartment for heating or cooling the sample solution could be built as shown in Figure 83. The diagram shows the top view of the cell compartment to illustrate the position of the excitation and emission detection beams. Also shown is the heating/ cooling jacket surrounding the cell compartment (shaded domain) and the IN/OUT ports. Not shown is the thermo- stated heating or cooling bath which cycles liquid through the jacket. This type of jacket is intended for use with the instrument based on the Cell Rotation Method. The -234- Figure 82. Jacketed sample cell for heating or cooling the sample solution. —235- \ \\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\ /m CHHII. 0 0 \\ \\\\X .COHHCHOm onEMM ecu mCHHooo Ho mCHHmoC How HCwEuHmmEoo HHoo onECw coonUMb .mo mesons 00000 0 0 0 0 0 0 00 0 00000000 0000 0000 000 0000 00000000000 0000000 0 0 0 \\\\\\\\\\\\\\\\\\\\\\\ \\\\\ 7 I \\ ‘\\ \\\\\\\\\\\\\§ 0. NE C 3 0% .. UH —236- jacket would be made from stainless steel, copper or brass to facilitate temperature equilibration. Adjusting the temperature of the cell compartment atmosphere and sample cell contents should be somewhat slower than use of a jacket making direct contact with the sample cell. H. Automated addition of solvent and solute and removal of solution is feasible with the arrangement shown in Figure 84. The diagram shows the position of both the excitation beam and the emission detection beam passing through the lower half of the sample cell. The addition and withdrawal of liquids would be accomplished by stainless steel (or teflon) tubing situated in such a way as to avoid any interference with the excitation or emission beams and to avoid contact with the cell walls during rotation. The normal rotation that takes place during a wavelength scan would provide the mixing necessary for uniform solute distribution. After a study is completed with a particular solute, several cell washing cycles could be made in preparation for the next study from the solvent reservoir. I. A flow-through sample cell to permit determination of total absorption corrected fluorescence could be constructed as shown in Figure 85. The diagram shows the position of both the excitation beam and the emission detection beams passing through the lower half of the sample cell. Also shown is the pathway of flow of sample passing through the cell. The path is tailored so as to -237— 101 Solvent 5 F‘ 5 Solute a T—-- JCNAA a. AHAAA‘» I‘ H.) “J v.4 /7 {id ~ 5 \L 00 jEM EX :: F t Figure 84. Automated addition of solvent and solute and removal of solution. -238- OUT IN 0’ , i I EM F4 EX R l 8 F2 EM Figure 85. Flow through sample cell to permit determination of total absorption corrected fluorescence. —239— prevent uneven flow and unwanted mixing. This type of flow cell is not intended for use with an instrument based on the Cell Rotation Method, but rather with an instrument based on fiber optics to obtain fluorescence measurements from the internal cell positions (1), (2) and (4) (note Cell Rotation Method). The amount of sample that would be held within the cell at any given time would be about 2 ml. This arrangement could be readily applied in—line to monitor column eluate in the purification of absorbing and fluorescing chemical species. Since such an instrument has the ability to monitor fluorescence measurement attenuations due to primary and secondary absorbance, absorbance corrected fluorescence can be calculated and output to identify the various chemical species. The instrument could also output both primary absorbance (determined spectrophotometrically or spectrofluoro- metrically) and secondary absorbance (determined spectro— fluorometrically). J. Introduction and maintenance of inert atmospheres in the sample cell compartment is possible with the arrange— ment shown in Figure 86. Occasionally it may be desirable to have an oxygen free atmosphere within the sample cell compartment. A tank of dry N2 or Ar could be hooked up to the cell compartment as illustrated. The input line selection would be made according to which gas is more dense, the inert gas from the attached tank or the -240- .ucwfiuummfiou Hamo meEmm wfiu Ca mwhmcmmosum pumcfl mo wocmcoucfimfi paw cofluodpouvcH .wm onsmflm —24l— atmosphere (air) in the laboratory. The two exit ports are incorporated to help flush the more and less dense gases from the chamber. All that would be necessary during the use of the instrument in anaerobic studies would be a slightly positive (1.01 atmospheres) pressure. If N2 is chosen as the inert gas it may be useful to pipe it through a bubbler containing a concentrated sulfuric acid solution before entry into the chamber to remove traces of water. K. Instrumentation based on the Cell Rotation Method could be made more convenient with the addition of compo— nents and accessories such as those shown in Figure 87. It is feasible that such an instrument could be operated from either a built-in all—in-one microcomputer, a portable computer (for temporary plug-in operation), or briefcase computer (for temporary plug—in operation). Rapid printing is possible with the addition of a dot- matrix printer (compared to the very Slow teletype). Long term storage of a large number of spectral scans would be possible with a hard—disk system. Entire compound libraries and the experimental results of other labora- tories could be stored in the same, accessible location. L. A convenient software display mode for kinetics would be possible using a repeat scanning instrument such as that based on the Cell Rotation Method or solid array detection. This instrument would permit on-line corrections of fluorescence for attenuations due to primary or -242— FLOPPV DISK DRIVE all-In-one microcomputer flatbed planer ergonomics dot-matrix printer bllclcuo computer hlrd-dluk system Figure 87. Instrumentation based on the Cell Rotation Method - future components and accessories. -243- secondary absorption or light scattering. Any spectral function can be determined in a repeating time period. Rather than new files being created for each scan, the data could be stored in series in a single file. A counter function built into the software would permit access to any particular scan of a set. When displaying a set or plotting a set each consecutive member (scan) of the set would be displaced along a 45 degree angle creating a pseudo z-axis for the time frame. A command similar to the CU command would allow selecting only a fraction of each member of a set to be displayed and expanded if necessary. The rapidity of the kinetics that could be made accessible by this approach depends on the range of wavelengths that would be scanned. The kinetics output would look as shown in Figure 88. M. Sample cell holders that can be readily exchanged in the current cell rotation instrument can be designed as shown in Figure 89. The object of this design for the cell holder is to provide a lock—in post for positioning on the stepper motor drive pin and a ball bearing seat for unhindered rotation. Such an arrangement would guarantee z-axis truing each time the cell holders were exchanged. The basic advantage of being able to exchange cell holders is to avoid undergoing the cell cleaning procedure between successive studies. A series of "used" sample cells could then be cleaned at one's convenience and ensure identical quality of cell cleanliness. A -244- KINETICS {>z P \ ~r4 U) \ C, . CD 4.) C1 F4 Wavelength Figure 88. A software mode for kinetics studies using an instrument based on the Cell Rotation Method. -245— /‘SAMPLE CELL BALL. BEARING TRACK HOLDERrx. . g 1 W l//////////// l, \ CELL3"”r- CELL HOLDER COMPARTMENT POST DIVIDER (UPPER) T: CELL fit HOLDER ' \\ POST ’ ‘. (LOWER) STEPPER MOTOR Figure 89. Sample cell holders that can be readily exchanged. -246- rack could be built to support a series of such cell holders. N. Quality control of Human Serum Albumin samples by comparison of relative fluorescence efficiency curves could be a rapid means of ascertaining purity. It has been reported that each molecule of native Human Serum Albumin can bind up to two globulin proteins and up to two fatty acid molecules. This protein has been associated with lipid transport. A preliminatry study using Human Serum Albumin samples from the Sigma Chemical Company has indicated that the relative fluorescence ,- efficiency curves can be employed to characterize the various states of purity (fatty acid and globulin free vs fatty acid free vs globulin free preparations). With further fine—tuning of these measurements and the analyses of Human Serum Albumin from other sources it may be possible to characterize all the permutations of binding globulins and fatty acids as shown in Figure 90. -247- I3 LIIVIJALIQ' ESIEIRJLTIMI JALIJI3IITD¢IIIVI . .OGB OFA ) . 1 GB . . 0 GB 0 FA 1 FA ) C 2 GB 0 GB 0 FA 2 FA - 1 GB C 2 GB : 1 FA ) 1 FA ) . 1 GB 2 GB ‘ 2 FA 2 FA Figure 90. Quality control of Human Serum Albumin samples by comparison of relative fluorescence efficiency values. APPENDICES APPENDIX A APPENDIX A SYSTEM PROGRAM COMMANDS This appendix includes details on use of the command Options from the following system programs: a. FLCAL; b. CALEDT; and c. FLUOR. The FLCAL program is designed to permit convenient calibration of the R-PVC, S-PVC and F-PMT detectors and normalization of their outputs to relative numbers of quanta. The CALEDT program is designed to allow editing of the calibration factors (look-up table FAC-l, PAC-2 and PAC-3 values) at any or all selected wavelengths. The FLUOR program is the main system operation and data manipulation program used to run the Cell Rotation Method instrument. It is in this section that each of the primary and secondary commands are explained in detail (an instruction manual of sorts). Also provided within this appendix are the following: a. full calibration scheme for the R-PVC, S-PVC and F-PMT detectors; and b. scheme for calibrating FAC-l and PAC-2 values, without the need to change the PAC-3 values. -248- —249— The FLCAL Program. 1) 2) 3) 4) 5) 6) 7) 8) 9) 10) ll) 12) l3) 14) 15) 16) 17) 18) 19) 20) 21) 22) 23) 24) 25) CA: TH: PA: IN: WE: WA: OP: RS: TX: AV: QB: ll: 17: G4: G3: OU: SM: GF: G1: G2: DE: SA: GE: / / / / / / / / / / / / / / / / / / / / / / / / / Calibration procedure Set threshold for F1 —F4 difference Sets number of points to average Set interval in % nm Set wavelength to input number Set to wavelength indicated Display of parameters Read F at input position Read S at input position Text input routine Average of a parameter over a range Total quantum efficiency ll-Point smoothing l7—Point smoothing Window to F1 -F4 difference Window to cell ratio Alter output parameters Set auto-smooth flag Input same geometric factor Input primary geometric factor Input secondary geometric factor Delay length Set X-axis scale Save a run Get run from disk 26) 27) 28) 29) 30) 31) 32) 33) PR: DI: CU: SC: RO: EM: EX: OS: \\\\\\\\ -250- Table printer Scope display routine Initializes cell position Scan selected monochromators Rotate cell Set up emission scan Set up excitation scan Return to 088 Monitor The CALEDT Program 1) 2) 3) ED: WR: ON: / / This command enters the "EDIT" mode for. FAC-l, FAC-2 and FAC-3 calibration fac- tors. The following series of prompts allow access to a particular factor value in one of the look-up tables: wavelength =? / Within the 250 to 550 nm range. FAC-l =# Y/N / Y allows input of new value. FAC—2 =# Y/N / Y allows input of new value. FAC-3 =# Y/N / Y allows input of new value. This is the "SAVE CHANGE" command. Unless this command is invoked, the changes made by the editing will not be stored in the CALTAB file. This is the command for sending a selected FAC look-up table to "UNITY". The three command Options are as follows: ON: 1 / Sets FAC-2 to 1's, divides FAC—l and FAC—3 by the old FAC-2. ON: 2 / Sets FAC—l to 1'5, divides FAC-3 by the old FAC-2. ON: 3 / Sets FAC-2 to 1'5, divides FAC-l by the Old FAC-2. -251- 4) MU: / This is the command used for "INTERNAL FAC LOOK-UP TABLE MULTIPLICATION". The three options are as follows: MU: l / Multiplies FAC—3 and FAC-1 by FAC—2. MU: 2 / Multiplies FAC—3 by FAC-1. MU: 3 / Multiplies FAC-l by FAC-2. 5) Control C / Returns from program subroutines to the monitor. 6) Control A / Returns from program (monitor or subroutines) back to the 088 monitor. The FLUOR Program. 1) Primary commands a) DI: / This is the "DISPLAY" command. This command plots a box—like graph on the video terminal of any wavelength dependent data. The Y—axis represents the (scaled) intensity of the output function and the X-axis represents the wavelength range. Tic marks divide the Y-axis maximum by a factor of 10 and divide the X-axis into 10 nano— meter intervals. / There is no limit to the type or num— ber of output functions which can be overlapped on the video terminal. Functions that are derived from the same file as well as different files can be overlapped for convenient comparisons. b) PR: / This is the "PRINT" command. This command prints out in tabular form any wavelength dependent data. The PRINTed output can be obtained either on the video terminal or the teletype. c) RT: / This is the "REAL—TIME" command. This command is used while the instrument is in operation. It allows selection of the wavelength, either excitation or emission, and will repeatedly out- put the requested wavelength dependent data (raw or calculated) about once (\u‘ d) e) f) AV: PL: OP: -252- per second. The video terminal is used with this command. The output will repeat until the command is terminated with the "CONTROL C" command to return the program back to the FLUOR monitor. This command cannot be used in a scanning mode, both the excitation monochromator and the emission monochromator must be locked. This is the "AVERAGE" command. The purpose of this command is to calculate the average intensity (unsealed) value of the requested wavelength dependent data between the X-axis pointers. The average intensity value across an entire spectrum is (generally) of limited usefulness, however the "OU:" command permits the resetting of pointers so that small regions of an entire spectrum can be averaged. The command is especially useful when the signals are low and the spectra noisy. This is the "PLOT" command. This command is used to plot graphs of any wavelength dependent data on the hard— copier. When the data output scaling factor is followed by a "RETURN" command the border box and tic marks are drawn together with the data generated curve. When the data output scaling factor is followed by a "z" command the border box and tic marks are not drawn, only the data generated curve is plotted. X-axis (wavelength range) compression or expansion is accomplished with the "XA" command. Y-axis (relative func- tion intensity) compression or expansion is accomplished by appropri- ate selection of the scaling factor. This is the "OPERATIONAL PARAMETER" command. This command displays the current settings of the following system factors and parameters: *All geometric factors (G1, G2, G3, G4 and GF). *Excitation/Emission wavelength settings. g) h) i) j) k) G1: G2: GF: G3: G4: -253- *Wavelength interval between the X-axis readings in multiples of % nanometers. *Points to average per stored reading. *Delay period in milliseconds after the sample cell finishes spinning to a new position. *Current position of the sample cell (1, 2, 3 or 4). *Threshold setting for the minimum allowable R1 —Sl 0=DF) value used in the relative fluorescence efficiency functions. *Pathlengths, "bx" and "b centimeters. This is the "G1 GEOMETRIC FACTOR CHANGE" command. Values selected must be positive (which may include decimals) or zero. To confirm a change in the G1 factor the "OP:" command will output the current (new) setting. This is the "G2 GEOMETRIC FACTOR CHANGE" command. Values selected must be positive (which may include deci- mals) or zero. To confirm a change in the G2 factor the "OP:" command will output the current (new) setting. This is the "GF GEOMETRIC FACTOR CHANGE" command. Values selected must be positive (which may include decimals) or zero. To confirm a change in the GF factor the "OP:" command will output the current (new) setting. This is the "G3 GEOMETRIC FACTOR CHANGE" command. Values selected must be positive (which may include decimals) or zero. To confirm a change in the GB factor the "OP:" command will output the current (new) setting. This is the "G4 GEOMETRIC FACTOR CHANGE" command. Values selected must be positive (which may include decimals) or zero. To confirm a y", in 1) m) n) TX: OU: DE: -254- change in the G4 factor the "OP:" command will output the current (new) setting. This is the "TEXT INPUT" command. This command is associated with a particular file name when activated. Following the initial command a string of 128 alphanumeric characters can be input for labeling purposes. Typically almost two full lines of text can be input and stored with a particular filename. The "CONTROL Z" command must follow the text string for filename association and to return the FLUOR program back to the monitor. A new text string can substitute for an old text string simply by writing another and storing the file with the "SAVE" command. In this manner the. new string writes over the Old string (permanently). This is the "OUTPUT POINTER RESET" command. This command permits selec— tive viewing of narrow regions of an entire spectrum. Both the starting and the ending pointers can be reset. Until the pointers are set back to their original positions all of the subsequent commands will acknowledge data within the new pointer settings. This command is very useful in con4 junction with the "AVERAGE", "PLOT" or "PRINT" commands. The new setting of the pointers only applies to the file currently being accessed. Retrieval of another file will return the pointers to what they were when the new file was stored. This is the "DELAY SETTING" command. This command permits setting of the delay period, in milliseconds, after the sample cell finishes spinning to a new position. Typically a delay of 200 milliseconds will bring the data sampling into the plateau region. o) q) r) XA: SA: GE: CU: —255— The problems associated with delay times shorter than 200 milliseconds are detailed in Chapter V (Automated Instrument for Method of Cell Rotation). This is the "X-AXIS SCALING FACTOR" command. With this command the X- axis (wavelength range) compression or expansion is accomplished. Since not all scans cover the same wave— length range this command makes it very convenient to thereafter display or hardcopy the spectra. This is the "FILENAME SAVE" command. This command permits the naming of a particular file (XXXXXX.XX) and subsequent storage on a designated storage disk or floppy. Any alpha— numeric input can be used in a filename. A Data life FD 34—9000—22625 single sided / single density 26 sector, 128 bytes/sec floppy can store 30 files occupying 16 blocks each. A new file can be stored over an old file by using the same filename and this storage command. This is the "FILENAME GET" command. This command permits retrieval of a designated file from storage on a floppy disk. There is no limit to the number of files that can be retrieved and sequentially overlapped via display on the video terminal or plot on a hardcopy. This is the "CURRENT CELL POSITION" command. Prior to initiating a scan it is necessary to inform the FLUOR program as to what position the sample cell is located in. If the sampling sequence begins in the wrong position the information stored (1R, 2R, 4R, ls, ZS, 48, 1F, 2F and 4F) will be nonsense. The cell can be initialized in positions 1, 2, 3 or 4. As long as the FLUOR program is not aborted the cell position will be automatically monitored. s) t) u) V) w) R0: SC: EM: BX: -256- This is the "ROTATE TO POSITION" command. Once the current cell position is set with the "CU:" command this command allows keyboard resetting of the cell position. The cell can be rotated into positions 1, 2, 3 or 4. This command is an efficient means of monitoring the R, S and F detector readings from any of the cell positions. This is the "ACTIVATE WAVELENGTH SCAN" command. This command activates the excitation and/or emission mono- chromators to initiate a wavelength scan. The nanometer interval between readings is preset by the "IN:" command in % nanometer increments. The scan can be run in either "SLOW" or "FAST" mode (for actual rates see Perkin Elmer Model 512 Spectro- fluorometer specifications given in the appendix). This is the "EMISSION MONOCHROMATOR POINTER SETTING" command. This command permits setting of the beginning and ending wavelengths the emission monochromator will scan between. Due to instrumental limitations (source output, quantum counter look-up table accuracy, detector response) the emission scans must be between, but not exceed, the 220 to 550 nanometer range. This is the "EXCITATION MONOCHROMATOR POINTER SETTING" command. This command permits setting of the beginning and ending wavelengths the excitation monochromator will scan between. Due to instrumental limita- tions (source output, quantum counter look-up table accuracy, detector response) the excitation scans must be between, but not exceed, the 220 to 550 nanometer range. This is the "STARTING POINTER SET" command. This command is employed to initialize the pointer for either the excitation or emission monochromator prior to a scan. From the time the X) y) WA: CA: IN: -257- pointer is set the FLUOR program monitors the wavelength of the scanning monochromator. This command will only be again required when either the FLUOR program is aborted or when a switch is made in the mono— chromator being scanned. This is the "MONOCHROMATOR RESET" command. Once the starting position of a scanning monochromator is set with the "WE:" command, this command is used to move the monochromator to a new setting. The FLUOR program will monitor the wavelength of the scanning monochromator even if this command is used repeatedly. This is the "k4 CALIBRATION" command which activates the routine to calcu- late and set the "k4" calibration factor. This factor is a constant for all of the wavelengths for a given setting of the detector amplifiers (R & S). Normally this calibration is conducted with the sample matrix in the cell (to minimize index of refrac— tion effects, internal reflections, cell wall absorption). This is the only calibration step conducted while in the FLUOR program, the rest of the calibration (prior 3 steps) is completed via the FLCAL program. This is the "SCAN INTERVAL SETTING" command. This command permits setting of the wavelength interval between readings while a monochromator is being scanned. The smallest interval is % nanometer. There is no limit to the largest interval that can be set, although 5 nanometer is usually maximum in order to still Obtain any spectral resolution. This is the "DF THRESHOLD SETTING" command. This command sets the lowest allowable threshold for the DF (=R -S) function which is part of the relative fluorescence efficiency function denominator. This command is mainly employed for the cosmetics of the extreme edges of a relative fluorescence efficiency curve (here the absorbance is typically too bb) cc) dd) ee) ff) SM: BX: BY: CO: QC: -258- low to permit meaningful calculations to be made since noise in the signals begins to dominate). This is the "AUTO-SMOOTHING" command. This command applies the Method of Golay ll-Point smoothing to a particu- lar wavelength dependent output func- tion. The following are the 11 factors applied to each consecutive data point throughout a scan: (-36/429), (9/429), (44/429), (69/429), (84/429), (89/429), (84/429), (69/429), (44/429), (9/429) and (-36/429). It is necessary to note that the first five and the last five points of the chosen data field are not smoothed due to the required centering of the smoothing routine. This is the "X-AXIS NEPHELOMETRY PATHLENGTH SETTING" command. This command allows for setting of the "bx" pathlength in centimeters. This value must be preset for use of the "NF:", nephelometric quantity, command. Since the position of the Optical viewing window is assumed to be constant, the pathlength value incor- porated is both system and wave- length independent. This is the "Y-AXIS NEPHELOMETRY PATHLENGTH SETTING" command. This command allows for setting of the "b " pathlength in centimeters. This va ue must be present for use of the "NF:", nephelometric quantity, command. Since the position of the Optical viewing window is assumed to be constant, the pathlength value incorporated is both system and wave— length independent. This is the "CONCENTRATION INPUT" command. This command allows input of a molar concentration for use with the "MA:", molar absorptivity, command. Decimal values are permitted as input. This is the "REFERENCE QUANTUM EFFICIENCY VALUE INPUT" command. This command allows input of a chosen reference quantum efficiency value to 99) hh) ii) 13') QR: Q0: PA: SF: —259- calibrate the "QR:" and "QO:" functions to obtain the total quantum efficiency by the comparative method. These values are typically selected from recent literature determinations of quantum efficiency by very exacting procedures. This is the "FR DETERMINED QUANTUM EFFICIENCY" command. This command employs a routine that calculates quantum efficiency utilizing the area underneath the FR curve. Since the routine has previously been calibrated through input of a chosen reference quantum efficiency value via the "QC:" command the QR value is norma— lized to this. This is the "FO DETERMINED QUANTUM EFFICIENCY" command. This command employs a routine that calculates quantum efficiency utilizing the area underneath the FO curve. Since the routine has previously been calibrated through input of a chosen reference quantum efficiency value via the "QC:" command the Q0 value is normalized to this. This is the "POINTS TO BE AVERAGED" command. This command allows selection of the number of data points to be taken and averaged (for each of 1R, 2R, 4R, ls, ZS, 45, 1F, 2F and 4F). It is common to set this value to 40 readings since this allows (approxi— mately) readings to be gathered over some 12 cycles of 60 Hz line noise. Since this source of noise appears to be highly regular it can readily be averaged away in this manner. To some extent the rate of a scan can be increased or decreased by resetting this value. This is the "GENERAL SCALE FACTOR INPUT" command. A number of the more elaborate routines in the FLUOR pro— gram require input of various scaling factors. This command permits any decimal number to be used as such a scale factor. Each time a new scale factor is entered using this command the old scale factor is erased. kk) 11) mm) nn) PP: BU: DU: DV: -260- This is the "SELECTED RELATIVE FLUORESCENCE EFFICIENCY INPUT" command. This command permits input and storage of a chosen RFE value for use in empirical determinations of the wavelength dependent window factors (note the "CY:"command) . The PP value is used as a constant at all wavelengths. This command permits any decimal number to be used as a chosen RFE value. This is the "BUFFER FORMATION" command. This command permits selection of either raw or calculated data (a single wavelength dependent output function) and creates a storage buffer. Subsequent commands such as "DV:" (for multiple derivatives) and "SM:" (ll-Point smoothing function) require prior buffering since the calculations will take place destroying the raw data storage. This is the "DUMP SELECT FILE INTO CALTAB" command. This command allows incorporation of a newly calculated FAC-l ("01:" command), FAC-2 ("CF:" command), or FAC-3 ("03:" command) set of wavelength dependent values into the CALTAB.FL file stored on the data floppy. This command is designed for tailoring of the look—up tables in the calibration sequence. This is the "MULTIPLE DERIVATIVE" command. This command will take any wavelength dependent buffered raw or calculated data and calculate the order of derivative selected. The "DV:" command will be followed by an integer (l, 2, 3, ... N) which tells the routine how many successive derivatives are to be taken. Note that for each derivative taken a % interval is lost on either end of a spectral scan. Prior use of the smoothing function, "SM:", is encouraged to avoid noise from becoming a dominant factor in the derivative spectra. Smoothing only works on data put into the buffer. Caution -do not attempt to smooth un-buffered data or it will be lost in the process. 00) pp) qq> rr) -261- Control C / This command will return the Control Z / This is the "TEXT STRING CLOSURE" FLUOR program from any primary or secondary command back to the FLUOR program monitor. This command is very useful in aborting an ongoing scan when a problem is detected (with the monochromators, a particular detector, source stability, etc.). The displaying of a scan that has been aborted in this manner will plot the Spectral data on the video terminal up until the wavelength the abort was made. command. After a text has been written to accompany a particular file, using the "TX:" command, this command is required to store 1 the text string with the file. After this command has been used i the program returns back to the ' FLUOR monitor. Without use of this command the text string will not be stored along with the file ("SA:" command). Control A / This command is known as the OF: "FLUOR PROGRAM EXIT". This command returns the computer back to the 088 monitor. This command accomplishes this whether or not a FLUOR program subroutine was operating. Note that the "k4" calibration factor is lost. This command is the "ZERO OFFSET" which sets the zero value to the middle (Y-axis) of the graph. The initial command is followed by a Y/N input which changes the range (scale) to +/- 500 instead of the normal range (scale) of +1000. The use of this offset is very useful in conjunction with the multiple deriva- tive command. Also note that a convenience exists which permits one spectral scan to be displayed at the middle of the graph, and a new spectral scan to be displayed at the bottom of the graph by a second use of the OF command (with N). -262- ss) LI: / This command "LINKS THE EXCITATION AND EMISSION MONOCHROMATORS" for light scattering studies. The command enforces that the correct f3 look-up table values are employed. Note that the study of light scattering with linked monochromators in the excitation mode, EX, will only use one value from the f3 look-up table assuming that the emission monochromator is not scanning. tt) RA: / This command allows a ratio to be determined from two wavelengths within a spectral scan. This "INTER- NAL RATIO" is set up as follows: RA: Prompt for function =? Prompt for starting wavelength.=? Prompt for ending wavelength =? Ratio==number given as decimal uu) IT: / This command determines the "INTEGRAL" from a selected spectral function. The OU command is used to set the starting and ending wavelengths between which the integration will take place. The integral is calcu- lated in the following manner: f fn(l.) fn(lf) y=f—1 I fn(l)dx = l + + ‘2 fn(lz) i 2 2 =i+1 where ( ) is the initial starting wavelength and ( ) is the final (ending) wavelength. This command permits easy comparison of areas below a peak in a spectrum or between different spectra. vv) CH: / This command sets up and computes a "CURVE STRIPPING" between any wavelength compatible secondary commands in the following manner: CH: / initializes chromphore stripping CMD-l: / secondary command for numerator CMD—2: / secondary command for denominator -263- Wavelength for ratio =? Ratio =(CMD-l)/(CMD-2) =decimal number In order to display the curve stripped spectrum it is necessary to use CH as a secondary command. ww) SO: / This is the "SCALE OUTPUT" command. This command scales all functions by this factor. Thus one can print out (using the PR command) functions multiplied by a scale factor. The format for the command is as follows: SO: Scale Factor =? (decimal number) Be careful! The scale factor entered by the SO command is a default factor for all functions. If in doubt, enter SO: SF =l.00. When the FLUOR program starts up initially, SO defaults to 1.00. 2) Secondary commands a) FO: / This command calculates and outputs the "TOTAL ABSORBANCE CORRECTED FLUORESCENCE INTENSITY FROM CELL POSITION (1)". This output accounts for the effects of both primary and secondary absorbance on the measure- ment of the fluorescence. The output is normalized to numbers of quanta in the wavelength range from 250 to 550 nm. b) F1: / This command outputs the "FLUO- RESCENCE INTENSITY FROM CELL POSITION (1)". The output is normalized to numbers of quanta in the wavelength range from 250 to 550 nm. c) F2: / This command outputs the "FLUO- RESCENCE INTENSITY FROM CELL POSITION (2)". The output is normalized to numbers of quanta in the wavelength range from 250 to 550 nm. d) F4: / This command outputs the "FLUO— RESCENCE INTENSITY FROM CELL POSITION (4)". The output is normalized to numbers of quanta in the wavelength range from 250 to 550 nm. e) f) i) j) FP: FS: 4A: 1C: 2C: 4C: —264— This command calculates and outputs the "PRIMARY ABSORBANCE CORRECTED FLUORESCENCE INTENSITY FROM CELL POSITION (1)". The output is normalized to numbers of quanta in the wavelength range from 250 to 550 nm. This command calculates and outputs the "SECONDARY ABSORBANCE CORRECTED FLUORESCENCE INTENSITY FROM CELL POSITION (1)". The output is normalized to numbers of quanta in the wavelength range from 250 to 550 nm. This command calculates and outputs the "SECONDARY ABSORBANCE CORRECTED FLUORESCENCE INTENSITY FROM CELL POSITION (4)". The output is normalized to numbers of quanta in the wavelength range from 250 to 550 nm. This command calculates and outputs the "FLUORESCENCE INTENSITY FROM CELL POSITION (l) CORRECTED FOR SOURCE FLUCUATIONS". The output is calculated by dividing the quantized F1 values by the quantized reference intensity values at each wavelength in the range from 250 to 550 nm. This command calculates and outputs the "FLUORESCENCE INTENSITY FROM CELL POSITION (2) CORRECTED FOR SOURCE FLUCTUATIONS". The output is calculated by dividing the quan- tized F2 values by the quantized reference intensity values at each wavelength in the range from 250 to 550 nm. This command calculates and outputs the "FLUORESCENCE INTENSITY FROM CELL POSITION (4) CORRECTED FOR SOURCE FLUCTUATIONS". The output is calcu- lated by dividing the quantized F4 values by the quantized reference intensity values at each wavelength in the range from 250 to 550 nm. k) l) m) n) 0) CP: CS: FR: Al: A2: / -265- This command calculates and outputs the "PRIMARY ABSORBANCE CORRECTED FLUORESCENCE INTENSITY FROM CELL POSITION (l) CORRECTED FOR SOURCE FLUCTUATIONS". The output is calculated by dividing the quantized FP values by the quantized reference intensity values at each wavelength in the range from 250 to 550 nm. This command calculates and outputs the "SECONDARY ABSORBANCE CORRECTED FLUORESCENCE INTENSITY FROM CELL POSITION (1) CORRECTED FOR SOURCE FLUCTUATIONS". The output is calcu— lated by dividing the quantized F5 values by the quantized reference intensity values at each wavelength in the range from 250 to 550 nm. This command calculates and outputs the "TOTAL ABSORBANCE CORRECTED FLUORESCENCE INTENSITY FROM CELL POSITION (l) CORRECTED FOR SOURCE FLUCTUATIONS". The output is calculated by dividing the quantized F0 values by the quantized reference intensity values, R1, at each wave- length in the range from 250 to 550 nm. This command calculates and outputs the "PRIMARY ABSORBANCE DETERMINED SPECTROPHOTOMETRICALLY PER CENTIMETER PATHLENGTH". This output is based on the quantized reference intensity values, R1, and the quantized sample (beam) intensity values, 81, obtained from along the excitation (primary) beam axis at each wavelength in the range from 250 to 550 nm. This command calculates and outputs the "PRIMARY ABSORBANCE DETERMINED SPECTROFLUOROMETRICALLY PER CENTIMETER PATHLENGTH". This output is based on the quantized fluorescence inten- sity values, Fl, obtained from cell position (1) and the quantized fluorescence intensity values, F4, Obtained from cell position (4) from along the excitation (primary) beam axis at each wavelength in the range from 250 to 550 nm. P) q) r) s) t) A3: T1: T2: T3: DF: —266- This command calculates and outputs the "SECONDARY ABSORBANCE DETERMINED SPECTROFLUOROMETRICALLY PER CENTIMETER PATHLENGTH". This output is based on the quantized fluorescence intensity values, Fl, obtained from cell posi— tion (1) and the quantized fluo- rescence intensity values, F2, obtained from cell position (2) from along the emission (secondary) beam axis at each wavelength in the range from 250 to 550 nm. This command calculates and outputs the "PRIMARY TRANSMITTANCE DETERMINED SPECTROPHOTOMETRICALLY PER CENTIMETER PATHLENGTH". This output is based on the quantized reference intensity values, R1, and the quantized sample (beam) intensity values, 81, obtained from along the excitation (primary) beam axis at each wavelength in the range from 250 to 550 nm. This command calculates and outputs the "PRIMARY TRANSMITTANCE DETERMINED SPECTROFLUOROMETRICALLY PER CENTIMETER PATHLENGTH". This output is based on the quantized fluorescence intensity values, Fl, obtained from cell posi— tion (1) and the quantized fluorescence intensity values, F4, obtained from cell position (4) from along the excitation (primary) beam axis at each wavelength in the range from 250 to 550 nm. This command calculates and outputs the "SECONDARY TRANSMITTANCE DETER- MINED SPECTROFLUOROMETRICALLY PER CENTIMETER PATHLENGTH". This output is based on the quantized fluorescence intensity values, Fl, obtained from cell position (1) and the quantized fluorescence intensity values, F2, obtained from cell position (2) from along the emission (secondary) beam axis at each wavelength in the range from 250 to 550 nm. This command calculates and outputs the "QUANTIZED DIFFERENCE BETWEEN THE REFERENCE AND SAMPLE BEAM INTENSITIES". This output is based on the difference v) W) x) FF: SQ: DR: FD: —267— between the quantized reference intensity values, R1, and the quan- tized sample (beam) intensity values, 81, obtained from along the excitation (primary) beam axis at each wavelength in the range from 250 to 550 nm. This command calculates and outputs the "QUANTIZED DIFFERENCE BETWEEN THE FLUORESCENCE INTENSITY VALUES OBTAINED AT CELL POSITIONS (1) AND (4)". This output is based on the difference between the quantized fluorescence intensity values, Fl, obtained from cell position (1) and the quantized fluorescence intensity values, F4, obtained from cell position (4) from along the excitation (primary) beam axis at each wavelength in the range from 250 to 550 nm. ‘ This command calculates and outputs the "QUANTIZED DIFFERENCE BETWEEN THE FLUORESCENCE INTENSITY VALUES OBTAINED AT CELL POSITIONS (1) AND (2)". This output is based on the difference between the quantized fluorescence intensity values, Fl, obtained from cell position (1) and the quantized fluorescence intensity values, F2, obtained from cell position (2) from along the emission (secondary) beam axis at each wavelength in the range from 250 to 550 nm. This command calculates and outputs the "QUANTIZED DIFFERENCE BETWEEN THE REFERENCE AND SAMPLE BEAM INTENSITIES CORRECTED FOR SOURCE FLUCTUATIONS". The output is calculated by dividing the quantized DF values by the quantized reference intensity values, R1, at each wavelength in the range from 250 to 550 nm. This command calculates and outputs the "QUANTIZED DIFFERENCE BETWEEN THE FLUORESCENCE INTENSITY VALUES OBTAINED AT CELL POSITIONS (l) and (4) CORRECTED FOR SOURCE FLUCUATIONS". The output is calculated by dividing the quantized FF values by the quantized reference intensity values, R1, at each wavelength in the range from 250 to 550 nm. y) 2) ab) ac) ad) ae) af) 4B: FM: 81: 82: S4: R1: R2: R4: -268- This command calculates and outputs the "SECONDARY ABSORBANCE CORRECTED FLUORESCENCE INTENSITY FROM CELL POSITION (4) CORRECTED FOR SOURCE FLUCTUATIONS". The output is calculated by multiplying the quantized 4C values by the secondary absorbance correction at each wavelength in the range from 250 to 550 nm. This command calculates and outputs the "FLUORESCENCE INTENSITY FROM THE CELL CENTER". This output is based on back—calculation for the effects of both primary and secondary absorbance on the measurement of the fluorescence intensity from cell position (1). The output is normalized to numbers of quanta in T the wavelength range from 250 to 550 nm. This command outputs the "SAMPLE BEAM INTENSITY WHILE IN CELL POSITION (1)". The output is normalized to numbers of quanta in the wavelength range from 250 to 550 nm. This command outputs the "SAMPLE BEAM INTENSITY WHILE IN CELL POSITION (2)". The output is normalized to numbers of quanta in the wavelength range from 250 to 550 nm. This command outputs the "SAMPLE BEAM INTENSITY WHILE IN CELL POSITION (4)". The output is normalized to numbers of quanta in the wavelength range from 250 to 550 nm. This command outputs the "REFERENCE BEAM INTENSITY WHILE IN CELL POSITION (1)". The output is normalized to numbers of quanta in the wavelength range from 250 to 500 nm. This command outputs the "REFERENCE BEAM INTENSITY WHILE IN CELL POSITION (2)". The output is normalized to numbers of quanta in the wavelength range from 250 to 550 nm. This command outputs the "REFERENCE BEAM INTENSITY WHILE (4)". The output is numbers of quanta in IN CELL POSITION normalized to the wavelength range from 250 to 550 nm. ag) ah) aj) ak) al) am) an) ao) ap) lS: ZS: 4S: 1R: 2R: 4R: 1F: 2F: 4F: SL: -269- This command outputs the "RAW SAMPLE INTENSITY WHILE IN CELL POSITION (1)". The output is presented in energy mode and not normalized to numbers of quanta. This command outputs the "RAW SAMPLE BEAM INTENSITY WHILE IN CELL POSITION (2) II. The output is presented in energy mode and not normalized to numbers of quanta. This command outputs the "RAW SAMPLE BEAM INTENSITY WHILE IN CELL POSITION (4)". The output is presented in energy mode and not normalized to numbers of quanta. This command outputs the REFERENCE BEAM INTENSITY CELL POSITION (1)". The presented in energy mode normalized to numbers of This command outputs the REFERENCE BEAM INTENSITY CELL POSITION (2)". The presented in energy mode normalized to numbers of This command outputs the REFERENCE BEAM INTENSITY CELL POSITION (4)". The ll RAW WHILE IN output is g and not quanta. ( ll RAW WHILE IN output is and not quanta. ll RAW WHILE IN output is presented in energy mode and not normalized to numbers of This command outputs the quanta. ll RAW FLUORESCENCE INTENSITY AS VIEWED FROM CELL POSITION (1)". The output is presented in energy mode and not normalized to numbers of This command outputs the quanta. ll RAW FLUORESCENCE INTENSITY AS VIEWED FROM CELL POSITION (2)". The output is presented in energy mode and not normalized to numbers of This command outputs the quanta. II RAW FLUORESCENCE INTENSITY AS VIEWED FROM CELL POSITION (4)". The output is presented in energy mode and not normalized to numbers of quanta. This command calculates and outputs a "MONITOR OF STRAY LIGHT BASED ON ABSORBANCES CALCULATED BOTH aq) as) at) PL: EB: TU: —27o- SPECTROPHOTOMETRICALLY AND SPECTRO- FLUOROMETRICALLY". The output is the difference between A1 and A2, with the assumption being that stray light that is not absorbed by the sample and medium will influence only the Al determination of primary absorbance. This command calculates and outputs the "PERCENTAGE OF STRAY LIGHT BASED ON ABSORBANCES CALCULATED BOTH SPECTROPHOTOMETRICALLY AND SPECTRO— FLUOROMETRICALLY". This output is calculated by dividing the difference between A1 and A2 by A2, and then presenting this value as a percentage. The assumption inherent is that stray light that is not absorbed by the sample and medium will influence only the A1 determination of primary ‘ absorbance. This command calculates and outputs the "MOLAR ABSORPTIVITY" of a chemical species. Before this command can be utilized, a molar concentration must be input by means of the CO command. This command should be applied with pure substances in a non-absorbing solvent matrix. This command calculates and outputs the "ERROR BAR BASED ON A SELECTED REAL-TIME FUNCTION". After the RT command is initiated, this command is called and followed by the selected wavelength dependent secondary command. This command is not employed in scanning mode. The final error bar determination will be made after 40 consecutive readings are taken and averaged. The high, low and average values are output. This command calculates and outputs "TURBIDIMETRIC (LIGHT SCATTERING) QUANTITIES". This routine is used in the dual monochromator scanning mode and calculates the light scattering analog of primary absor— bance based on the quantized reference beam intensity, R1, and the quantized sample beam intensity, $1, in the au) av) aw) ax) NF: P1: P2: P6: -271- wavelength range from 250 to 550 nm. Typically, the scanning monochromators will be set to the same wavelength in order to Obtain maximal light scattering (Rayleigh). This command calculates and outputs "NEPHELOMETRIC (LIGHT SCATTERING) QUANTITIES". This routine is used in the dual monochromator scanning mode and calculates the light scattering analog of source corrected fluorescence from cell position (1): (Fl)/(Rl). This calculation is available in the wavelength range from 250 to 550 nm. Typically, the scanning monochromators will be set to the same wavelength in order to obtain maximal light scattering (Rayleigh). Prior to use of this . command the X-axis and Y-axis dis- a tances, BX and BY, must be input which are dependent on the internal K cell position (1). This command calculates and outputs "RELATIVE FLUORESCENCE EFFICIENCY" based on the following ratio: (FS)/(DF). This calculation can be Obtained in the wavelength range from 250 to 550 nm. Both of the values used to calculate the ratio have been normalized to numbers of quanta. This command calculates and outputs "RELATIVE FLUORESCENCE EFFICIENCY" based on the following ratio: (Fl)/(DF). This calculation can be obtained in the wavelength range from 250 to 550 nm. Both of the values used to calculate the ratio have been normalized to numbers of quanta. This command calculates and outputs "RELATIVE FLUORESCENCE EFFICIENCY" based on the following ratio: (FS)/(FF). This calculation can be obtained in the wavelength range from 250 to 550 nm. Both of the values used to calculate the ratio have been normalized to numbers of quanta. ay) az) ba) bb) P9: 1D: 2D: CX: -272- This command calculates and outputs "RELATIVE FLUORESCENCE EFFICIENCY" based on the following ratio: (4A)/(DF). This calculation can be obtained in the wavelength range from 250 to 550 nm. Both of the values used to calculate the ratio have been normalized to numbers of quanta. This command calculates and outputs the "DIFFERENCE CURVE FROM FLUOROPHORE ABSORPTION CURVE STRIPPING" that is based on the following function: 1D =(Al) -(FR)(SF). The quantity SF is a scale factor used to match magnitudes of the primary absorbance, A1, curve with the absorbance and source corrected fluorescence profile obtained at cell position (1). The 1D output is actually the spectral profile of the system chromophores. This function is applicable in the wavelength range from 250 to 550 nm. This command calculates and outputs the “DIFFERENCE CURVE FROM FLUOROPHORE ABSORPTION CURVE STRIPPING" that is based on the following function: 2D =(A2) -(FR)(SF). The quantity SF is a scale factor used to match magnitudes of the primary absorbance determined spectrofluorometrically, A2, with the absorbance and source corrected fluorescence profile obtained at cell position (1). The 2D output is actually the spectral profile of the system chromophores. This function is applicable in the wavelength range from 250 to 550 nm. This command calculates and outputs the "IDEAL QUANTUM COUNTER LOOK-UP TABLE" based on the following function: CX =[(F0)em(SF)]/[(1R)ex(fl)ex(A2)] where FO, SF, lR and A2 are as defined previously. The (Fl)ex values are the look-up table matching the spectral response curves of the reference PVC and sample PVC detectors as stored in the CALTAB file. Compounds such as rhodamine b are used here since they possess an In") . l FLT .' ‘0 .‘I‘ if): :9 ’T W . ‘ I. ) I“ ;‘ l‘ I l (IL , .2”) l a I) I) !| lfl'T w % g) ' ‘Y bc) bd) be) bf) CY: BU: CN: IT: -273- absorbance profile from 250 to 550 nm. Note that for determination of this function it is necessary to have a continuous and meaningful (not noise dominated) A2 curve. The A2 primary absorbance curve is used instead of the Al primary absorbance curve in order to minimize the effects of stray light not absorbed by the sample. This command calculates and outputs the "WAVELENGTH-DEPENDENT WINDOW FACTOR" based on a chosen relative fluorescence efficiency value. The CY function can be expressed as follows: or = (FS)em/[ (1R)ex + + " é $7 Law ['1 g”. 2.7K V (P05) (P05) )3: ">1. 4. g; flunk - (P05) (NEG) (P05) (P05) Figure 92. —281- Analog to digital conversion circuitry. The Analogic MP—2112 operates at high speed, 12-bit conversions are completed in less than 7 microseconds providing a throughput speed of up to 140,000 conversions per second. Allowing up to 3 microseconds for the multi- plexer and the sample—and—hold amplifier to settle in conjunction with the 7 microseconds, the resulting throughput rate is 100 kHz. Outputs available include both parallel and serial. -282- 2. IF:— 9.3 2.21... IC 930 van a m u a 2.5.. €3.50 m. a M. a w w (2( Jain» w. w _ H w . a 5» had no. 1vw _ um . mum o z. ...:a m w . M w. _ M a H d z. . 3 . 8 _ I. I _ m u u .... n a 2. 43.21.: . u _ M i _ H . . o o o I .0 N. W I s K0P(¢4nzou ra3h04 , ho< ua2(x .5920 F in ...xu .50 v3.0.5 ...Zutxao Uzi >455.» IIUOJU UZ.>(JQ UUZUKULUK muzucuiuc thnuu 1 20.20:: T . .uz... - “w” - A) .52: mmeamyzou .5th 8. @352 had 82.....me Saar Figure 93. —283- Four Channel CMOS Multiplexer. The MXD-409 Analog Multiplexer by Datel Systems, Inc., is a 4 channel monolithic device manufactured with a dielectrically isolated complementary MOS process. Circuits incor- porate analog and digital input protection which safe- guards the units from both overvoltage and loss of power. The digital inputs are DTL/TTL/CMOS compatible and address the proper channel by means of a 2 bit binary code. Use of break-before—make switching to insure that no two channels are ever momentarily shorted together. Transfer accuracies of 0.01% can be achieved at channel sampling rates up to 200 kHz and over +/- 10 V signal ranges. The power consumption is 7.5 mW at standby and 15.0 mW at 100 kHz switching rate. The power supply ranges from +/— 5 V to +/— 20 V. -284- ®®©©©©9® @J\ T EDP—.30 @TI. .. A - N @Illl a FDQZH M Olll .. @ O @TIIL WEUFAS @vlllll .53me onSE mMNmeHPJDZ mOZU Jug/EEO xv —285- Figure 94. Pulse fitting circuitry for stepper motor control. The circuit is an interface between the I/O port of the pdp 8/e computer and the power supply of the stepper motor. The timing (RC) circuit is set for 70 Us for each pulse. Key: 74—121 =one shot chip; SK—3025 = PNP transistor of l A capacity; and all resistors are of k W capacity. -286— m mmsz SHEEP x oh .Saza U 22> Ill<<<<< . - .C. M ._. SE28 $38 8 @ O O @ eokozkmwgmkn «H x .E E. a .50 PDO> 9 © 9 'U‘X'S haueu wzFE ”SEE Figure 95. -287— Modified M1709 Omnibus Interface Foundation Module. The module is a general interface card that allows the user to interface a variety of instruments and accessories to the pdp 8/e computer. Shown is the custom design of the M1709 used with the cell rotation instrumentation. The Omnibus interface logic provided for by the module includes that for bus drivers/ receivers, device selectors, and interrupt/skip circuitry. -288- M1709 OMNIBUS INTERFACE FOUNDATION MODULE A5 MOOlFlED no on no 04 HO 0‘ H0 06 MD 07 D INTERNAL 1’0 no 00 (0:. 1’0 nus: DATA socxt‘r, P-n Tuscan: no on um» soucf,P-| MD to 1»? new no u ”up Tn co INIT :1 DATA DD DATA OI DATA oz, DATA 03 DATA 94 DATA 0‘ DATA 0‘ DATA 07 DATA 06 +3 V DATA os sounct DATA :0 DAYA H APPENDIX C SPECIFICATIONS FOR SYSTEM COMPONENTS ~289— -290- "/ Ea}, .AQEE<::,:: ./122 SKEWED POSITION 0F SAMPLE CELL V L . J ///'///2' WW IDEAL ORTHAGONAL Figure 96. Skewing of the sample cell. The comparison shown is of a sample cell a few degrees off from being square with the excitation beam and the emission detection beam versus the cell in a position orthagonal with those beams. The problem is that a Single step away from orthagonality, +/- 1.8°, causes significant attenuation of the fluorescence signal detected of about 3% and three steps, +/— 5.4°, causes severe attenuation of more than 10%. Figure 97. -291- CELL POSITION 2 I V 1‘ CELL POSITION 1 ORTHAGONAL MISALIGNMENT BETWEEN POSITIONS Misalignment problem which can occur even though the cell positions are orthogonal to the excitation and emission detection beams. Cause of such a misalignment is either the cross-hairs of the excitation and emission beams are not intersecting on the diagonal of the sample cell or Z—axis truing is properly completed. -292- \ \ \ I I I \ \ \ \ I I I \ \ I I \ \ \ ---------J I I \ I ‘I ‘- A I I I K-l.-- I \ “I ‘ L--\.--l--J \ N I \ EM EX REGIONS 0F OBSERVATION Figure 98. Geometric view of the three observation windows utilized in the Method of Cell Rotation. The three cell position volume elements shown within the boundaries of the sample cell are the 3-D regions of intersection between the exci— tation and emission detection beams. Height of the three cell positions is readily adjustable by the sample cell platform support post - set beams no closer than 6 mm to the top surface of the sample solution (due to swirling and turbulance at the interface) and no closer than 4 mm from the bottom surface of the sample cell. Interference (scattering and/or reflection) between the beams and sides of the cell walls is detectable if the outer edges of the collimated excitation beam and the emission observation windows are closer than 2 mm. Figure 99. —293- Wavelength functionality of the source, monochromators, sample cell and detectors of the Cell Rotation System versus that of other spectroscopic instruments on the commercial market. The CRM instrument components include the following: source =150 W Xenon lamp with continuous output; monochromators==Czerny—Turner mount with aperture value of F3.5, diffraction grating has 600 lines/mm and is blazed at 300 nm; sample cell =quartz, with all faces optically matched; detectors =photovoltaic cells for the reference and sample beams, photomultiplier tube for fluorescence beam; window ports =quartz for both excita— tion and emission; wavelength functionality of the CRM instrument =prime utility in ultraviolet and visible regions of the electromagnetic spectrum, due to composite of above selected components. —294- Wavelength, nm 100 200 Spectral legion (a) Source: 400 700 1000 2000 4000 IR IR 700010.000 20,000 Xenon lamp or Contmuous Tungsten lamp Nernst glower + Y wtre Globat Hollow cathode Discontinuous (b) Wavelength Conttnuous 3000 Tunes/mm Dtscontinuous (C) Matertals for cells, Windows, & lenses (6) Transducers Photon detectors eat detectors Fused silica or quartz Glass NaCI prism Grattngs with various number at lines/mm lnterlerence Interference filters Glass absorptton LiF SIIlca or TlBr—TII Photocell (volts) or (ohms) 40,000 IR AV. ...” -295- l I l I Figure 100. Comparison and overview of the output capabilities of the first system that integrated both a spectrophoto— meter and a spectrofluorometer to operate under the Cell Rotation Method with the current version of the CRM instrument. A. First CRM instrument - documented in the M.S. Thesis by Karlis Adamsons entitled: "The Method of Cell Rotation for Computer Based Correction of Fluorescence Measurements for Attenuations Due to Primary and Secondary Absorption", Michigan State University, Chemistry Department, 1982. B. Current CRM instrument - documented in this Ph.D. Dissertation. -296— J‘mzqanfip Srsc'fxo mayo” ETER. :5 pflCTROFI-UORO M ETP—R 5&0:prth PRIMARY Afisogm’loN fiySORpMICE. GoAREGTEP Come 675;) S magma: Fwoxfircarld 11:76:53qu SPECTRO moron STE R ,5 PECTROF L uoRoM 675R PRIMARY ‘ «‘5‘ N ApsoRp-rtoxt A3501: pp: (:45 CORRECTEP CoAREG'rEp L E CE C F U08 5 N 5 FLUORESCENCE? H fifiSoR P7110)! C Mo; OD I“) 5&1)»de CURVE E LL ROTATION ETERMHJEp STRIPPII‘“; SPECTxof’woxoygmcbIL}, BEE/VINE QUANTUM EFFICIEHCIE5 ,Strl GLE 0% M U L‘fII’L E . “W“ OTTO... 0 ’5 P £61,105”? EFFICIENCY 5 EC ONPA RY on Ime’rRld & So): was: Eggs Lo MEIR! (1 QETEREMEP Sficfkofnvoxpngfgichu‘t? MEASUREMENTS -297— TABLE XVI. Specifications for the Perkin-Elmer Model 512 Spectro- fluorometer Components Employed in the CRM Instrument. I. General Specifications for the Main Unit. II. III. 1. N 7. 8. 9. 10. 11. 12. l3. 14. 15. Monochromators - excitation and emission. a. Optical system — Czerny-Turner mount, aperture F3.5. b. Diffraction grating — 600 lines/mm, blazed at 300 nm. Spectral bandpass - 3, 10, 20 nm. Wavelength range. a. Emission measurement — 200 to 900 nm. b. Absorption measurement — 250 to 640 nm. Wavelength accuracy - i1 nm. Wavelength reproducibility - within 0.2 nm. Wavelength backlash - when turning from long wavelengths to short wavelengths, within 0.5 nm. Scan speed - 60, 120, 240, 480 nm/min. 1 Light source - Xenon lamp 150 Watt. Detector for fluorescence — Photomultiplier R-446F. Photometric system — direct energy mode. Sensitivity - 0.1, 0.3, l, 3, 10, 30, 100 fold amplification. Response - fast, medium, slow. Dimensions — 67 cm wide, 39 cm deep, 27 cm height. Weight - 54 kg. Signal output — 10 mv for potentiometer recorder or 1 v for digital voltmeter. Xenon Lamp Power Supply 1. 2. 3. Constant-current (at 7.5 A) pulse ignition type. Dimensions - 25 cm wide, 29 cm height, 35 cm deep. Weight - 22 kg. Fluorescence Mode Specifications. 1. Stray light. a. Excitation monochromator - 1% at 250 nm. b. Emission monochromator - 2% at 350 nm. Sensitivity - The Raman band of water will show a 25% pen deflection under the following conditions: high voltage =700—800 V; sensitivity =X100; energy output setting; excitation wavelength =350 nm; slit =10 nm/ 10 nm. ..n ..ITEII II) ll) . It It Ill It I —298- TABLE XVII. Stepper Motor Specifications. II. Stepper motor in the Cell Rotation Device. A. B. IO'UOZZL'INf—IHUJOWEIUO ‘ Model number - 23D-6102. Accuracy — 3% non-cummulative. Resistance / 0-5.l Ohms. Rated voltage - 5.1 Volts. Current per phase - 1.0 Amp. Inductance per phase - 10.0 Millihenries. Time for single step — 2.5 Milliseconds. Holding torque — 53 Oz. In. Maximum running torque — 35 Oz. In. Detent torque — 5 02. In. Maximum thrust load — 25 lbs. Maximum overhang load — 15 lbs. Rotor inertia — 87 gm. cm . Weight - 20 02. Length - 2.0 In. Degrees per step - l.8° in full step mode. Source voltage - 28 Volts. Stepper motors for monochromator control. 0 D'UOZZF‘XCIHHIOWUJUOWIP‘ Model number - HS-25 (M062-FC03). Accuracy — 3% non-cummulative. Resistance / 0-3.4 Ohms. Rated voltage - 5.3 Volts. Current per phase - 1.6 Amp. Inductance per phase - 8.6 Millihenries. Time for single step — 3.5 Milliseconds. Holding torque - 100 02. In. Maximum running torque — 65 Oz. In. Detent torque - 8 Oz. In. Maximum thrust load - 25 lbs. Maximum overhang load - 15 lbs. Rotor inertia - 0.08 Lb. In.2. Weight — 2 Lbs. Length - 2.0 In. Degrees per step — 1.8° in full step mode. Source voltage — 24 Volts. -299- TABLE XVIII. Specifications for the Reference (R-PVC), Sample (S—PVC), and Fluorescence (F-PMT) Detectors. II. Photovoltaic Cells (for reverence and sample beam detection). A. Hamamatsu model s-l337-10lO—BQ. B. Spectral response — 200 to 1150 nm. C. Response linearity — l ><10'12 to 1 >