lllllllllllHlllllulmulmunnumum.................. . 0616 3862 l 3 1223“;a a! Michigan State University fl This is to certify that the thesis entitled THE METHOD OF CELL ROTATION FOR COMPUTER BASED CORRECTION OF FLUORESCENCE MEASUREMENTS FOR ATTENUATIONS DUE TO PRIMARY AND SECONDARY ABSORPTION presented by Karlis Adamsons has been accepted towards fulfillment ‘ of the requirements for 1 M. S. ,zfi v i degree in W i i Major professor Date fl./1)/9£Z } 0-7639 OVERDUE FINES: 25¢ per day per item RETURNING LIBRARY MATERIALS: -——_.._._____ Place In book return to remove charge from circulation records COPYRIGHT BY KARLIS ADAMSONS 1982 © 1982 KARLIS ADAMSONS All Rights Reserved THE METHOD OF CELL ROTATION FOR COMPUTER BASED CORRECTION OF FLUORESCENCE MEASUREMENTS FOR ATTENUATIONS DUE TO PRIMARY AND SECONDARY ABSORPTION By Karlis Adamsons A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Chemistry 1982 W: b: he CE dz (377‘7252/ ABSTRACT THE METHOD OF CELL ROTATION FOR COMPUTER BASED CORRECTION OF FLUORESCENCE MEASUREMENTS FOR ATTENUATIONS DUE TO PRIMARY AND SECONDARY ABSORPTION By Karlis Adamsons An improved computerized right-angle spectrofluorometer capable of automatically implementing primary and secondary absorption corrections was constructed. The instrument incorporates a unique approach utiliz- ing off center cell rotation to allow regulation of the thickness of the sample solutions through which the excitation and emission beams pene- trate. Knowledge of the transmittance as a function of pathlength along both the excitation and emission optical axes permits determination of fluorescence Signal attenuation caused by sample absorption. Equations have been determined and programs written to allow calculation of the correction factors to be done in real time. Once the factors are calculated, they are automatically instituted to generate fluorescence data corrected for these absorption effects. Successful primary absorption corrections were obtained for qui- nine sulfate solutions in the concentration range 1x10—8 to 1x10—5 M with 10 absorbances 7x10-5 to 7x10-2. Successful secondary absorption corrections were obtained for solutions containing quinine sulfate, 1x10'5 M, and fluoroscein over the concentration range 1x10_6 to 1x10_u M with 20 absorbances 6x10.3 to 2.3x10-1. I yet an this h the me were h nature the un Dedication The most beautiful thing we can experience is in the unveiling of yet another artistic masterpiece from nature's museum. The success of this work is dedicated to all of those who provided me the freedom and the means for such an experience in scientific discovery. Witnesses were we to the birth of a new means for looking at the finer details of nature's composition and brush strokes. With new vision the extent of the unveiling we are about to encounter is unknown... ii scie from tPUU not accm canm is m to h Scier sati: discc Sell. from assoc achie Acknowledgements Credit must be primarily extended to man's development of the scientific method and, thereby, the means of separating our fantasies from nature's truths. The method is the key that will unlock these truths, but only to an open mind. As long as one is only biased it does not make any difference, because if one's bias is wrong a perpetual accumulation of experiments will perpetually annoy one until they cannot be disregarded any longer. They can only be disregarded if one is absolutely sure ahead of time of some precondition that science has to have. In fact it is necessary for the very existence of truth in science that open minds exist which do not allow that nature must satisfy some preconceived conditions. Credit is due also to the open—minded individuals who helped me in discovering the key for myself: Andrew Timnick, Jack Holland and John Sell. The relentless programming of Mike Reid and the words of insight from Bill Franz are gratefully acknowledged. The closeness of the association between us all has been the catalyst to the successes achieved and those only now dreamed of. TABLE OF CONTENTS LIST OF TABLES . . . . . . . . . . . . . . . . . . LIST OF FIGURES . . . . . . . . . . . . . . . . DEFINITION OF SYMBOLS CHAPTER I — INTRODUCTION . CHAPTER II - HISTORICAL A. Problems - 1. Quantitative FluoreScence Measurements . 2. Qualitative Fluorescence Measurements . . 3. The Effects of Sample Absorption on Other Forms of Information Derived From Fluorescence Measurements . . . . . . . . . . . . B. Conventional Methods 1. Sample Dilution 2. Optimum Choice of Excitation and EmissiOn Wavelengths . . . . . . . . . . . . 3. Detection Geometries . . a. Transmission Geometry . b. Front Surface Geometry c. Right-Angle Geometry 4. Designs of Sample Cells 5. Two Photon Excitation C. Mathematical Expressions 1. Absorption Corrections for TranSmisSion Geometry . 2. Absorption Corrections for Front Surface Geometry. 3. Absorption Corrections for Right-Angle Geometry CHAPTER III — CORRECTIONS FOR RIGHT- ANGLE GEOMETRY — A Detailed Examination . . . . . . . A. Introduction . . . , , 1. Need for Both Absorbance and Fluorescence . B. Theory . iv L 2. 3. d. SDQOU'W H: The Moving (Vibrating) Mirror Method . a. . . . . b. 0. Geometry Basic Assumptions . . . The Beam Intensity as a Function of Measured Quantities . Emission Radiation (FluoreScence) in Terms of Measured Quantities and the Observation Window . . Dependence of Corrected FluoreScence on Absorption (Two Approaches) . . . . The Primary Correction Factor . Limitations . . . . . . . . The Cell Shift Method Geometry . Basic Assumptions . . The Attenuation By Primary Absorption . The Attenuation by Secondary Absorption . Absorption Effects on the Measured Fluorescence Signal . The Correction Factors . A Limitation of the Secondary Absorption Correction . . . . Method of Cell Rotation ”)0 0-0 0‘9.) Geometry Basic Assumptions . . Primary Correction Factor . Secondary Correction Factor . . . . . Combined Correction Factor Satisfying the Assumptions CHAPTER IV — AN AUTOMATED INSTRUMENT TO CORRECT FLUORESCENCE MEASUREMENTS FOR PRIMARY AND SECONDARY ABSORPTION EFFECTS VIA THE METHOD OF CELL ROTATION . . . . . . OUJID Introduction Theory . . . . Instrumental . . . . . . . . . . . . . . . . . . . . 1. Overview . . . . . . . . . . . . . Data Treatment . . . . . . . . . . . . . . . . 3. The Cell Positioner . . . . . . 4. Detection Field Characteristics . . . 5. Detection Window Calibrations 6. Noise Analysis . Results of Correction Factor AnalySis . . . . 1. Study of Primary Absorption . . . . . . . . . . 2. Study of Secondary Absorption . . . . . . . . . CHAPTER V - CONCLUDING STATEMENTS A. B. Summary . Future Work . . . Chemical Systems . Instrumental Improvements 1. 2. 46 46 49 APE APE APP REF APPENDIX A - System Interfaces APPENDIX B - PE Model 512 Specifications Rapid Syn Stepper Motor Specs . APPENDIX C - Sample Cell Cleaning Procedure Vessel Prep. For Sample Storage . Reagents Used . . . Proper Selection of Slit Widths . APPENDIX D - List of I/O Instructions Complete Program Listing REFERENCES . vi Page 157 169 171 172 172 173 1711 175 178 . 2A6 Table Table Table Table Table Table Table Table Table Table Table Table Table Table Table Table Table Table Table Table Table Table 1. \OGDNOUTJ: LIST OF TABLES Activation and Deactivation Processes . . Instrumental Variables . . Photophysical Variables . Effect of Primary Masking . . Effect of Secondary Masking Quartz Sample Cuvette Cleaning Procedure Proper Selection of Slit Widths Rapid Syn Stepper Motor Specifications Perkin-Elmer Model 512 Specifications I/O Instructions List of Reagents Page 111 111 172 1711 171 169 175 173 Figur Figur Figur Figur Figur Figur Figup Figure Figup Figure Figur LIST OF FIGURES Page Figure 1. Schematic Diagram of Activation and Deactivation Processes for Molecules . . . . . . . . . . 3 Figure 2. Fluorescence Radiant Flux as a Function Of the .1 Penetration Distance of UV-VIS Radiation over a. Hypothetical Series of Increasing Fluorophore Con— centrations . . . . . . . . . . . . l 15 Figure 3. Apparent Fluorescence Emitting Region as a Function of Fluorophore Concentration . . . . . . . . 17 Figure A. Effect of Primary Absorption Along Excitation Axis with Respect to Apparent Luminosity . . . . . . 19 Figure 5. Effect of Secondary Absorption Along Emission Axes with Respect to Apparent Luminosity . . . . . . 21 Figure 6. Detection Geometries . . . . . . . . . . . 30 Figure 7. Instrumental Components and Their Arrangement for the Moving Mirror Method . . . . . . . . . 40 Figure 8. Instrumental Components and Their Arrangement for the Cell Shift Method . . . . . . . . . . #2 Figure 9. Geometrical and Optical Configuration for the Collec— tion of Fluorescence Radiation by the Moving Mirror Method . . . . . . . . . . . . . A8 Figure 10. Limiting Conditions for the Fluorescence Observation Angles for Point Sources Across the Sample Cell . . 55 FiguPe 11. Geometry for a Right Angle Spectrofluorometer with 6 . . 2 a Square Cell viii Figt Figt FigL Figt Figu Figu Figu Figu Figu Figu Figu Figu Figu Figu Figu Figu Figu Figu Figu Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure _| 13. 1M. —I 2. 5. Regions of Observation for the Method of Cell Rotation . . Attenuation of Radiation Cell Positions Available Using the Cell Rotation Method . . . . Block Diagram of Instrumentation Used in the Cell Rotation Method. . . . . . . . . . . Flowchart of Physical Devices Employed in the Cell Rotation Method. . . . . Stepper Motor and Lock-in Platform Post and Cell Holder Platform . The Cell Holder. Flowchart of System Program Positions of Emission Detection Fields . Photograph of Cell Positioner as Employed by the Cell Rotation Method . . . Elliptical Cell Rotation Cell Displacement in X & Y—Dimension as a Function of Lead Screw Rotation . . . . . . Vertical (2. Axis) Truing Stepper Motor Platform Alignment Tool Study of Detection Field Using Primary Masks . Study of Detection Field Using Both a Primary and Secondary Mask . Calibration of Emission Window Calibration of Excitation Window . Potential Misalignment Problems Encountered During Tune-Up in the Cell Rotation Method . . . . . ix Page 64 68 76 79 81 83 85 87 9O 92 95 97 99 102 104 107 109 114 116 122 Fig Figl Figl Fige Figu Figu Figu Figu Figu Figu Figu FiEu: Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Exploded View of Noise Monitored at Various Output _Voltage Levels . Signal to Noise Ratio for Full Range of Analog to . Digital Converter Output Reproducibility of Cell Position One with Multiple Rotations . . Instrumental Output With Concentration for Quinine Sulfate . . . . A. Range 1 x 10 '9M to 1 x 10'8M B. Range 1 x 10_ M to 1 x 10:gM C. Range 1 x 10 M to 1 x 10 M D. Range 1 x 10_ M to 1 x 10:2M E. Range 1 x 10 M to 1 x 10 M Quinine Sulfate - Fluorescence Output With Concen- tration As Detected in Four Cell Positions Study in Linearity of Primary Absorption with In— creasing Quinine Sulfate Concentration . Quinine Sulfate — Fluorescence Emission Scan as Detected in Four Cell Positions and Absorption Corrected . . . . . Study in Linearity of Secondary Absorption With Increasing Fluorescein Concentration . . Fluorescence Emission Scan for Mixtures of Quinine Sulfate and Fluorescein . . . . . . A. Quinine Sulfate 1 x 10:2M Fluorescein 2 x 1O_5M B. Quinine Sulfate 1 x 1O_6M Fluorescein 6 x 1O_5M C. Quinine Sulfate 1 x 10_ M Fluorescein 2 x 10 M Modified M1709 Omnibus Interface Foundation Module . Analog To Digital Conversion Circuitry . Four Channel CMOS Multiplexer Circuitry Spectrofluorometer Output Amplification Circuitry Page 124 126 128 131 135 137 139 141 1AA 158 160 162 164 Figure Figure Page Figure H5. Pulse Fitting Circuitry Between PDP 8/e and Power Drive of Stepper Motor . . . . . . . . 166 Figure A6. Ozone Exhaust System . . . . . . . . . . 168 (0) LIST OF SYMBOLS Herein refers to quantized energy equal to the product of Planck's constant and the frequency of the radiation. is Planck's constant. Refers to the frequency of radiation. Known as fluorescence quantum efficiency and is defined as a ratio of the total energy emitted to the total energy absorbed as a function of time and independent of excitation wave- length. is the fluorophore concentration is the fluorescence radiant flux is the incident (excitation beam) radiant flux is the molar absorptivity. is the cell pathlength in centimeters. i a product of three constants: e , Q and b. is the geometrical factor dependent on the effective solid angle viewed by the detector. is the response characteristic of the detector varying with wavelength. is a product of five contants, f(e), g(7\),€,§, and b, for a particular set of experimental conditions. is the concentration level of a fluorophore above which nonlinearity is observed in a plot of ¢ with C . is the molar absorptivity at a specific wavelenggh of excita- tion. is the excitation radiation or source of said radiation. is the emission radiation, fluorescence radiation in particu- lar. are distances of the fluorescence detector axis from the inner surface of the excitation cuvette face; Xe; is the shorter penetration distance, X13213 the longer penetration distance. are distances of the excitation beam axis from the emission port cuvette face; chis the shorter penetration distance, T5 is the longer penetration distance. is the distance between the focal points of fields of detec- tion along either the excitation or emission optical axes; refers to distance between foci of positions 1 & A, 2 & 3, or 1 & 2 and 3 & 4. is an ideal (non-existing) cell position which is attained by correcting for primary and secondary absorption attenuations. (I) xii (1) (2) (3) (4) is a position refered to in the method of cell rotation possessing the least primary absorption and the least secon— dary absorption with regard to other attainable cell posi- tions. is a position refered to in the method of cell rotation possessing the least primary absorption and most secondary absorption with regard to other attainable cell positions. is a position refered to in the method of cell rotation possessing the most primary absorption and the most secondary absorption with regard to other attainable cell psotions. is a position refered to in the method of cell rotation possessing the most primary absorption and the least secon- dary absorption with regard to other attainable cell posi— tions. is the wavelength of excitation radiation. is the wavelength of emission (or fluorescence) radiation. refers in general to the distance of the detection axis from the inner source excitation face of the sample cell. refers to the component of radiant flux being detected in a slice of solution volume perpendicular to the propagation axis of excitation. is the number of component slices of detected radiant flux across the emission window. is an idealized component slice of detected radiant flux across the emission window; here the radiant flux is assumed identical for each Slice regardless of penetration distance. is the sample transmittance at a specified wavelength. is equal to the ratio xd/b, a dimensionless emission window parameter. is equal to the ratio xB/b, a dimensionless emission window parameter. is equal to the ratio HK/b’ a dimensionless excitation window parameter. is equal to the ratio Yg/b, a dimensionless excitation window parameter. is equal to the ratio Yi/b, a nonspecific dimensionless excitation window parameter. is the fraction radiant flux in each slice of the detected emission beam; it describes the ratio¢/¢. refers to the difference in magnitude betaeen W.‘ and Wfi, Wg-Wgc refers to the difference in magnitude between exand 93; e} —€3¢‘. is the fluorescence radiant flux detected at UJhc is the fluorescence radiant flux detected at tLfle refers to the absorption of excitation radiant energy as a function of penetration distance along the detected excita— tion propagation axis. is the geometric factor required to extrapolate the absorp- tion of excitation radiation from the inner cell surface to the focus of position one. is the detected fluorescence radiant flux at position one. xiii 550f1 is the detected fluorescence radiant flux at position one corrected for primary absorption. is the fluorescence radiant flux detected at €3.¢. is the fluorescence radiant flux detected at €15. refers to the absorption of emission radiant energy as a function of penetration distance along the detected emission propagation axis. is the geometric factor required to extrapolate the absorp- tion of fluorescence radiation from the inner cell surface to the focus of position one. is the effective field generated emission beam radiant flux prior to penetration of solution toward the emission port. is the concentration of the secondary absorber, either the fluorophore itself or another chromophore. is the detected fluorescence radiant flux at position one corrected for secondary absorption. is the correction factor derived frou1 primary absorption measurements; (GFMAA o is the correction factor derived from secondary absorption measurements; (GFNAA o is the detected fluorescence radiant flux at position one corrected for both primary and secondary absorption. is the detected fluorescence radiant flux at position two. is the detected fluorescence radiant flux at position four. refers to quinine sulfate is the ratio of detected fluorescence signal to the back— ground noise. em Gel Che CHAPTER I INTRODUCTION When a beam of electromagnetic radiation passes through a material or sequence of different materials (i.e., the sample and its containing vessel), its energy may be channeled into a variety of processes (Figure 1). Part of the radiation will be absorbed and transduced into whatever system dependent deactivation processes (Table 1) are in effect, part will be reflected, part will be transmittefi, and a part will be scattered in various ways (i.e., Tyndall, Rayleigh and Raman scatter- ing). Absorption takes place in discrete units of quanta, the energies of which are equal to the product hJI, where 3’ is the frequency of the radiation and h is Planck's constant. Quanta of visible or ultraviolet radiation are of sufficient energy to raise a molecule to an excited electronic state, from which this energy may be converted into rotation- al, vibrational or kinetic energy (i.e., heat) or into chemical energy (i.e., photochemical reactions, aggregation phenomena) or part of the energy may be re—emitted as quanta of lower energy (i.e., as fluores- cence or phosphorescence). For analytical applications, fluorescence has been the most uti— lized of the molecular deactivation processes due to its facility for characterizing excitable chemical systems and its remarkable Figure 1. Schematic Diagram of Activation and Deactivation Proces- ses For Molecules Key: (1) (2) (1|) (5) (7) Activation step, excitation of singlet ground state, So, to the first excited singlet state, S*. Radiationless deactivation of 8* Radiational deactivation of 8* (fluorescence) Intersystem crossing S* to the ground triplet state, To Intersystem crossing To —# 5* Radiationless deactivation of To Radiational deactivation of To (phosphores- cence) ENERGY DIAGRAM FIGURE 1 Table 1. Activation and Deactivatioh Processes1 Rate . . Type t Reactan s Constant Products Description (Figure 1) S + hlfa ka 8* Excitation Ste 1 O -—-—-—-’ p 3* + SO ksq 280 Quenching due to 2 _.....__..— Collision with So 5* + Y ksy So + Y Quenching due to 2 ' Collision with Y S* kSp Products Photochemical 2 " Reaction 8* + So ksd (So) Dimerization 2 ______4. 2 8* + Y ksf So + Y* Forster-type 2 “‘—-‘* intermolecular energy transfer 5* kf So + hJIf Fluorescence 3 ———>- 8* ksi To Intersystem A " crossing (Spin forbidden) To kti 8* Intersystem 5 ' crossing (spin forbidden) To + So ktq 280 Quenching due to 6 ' collision with S To + Y kty So + Y Quenching due to 6 _" Collision with Y To ktp Products Photochemical reaction 6 To + So ktd (So)2 Dimerization 6 k To + Y tf SO + Y Forster-type 6 ‘—-——‘*' intramolecular To kp So + hlfp Energy Transfer -———-*- Phosphorescence 7 Symbols defined: So singlet state of molecule; To = ground singlet state of molecule; 8* : lowest triplet state of molecule; h = planck's constant;37a = frequency of absorbed radiation;35f = frequency of fluorescence radiation;)§p = frequency of phosphorescence radiation; Y = solvent or impurity molecule; Y* = excited nonluminescing solvent or impurity molecule; (so)2 = non-luminescing dimer. 5 sensitivity. The initial excitation or absorption of radiation by a molecule in solution is dependent on the energy difference between its electronic states. In general, the molecules will absorb a narrow band of radiation the wavelengths of which are equivalent to the energy level separation between two electronic energy states and associated vibra- tional and rotational energy levels. Since for each electronic state there are many vibrational and rotational levels, an absorption band is observed instead of absorption at a single wavelength. Scientific literature over recent years spanning a wide variety of areas such as trace metal determination, analyses for traces of organic materials, and particularly for determining trace constituents of bio- logical systems, attests to the ever increasing interest in and impor— tance of fluorescence. Instrumentation for the measurement of fluorescence has evolved ‘over the last several decades to where it now routinely provides a means for obtaining accurate qualitative and quantitative information about these molecules. In brief, fluorometry, no matter how simple or complicated the instrumentation, consists of three basic components: a source of radi— ant energy with which to irradiate (excite) the sample; a sample holder; and a detector to observe the fluorescence emitted by the sample. The complexity of the instrument depends on the degree to which the frequen- cy and radiant flux of the exciting radiation need be controlled, the egree to which specific frequencies of fluorescence can be selected and he sensitivity and precision with which the selected fluorescence adiant flux can be measured. Table 2a. Instrumental Variables A. B. C L’HU Source Stability Source Spectral Distribution Excitation Monochromator 1. Intensity Variation 2. Bandwidth Control 3. Stray Excitation Radiation Cell Geometry Emission Monochromator 1. Intensity Variation 2. Bandwidth Control 3. Stray Excitation Radiation Detector 1. Sensitivity 2. Linearity 3. Stability 4. Spectral Response Amplifier 1. Stability 2. Linearity Read Out 1. Stability 2. Linearity Table 2b. Photophysical Variables A. MUG Quantum Efficiency 1. Radiationless Collisions 2. Internal Conversion 3. Phosphorescence Absorption 1. Primary 2. Secondary Reflections and Refractive Index Light Scattering Re-Emission In filter fiuorometers, selection of the frequency is made by inserting appropriate filters in the beams of the exciting and fluores- cence radiation. If a monochromator is used to select the frequency of either beam, the instrument by definition becomes a Spectrofluorometer. It can then be used to measure the spectral distribution of the fluorescence radiation (i.e., "fluorescence emission Spectrum"), or the variation of fluorescence radiant flux with frequency of the exciting radiation (i.e., "Fluorescence excitation Spectrum"). Spectrofluorometry has the advantages over spectrophotometry of being potentially far more analytically sensitive and providing two spectra as criteria for identification and characterization instead of one. It has the obvious limitation that not every absorbing substance fluoresces. Molecules that fluoresce do so with a characteristic quantum efficiency,§, defined as a ratio of the total quanta emitted to the total quanta absorbed per unit time and is independent of the excitation ‘wavelength. § _ Number of quanta fluoresced _ Number of quanta absorbed Where both the quantity in the numerator and denominator are measured in a given time frame for a specific emission and excitation transition, respectively. The relationship between fluorescence measured and the fluorophore concentration, Cf, is given as: nd expanded, ¢f=§.¢o e1—e'eb°r) <1) 2 3 = é .¢O' (1-1+ Ebcf, 13%f) + (egff) -...) (2) Where :jéf = fluorescence radiant flux;flé)= incident radiant flux; €.= molar absorptivity; b = pathlength of cell; Cf = molar concentration of fluorophore. For dilute solutions in which only a small fraction of the exciting radiation is absorbed (€bc is small and only the first term in Equation f 2 is significant): ¢f=k'¢o'cf ‘ (3) Where k represents the product of the constants §, 6 and b. A plot of ¢§ vs Cf should yield a straight line. The linearity, however, breaks down at higher concentrations when ebcf.)’0.01 and higher order terms in Equation 2 become significant. Table 2 provides a listing of instrumen- tal and photophysical factors which can account for observed deviations from linearity. The above equation demonstrates ¢§ is directly related to ya. To 1 obtain higher analytical sensitivity, 9% can be increased. With modern Uphotomultipliers and amplifiers the detection of very low concentra— Ztions of fluorescent substances is feasible. A fundamental distinction igof Spectrofluorometry from spectrophotometry becomes apparent in that «for the latter the detection limit is set by the minimum detectable difference in radiant flux between the incident and transmitted radia— étion and extremely precise measurements of radiant flux are required to iattain as high a sensitivity. Furthermore, in spectrophotometry, a “ratio ¢/¢B is evaluated. Increasing ¢o will increase ¢ in a proportion- }ate amount so that the ratio is constant for a particular concentration. Outside of instrumental factors quenching and inner filter effects (i.e., primary and secondary absorptions) are the major influences on the accuracy of quantitative fluorometric measurements. Lack of appre- ciation of the elementary principles governing these two effects, or confusion between the two, has in the past led to the use of unsatisfac- tory equipment or procedures and to the incorrect interpretation of fluorescence measurements. Quenching includes all those processes that result in lowering the quantum efficiency from that of the isolated ideal state by diverting radiant energy absorbed by the molecule into channels other than the fluorescence emission process. Primary absorp- tion involves absorption of the exciting radiation by the fluorophore or other chromophores in the background matrix. Secondary absorption involves absorption of the fluoresced radiation either by self—absorp— tion due to partial overlap of the absorption and emission bands or by absorption by other chromophores. The combined effects of the absorp- tion processes have been called the innter filter effects in the period prior to clear definition of causation. This term is still used by some authors. Over recent years, investigators have evaluated these inner filter effects and have, to varying degrees, succeeded in compensating for the associated attenuations. Historical aspects leading up to the current state—of—the-art instrumentation, optical geometries and data correc— tion procedures follow. The goal of this present work was to design an improved computer- ized instrument to make accurate and correct corrections in fluores- cence measurements for primary and secondary absorption effects and to 1O evaluate its performance. To this end, a novel method employing cell rotation was developed which allows regulation of the thickness of sample through which the excitation and emission beams pass. CHAPTER II HISTORICAL A. Problems A lucid and exhaustively detailed account of the problems caused by excessive sample absorption, the conventional methods applied in deal- ing with excessive sample absorption, and the mathematical expressions used to correct spectra attenuated by primary and secondary absorptions is compiled in Christmann's Ph.D. dissertationz. Inner filter effects diminish the quality, and therefore, the 1 usefulness of the fluorescence data obtained from a particular chemical system. Both quantitative fluorescence measurements and qualitative spectral determinations are adversely affected. The discussion will focus individually on these aspects of Spectrofluorometry. 1. Quantitative Fluorescence Measurements. The analytical versatility of fluorescence Spectroscopy is due not nly to its inherently high sensitivity, but also to the relationship etween observed fluorescence and fluorophore concentration in ideal ituations. This point is made in many current analytical textbooks hat include even rudimentary descriptions of the processes of photo- uminescence. This relationship can be represented as follows: 12 psi. = f(e).gex).¢o.§. (1—e'eb°) = f(e).g(7\).¢o.§~x 2 3 n [1414.3 ebc + (2.32€!bc) _ (2.3!ebc) + + (2.3n61bc) ) 1 = f(e).g(7\).¢o.§-x 2 3 n I: (2.3ebc — (23290) + (2.36bc) + + (2.3ebc) 1 (L1) 3! n! Where f(e) is the geometry depending on the effective solid angle viewed by the detector, g(7\) is the response characteristic of the detector varying with wavelength, and 6 , b and c have their usual Beer's law connotations. When the fluorophore is very dilute (i.e., when the primary absorbance of the solution is less than 0.011, only the first term of the power series is significant. The expression reduces to the linear form: 551. = f(e).g(7~).¢o.<§.€.b.o. (5) : K'. ¢O.C. (6) Fluorescence radiant flux is a linear function of concentration and of anident radiant flux. The term K' for a particular set of experimental .onditions is equal to a collection of constants, f(e).g(?\).§.€.b. At higher fluorophore concentrations it becomes necessary to in— lude additional terms from the power series. Examination of the xpanded equation Shows that the terms alternate in sign. As more terms re evaluated (for higher concentrations), those which are negative egin to influence the overall value significantly. 13 The concentration level above which non-linearity becomes apparent :an be determined by: C _ 0.01 maximum ' exb (7) Where Ex is the molar absorptivity at the wavelength of excitation and b is the sample pathlength along the axis of excitation. For establish- ing a standard curve it is important to determine the fluorophore concentration at which the fig vs. Cf plot becomes significantly non— linear for the accuracy desired. At sufficiently high concentrations the fluorescence radiant flux reaches a peak and then decreases rapidly. This observation1 demon- strates that fluorescence radiant flux is a function of the penetration distance of the exciting radiation and increasing fluorophore concen- tration (Figure 2). Recently investigators have described this pheno- menon as an apparent shifting of the most intensely fluorescing region ‘Of the sample solution toward the excitation face of the cell as the ipenetration depth of the exciting radiation is reduced by increased Jabsorption (Figure 3). It has been shown that the position of the {geometrical detection window is directly related to the curvature of the ifluorescence response1o. Departure from linearity is mainly due to aprimary absorption (Figure A). Other key processes involved are self- :quenching and secondary absorptions (Figure 5). The primary absorption process is responsible for supplying energy Ethat is subsequently reemitted as fluorescence radiation (Table 1). Its Molecular basis had been first identified in the early thirties. Later 1 _ studies11 13 further described these forms of energy transition, both 1A .chMpmapcwomoo oumflcoacopqfl m>ammooosm on swsoenpmAmv emepqoocoo >Lw> Amy “sovcflz scammflEo mmoeom coepmscoppm mu macaumcpzoocoo ecosqoeosam m: o oocmpmwa GOHpMLpocom one we 20H m Lo>oc0fiucflcmmmH>e>D m pOGSh .m mm “compmscouum $u caoapxm .cop canopoouop o: .musaflp mam> m "mom A V HmmmcoeH no moHLom Hecapospoazm xzam pcmficmm oocoommLOSHm .N maamHm 1..._ AZ 3 4 II n 19 FIGURE 4 20 g Emission Axis With Res- Figure 5. Effect of Secondary Absorption Alon pect to Apparent Luminosity Top = Reference medium (negligible secondary absorption) Bottom = Sample medium (detectable secondary absorption) Y1,Y2 = distances of excitation beam axis from the emission port cuvette face Excitation Source Radiation Emission Radiation Key: EX EM 21 FIGURE 5 22 theoretically and experimentally. It was not until the seventiesg’11 that an instrument was developed to correct fluorescence measurements conveniently for this attenuation. Prior to the advent of correction procedures, accurate analysis could only be attained by working with dilute solutions where the attenuation caused by primary absorption was negligible. However, the limitation of measuring only dilute solutions is not a universal remedy. This point will be further discussed in the section under conventional methods. The secondary absorption processes include self—absorption and absorption of fluorescence by other chromophores in the matrix. Both processes will cause attenuation of the ¢f vs. C analytical curve. f Self—absorption is a result of overlap between a molecule's absorption and emission bands. The effect is noticeable when the fluorescence is measured on the short wavelength side of an emission band so that the fluorophore itself may absorb its own fluorescence radiation. This attenuation is observed as an exponential decrease of the relative portion of radiation transmitted by the solution at the emission wavelength with increasing concentration of the fluorophore1u. Usually fluorescence is measured outside of this region of the spectrum unless it is necessary to use this region to avoid more severe interferences ‘ from other absorbing species. Fluorescence absorption by other solu- . tion components depends on their respective concentrations and overall ' characteristics of the absorption spectra. When possible the back- : ground matrix should be prepared without inclusion of such component ispecies. If they cannot be eliminated and these effects are signifi- .cant, then a correction for the attenuation must be applied. 23 Performance of routine fluorescence analyses requires that the calibration (standard) curve is prepared with a set of standards possessing an identical background matrix. If this practice is not observed, there is no compensation made for the different degrees of primary and secondary absorption encountered15’16. 2. Qualitative Fluorescence Measurements Fluorescence and ultraviolet-visible spectrometry provide similar information regarding qualitative analyses. The excitation Spectrum of a fluorescent molecule theoretically is Similar to its uv-vis absorp- 17,18 tion spectrum This signifies that fluorophores may be character— ized by direct comparison to universally available uv-vis standard spectra reference files such as those published by Sadtler Research19 and APIZO. In theory, the emission spectrum should be a mirror image of the excitation spectrum. However, since an excitation spectrum is normal- ized with respect to energy along the wavelength or wavenumber axis and that the emission spectrum is not, severe distortions from the expected may result17’18. Also when complex transitions are involved, as in the case of quinine sulfate, emission Spectra will not be a mirror image of excitation spectra. Instrumental factors such as the variation of the source energy with wavelength and variations in the Spectral sensitivity of the detection system distort the fluorescence excitation and emission spec- tra12’13’21-26. Several commercially available spectrofluorometers correct for these effects. 24 Photophysical factors such as primary and secondary absorption can result in severe spectral distortions12. In dilute solutions of primary absorbance of less than 0.01 these factors are usually negligible. An exception would be a system containing a strong secondary radiation absorbing species which is transparent to the excitation radiation. As primary absorption begins to exceed 0.01 a decrease in the fluorescence excitation spectrum relative to the uv-vis absorption spectrum is observed27. As was noted in making quantitative measurements, even higher increases in absorption will reduce the effective penetration distance of uv-vis radiation through the media (Figure 2). This will cause the ¢f vs. C plot to pass through a maximum and proceed to bow f down toward the concentration axis. This may grossly distort the excitation spectrum further departing from it's expected "true" charac- teristics. Attenuations caused by the absorption of emission radiant energy will incorporate a negative error in relative quantum yield determina— tions employing area integration techniques26. Self-absorption can be differentiated from other chromophores 1 since the loss of radiant flux will be greater on the short wavelength 2 side of the fluorescence emission spectrum. Secondary absorption by the background matrix will reduce the 1 observed fluorescence emission. The matrix components will affect the 1 emission spectrum only in the spectral regions where they themselves absorb. In this case quantum yields will also be low. 25 3. The Effects of Primary and SecondarygAbsorption on Other Forms of Information Derived From Fluorescence Measurements Thermodynamic quantities and associated chemical equilibrium con- stants have been extracted from fluorescence data28. Unfortunately, these data, unless corrected for both instrumental and photophysical errors, will give erroneous values for such quantities. Calculations of this nature assume linearity in the Q? vs. Cf interrelationship through— out the concentration range under study. The most highly regarded determinations of these quantities2 involved preparation of calibration curves to encompass the concentration range. In all cases, background matrices were matched to that of the sample. If there are absorption changes, the fluorescence data collected over time as a reaction proceeds must be continually corrected. In a monitored reaction one must be aware of the potential for a changing background matrix. These changes must be accounted for prior to evaluation of rate constants and activation parameters used in estab- lishing system specific rate laws. In recent years correction factors for primary and secondary absorption have been applied to static 2,9,29,30 jsystems An automatic and rapid absorption correcting instru- ment adaptable to repetitive scanning of dynamic chemical systems has inot yet been reported. Until the advent of such instrumentation, the e. 1 1 l meaningfulness of these quantities should be viewed with suspicion. 26 B. Conventional Methods Various approaches, both in experimental procedure and in instru- mentation, have been devised in overcoming or correcting for primary and secondary absorption. The following methods will be discussed: sample dilution; optimum choice of excitation and emission wavelengths; detec- tion geometries (i.e., transmission, front-surface, right-angle (Figure 6); non-typical designs of sample cells; and two-photon excitation. Limitations encountered in each method will be emphasized. 1. Sample Dilution A common "textbook" approach for minimizing excessive sample ab— sorption was to dilute the sample until the linear portion of the ¢f 17’31. For many chemical systems, this was with Cf curve was attained the easiest method for reducing the attenuations due to these absorban- ces. This remedy was not found universally applicable. Chemical systems containing a fluorophore at very low concentrations and a highly ‘ absorbing background matrix are an example. Further dilution of such a system must encounter the trade-off of decreasing Cf below detection L sensitivity of the instrument and never attaining the linear region of ‘ the calibration curve. Also, since a wide variety of chemical Species U are concentration dependent (i.e., possible conformational changes, 5 alterations in intermolecular or intramolecular bonding, formation of ;:different solvent structures) unidentified chemical events can reduce Lthe concentration of the fluorescing species or may cause other signifi- “cant changes in fluorescence. As a result, errors of unknown magnitude are introduced into the measurements. s... 27 As simple as dilution appears, extreme care must be exercized to avoid contamination of the sample by the dilution procedure. Purity of all media used in dilution must be ascertained and an identical back- ground matrix should be included. These precautions will give more confidence to the resulting measurements. 2. Optimum Choice of Excitation and Emission Wavelengths The use of filters and monochromators has made possible the selection of narrow bandwidths of either the excitation or emission radiation. The chosen excitation or emission wavelengths are those at which primary and secondary absorption interferences are minimal. This approach is convenient with commerically available spectrofluorometers since they have both an excitation and an emission monochromator. Problems with this approach arise when the sample complexity precludes the existence of such wavelengths where it may be impossible to find wavelengths where primary and secondary absorption effects are ‘negligible. Where one chromophore does not absorb, another may. The ‘absorption and emission characteristics of other fluorophores may cause spectral interferences. A fundamental limitation is in the reduction of sensitivity by selection of wavelengths shorter or longer than the excitation or Hemission maxima. Wavelengths should be chosen to minimize Rayleigh scattering. The «magnitude of the scatter peak depends upon the absorption characteris— Ztics of the solvent and sample, the size and number of‘ particles Ssuspended, and the wavelength of the exciting radiation. r1 28 Also wavelengths that encompass intense Raman scattering Should be avoided. Raman scatter is the result of interaction of photons with the vibrational energy levels of solvent or solute molecules which results in inelastic scattering of photons of lower or higher energy. A blank is used to determine the radiant flux of the Raman bands which can be subtracted from the sample fluorescence radiant flux. Often it is possible to minimize Raman interferences 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 reduces sensitivity to some extent, but in the process moves the Raman band toward shorter wave— lengths, thus reducing its interference with the emission band of interest. Many commercial double beam spectrofluorometers subtract fluores- cence radiation from a reference solution containing only the back- ground matrix from the sample solution. This will eliminate the scatter bands and the effect of matrix chromophores and fluorophores which do not interact significantly with the fluorophore of interest. 3. Detection Geometries Cell geometry describes the cell orientation as it relates to the 3excitation beam and emission optics. The evolution. of geometries applied to account for the absorption artifacts will be briefly presen- ted here and with regard to applied correction factors in the following section. Figure 6. Detection Geometries A. Transmission B. Front Surface C. Right Angle 29 30 G) W “W R, (TRANSMITTED) ................ WW SAMPLE BECFLUORESCENCE) Weevil???“ ‘10 § © he. (FLUORESCENCE) A. (EXCITING) 45° R. “as? a, (FLUORESCENCE) x. (REFLECTED) FIGURE 6 31 a. Transmission Geometry The arrangement in transmission geometry is shown in Figure 6a. Fluorescence is viewed along the same axis that the excitation radiation propagates. Generally an emission monochromator is required to isolate only fluorescence wavelengths. The advantage, aside from the simplified instrumental set—up, is that attenuation of of measured fluorescence due to primary absorption is much less pronounced than in instruments using a right-angle geome- try32. The actual primary absorption over a constant.distance, 45d, is unchanged between geometries, but as this attenuation increases and, subsequently, the maximal (apparent) fluorescing region compresses toward the source of exciting radiation a dramatic difference between the two geometries becomes apparent. In the right—angle mode of detection the region of maximal fluorescence on increasing primary iabsorption will be at the entrance cell wall for the excitation beam from where it is virtually impossible to isolate emission radiation. With the transmission arrangement all emitted radiation within the detection window is monitored. Negative deviations from the ideal linearfif and Cr plot are thus reduced in the ideal case, dependent only on the inverse square loss of the emitted radiation as its origins become more remote from the viewing detector. The major drawback to transmission geometry remains, however, the quality of the emission optical systems needed for viable separation of stray light from the desired fluoresced radiation. A disadvantage relative to right angle detection is the apparent increase in the effect of secondary absorption32. This is caused by an 32 increase in the average pathlength traversed by the fluorescence radia- tion in the transmission geometry. This average is further increased if 33 the primary absorption is high . The reason, discussed in the problems section, for this observation is depicted in Figures 2 and 3. b. Front Surface Geometry The arrangement in front-surface geometry is shown in Figure 6c. Fluorescence is viewed at a 450 (or smaller) angle of reflection from the axis of propagation for the excitation radiation. Frontal illumination has the advantage of permitting the measure— ment of the emission spectrum of a solution containing a high concentra- tion of a fluorescent substance. Obtaining such a spectrum with right- angle or transmission geometries may be impossible since in very concentrated solutions a large portion of the exciting light is absorbed close to the region of entry into the cell. For concentrated systems, this geometry is the only feasible way of keeping the fluorescing region in the detection window. This greatly reduces the primary and secondary absorptions that are encountered by further penetration of the solu- tion. However, this approach only reduces the magnitude of these absorptions, they are not eliminated. Various investigators have demonstrated that, indeed, secondary absorption can be quite high with 33.35. some systems Later, suggestions were made for exciting only at absorption maxima and thereby reducing the high concentration of fluor- ophore required17’35. When dilute solutions of a fluorophore are measured, the observed fluorescence is proportional to concentration and an excitation spec- rum unattenuated by primary absorption is recorded. At higher concen- rations where primary absorption begins to attenuate the ¢fwith Cf 33 >lot, distortions appear in the excitation spectrum. The major disadvantages inherent in this mode of detection are high :econdary absorption effects, an increase in the probability of quench- .ng because of the high concentrations, significant distortions of the 32’3” and emission spectra due to re-emission of absorbed radiation 'eduction of fluorescence if analyte association occurs. In addition, [on-orthagonality between excitation and emission beams and their res— )ective cell faces can induce severe polarization artifacts and multi- >le internal reflections. c. Right—Angle Geometry The arrangement in right-angle geometry is shown in Figure 6b. Fluorescence is viewed at an angle of 900 to the propagation axis of the Excitation radiation. , The major advantage of right-angle detection, where the detector onitors only the illuminated liquid and not the illuminated cell face, s that interference by stray excitation radiation arising from either eflections at the cuvette faces or any fluorescence of the cuvette tself is minimized. A limitation is the fact that the excitation radiation has to pass hrough a depth of solution, Ad, before reaching the region viewed by he detector where its effective radiant flux may be reduced by primary sorption. This arrangement is suitable for moderately absorbing lutions, which generally refers to dilute solutions of the fluoro— ore. Considerably larger concentrations of a transparent solute in e background matrix can be tolerated with no adverse effect. 34 4. Designs of Sample Cells The normal cell employed with commercial spectrofluorometers has dimensions of 1 cm x 1 cm x 4.5 cm height. Occasionally instruments using cells of different dimensions have been reported. Absorption interferences can be reduced by taking advantage of shorter pathlengths since primary absorption is proportional to dis— tance of solution traversed. In many cases, the analysis can be confined to the linear portion of the ¢§.with Cf curve36. In addition to absorption, deviation from linearity may be caused by light scatter— ing and fluorescence of the cell walls when right—angle geometry is applied. Care must be taken that the detector monitors only a region within the solution, otherwise large distortions may result. Fiber optics have been used as channeling devices for both excita- tion and emission radiation. The fiber optic bundles are attached directly to the faces of the cuvette. This approach has been observed 37 to account for a three-fold decrease in primary absorption artifacts 5. Two-Photon Excitation A novel method has been developed to eliminate virtually all interference of primary absorption38. In principle, photons possessing half of the energy required for absorption are passed through the media containing the fluorophore. When two such photons impact on a fluoro- phore molecule simultaneously, they are absorbed and the system suc- cessfully reaches its first excited singlet state. The investigators have found that very concentrated solutions could be monitored without excess absorption artifacts. 35 Disadvantages2 include considerably more expensive and complex instrumentation, continued presence of secondary absorption effects, and the requirement for a laser source to increase the probability of simultaneous impact of low energy photons. The method provides an interesting, although impractical for routine analyses, approach to the problem of primary absorption. C. Mathematical Corrections The objective of the conventional methods described in the last section was to eliminate artifacts due to excessive absorption. None of the instrumental arrangements was found to be a universal remedy. A direct means of eliminating these attenuations may prove to be elusive, but an indirect means has already been introduced. If either the primary or secondary absorption can be described by a mathematical expression, then a correction factor can be derived and applied to the fluorescence measurements. 1. Absorption Corrections for Transmission Geometry Crude attempts to describe primary absorption were made as early as ‘193039. Little success was achieved. The expressions used were later 40,41 , more found to be simplistic and incomplete. Soon after in 1938 elaborate expressions were derived in terms of primary and secondary absorbances. It was later deduced that these were based on an incorrect iassumption. All of the excitation radiation absorbed does not, as the expressions indicated, contribute to the emitted fluorescence. Back- ground matrix components sometimes absorb and may not fluoresce. In W94442 similar equations were derived. This time agreement was satis- [ factory in the comparison of calculated and measured fig. values. I i 36 However, the systems were composed of a single fluorophore in a non— absorbing solvent. In 197343 21 strictly theoretical work appeared accounting for these absorptions, but that an accurate compensation for the factors would require homogeneous, monochromatic and collimated excitation radiation and monochromatic and collimated fluorescence radiation. 2. Absorption Corrections for Front-Surface Geometry For front-surface geometry the first expressions to account for both primary and secondary absorptions appeared in 193840,41. Similar descriptions of these effects appeared later in 1951”” and again in 197318. ”548 a None provided experimental evidence. From 1956 to 1959 series of papers presented very elaborate corrections and experimental verification. Effects such as multiple reabsorptions and re-emissions were considered and incorporated into the correction schemes. In 1961).19 these expressions were adapted to account for the apparent receding of the maximal fluorescing region from the front face on dilution. Limita— ‘tions at the detectable extremes of fluorophore concentration were jdiscussed. Other work reported on corrective expressions for this geometry was concerned with correcting fluorescence quantum yields32’50-53. 3. Absorption Corrections for Right:Angle Geometry The first theoretically derived expressions to account for primary and secondary absorption appeared in 1938u0. They were later found to be too simplistic. They failed to include the possibility of absorbing ‘species other than the single fluorophore. Also, the geometrical models \ lused failed to describe accurately the actual experimental arrangement. 37 During the early fifties renewed interest in this area was awak- ened. One theoretical paper1M was presented that based all of the fluorescence as originating from a point source. Experimental evidence 55’56 and offered more was lacking. Two more papers quickly followed elaborate expressions. Both ignored the excitation and emission window dimensions, critical in avoiding attenuations by the cell walls. The late fifties offered a theoretical description «of primary absorption based only on the solution (sample) absorption of excitation radiation and dimensions of the emission window27. Both the fluorophore and it's background matrix were considered. In the early sixties59 a more extensive treatment of derivations based on this measurement geometry was published. Both calculated and analytically measured curves of‘¢%.with Cf from several chemical systems were in agreement below a primary absorbance of 0.5. Large deviations ‘ were found to occur at higher concentrations. Inaccuracy in defining the applied detection geometry and failure to incorporate secondary ; absorption effects were limitations in extending plot linearity into : higher concentrations. Further documented attempts at experimental verification were not 57 11,58 to follow until the early and mid seventies Finally an accurate lexplanation of the validity of this approach had surfaced. Holland9 .found primary absorption to be independent of the nature of the absorb— ing species and the excitation and emission wavelengths. A computer {centered spectrofluorometer (capable of making simultaneous absorption _and fluorescence measurements) was developed for testing a newly devel— oped mathematical model, more detailed than the ones previously pro- \posed, that was derived to account for this absorption. This provided 38 the basis for calculating the required correction factor. Holland, 11’29 found that this model resulted in primary- Kelly and co-workers absorption-corrected fluorescence that was linear with the fluorophore concentration in solutions with total absorbances as high as 2.0. However, a limitation of this approach was not including a correction factor for the effects of secondary absorption. In the mid sixties60 a theory that developed expressions for primary and secondary absorption as a function of observed signal to noise ratio in photoluminescence spectrometry was formulated. Investi- gators2 later questioned the validity of the expressions since they were partly derived from another theory describing self—absorption in flame photometry. 43 A comprehensive theoretical work in the early seventies appeared to account completely for the geometry, all components responsible for both primary and secondary absorptions, and reflections within the sample cuvette. Soon afterwards, experimental verification was forth- coming61. The above theoretical expressions were adapted to procedures :requiring fluorescence measurements to be made at three cell positions. :Here the sample cell was manually shifted with respect to the excitation | \ ‘and emission optical axes. The positions are illustrated in Figure 8. *A correction factor was calculated for the attenuation of the fluores— flcence signal as a function of distance along either axis. This 2,61 ;procedure was called the "cell shift method" . Practical use of this ‘method was severely limited by problems in the manual re-positioning of ,the sample cell66. Over the last two years, a series of publications appeared by ‘Christmann and co-workers3o’62’63 offering a detailed critical 39 ooa>om coapmuom owmaH Lopnsoo savanna moammmm :OHmeflHHoo soammfism :8 Susan Haoo oomogomom oz on» Low pcoao cmnp< Lfiosa one mucoqoasoo 40 N. ”#50; fickaiommUOZOE MOPoz aoo Hmoflu O was HmUHHumEomw .m ouswfim 48 m memzu m0hmm<2Ea mzmm wz_mDoOn_'\ mommzz 4‘————————‘—————————————————-————————______________________f::::]III'FV 49 (3) For the duration of‘ any observation period, the quanta fluoresced by any absorbing fluorophore species are linearly related to the quanta absorbed by that species F .... s was) Where Q = quantum efficiency and k" = geometric and instrumental constant. (4) The ratio of the fluorophore absorbance to the total absorbance in the cell is equal to the ratio of the quanta, Qf, absorbed by the fluorophore to the total quanta absorbed, QT. (5) A fixed fraction of the fluorescence radiation generated within the observation window is viewed by a detector with uniform sensitivity. F measured = k"" F window (6) Only the fluorescence of a single fluorophore is measured and any absorbance of this fluorescence is negligible. (7) The effects of scattered light, refractive indices, and anisotropic characteristics are assumed to be negligible. c. The Beam Intensity as a Function of Measured Quantities Assuming that the excitation beam is attenuated exponential- ly, the radiant flux at any point on the beam axis in the cell may be represented as a function of the distance from the point of entry. . f4 ‘5ng ‘v: «~ 5O d¢= -¢€cxdx In measuring absorption, the beam usually passes through the entire cell, and thus x is equal to the pathlength b. If a partial penetration of the cell is considered, the following expression can be derived11: X ¢x = Re (BlnT) = RT(X/b) Substitution of x/b by ax, the fractional distance across the cell, produces: 92‘, = RI‘“’ representing the radiant flux of the beam at any fraction of the cell length,CU', from the plane of entry. d. Emission Radiation (Fluorescence) in Terms of Measured Quantities and the Observation Window. Since F is assumed equal to the product of a constant and the quanta absorbed within the observation window, and that the fraction of the total quanta absorbed by the fluorophore is Af/(Af + Ac), the derived expression for fluorescence becomes: 012 an 2.3 K Af R (T — T ) lnT e. Dependence of Corrected Fluorescence on Absorption. (Two Approaches) (1) Absorption-corrected fluorescence may be represen- ted as the sum of segments Aid wide across the window from «’1 to (0'2. Here it is assumed that the source-corrected fluorescence ratio FV¢% 51 actually describes the limiting condition as sample absorption approaches zero. The following expressions were presented: F = Fm F (41 +501) 1lie 00 -—- + + . . . + ——— 75011 («J1 +Au1) 75.12 out. = 23K Af-Zw Aux («f-U) :2.3KAf 12 (2) Absorption-corrected fluorescence may also be defined as the observed fluorescence divided by the average radiant flux of the excitation beam across the window from 1 to 2. The following expressions were reported: “’1 ”2"”1’ 1 1 | = F lnT (”z—v.13) 1 2,...) R(T (“f-W) =2.3KAf 21 (3) Both approaches led to the same solution. The Aw value will be a constant for any system where the observation and detection geometry are not changed, therefore: 52 The important implication here is that fluorescence values corrected in the above manner will be linear with the absorbance of the fluorophore even if one or more chromophores are present. f. The Primary Absorption Correction Factor The absorption correction factor is defined as: w’... lnT (2 «{) (If U (T 2—1‘ 1> and the corrected fluorescence becomes simply: f F Fco ' (R) x fa Note that a similar correction factor had been introduced earlier27. g. Limitations Use of the determined correction factor, fa, was considered valid only if the assumptions used in its derivation are reflected in the chemical and experimental measurement conditions. Assumption (1) is found valid from consideration of their instrumental arrangement. Assumptions (2), (3), (4), (6) and (7) could be satisfied by the correct choice of fluorophores and chromophores. The remaining assumption, (5), required further consideration. Ideally, all lateral information along the excitation beam axis between X1 and X2 should reach the detector. However, such a goal cannot be reached without geometric modifications due to emission ‘monochromator slit orientation. The approach used was suited to the .case where the slit height of the monochromator is larger than the horizontal dimension of the fluorescence window. Here, this end was 53 accomplished by a 900 rotation of the fluorescence cell image through the use of front surface mirrors. As mentioned in introducing the applied geometry, a 1:1 image ratio was attained at the entrance slit to the monochromator. The second requirement assumed that the fluorescence must be observed at a fixed observational angle at any point across the fluores- cence window. This goal was closely approached by placement of a second mask, with the same window dimensions,uf, as the window in the primary mask, at a distance, d, from the source of fluorescence as shown in Figure 10- The e and s angles are represented as the limiting values for the observation angles across the fluorescence window. The differences between 9 and 6 for various d values were expressed as relative percent error. Ideally, it would be desired to collect only radiation which is parallel to the optical axis. However, the large secondary mask ‘ distance would be prohibitive and greatly reduce efficiency in radia- 1 tion collection. A compromise was reached allowing a maximum error of 1 1%. This resulted in a secondary mask distance of 4.0 cm. The error in { observation angles across the cell was small while permitting the 1 1 passage of a reasonable amount of fluorescence to the detector. A The last requirement inherent in assumption (5) dealt with the iuniformity of the detector sensitivity. Once this was ascertained as iisatisfactory, all of the requirements of this assumption were ful- 1 filled. With use of the proper fluorophores in conjunction with the 1 instrumental configuration outlined, correction of raw fluorescence .{data could be found to give an extended linear relationship between ,,fluorescence and the fluorophore absorbance. Evidence presented demon- .istrated that the absorption-corrected fluorescence is linear with 1 54 Figure 10. Limiting Conditions for the Fluorescence Observation of Fluorescence Radiation by the Moving Mirror Method ...-,‘.'. I ‘-“'.'..- '5" . .4 - " {3'15} I SECONDARY ' J MASK L i l ’1 EXCITATION BEAM r PRIMARY MASK .~-..' .,...._. ...--,_. .'..'.¥" ....... .._-... , .o .‘ ‘ .- n ... .. .,'..... . - .-. . l \f SECONDARY MASK EXCITATION BEAM FIGURE IO 56 fluorophore absorbance, up to a solution absorbance of 2. 3. The Cell Shift Method a. Geometry The geometry used in this method is illustrated in Figure 11. A square cell of internal dimensions b x b cm was employed. The minimum and maximum pathlengths (cm) for absorption of exciting radiation at B.are Xc‘and RB, respectively. The minimum and maximum pathlengths (cm) for absorption of fluorescence radiation at k" and Yea and yfi, respectively. b. The Basic Assumptions30 In addition to the cell geometry, it was intially convenient to nake the following assumptions. (1) The excitation beam is homogeneous, collimated, and nonochromatic with a wavelength ?\- (2) The emission beam is collimated and monochromatic vith wavelength A ’. (3) Fluorescence photons which are absorbed in the cell ire not re-emitted by the sample. (4) Scattered light, refractive index effects, and re— ‘lections within the cell are negligible. (5) The sample is homogeneous and contains a single ‘luorophore although other chromophores may be present. (6) Both primary and secondary absorption processes bey Beer's Law. 57 c. The Attentuation by Primary Absorption A derivative form of the correction factor determined by Holland and coworkers11 for primary absorption was applied. However, the usefulness of such corrected measurements was limited to where the absorbance at the excitation wavelength is not higher than 2.0 A. The problem deduced was that the primary absorption-corrected fluorescence is directly proportional to the fluorophore concentration only if the sample is completely transparent to the emitted fluorescence. Because many real samples contain components which absorb the fluorescence of the analyte, consideration was given to the attenuation caused by secondary absorption. d. The Attenuation by Secondary Absorption The fluorescing volume of solution, which is viewed by the detector was represented as a collection of n parallel and equally spaced plane sources of light, each at a different distance yi (cm) from the emission face of the cell. Each of these planes contributes a component of radiant flux to the emission beam such that beam flux at the cell face can be given as: ¢=Zj¢i In a case where secondary absorption is negligible and if assumptions (1), (2) and (5) are valid, each plane contributes an equal component to the emission beam. But, if secondary absorption occurs, the power contributed from each plane is attenuated in accordance with the Beer- Lambert law. The steps in the derivation of attenuation by secondary absorption 58 are detailed in the 1980 paper by Christmann and coworkers3o. The 'esult obtained was: lim _ T93 41‘9“ new ‘ AelM? This indicated that the fraction of radiant flux in the emission beam :ransmitted to the cell wall is an explicit function of sample transmit- :ance at the emission wavelength‘bJ and excitation window parameters an and Sec. e. Absorption Effects on the Measured Fluorescence Signal It was found that actual measurements required that condi— ;ions of assumptions (1) and (2) must be relaxed. In instrumental set— zp the excitation and emission beams are nearly collimated over small iistances, but never truly monochromatic. f. The Correction Factors When the spectral bandpasses for excitation and emission are Jade sufficiently narrow, the sample transmittances at the respective mvelengths ).and‘}d do not vary significantly over the particular andpasses. The expression for the fluorescence signal current was ;iven as: «f «lb; lf‘pa = 2303 K Ark lnTk £3 (T‘k') 3-(TX')G°‘ ,ffooo (9.5 -eo<.) lnTx' 59 All wavelength dependent factors are included within the integral. In the case where Thand TA' go to unity, the fluorescence signal becomes ,-.,,,.f./... totally absorption free: i = 2.303 K A 0 f8 “If The relationship between éfpa and iothen becomes: 10 = (“é-UuMnTs, (eg -eo<)lnT5,' _ i (T,)“’13 4n. )‘”’ (T we»?! -(T 50°04 fpa = fa1' fa2' lfpa Where fa1 is the primary absorption correction factor and fa2 is the secondary absorption correction factor. g. A Limitation of the Secondary Absorption Correction When the absorbed fluorescence is re-emitted by the sample, this type of correction breaks down in a violation of assumption (3). This was considered to be a potentially serious limitation of this secondary absorption correction because many fluorophores have highly overlapping absorption and emission Spectra, and thus absorb and re- emit their own fluorescence. Solutions in which the re-emission of absorbed fluorescence radia— tion is negligible permit correction of right-angle fluorescence mea— surements for secondary absorption interference. When both primary and secondary absorption correction factors are applied, totally absorp— tion-free fluorescence information is generated. ‘. i 6O 4. Method of Cell Rotation a. Geometry A diagram of right-angle geometry is shown in Figure 11. The fields of detection are shown for both the emission axis (top) and excitation axis (bottom) of Figure 12. For the following mathematical treatment it is assumed that the sample cell wall is transparent to the radiation, and of negligible thickness. b. Basic Assumptions (1) The excitation radiation is homogeneous in wave- length, 61, and is collimated. (2) The emission radiation is observed as a collimated monochromatic beam of fixed dimensions with wavelength,é»2. The width of the window of emission observation is much less than the total cell dimension (width) such that distinct slices with varying cell positions are obtained. (3) Photons of emission radiation which are absorbed by the sample solution will not be re-emitted. (4) Effects due to scattering of excitation radiation, changes in refractive index, and internal reflections are all negligi- ble. (5) Samples are composed of a single fluorophore, but may contain other chromophores. (6) The cuvette walls are matched in transmission characteristics for both excitation and emission radiation, and the walls parallel to the viewing optics do not affect the observed fluores- cence. 61 Figure 11. Geometry for Right-Angle Fluorometric with a Square Cell of Internal Dimensions b x b cm. Key: X0: X5 ‘9: pathlength (cm) for absorption of exciting radiation at nm, minimum detected in win- dow. pathlength (cm) for absorption of exciting radiation at nm, maximum detected in win— dow. pathlength (cm) for absorption of fluores- cence radiation at 'nm, maximum detected in window. pathlength (cm) for absorption of fluores- cence radiation at 'nm, minimum detected in window. Xu/b )QB/b Yx/b Y13/b 62 fl rX L——>. <9 V EX FIGURE 11 63 Figure 12. Regions of Observation for the Method of Cell Rotation A. Along emission axis. B. Along excitation axis. 64 EMISSION AXIS EXCITATION AXIS @ FIGURE 12 65 (7) Both primary and secondary absorption processes bey Beer's Law. 0. Primary Correction Factor The top half of Figure 13 shows a typical case where primary bsorption is manifested. Two illuminated slices are detected, one at 03‘ and one at 01:3 . The change in absorbance between OJ; and 01'}; is efined as follows: 15.. A A = l — l 2 My?” ) ugly? = ln(¢w.¢) - ln(¢w,5) - 1n(£§—15 _ 55% he calculated change in absorbance between the entry plane and “‘(is btained as follows: QJ’ AA = AA ( d 1 2 “59-0,?“ : 111% ) fifianz n this manner it is possible to calculatefig. Since the fluorescence adiant flux is directly proportional to the incident radiant flux of He excitation radiation, according toféf = k Ciéy then the following alationships can be given: o%_oc xii—ms (5%?)ac , Egg)” 66 This allows correction for primary absorption according to the follow- ing expression: 0J« (———)- A ¢cr1° = 75r(1)'e “13'wa 2 Where ¢cf1° 13 primary absorption corrected fluorescence and ¢f(1) 13 the uncorrected fluorescence reading at position (1). The above correction factor is determined while holding secondary absorption constant. This situation occurs between positions 1 and 4 of Figure 12. d. Secondary Correction Factor The bottom half of Figure 13 shows a typical case where secondary absorption is manifested. Two illuminated slices of volume are detected, at fractional distances of eac and as. The change in absorbance between these planes, ex and 3%, may be defined as follows: . ' ¢$ AxA. = 1n§§9 ) - 1n ) 2 Gfi Soc : lnwefi) - ln(¢e°c) : lné‘Z—f—c) The calculated change in absorbance between the exit plane (since Yea: (b-¥5)) and as is obtained as follows: 1 V efi AA =AA (——_——-) 1 2 a“. a» Figure 13. 67 Attenuation of Radiation A. Along excitation axis B. Along emission axis 68 ex EMISSION 13 FIGURE 938 zo_.r<_oE.zm no mza._.zw owk<40a9 cam: ma Louos one IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII p02 Longbow .NF onsmflm smomumam cHIxooq one Lo .xflocoaam 02» CH oanmaam>m can once Essfiesam some m ma woman Losoa ose 83 D manor. ZQOLH POSITION FLUORESCENCE r PoSrfloN ‘4 SCAN V MONITOR 4 V b V PosnioN a SCAN FIGURE 20 SCAN 0R DISK RETRIEVE ‘ FROM DISK I SCAN Figure 21. Key: 91 Positions of Emission Detection Fields. 1 2. 3. )4 0 Least Primary Absorption, least secondary absorption. Least Primary Absorption, most secondary absorption. Most primary absorption, most secondary absorption. Most primary absorption, least secondary absorption. Corrected for attenuations by primary and secondary absorptions. 92 EMISSION hug, DETECTION iary absorption. iary absorption. ary absorption. @ dary absorption. _«y and secondary EXCITATION SOURCE EXCITATION hi}: TRANSMITTED FIGURE 21 93 3. The Cell Positioner This device, detailed in Figures 17-19, and photographed in Figure 22, was designed to provide a very rapid and highly reproducible means of automatically correcting for both primary and secondary absorption effects. The durable construction and the choice of stepper motor was to allow continual service over long periods of time without the need for re-alignments. The positioner has a range for tsd between 0.2 cm to 1.0 cm along either of two perpendicular axes. This permits a large choice in the rotation patterns, both in magnitude and in ellipticity. While holding the chosen major axis constant for Ad and decreasing Ad along the minor axis one may readily obtain rotation patterns as shown in Figure 23. The cell displacement along either axis is a function of the respective lead screw rotations, and is found to be highly reproducible. This is shown in Figure 24. This figure demonstrates the linearity and accuracy of the posi- tioner along either of the Optical axes. Vernier calipers were employed to ascertain cell displacements in X and Y dimensions from the station- ary outer walls of the cell holder platform. These displacements were plotted against lead screw revolution. Linear regression gives a correlation coefficient (essentially) equal to unity in both cases. The precision of the positioner was determined by measuring A/D converter output after each of five successive 360o rotations and averaging to obtain the error distribution. Possible shifting of the average was determined by several sets of five successive readings taken consecutively. The error bars between sets did not alter in magnitude and differences between the averages were negligible. Figure 22. 94 Cell Positioner as Employed by the Cell Rotation Method I NN mane; \x. Home. s wx..\.s\s$\\\ . x... \ 95 Figure 23. 96 Elliptical Cell Rotation All measurements reported in this thesis were conducted with larger or smaller circular rotation patterns. Other possible rotation patterns include ellipses with major axes along either the X- or Y- direction. Extremes of ellipticity would allow study of either the primary absorption or secondary absorption alone. 97 ELLIPTICAL CELL ROTATION FIGURE 23 Figure 24. 98 Cell Displacement in X— and Y-Dimension as a Function of Lead Screw Rotation Off center positioning and securing of the cell holder on the post platform is accomplished by perpendicular sets of 8-32 brass lead screws. Circular rotation patterns pos- sessing AId values anywhere from 2 mm to 10 mm can be set by matching lead screw revolutions. Note that in a circular pattern Ad primary is equal to Ad secondary. IN CENTIMETERS DISPLACEMENT l.2 |.0 0.8 0.6 0.4 0.2 LE I.0 0.8 0.6 0.4 0.2 - X - AXIS 3 6 9 I2 LEAD SCREW REVOLUTIONS - Y - AXIS 3 6 9 12 LEAD SCR EW REVOLUT IONS FIGURE 24 100 The sample cell is held by eight nylon screws against the four beveled edges as shown in Figure 19. The cell is suspended vertically with the four slots of the holder centered. The walls of the slots are 1/64 inch in from the inner faces of the cell. Several thin coats of ultra flat black paint were used to minimize reflections. The rugged design of the holder was intended to prevent transferred distortions to the cell edges while being locked in position. The post cell holder platform is tightly held on the post by one brass 8-32 screw. The post is countersunk into the base of the platform to aid in looking the two components together. The lower end of the post contains a cylindrical opening, the inside diameter of which is 0.003 inch larger than the outside diameter of the stepper motor drive rod. The six lock screws are arranged in two sets of three as diagrammed in Figure 18. They are used both to align vertically the post, and subsequently the sample cell itself, and to secure the post on the rod. The lack of vertical truing can lead to a 2 to 3 fold decrease in the detected fluorescence signal, as shown in Figure 25. The stepper motor is tightly locked on the heavy aluminum platform by a series of eight 8-32 screws as shown in Figure 17. The platform possesses an extension that permits use of a clamp device to attach it to the instrument table. An alignment tool has been designed to aid in rapid initial positioning of the entire stepper motor platform. This tool is shown in Figure 26. Both the stepper motor and platform, readily fit into the lower half of the Perkin-Elmer Model 512 spectro- fluorometer cell compartment. ‘4.... _A:._,. : la 101 Figure 25. Z Axis Truing The cell holder post is designed to allow fine tuning along the vertical axis. In a rotating system, this is critical to aid in preventing a forward and backward wobble of the cell surfaces perpendicular to the excitation and emission optical axes. The post can be raised or lowered to accomodate different volumes of solution. A shift of A inch is possible with maintenance of all six lock-in screws. 102 L FORWARD VERTICAL BACKWARD MAX RADIANT FLUX 1 .14- .I6 .11 .06 O .06 .17. as .14 DEGREES OFF VERTICAL AXIS FIGURE 25 Figure 26. 103 Stepper Motor Platform Alignment Tool This device permits rapid alignment (coarse) of the field of detection within the cell. Once the approximate posi- tion is reached the stepper motor platform is locked onto the bench. In the secured state any vibrations encountered by the cell following a rotation are minimal. 10M STEPPER jmnamwmw MOTOR PLATFORM FIGURE 26 105 A shield to prevent access of stray external radiation divides the upper and lower halves of the cell compartment. A slot in the shield facilitates the post positioning in alignment of the cell with the optical axes. A black sliding disk is secured to the post over the slot to keep unwanted radiation from penetrating to the top half of the cell compartment. The specifications for the stepper motor are provided in Table 6 (Appendix). The rotation rate is 2.5 msec per 1.80 or 125.0 msec to shift between adjacent cell positions (900 apart). The stepper motor is rotated counterclockwise by a preset sequence of fifty pulses for each 900 turn. These pulses proceed through an interface that increases pulse width (Figure #5, Appendix) for compatibility with the power translator (Superior Electric Company, Slo—Syn Translator Model STM- 1800). H. Detection Field Characteristics Study of the narrowness of the field of detection was completed. Both primary and secondary masks were used at a separation of 3 cm. The thickness of the primary masks was 1/32 inch. The thickness of the secondary mask was 1/8 inch. Primary masks were attached 3/8 inch from the outside surfaces of the cell walls flush with the outer walls of the cell holder as shown in Figure 27. They were secured in place with rubber cement. Each mask covered % of the surface area and had been painted ultra flat black to absorb most of the incident radiation. A single secondary mask was placed in the emission port filter clip 5 Shown in Figure 28- This mask possessed a milled & inch vertical m '—.i.'i‘—a—HL:.‘ ‘— Figure 27. 106 Study of Detection Field Using Primary Masks The masks were 3/8 inch from the outer faces of the sample cuvette and they extended halfway across the cell as shown in each of the four positions. They were painted ultra flat black to absorb most of the incident radiation. The differences between measurements made with the masks and without them were negligible. This supports the belief that the field of detection is quite narrow. FIGURE 27 Figure 28. 108 Study of Detection Field Using Both a Primary and Secondary Mask The primary mask was situated 3/8 inch from the outer face of the sample cuvette while in position one. The secondary mask was placed in the emission port filter clip. The primary mask extended across half of the cell face in both cases. The secondary mask was centered on the detection "hot Spot" of the parabolic mirror. A verticle slot of A inch width was not found to apprecia- bly attenuate the measured signal. This further sup— ports the contension that the field of detection is very narrow. 109 FIGURE 28 110 slit which was centered on the detection "hot spot" of the recessed parabolic mirror. This mask was also painted ultra flat black. Table 3a illustrates the effect of the primary masks. A comparison of measurements made with and without the masks indicate negligible differences. This demonstrates that no attenuation is evident and that the masked region of the solution has no emitted radiation reaching the fluorescence detector. Table 3b illustrates the effect of the secondary mask. Providing that the verticle slot is A inch (or wider) and is centered on the "hot spot", the differences observed with and without the mask are negligi- ble. Inclusion of both primary and secondary masks verifies that the field of detection is quite narrow. This provides a basis for the initial assumption that the emission radiation is observed as a very narrow bandwidth and collimated beam with a fixed limited angle of admittance. 5. Detection Window Calibrations The emission window was calibrated to determine the maximum allow- able A.d between positions one and four, two and three. The chosen positions must be such that no significant attenuation is encountered by having the field of detection interact with either cell wall. Measure- ment of changes in fluorescence radiant power across the cell interior were made in increments of 0.5 mm. The sample studied was 1x10-6 M quinine sulfate in a background matrix of 0.1 N H280” prepared with house distilled water. This sample was chosen because very little attenuation of the excitation beam occurs. The solution was excited at 250 nm and the emission was monitored 111 Table 3a. Effect of Primary Masking Cell Positions Conc. Q.S. gggflg (1) (2) (3) (4) 1 x 10"6 M No 365 368 308 310 1 x 10'6 M Yes, 10 362 367 310 310 2 x 10‘6 M No 718 713 559 562 2 x 10'6 M Yes, 10 720 717 560 564 ...)...”- as». Table 3b. Effect of Secondary Masking Cell Positions ,3...;€3 3.14:. Conc. Q.S. Mask (1) (2) (3) (4) . 1 x 10‘6 M Yes, 10 360 365 306 307 1 x 10'6 M Yes, 1°+2O 360 362 306 309 2 x 10‘6 M Yes, 10 720 718 565 562 2 x 10'6 M Yes, 1°+2° 716 716 560 557 Note: Quinine Sulfate in 0.1 N 32304 Sensitivity 0.1x Slits EX/EM 10/10 nm Ref. Attenuation Control 600V (Adjusts reference radiant power to use full range of A/D converter Output for largest analyte peak, 0000 to 4096). Fri-,1»— 112 at 460 nm. The fluorescence profile along the emission window is shown in Figure 29. To avoid attenuations positions were chosen 3mm removed from either cell wall. This led to the routine use of a H mm cell centered Ad value along this axis. The excitation window was calibrated to determine the maximum allowable 13d between positions one and two, three and four. In this case, the chosen positions must be such that negligible attenuations are encountered by having the excitation beam interact with either the front or back cell surfaces. The fluorescence radiant flux was measured at increments of 0.5 mm across this window. At each of these increments the emission window axis was also calibrated from wall to wall. On completion a full matrix of values for the excitation/emission window mapping were obtained. The fluorescence profile along the excitation window is shown in Figure 30. It became apparent that a 4 mm cell centered (Ado value was also the maximum along this axis. The above mapping was generated with the cell originally in position one. Rotations of the cell within the cell holder were made to study the quality of the matching between the four vertical cell surfaces. Minor differences within 2 mm of the cell walls were detected. Otherwise all four cell surfaces were matched. If signifi- cant differences were to exist then the correction procedures used would have to account for the transmission differential for two adjacent surfaces after each cell rotation. Use of a circular rotation pattern resulting in d's of H mm avoids the need for such two surface matching corrections. 113 nrmwxm was» macaw pd. Lou nonopcoo So :6 no ooaono 02.330.» on» on pod mafia: :00 on» on one? oogmzcopflw Bo m : >2 60 F s3 Eo P mo macamcosflc mm: Sec 5.3an one .mHHmz :00 songs on» a «C 00:96? mo nowpoca m mm and unmanned monoomososfiw popoopoc 0:» 3230.32 p03 05. IIIIIIIIIIIIIIIIII Zoo oaasmm sounds noammaam . mm ogsmflm 5.6.. mm mmmeZfifl:z Z_ I. N n v maze; 44<3 nnwo 20mm m02_m - - ALL ~ x<2- BONBOSEHODWJ AlBNElNI 115 .maxm was» wcoam paw pom vosmpsoo 80 :.o mo ooaozo osflpaon on» on pea maams Haoo on» on omoao macapmsnoup< .20 m.: >9 So F an Bo F «0 meowmnoaflo mm: HHoo Masada one .nHHm: Haoo gonna on» Gosh consumflp mo soaponsm m mm XSHM pnmflcmn monoomoLOSHm oopoopop on» mpsomosqon poaa och IIIIIIIIIIIIIIIIIIIIIIIIIII Haoo easemm seems: schemesoxm .om seamen 116 Om mesa... EMEEEAJE z. I .33; jmo 20E mozima o _ m n v m w m m _ — a A I- — a a I- u - JJIFIFJ BOOZE ZOC.zoo D\< 4OO ZOO 100 FIGURE 32 11111 1 «011.1119: 111, .01. g Figure 33. 125 Signal to Noise Ratio for Full Range of A/D Converter Output These ratios are independent of sample solutibn measured; H 0, ethanol, 0.1 y H SO , 1.0 N H 304’ quinine sulfate in acidic media were monitored for SgN characteristics. The correlation coefficient of the 0.1 N H SO data is 0.9933 indicating a linear rise in S/N with voltage. 126 072m “.552 OH .._zoo o\< 23456789IO VOLTS FIGURE 34 129 was possible down to 1x10-9 M. Figure 35 shows in plots A through E the extent of the above detectable linear range. Primary absorption differences for quinine sulfate over a range of concentrations is shown in Figure 36. Significant primary absorptions are encountered at 1x10"7 M and above. Only two curves are apparent even though measurements were conducted at all four cell positions. This is the case since excitation at 250 nm is absorbed by the sample, but the emission at U60 nm is not absorbed. As was expected there is no secondary absorption thus matching position 1 and 2 readings and position 3 and 4 readings. A plot of the logarithmic difference between the window edge positions along the excitation optical axis with concentration is shown in Figure 37. The plot is observed to be linear over the range of concentrations measured. Since this expression, (ln(F¥/§%)1 = ln (¢}/9g)u, appears to describe adequately the attenuation of incident excitation radiation with distance, a correction factor can be deter- mined. An emission scan of 1x10.6 M quinine sulfate in 0.1 N H230,4 in four cell positions is shown in Figure 38. Scans in position 1 and 2 are well matched, as are scans in positions 3 and 4. Application of the correction routine for primary and secondary absorptions resulted in curve 0. 2. Study of Secondary Absorption A plot of the logarithmic difference between the window edge positions along the emission detection axis with concentration is shown in Figure 39. The plot is linear over the range of concentrations Figure 35. 130 Instrumental Output with Concentration for Quinine Sul— fate A. 1 x 10:3 to 1 x 10:? M B. 1 x 10_7 to 1 x 10_6M C. 1 x 10_6 to 1 x 10_5 M D. 1 x 10__5 to 1 x 10-4 M E. 1 x 10 to 1 x 10 M Specifications: Solvent 0.1 N H SO” Excitation Wavelength 350 nm Emission Wavelength 450 nm Reference Attenuation 600 V Sensitivity 0.1x Ratio Mode Slits 20/20 nm MILLIVOLTS 099.090.09.0r MILLIVOLTS 131 POSITION (D ‘ I 41 L I l J 41 T ' 1 I T 1 j .e‘-I_li- 4 5 6 7 8 9 IO QUININE SULFATE CONCENTRATION M> omo :OHpmsqmpu< mosouomom a: co: nuwcmam>mz qoflmwflam e: omm sumswmm>m3 soapmufioxm zom m 2 To pumiom :Hcfiso :oHmepcmoqoo z olor x F mummasm O "mGOHpNOflMHoQO .cwuomgpoo GOAQ Lomn< I mammasm wcflsflzo .om mgsmflm a :mom coflwmwsm mocmommLosam 135 mm minor. .10. x z 20.252328 0.0 to No .6 3.0 mod 0.W. _ . . _ _ _ _ _ _ _ _ . ..o.. 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Ihozmgm><3 .5: 00m Omv Owe 03V qu 00v zc5pm .mm 25mg 141 mm mmawl Z_momm¢034m T O_ x E O_ m m N. m m w m N a _ — _ - .5 mg 12m A mwmxxm of z_ V..omf z _.o@ Efiém mz_z_:d z T916 x522 ozooaoxomDDHm md O._ L”. O. m m.N ltd/NW “(a/3W6 - 142 measured. The expression, ln(¢§/9g)1 - 1n(F¥/9€)2, describes the attenuation of radiation at the emission wavelength with distance. From this a correction for secondary absorption can be determined. A series of scans were conducted for mixtures of quinine sulfate and fluorescein as shown in Figures 40A, B, and C. In each case, the correction routine for primary and secondary absorption was applied to generate curve 0. azw“ '1... . ”a l 1N3 as o_\or mpflam wee: onpmm x r.o spfl>flpflmaom > ooo coflpmscmpp¢ ooszmmom a: oomuoo: cmom :onmmusm as mom camcoaw>m3 coflumpfioxm :ommm z P.o pcmsaom "mGOHQNOHMAOOQm E OF x N :floomOLosam orom F «unmasm massage mu: OP x o camomwposam or m _ mammasm massage ml: or x N cHoommLoSHm 2 much m P mpmeasm massage Z Z IlI‘II‘l IIIIIIIIIIIIIIIIIIIIIIII :fioomogosam new mummasm moflzfiso mo mOLprHz Low :mom scammflem oozmommposam .o-<3 E: 00m omv Omv 03v ONG 00¢ - ‘I q u a - — d - a 1. I T 0...] .d I . I . .... l. .............o...... I... I .......a .... T .....u... 1 I ....... .... I. ........ .o. ............ . p — - ZEommmODIE ea mbfimDm m_Z_Z_3d 25m ZQmmzzm z< Om - CO— and .LNVICIVU C13 Zl WVWUON 145 QMDZEZOU 0v MEDGE IHGZ mm m ><3 4:. 00m 00¢ 00¢ 03 ON? - d - — - u q 4 G - Z_m_0@mm03I_n_ ea mIZOZDm mZ_Z_30 25m 20_mm__>_m_ z< 0m 00. Xn'Id lNVI 0V8 CIBZI'IVWUON 146 DMDZFZOU 0¢ MEDOE Ikmzm4w><3 Ea 00m 00¢ 00¢ 0¢¢ 0m¢ - A I . © l 0m Z_m0mm103I_I._ 9,2 mbfiISm mz_Z_Dd 25m 20_mm__>_m_ z< 00. XD‘H lNVIOVH C13 Zl-IVW tION CHAPTER V CONCLUDING STATEMENTS A. Summary The original goal of this research has been realized. An instru- ment interactive with the laboratory PDP 8/e computer has been construc- ted to correct automatically for attenuation of the detected fluores— cence signal caused by the sample's characteristic primary and second- ary absorption. The instrument's ease of operation and overall perfor- mance have been evaluated. At the outset of this project, emphasis was placed on ease of operation, reliability, precision, accuracy, rapid data acquisition, rapid spectral correction, and the adaptability for conversion of commercially available spectrofluorometers into absorption correcting units without the need for costly reconstruction. Design of an integral system was implemented. The flowchart of all physical devices employed in the computer controlled absorption cor— recting spectrofluorometer is shown in Figure 16. Most of the devices involved are readily available in typical, modern analytical labora- tories. Components such as the stepper motor and its power supply are commercially available. The circuitry required to adapt the computer's stepper motor command pUlses to the power drive of the moto; is straight 148 forward and inexpensive as shown in Figure M5 (appendix). The heart of the correction system, the cell positioner, can easily be constructed from diagrams detailing the three sections shown in Figures 17-19. A spectrofluorometer incorporating right-angle geometry was selec— ted as most appropriate since sample transmission at both the excitation and emission wavelengths can be accurately computed from fluorescence monitored at the three cell positions thus obviating the need for a second instrument. The cell positioner was designed to meet the requirements for the necessary data acquisition in computation of and correction for primary and secondary absorbance. The present arrangement has a preset range for Aid between 0.2 to 1.0 cm along either the excitation or emission optical axis. 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A; com EDUMEU Zopduifimz < 165 m no one encumamon Han pmflmnenp mzm n mNOMImm .pono one u em—Ine .eueoeaeo one: .eufloeneo use e noes no "how .omana noeo non m oe mo onHe> e now new we pesoneo nomv wnflseu one .nooos someone on» eaonsm nozoo onp one nopsoEoo o\m mom on» no anon o\H on» noozaon oooemnopne we pesonflo mHne pasonflo enop.e mango .ms apnoea W6 m¢ mane; JOMEOU MmBOm moPOE ammmmyrm. O._. HDLPDO PDQ > mdon TTOZZ 17X“3 AHM. u mason muons. x SH .Saza v 2. > 0 0 ® 0 @ S: I .E 9 © 0 Figure 46. 167 Ozone Exhaust System This system was designed to eliminate the buildup of ozone in our laboratory when two spectrofluorometers are operating concurrently. The ozone is pumped beneath the floor into a drainage line. Since the half-life of ozone is about 175, this method effectively reduces any poten- tial hazards. 168 I r" 03 03 I INSTRUMENT 7 r o. 3’ 03 I ‘ L 03 03 I INSTRUMENT _ . 1 ® 03 03 L O. PUMP 03 03 e, FLOOR ' 7///////////////////‘ DRAIN FIGURE 46 APPENDIX B 169 Table 7. Model 512 Specifications The Model 512 Fluorescence Spectrophotometer Specifications I. General Specifications a. Main Unit 1) Monochromators... Optical system Diffraction grating: Excitation and emission Czerny-Turner mount, aperture value F3.5 600 lines/mm, blazed at 300 nm 2) Spectral bandpass 3, 10, 20 nm (changeable in 3 steps) 3) Wavelength range Emission measurement 200 to 900 nm and zero order Absorption measurement 250 to 640 nm 4) Wavelength accuracy: :1 nm, zero order: :3 nm 5) Wavelength reproducbility: Within 0.2 nm 6) Wavelength backlash: Wavelength backlash, when I turning from long wavelength 2 to short wavelength: within I 0.5 nm 1'} 7) Scan speed 60, 120, 240, 480 nm/min 4 8) Light source Xenon lamp 150 W 9) Detector . Photomultiplier R-446F 10) Photometric system : Direct (ENERGY) Ratio recording Subtraction mode %T mode Absorption mode 11) Sensitivity 0.1, 0.3, 1, 3, 10, 30, 100 (selectable in 7 steps) 12) Response Fast, medium slow (Changeable in 3 steps) 13) Dimensions 67 cm W x 39 cm D x 27 cm H 14) Weight 54 kg 15) Signal Output 10 mv for potentiometer recorder b. Xenon Lamp Power Suply 1) Constant-current (7.5 2) Dimensions : 3) Weight . II. Fluorescence Mode Specifications 1 ) Stray light 2) Sensitivity 1 volt for digital voltmeter A) pulse ignition type 25 cm W x 29 cm H x 35 cm D 22 kg 1% 2% Excitation monochromator = at 6 = 250 nm Emission monochromator = at 5:: 350 nm The Raman band of H 0 will show a 25% pen deflection under the following conditions: High voltage = 700-800 V Sensitivity = X100 Energy mode Excitation wavelength = 350 nm Slit = 10 nm Emission Slit : 10 nm 170 Table 7 (cont'd) Response : medium Noise level = within 5% or less of full scale (Make measurement at wavelength at Raman Peak) 3) Stability in Ratio (Power must be on for at least 30 Mode: minutes) Drift is 1%/hour under the following conditions. 1. Diffuser plates should be in both sample and reference beams. 2. Wavelength of excitation should be 400 nm, slit : 10 nm. 3. Wavelength emission : 400 nm, slit = 10 nm III. Subtract Mode Specifications 1. Flatness of zero baseline 1% under the following conditions: 1. 1 ppm quinine solution placed in both beams. 2. excitation = 350 nm, slit = 10 nm. 3. emission - 380—580 nm scan, slit = 10 nm 2. Subtract Dial Magnification: More than 5 times IV. Transmission Mode Specifications 1) 100% Line flatness : +2% from 250 to 640 nm 2) Stability : 1% drift/hour at 100 T; 0.1% drift/hour at 0%T (Measurement should be done at 30 minutes after power turn-on). 3) Photometric Accuracy: : 10.77%T ABSORBANCE: :0.01 from 0 to 0.5A, 10.03 from 0.5 to 1.0A. 171 Table 6. Stepper Motor (Rapid-Syn) Specifications Model Number Accuracy Resistance/ 0 Rated Voltage Current per phase Inductance per phase Time for single step Holding Torque Maximum Running Torque Detent Torque Maximum Thrust Load Maximum Overhang Load Rotor Inertia Weight Length Degrees per step Source voltage 23D - 6102 3%nmmumflafiye 5.1 ohms 5.1 V 1.0 amp 10.0 milli henries 2.5 milli sec 53 oz in 35 oz in 5 oz in 25 lbs 15 lbs 87 gm cm 20 oz 2.0 in 1.80 (full step mode) 28 V APPENDIX C 172 Table 4. Quartz Sample Cuvette CleaninggProceudre I. Aqueous Solutions A. Rinse cell thoroughly with lukewarm tap water. B. Give final rinse with chemically pure distilled water. C. Occasionally it may be necessary to cleanse first with detergent solutions or similar liquids and 3-5% aqueous hydrochloric acid. II. Aqueous Protein Solutions A. Soak cell in solution of 0.5% hydrochloric acid (pH 1-2) and pepsin (1g/100 ml). Soak overnight if necessary. B. Follow procedure for aqueous solutions. III. Organic Solutions A. Rinse cell with suitable water soluble orgfanic solvent such as acetone, dioxane or alcohol, possibly mixed with lye or acid. B. Follow procedure for aqueous solutions. IV. Heavy Metal Traces A. Wash cell with tap water. B. Dip cell into clean lukewarm concentrated nitric acid (aqua regia if appropriate). C. Give final rinse with chemically pure, distilled water. V. Preservation of Cuvette Windows A. Follow the appropriate procedure above for the type of sample solution run in the cell. B. Remove all traces of water by using a suction pump (aspirator). C. Dip the cell into chromosulfuric acid (a saturated solution of potassium bichromate in pure concentrated sulfuric acid) at a temperature of 35°C. Let soak if necessary, for no more than one hour. D. Remove cell from acid and allow residual acid to drip from cell, or remove it by suction. Leave cell in open air to allow humidity in air to dilute any residual acid in cell corners. E. Rinse cell thoroughly with lukewarm tap water. F. Give final rinse with chemically pure distilled water. G. Allow to dry in dust free air. H. Polish outside of cell windows wiht optical cleaning tissue free of chlorine or acid traces. I. Store cell in dust free location until next use. Table 9. 173 Reagents List Sulfuric Acid - Mallinckrodt. Lot KLMK Ethanol - Anhydrous from MSU Chem. Stores Fluorescein (sodium salt also known as uranine) - Fisher Scientific Company; laboratory grade Quinine Sulfate Dihydrate - Aldrich, high purity lot LD Distilled H20 - house 174 Table 5. Proper Selection of Slit Widths Excitation Emission Purpose Slit Slit Comments Excitation Both of the slit widths Spectra Narrow Wide can be wide for measuring samples possessing broad Emission peaks Spectra Wide Narrow For obtaining high S/N Quantitative ratio the slit widths Analyses Wide Wide should be as wide as possible. Table 175 8. List of I/O Instructions Information for the use of FLROT (11-30-81) Start-Up 1. The core image file is on the disk marked "SYS:" in pencil. This disk goes in the left slot of the floppy disk drive. 2. With the disk in the drive, boot the system by loading Under FLROT 7600 octal in the switch register, press the clear button on the front panel (this should cause the drive to click), and then press the continue button on the panel. 3. The computer should cause the terminal to respond with A "." which indicates that 08—8 is active. 08-8 1. To load the core image file of FLROT.PA from 08—8, type: R FLROT The computer should respond with a (bell? "*". When this happens the FLROT program is in control of the computer. 1. Whenever the computer responds with a "? 197 00621 1303 TAD NEG3 193 00622 7640 SZA CLA /IS IT A ? 199 00623 7410 SKP 200 00624 5255 JMP SPC CHR /TREAT SPECIALL 201 00625 4710 READ2N. UMS I INCHAR IGET SECOND vCHARACTER 202 00626 3301 DCA TMC HR2 VE 203 00627 1301 TAD TMCHR2 204 00630 1302- 'TAD CLF 205 00631 7440 SZA /IS IT A LF? 206 00632 1303 TAD NEG3 207 00633 7640 SZA CLA /IS IT A CR? 208 00634 7410 SKP 209 00635 5261 UMP SPCR2 /TREAT SPECIALLY 210 00636 130 TAD TMCHR2 /GET SECOND CHARACTER 211 00637 7417 SHIFTR. LSR /SHIFT SIGNIFICANT BITS TO M0L 212 00640 0005 5 213 00641 7200 CLA 214 00642 1300 TAD TEMCHR /GET FIRST CHARACTER 215 00643 0306 AND KK77 IMASK 6 BITS OUT 216 00644 7413 SHL /SHIFT BACK TO Ac 217 00645 0005 215 00646 3704 DCA I STARTI /STORE IN STRING 219 00647 2304 152 STARTI IBUMP STRING POINTER 220 00650 230 52 NDOCHR /DECREMENT #DF HORDS LEFT 221 00651 5214 UHP READIN /IF ROOM READ NEXT CHARACTER 222 00652 7201 ENDSTR. CLA IAC /STRING FULL 223 00653 3704 DCA I STARTI /PUT A ONE AT THE END OF STRING 224 00654 5605 UMP I PACKER /RETURN 225 00655 1300 SPCCHR. TAD TEMCHR /GET CHAR 226 00656 3301 DCA TMCH R2 /ST0RE IN SECOND CHAR 227 00657 3300 DCA TEMCHR /CLEAR FIRST CHAR 223 00660 5236 UMP SHIFTR—I ISTDRE IN STRING 229 00661 7300 SPCR2. CLA CLL /SPEC CHAR LONER HALF 230 00662 1300 TAD TEMCHR /GET UPPER HALF 184 /FLOURESCENCE PROGRAM 11/19/81 PALS-VIOA N0 DATE PACE 5-1 . 1 231 00663 0306 AND KK77 IMASK OUT SIGNIFICANT'DIGITS ‘ 232 00664 7006 RTL 233 00665 7006 RTL 234 00666 7006 L 235 00667 3704 DCA I STARTI /STORE IN STRING 236 00670 2304 I52 STARTI /BUMP STRING POINTER 237 00671 1301 TAD TMCHR2 IGET SECOND CHAR 238 00672 3300 DCA TEMCHR /PUT IN FIRST CHAR 239 00673 2305 I52 NOOCHR IDECREMENT “OF NORDS LEFT 240 00674 5225 JMP READ2N IIF ROOM READ NEXT CHAR 241 00675 1307 TAD CCIOO /APPEND STRING ENDER 242 00676 3704 DCA I STARTI ISTORE IN STRING 243 00677 5605 JMP I PACKER /RETURN 244 00700 0000 TEMCHR. 0 245 00701 0000 TMCHR2. 0 246 00702 7766 CLF. -12 247 00703 7775 NEGS. -3 248 00704 0000 STARTI. O 249 00705 0000 NOOCHR. O 250 00706 0077 KK77. 77 251 00707 0100 CC100. 100 252 00710 0711 INCHAR. CHINPT 253 l 254 /CHARACTER INPUT ROUTINE 255 256 00711 0000 CHINPT. 0 257 00712 6031 KSF 258 00713 5312 JMP .-1 259 00714 6036 KRB 260 00715 6046 TLS 261 00716 5711 JNP I CHINPT /FLOURESCENCE 289 20062 290 20063 291 20064 292 20065 293 20066 294 20067 295 20070 296 20071 297 20072 298073 BY ABORTS) 299 20074 300 20075 301 20076 302 20077 303 20100 304 20101 305 20102 306 20103 307 20104 308 20105 309 20106 310 20107 311 20110 312 20111 313 20112 314 20113 315 20114 316 20115 185 PROGRAM 11/19/31 PALS—VIoA NO DATE PAGE 6 /FL11 FLUORO 7/19/73 (A 0002 FIELD 2 UTO SMOOTHING ADDED) / CODE FIELD DECLARATION: 0020 CODFLD=20 0005 *5 7400 FLIN. 7400 /FLOATING IN 7200 FOUT. 7200 /FLOATING OUT 0007 :7 /THIS MUST BE 7 5600 FLTPT. 5600 /START OF FLOATING POINT INTERPRETTER 0020 *20 0000 EMFLG. 0 /ON EM THIS IS I 0000 EMFLG2. 0 ISAME AFTER SCAN 0000 START. o /STARTING MONOCHRDMETER PARAM 0000 START2. 0 0000 END. 0 /ENDING WAVE VALUE « 0000 END2. 0 ~ '0 0000 MORx. o /FIXED MONOCHROM wAVE VALUE ‘3 0000 MORX2. 0 i 0000 HAVE. 0. y“ 0000 NOVAL. 0 /4 OF DATA POINTS 0000 NLCH. 0 ITEMP WAVE CHANNEL HOLDER 7773 5.--5 0000 TEMP1. o 0000 TEMP2. 0 0000 TEMPS. 1453 SPARSE. SPASE /TYPE THO SPACES 0062 *62 /LEAVE HOLE FOR FLOATING POINT 0000 TEMP4. 0 0000 TEMP5. 0 0000 TEMP6. 0 0042 EXCH. 42 /EXCITATION CHANNEL 0043 EMCH. 43 /EMISSION MONOCHROM CHANNEL 0540 ROUND. OFF /ROUND OFF ROUTINE 1050 A2D. ADAVER /A—D ROUTINE 4000 P4000. 4000 4722 INPUT. ENPUT /GENERAL CHAR INPUT 0200 MONIT. MONITR /POINTER TO MONITOR (MAv BE CHANGED 0200 MDNSAV. MONITR /PERMANENT POINTER TO MONIT 0004 INTVAL. 4 /uAVE LENGTH OR HAVENUMBER INTERVAL 0004 INTVL2. 4 4000 NUMDUT. DECPRT /SINGLE PREC DIG OUT 3340 NUMIN. SINGLE /SINGLE PRECISION INPUT 4063 TYPIST. TYPSTG /PACKED CHAR STRING PRINTER 0317 PR1NT1. TTOUT IGENERAL CHAR PRINTER ~0342 IITYCR. TYCR /CR. 0347 YORN. YESND 0600 NULE1. NULE /NAVE NUMBER TO LAMBDA 0271 TTOINT. KBRK /INTERRUPT ON Ac 0000 R1. 0 0000 F1. 0 0000 S1. 0 0000 R2.o 0000 F2. 0 0000 S2. 0 7766 N12. —12 186 /FLOURESCENCE PROGRAM 11/19/91 PALS-V10A NO DATE PAGE 6"! 317 20116 0747 GEXSET. EXSETG /"EXCITATIGN=" 313 20117 1310 GEMSET. EMSETG 319 20120 0740 0START. STARTG /"START=" 320 20121 0744 GEND. ENDG 321 20122 1470 SETUP. UPSET /SETUP FOR OUTPUT 322 20123 1601 GOTCHA. GOTCH1 /GET RSF FOR OUT 323 20124 4447 OUTSTG. STIGMA /STRING 0F OUTPUT UOPTIONS 324 20125 3363 RFLOT. RFL 325 20126 3360 SFLOT. SFL 326 20127 3366 FFLOT. FFL 327 20130 3560 BUFI. BUFF1 329 20131 0756 ONEFLT. FLUTEl /FLOATING POINT 1 g 329 20132 3566 BUF3. BUFF3 4% 330 20133 0004 P4. 4 i1 331 20134 0213 ENTURP. INTPRT /INTERPRETTER FOR COMMAND OPTIONS 332 0057 NLA = 7 /LAST CHAR ON FLOATING NUMERICAL INPUT 333 20135 7526 NASTY. -252 334 20136 0012 P12. 2 335 20137 3522 YUNORM. UNORM /UNNORMALIzE 336 20140 332 NORM. NORMAL /NORMALI2E 337 20141 5200 DRPLOT. PLOTx /PL0TTERDRIVER 333 2 2 33 NEGA1. NEGII /ROUTINE TO CIA FOR NU AND NOP FOR LE 3(UAVELENGTH=LE) 20143 4333 LINCOM. LINKER /" T0 CML FOR NU AND NOP FOR LE (HAV ENUMBER= NU) 20144 0334 TYQM. GMGM /TYPE 9 341 20145 3563 BUF2. BUFF2 342 20146 0310 P310. 310 /200D 343 20147 4460 DIAG. DINOG /PRINT MESSAGES CONCERNING BACKGROUND. ART. PM. ETC 344 20150 0207 BELL 207 345 20151 1317 USEREG. REGUSE /ROUTINE TO OUTPUT A CONTROL US TO RETURN TO TEXT MODE 346 20152 3627 x0FFI. XOFF /OFFSET BUFFER 347 20153 2407 LOGT. LOGTT /LOG(R/S) 348 20154 0001 SMFLG. 1 /AUTO SMOOTHING FLAG 349 20155 5042 SMITCH. SMUST2 350 20156 0000 FTEMPo. 0. 0. o 351 20157 0000 352 20160 0000 353 20161 0000 FTEMP1. 0. 0. o 354 20162 0000 355 20163 0000 187 /FLOURESCENCE PROGRAM 11/19/81 PALS—VIOA NO DATE PAGE 7 356 0200 *200 357 20200 4551 MONITR: JMS I USEREG 358 20201 4342 S TYCR IPRINT CR. LF 359 20202 1150 TAD BELL 360 20203 4317 JMS TTOUT 361 20204 1311 TAD ASTR 362 20205 4317 JMS TTOUT /FRINT l 363 20206 1212 TAD CMNDS /POINTER TO L1ST OF COMMAND OPTIONS 364 20207 4213 JMS INTPRT /GO TO THE INTERPRETTER 20210 0000 0 /THIS LDC HILL HAVE THE COMMAND FOUND BY THE I NTERPRETTER 366 20211 5610 JMF .- 367 20212 4400 CMNDS. CMDSTG /POINTER TO LIST OF OPTIONS (THIS M UST BE SET BY USER) ‘ ' / 369 20213 0000 INTPRT; 0 /INTERPRETTER. INPUT AND PROCESS A COM 370 20214 3304 DCA LOCLOC IETER WITH POINTER TO OPTION STRING 371 20215 3307 DCA CHRPR /CLEAR COMMAND HORD FOR PREMATURE DELI 372 20216 4257 UMS CHARIN /GET THE FIRST COMMAND CHAR 373 20217 0310 AND P77 374 20220 7106 CLL RTL /MOVE TO LEFT HALF 375 2022 7006 RTL 376 20222 7006 377 20223 3307 DCA CHRPR 378 20224 4257 JMS CHARIN IGET SECOND CHAR 379 20225 0310 AND P77 380 2022 1307. ,TAD CHRPR 381 20227 '3307 DCA CHRPR 382 20230 4257 JMS CHARIN 383 20231 5230 JMP .—1 /KEEP READING AND ECHOINO UNTIL DELIMIT ER CR OR . 384 20232 4437 JMS I SPARSE /TYPE A FEW SPACES AFTER * 385 20233 1304 DELIMR. TAD LOCLDC /A DELIMITER HAS FOUND 386 20234 3305 DCA TEMPRM 387 20235 1705 CHECKI. TAD I TEMPRM 388 20236 7440 SZA /GET A COMMAND NORD FROM THE OPTION STRIN 389 20237 5243 . 390 20240 4334 ERRERR. JMS GMGM /END OF COMMAND OPTION STRING SI CNALED BY A 0 391 20241 4342 JMS TYCR /PRINT 7 CR LF 392 20242 5215 JMP INTPRT+2 /AND TRY AGAIN 393 20243 7041 CIA /MAKE COMMAND NEGATIVE 394 20244 1307 TAD CHRPR /AND SEE IF IT EGUALS ENTERED COMMAND 395 20245 7650 SNA CLA 396 30246 5252 JMP .+4 97 20247 2305 ISZ TEMPRM /THESE COMMANDS DON’T MATCH SO MOVE TO NEXT COMMAND 393 20250 2305 152 TEMPRM 399 20251 5235 JMP CHECK 400 20252 2305 ISZ TEMPRM IFDUND CORRECT COMMAND NDRD TED COMMAND 401 20253 1705 TAD I TEMPRM IGET ASSOCIA 402 20254 3613 DCA I INTPRT /PLACE COMMAND AT RETURN ADDRESS 403 20255 2213 ISZ INTPRT /INCREMENT RETURN ADDRESS 404 20256 5613 JMP I INTPRT /RETURN 405 20257 0000 CHARIN. O /READ AND ECHO CHAR. LOOK FOR CR OR : AS DELIMITERS 406 20260 4777' JMS ENPUT /INPUT A CHAR AND TYPE IT 407 20261 1314 TAD N272 /IS IT A COLON 408 20262 7450 SNA __ § 409 20263~ 5232 JMP DELIMR-lr-IYES 410 20264 1315 TAD P55 /NO: IS IT A CR /FLOURESCENCE 411 20265 412 20266 413 20267 INPUT 414 20270 415 20271 416 20272 417 20273 418 20274 419 20275 420 20276 421 20277 422 20300 423 20301 424 20302 425 20303 426 20304 427 20305 429 20306 429 20307 430 20310 431 20311 432 20312 433 20313 434 20314 435 20315 436 20316 437 20317 438 20320 439 20321 440 20322 441 20323 442 20324 443 20325 444 20326 445 20327 446 20330 447 20331 448 20332 449 20333 450 20334 451 20335 452 20336 453 20337 454 20340 455 20341 456 20342 457 20343 458 20344 459 20345 460 20346 461 20347 N+1 462 20350 463 20351 464 20352 465 20353 /FLOURESCENCE 466 20354 467 20355 468 20356 469 20357 470 20360 471 20361 472 20362 473 20363 474 20364 475 20365 476 20366 188 PROGRAM 11/19/51 PALS—V104 NO DATE PAGE 7-1 7650 SNA CLA 5233 UMP DELIMR 1306 TAD FCHAR /NOT DELIMITER. RETURN HITH CHAR UUST 5657 UMP I CHARIN 0000 KBRK. 0 /INPUT AND INTERRUPT SERVICER 6031 KSF 5671 UMP I KBRK /NO INPUT 6036 KRB 3306 DCA FCHAR 1306 TAD FCHAR 1316 T60 CNTRLC /—CODE FDR Ac 7650 SNA LA 5473 UMP I MONIT 1306 TAD FCHAR /GET THE CHAR AND RETURN 5671 UMP I KBRK 000 LOCLOC. o 0000 TEMPRM. 0 0000 FCHAR. o 0000 CHRPR. o 0077 P77.77 0252 ASTR. 252 /4 0215 CR215. 215 /CR 0212 LF212. 212 /LF HM WH.QH thN 0055 P55. 55 /COLON-CR 7575 CNTRLC. —203 /-CODE FOR “C 0000 TTOUT. 0 /SUBROUTINTE TO PRINT A CHAR 6046 TLS 6041 TSP 5321 UMP —1 7041 CIA /CHECK FOR CR 1312 TAD CR215 7640 52A CLA 5331 UMP .+3 1313 TAD LF212 5320 UMP TTOUT+1 4271 UMS KBRK /LOOK FOR ABORT 7200 CLA 5717 UMP I TTOUT 0000 GMGM. o 4342 UMS TYc 1341 TAD GM /PRINT A 7 4317 UMS TTOUT 5734 UMP I GMGM 0277 GM. 277 0000 TYCR. o 7200 CLA /TYPE CR. LF 1312 TAD CR215 4317 UMS TTOUT 5742 UMP I TYCR ' 0000 YESNO. 0 /YES OR NO ROUTINE: NO=RETURN. YES=RETUR 7201 CLA IAC 1347 TAD YESNO 3363 DCA YUP 1347 TAD YESND PROGRAM 11/19/31 PALE-VIDA ND DATE PAGE 7—2 3365 DCA NOPE 1361 TAD YESTRG 4213 UMS INTPRT 0000 5757 UMP I .-1 0362 VESTRG. .+1 3105 3105 /YES 0000 YUP. o 617 1617 /NO 0000 NOPE. o 0000 o /STRING ENDER 189 /FLOURESCENCE PROGRAM 11/19/91 PALS—VIDA NO DATE PAGE 9 477 20377 4722 478 0400 4400 479 20400 1264 CALIB. TAD 0200 /SET UP MONOCHROMETER CALIBRATION 430 20401 4501 UMS I TYPIST xPRINT "SET TO 200" :31 20402 4472 UMS I INPUT 2 20403 7300 CLA CLL ARD INPUT /UAIT FOR TTO RESPONSE AND THEN DISC 483 20404 1065 TAD EXCH /ExCITATION CHANNEL 484 20405 4234 UMS CAD /INPUT D1 RELATING TO LE1 (200NM) 495 20406 3266 DCA Ex01 486 20407 1066 TAD EMCH /EMISSION CHANNEL 487 20410 4234 UMS CAD ASS 20411 3270 DCA EMD1 459 20412 4503 UMS I IIT CR 490 20413 1265 TAD 0650 /SET TO 650 491 20414 4501 UMS I TYPIST /PRINT 492 20415 4472 UMS I INP 493 20416 7300 CLA CLL 4 494 20417 1065 TAD EXCH 495 20420 4234 UMS CAD /GET 02 FOR LE2 496 20421 7041 CIA 497 20422 1266 TAD EXD1 /D2-D1 492 20423 7041 CIA 499 20424 3267 DCA EXD2DI 500 20425 1066 TAD EMCH 501 20426 4234 UMS CAD 502 20427 7041 CIA 503 20430 1270 TAD EMD1 504 20431 7041 CIA 505 20432 3271 DCA EMD2D1 506 20433 5473 UMP I MONIT /DONE 507 20434 0000 CAD. 0 /A—D INPUT AND AVERAGE 505 20435 7000 NOP 744444 ADSC /SET CHANNEL FROM AC 509 20436 7300 CLA CLL 510 20437 3034 DCA TEMP1 511 20440 3035 DCA TEMP2 /CLEAR Low AND HIGH SUMMATION REGISTER S 512 20441 1033 TAD N5 513 20442 3036 DCA TEMP3 514 20443 7000 NOP /4*444 ADSF /HAIT FOR CHANNEL CHANGE 515 20444 7000 NOP /4446* UMP .-1 516 20445 4470 UMS I A2D /GET A TIME AVERAGED A—D VALUE 517 20446 7100 CLL 515 20447 1035 TAD TEMP2 /LEAST SIG 519 20450 3035 DCA TEMP2 520 20451 7430 82 521 20452 2034 152 TEMP1 /CARRY TO MOST SIG 522 20453 2036 182 TEMP3 /CDUT 523 20454 5245 UMP ,—7 524 20455 1035 TAD TEMP2 /GET ANSUER 525 20456 7421 MGL 526 20457 1034 TAD TEMP1 527 20460 7407 DVI 528 20461 0005 5 529 20462 4467 UMS I ROUND /ROUND AND GET ANSUER 530 20463 5634 UMP 1 CAD 531 20464 0556 0200. 00200 IFLOURESCENCE PROGRAM 532 20465 0564 533 20466 1742 534 20467 3616 535 20470 2553 536 20471 3731 537 20472 0000 TAL NUMBER FOR 538 20473 7425 EL 539 20474 0000 540 20475 7407 541 20476 7020 542 20477 4467 543 20500 1302 544 20501 5672 INTECEPT 545 2 0000 PSEUDO HAVELENGTH 546 20503 7201 547 20504 3020 548 20505 1271 549 20506 3274 550 20507 1270 551 20510 3302 552 20511 1116 553 20512 3064 554 20513 1120 555 20514 4501 556 20515 4737 557 20516 3022 558 20517 1121 559 20520 4501 560 20521 4737 561 20522 3024 562 20523 1064 563 20524 4501 564 20525 4737 565 20526 3026 566 20527 5473 567 20530 3020 568 20531 1267 S 569 20532 3274 570 20533 1266 571 20534 3302 572 20535 1117 573 20536 5312 574 537 0624 ROUTINE 5 5 20540 0000 ER DIVISOR) 576 20541 3355 577 20542 7344 578 20543 1340 579 20544 3372 580 20545 1772 581 20546 7110 582 20547 7141 583 20550 1355 584 20551 7701 585 20552 7430 586 20553 7001 190 11/19/81 PALE-VIDA NO DATE PAGE 8-1 0650. 00650 EXDI. 1742 EXD2D1. 3616 ICALIBRATION CONSTANTS EMD1. 2553 EMD2D1. 3731 LE2DIG. 0 /CHANGE UAVELENGTH TO CORRESPONDING DIGI MGL MUY /COMPARISON WITH A-D MONOCHROMETER CHANN D2D1. 0 /SET BY EM OR EX ROUTINES DVI 7020 /(650-200)*8 JMS I ROUND TAD D1 JMP I LE2DIG IRETURN HITH ANSHER AFTER ADDING D1. 0 /A-D VALUE CORRESPONDING TD 200NM WHICH IS 0 a EM. CLA IAC /SET FOR EMISSION .1 DCA EMFLG ISET EMSISSION FLAT i TAD EMD2D1 /SET UP CALIBRATION CONSTANTS ‘1 DCA D2D1 TAD EMD1 DCA D1 TAD GEXSET /"EXCITATION=" EITHER. DCA TEMP6 TAD GSTART ISTART= UMS I TYP T UMS I PARM1 /NU OR LE INPUT ROUTINE DCA START /SET STARTING HAVE PARAMETER TAD OEND IASK "END=" _ JMS I TYPIST UMS I PARM1 DCA END TAD TEMP6 UMS I TYPIST /ASK FOR HAVE VALUE OF FIXED MONO UMS I PARM1 DCA MORX UMP I MONIT /DONE EX. DCA EMFLG /CLEAR EM FLAG TO SET FOR EMISSION TAD EXD2D1 /SET UP MONCHROM CALIBRATION CONSTANT DCA D201 TAD EXDl TAD QEMSET lASK "EMISSION=" JMP EITHER PARM1. LEIN IOR NUIN HAVE LENGTH OR NUMBER INPUT OFF. 0 /ROUND OFF DIVISION ROUTINE (UMS RIGHT AFT DCA REMAIN ISAVE REMAINDER CLA CLL CMA RAL /—2 TAD O F DCA REMAN2 /POINTS TO DIVISOR TAD I REMAN2 CLL RAR /DIVISOR BY 2 CIA CLL TAD REMAIN CLA MGA SZL IAC /ROUND OFF 20554 20555 20557 20560 20561 20562 20563 20563 20564 20565 20566 20567 20570 20571 20571 20572 JMP I OFF REMAIN. 0 00200. TEXT "SET TO 200” .-1 1 /STRING ENDER 00650: TEXT “SET TO 650" *.-1 1 REMAN2; 0 192 IFLOURESCENCE PROGRAM 11/19/31 PALE—v10A NO DATE PAGE 9 606 0600 «600 607 20600 0000 NULE, o /cHANGE HAVENUBER IN Ac T0 HAVELENGTH 608 20601 3220 DCA NULE2 609 20602 1220 TAD NULE2 610 20603 7425 MeL HUY 611 20604 3100 3100 /1600 IN OCTAL (04200) 612 20605 3034 DCA TEMP1 luAVELENGTH IS 8*(LE—200) wITH LE IN NANOHETERS C:13 20606 7501 MGA /HAVE NUMBER IS 8*NU NITH NU IN 100 INVERSE 614 20607 7141 CIA CLL /DOUBLE PREcIsIoN SUBTRACT 615 20610 1071 TAD P4000 /LEAST SIG PART OF 64*10E5 616 2 1 7421 MoL /PoRMALA IS 64*10E5*NUMBER-B*200*NUMBER ALL DIVIDED av NUMBER ' 617 20612 103 TAD TEMP1 618 20613 7040 CMA 619 20614 7430 SZL . 620 20615 7001 IAc ” 621 20616 1223 TAD P3032 / MOST SIG PART OF 64E5 I 622 20617 7407 DVI ’ 623 20620 0000 NULE2 624 20621 4467 UMS I ROUND /R0UND OFF AND GET ANswER 625 20622 5600 UMP I NULE 626 20623 3032 P3032: 3032 627 20624 0000 LEIN. 0 /INPUT UAvELENGTH AND SEE IF INEOUNDS 628 20625 4500 UMS I NUMIN /INPUT AN INTEGER HAVELENGTH IN NM 629 20626 1265 TAD N310 ISUBTRACT 200 ILONER BOUNDS 630 20627 7510 SPA 631 20630 5242 UMP LERR /ERR0R 632 20631 3244 DCA NUIN 633 20632 I244 TAD NUIN . 634 20633 1266 TAD N702 /—450 T0 MAKE UPPER LIMINT 650 635 20634 7740 SMA CLA SZA 636 20635 5242 UMP LERR 637 20636 1244 TAD NUIN /OK 630 20637 7106 CLL RTL /50 MULT BY 5 639 20640 7004 RAL 640 20641 5624 UMP I LEIN 641 20642 4544 LERR. UMS I TYGH /ERR0R 642 20643 5225 UMP LEIN+1 /TYRY AGAIN 643 20644 0000 NUIN. 0 /INPUT HAVE NUMBER 644 20645 4500 UMS I NUMI 645 20646 3224 DCA LEIN 646 20647 1224 TAD LEIN 647 20650 1267 TAD N232 /—154 LONER LIMIT 648 20651 7710 SPA CLA 649 20652 5263 UMP NERR /T00 SMALL 650 20653 1224 TAD LEIN 651 20654 1270 TAD N764 /—5oo UPPER LIMIT 652 20655 7740 SMA SZA CLA 653 20656 5263 UMP NERR 654 20657 1224 TAD LEIN /0K 655 20660 7106 CLL RTL 656 20661 7004 RAL /SCALE UP BY 5 657 20662 5644 UMP I NUIN 658 20663 4544 NERR, UMS I TvoM /ERR0R 659 20664 5245 UMP NUIN+1 /TRY AGAIN 660 20665 7470 N310. —310 /200 193 /FLOURESCENCE PROGRAM 11/19/81 PALB—VlOA NO DATE PAGE 9-1 661 20666 7076 N702. ~702 /-450 662 20667 7546 N232. -232 /-154 663 20670 7014 N764. ~764 664 20671 1120 OUTRNG: TAD GSTART /SET NEH OUTPUT LIMITS 665 20672 4501 UMS I TYPIST /ASK "START=" 666 20673 4737 UMS I PARM2 /LEIN OR NUIN 667 20674 3034 DCA TEMP1 668 20675 1034 TAD TEMP1 669 20676 7141 CIA CLL 670 20677 1022 TAD START 671 20700 7450 SNA 672 20701 5305 2011 UMP OK11 /SNL CLA FOR NU 673 20702 4543 JMS I LINCOM 674 20703 7630 202. SZL CLA ISNL CLA FOR NU 675 20704 5271 JMP OUTRNG /ERROR ASK AGAIN 676 20705 1034 OKIII TAD TEMP1 677 20706 3023 DCA START2 678 20707 1121 TAD GEND /“END='I 679 20710 4501 JMS I TYPIST 680 20711 4737 UMS I PARM2 681 20712 3034 DCA TEMP1 682 20713 1034 TAD TEMP1 : 683 20714 7141 CIA CLL 9 684 20715 1024 TAD END .3 685 20716 7450 A 686 20717 5323 JMP OK12 687 20720 4543 UMS I LINCOM 688 20721 7620 203: SNL CLA /OF SZL CLA FOR NU ‘ 689 20722 5307 JMP OK11+2 /ERROR ASK AGAIN I 690 20723 1034 OK121 TAD TEMP1 /OK DCA D'1 691 20724 3025 2 692 20725 1021 TAD EMFLG2 693 20726 7650 SNA CLA 694 20727 5332 UMP .+3 695 20730 1116 TAD OEXSET /TYPE “EXCITATION=" 696 20731 7410 SKP 697 20732 1117 TAD GEMSET 698 20733 4501 JMS I TYPIST /ASK FOR NEW VALUE OF FIXED MONOCHR OH 699 20734 4737 JMS I PARM2 700 20735 3027 DCA MORX2 701 20736 5473 UMP I MONIT 702 20737 0624 PARM2. LEIN /OR NUIN 703 20740 2324 STARTG: TEXT "START=" 706 20743 0000 707 0743 #.-1 708 20743 0001 1 709 20744 0516 ENDQI TEXT "END=” 710 20745 0475 711 20746 0000 , 712 0746 *.-1 713 20746 0001 1 714 "0747 0530 EXSETG: TEXT ”EXCITATION= ” 715 20750 0311 /FLOURESCENCE PROGRAM 11/19/61 PALS-V10A NO DATE PAGE 9—2 716 20751 2401 717 20752 2411 718 20753 1716 719 20754 7540 720 20755 0000 721 0755 ‘.-1 722 20755 0001 1 723 20756 0001 FLUTE1. 1 724 20757 2000 2 00 725 20760 0000 /FLOATING POINT: 1 /FLOURESCENCE 726 727 21000 728 21001 729 21002 730 21003 AGE 731 21004 732 21005 733 21006 734 21007 735 21010 ATER CHANGES 736 21011 737 21012 738 21013 739 21014 740 21015 741 21016 742 21017 743 21020 744 21021 745 21022 746 21023 747 21024 748 21025 749 21026 750 21027 751 21030 752 21031 753 21032 754 21033 755 21034 756 21035 757 21036 758 21037 759 21040 760 21041 761 21042 762 21043 763 21044 764 21045 765 21046 766 21047 767 21050 768 21051 769 21052 770 21053 771 21054 772 21055 773 21056 774 21057 775 21060 776 21061 777 21062 778 21063 779 21064 194 PROGRAM 11/19/81 PALE-VIDA NO DATE PAGE 10 1000 i1000 7240 SCAN. CLA CMA ISCAN AND COLLECT DATA 3010 DCA 10 3031 DCA NOVAL /DATA POINT COUNTER 1022 TAD START /MOVE SCAN PARAMETERS TO PERMANENT STOR 3023 DCA START2 1024 TAD END 3025 DCA END2 1075 TAD INTVAL 3076 DCA INTVL2 /MOVE ALL PARAMETERS TO STORAGE SO L HILL 1026 TAD MORX INOT FOUL UP OUTPUT 3027 DCA MORX2 1020 TAD EMFLG 3021 DCA EMFLG2 1022 TAD START 3030 DCA HAVE 1155 TAD SMITCH /SET RETURN ADDRES FOR AUTO SMOOTH 3073 DCA MONIT 1020 TAD EMFLG 7650 SNA CLA 5226 UMP .+3 1066 TAD EMCH IEMISSION CHANNEL 7410 SKP 1065 TAD EXCH 3032 DCA HLCH ISET MONCHROM CHANNEL 4720 SCAN2: UMS I DATAIN /BIG ENOUGH. TAD DATA 1030 TAD HAVE 7141 CIA CLL 1024 TAD END 7450 SNA 5646 UMP I FNSCN /FINISHED 4543 UMS I LINCOM 7620 2041 SNL CLA /OR SZL CLA 5646 UMP I FNSCN IDONE 1075 TAD INTVAL 4542 UMS I NEGA1 /EITHE NOP FOR LE OR CIA FOR NU 1030 TAD HAVE 3030 DCA HAVE 5230 UMP SCAN2 /REPEAT 1124 FNSCN: DONEO 1200 GFIN. GFING /FINISHED 0000 ADAVER. 0 /A TO 0 ROUTINE 7300 CLA CLL 1303 TAD PP4 7041 CIA 3017 DCA 17 3062 DCA TEMP4 3063 DCA EMP5 1321 KLOCK; TAD DELTIM /8ET CLOCK FOR TIME INTERVAL 3177 DCA 177 6532 ADCV 6531 ADSF 5262 UMP -1 7200 CLA 6534 ADRB /FLOURESCENCE 781 21066 782 21067 783 21070 784 21071 785 21072 786 21073 787 21074 788 21075 789 21076 790 21077 791 21100 792 21101 793 21102 794 21103 795 21104 796 21105 797 21106 798 21107 799 21110 800 21111 801 21112 802 21113 803 21114 804 21115 805 21116 806 21117 807 21120 808 21121 809 21122 810 21123 811 21124 812 21125 813 21126 814 21127 815 21130 816 21131 817 21132 818 21133 819 21134 820 21135 821 21136 822 21137 823 21140 824 21141 825 21142 826 21143 827 21144 828 21145 829 21146 830 21147 831 21150 832 21151 833 21152 834 21153 835 21154 /FLOURESCENCE 836 21155 837 21156 838 21157 839 21160 840 21161 841 21162 842 21163 843 21164 844 21165 845 21166 846 21167 847 21170 21171 195 PROGRAM 11/19/81 PALS-VIoA NO DATE PAGE 10-1 7100 CLL 1062 TAD TEMP4 3062 DCA TEMP4 7430 SZL 2063 182 TEMP5 ICARRY TO HIGH PRECISION 2177 152 I77 /TIMING LOOP 5273 UMP .-1 /HAIT ON CLOCK 2017 152 17 5257 UMP KLOCK 1062 TAD TEMP4 7421 MGL 1063 TAD TEMP5 7407 DVI /AvERAGE 0004 PP4. 4 4467 UMS I ROUND 7001 IAc 7440 SZA 5315 UMP .+6 /NOT SATURATED 1150 TAD BELL /UH OH. SATURATED. SOUND ALARM 6041 TSF 7410 SKP 6046 TLS 7200 CLA 7041 CIA /RESUBTRACT I 7140 CMA LL 5650 UMP I ADAVER 123 DATAIN, INDATA 4647 DELTIM. 4647 /2NM/SEC ON THE PERKIN ELMER 0472 LEDIGI. LEeDIG 1124 7201 DONEo. CLA IAC 4777' UMS TURN 4503 UMS I IITYCR 1371 TAD ORESET 4501 UMS I TYPIST 4472 UMS I INPUT 7200 CLA 3010 DCA 10 3031 DCA NOVAL 1022 TAD START 3030 DCA HAVE 1342 TAD DONEI—I 3246 DCA FNSCN 5230 UMP SCAN2 1143 ONEI 7325 DONEI. CLA CLL IAC CML RAL 4777' U TURN 4503 UMS I IITYCR 1371 TAD GRESET 4501 UMS I TYPIST 4472 UMS I INPUT 7201 CLA IAc 3010 DCA 10 3031 DCA NOVAL 1022 TAD START PROGRAM 11/19/81 PALS-V1OA NO DATE PAGE 10—2 3030 DCA HAVE 1361 TAD DONE3—1 3246 DCA FNSCN 5230 UMP SCAN2 162 DONE3 7200 DONE3. CLA 4777' UMS TURN 1323 TAD DONEO—1 3246 DCA FNSCN 1247 TAD GFIN 4501 UMS I TYPIST 4 UMP I MONIT 5 73 1207 GRESET: GORSET /FLDURESCENCE 849 21177 850 851 21200 852 21201 853 21202 854 21203 855 21204 856 21205 857 21206 858 859 21206 860 21207 861 21210 862 21211 863 21212 864 21213 865 21214 866 21215 867 21216 868 21217 869 21220 870 2122 871 872 21221 873 21222 874 21223 875 21224 876 21225 877 21226 878 21227 879 21230 880 21231 881 21232 CI. 21233 883 21234 884 21235 885 21236 886 21237 887 21240 888 21241 889 21242 890 21243 891 21244 892 21245 893 21246 894 21247 895 21250 896 21251 897 21252 898 21253 899 21254 900 21255 901 21256 902 21257 196 PROGRAM 11/19/81 PALS—V1OA N0 DATE PAGE 11 1222 1200 *1200 0015 GFING. 15 0012 12 0611 TEXT "FINISHED" 1611 2310 0504 0000 1206 *.-1 000 1 2205 GORSET. TEXT "RESET MONOCHROMATORS" 230 2440 1517 1617 0310 2217 1501 2417 2223 0000 1221 *.-1 0001 0000 TURN, O 6213 CDFCIF 10 4627 UMS I ROCEL 7200 CLA 5622 JMP I TURN 3067 ROCEL. ROCELL OOOO INDATA. 0 /DAT COLLECTION SUBROUTINE 7326 CLA CLL CHL RTL /SET HULTIPLEXER CHANNEL 6533 ADSC 7200 A 1115 TAD N12 /AVERAGE 10 OF THEM 3013 DCA 13 3111 DCA 81 /CLEAR SUMMATION REG 3114 DCA 82 4470 SMPNT. UMS I A2D 7100 CLL /SUM UP 1111 TAD 1 3111 DCA S1 7430 SZL 2114 152 52 2013 152 13 /COUNT DATA POINTS 5240 UMP SMPNT 1111 TAD 51 7421 MGL /NDN AVERAGE 1114 TAD 82 4270 UMS SAVOR 2010 182 10 2010 152 10 2031 182 NOVAL ICOUNT NUMBER OF COMPOSITE POINTS 1010 TAD O 1266 TAD N2775 /LIMIT OF DATA STORAGE 197 /FLOURESCENCE PROGRAM 11/19/81 PAL8-V10A NO DATE PAGE 11-1 904 21261 7710 SPA CLA 905 21262 5630 JHP I INDATA 906 21263 1267 TAD OFULL /DATA TABLE FULL 907 21264 4501 JHS I TY T 908 21265 5473 JHP I MONIT 909 21266 5003 N2775: ~2775 910 21267 1300 GFULLI GGFUL 911 21270 0000 SAVOR. O 9.: 21271 7407 DUI 913 21272 0012 12 914 21273 4467 JMS I ROUND 915 21274 6231 CDF 30 /AVERAGE AND STORE 916 21275 3410 DCA I 10 917 21276 6221 CDF CODFLD 918 21277 5670 JMP I SAVOR 919 21300 0015 OGFUL. 15 921 21302 2401 TEXT "TABLE FULL" 927 1307 i.-1 1 929 21310 0515 EMSETG: TEXT "EMISSION= " 935 1315 *.-1 936 21315 0001 1 937 21316 0237 237 ICODE FOR CONTROL US 938 21317 0000 REGUSE. O CLA 940 21321 1316 TAD .-3 941 21322 4502 JMS I PRINTI 942 21323 5717 JHP I REGUSE 943 0055 *55 944 20055 0000 0 /SET 50 NO CR 0N OUTPUT 945 20056 0000 O 946 7345 #7345 947 27345 4502 JHS I PRINT1 /CHANGE FLOATING POINT OUTPUT 948 27346 7000 949 27347 7000 NOP 950 7170 *7170 951 27170 7441 —337 /TO CHANGE RUBOUT TO BACKARROH FOR CONSISTENC Y 952 27171 0122 337-215 953 7144 *7144 954 27144 4472 JHS I INPUT 955 27145 7000 NOP 956 27146 7000 NOP IOVERLAY OF FLOATING INPUT 957 7150 *7150 958 27150 7200 CLA /FLOURESCENCE PROGRAM 11/19/81 PALE-VloA NO DATE PAGE 11-2 959 27151 7000 NOP /INPUT ALREADY HAS BEEN ECHOED /FLOURESCENCE 960 961 21400 962 21401 963 21402 964 21403 965 21404 966 21405 967 21406 968 21407 969 21410 970 971 21411 972 21412 973 21413 974 21414 975 21415 976 21416 977 21417 978 21420 979 21421 980 21422 981 21423 982 21424 983 21425 984 21426 985 21427 986 21430 987 21431 988 21432 989 21433 990 21434 991 21435 992 21436 993 21437 994 21440 995 21441 996 21442 997 21443 998 21444 999 21445 1000 21446 1001 21447 1002 21450 1003 21451 1004 21452 1005 21453 1006 21454 1007 21455 1008 21456 1009 21457 1010 21460 1011 21461 1012 21462 1013 21463 21464 198 PROGRAM 11/19/31 PALS—V10A NO DATE PAGE 12 1400 41400 4263 PRINT, JMS GOODIE /GET THE OUTPUT PARAM 4503 PRINTU. ans I IITYCR 4547 UMS I DIAG 5232 JMP PRIN3—3 /JUMP PAST THIS ISLAND oooo ADVIS. o 1120 TAD 0START /PRINT ASSOCIATED DATA 4501 one I TYPIST 1023 TAD START2 4322 JMS UNDO 0077 PRTDEC=NUHOUT 4253 JMS SPASE /TYPE SOME SPACES 1121 TAD GEND 4501 JMS I TYPIST 1025 TAD END2 4322 JHS UNDO 4253 JMS SPASE 1021 TAD EMFLGZ 7650 SNA CLA 5224 HP .+3 1116 TAD GEXSET /EM SCAN 7410 SKP 1117 TAD GEHSET /Ex SCAN 4501 UMS I TYPIST 1027 TAD MORx2 4322 JHS UNDO 3010 DCA Io /CLEAR LINE COUNTER 5604 JHP I ADVIS 4522 JMS I SETUP /SETUP R.S.F POINTERS 4523 JHS I GOTCHA /GET FIRST RSF VALUES 5240 JMP .+4 4523 PRIN3. JMS I GDTCHA 2016 182 16 5247 JHP .+10 4503 JMS I IITYCR /ONLY 4 PER LINE SO GIVE CR. LF 2010 152 10 1010 TAD 10 4477 one I PRTDEC IPRINT LINE NUMBER 4253 JMS SPASE I257 TAD N4 3016 DCA 16 4666 JMS 1 PRUNE /DO THE CONVERSION AND OUTPUT IT 4406 JMS I FDUT 4253 JMS SPASE 5235 JMP PRIN3 oooo SPASE, 0 ISPACE PRINTER 1260 TAD SFASG 4501 JMS I TYPIST 5653 JHP 1 SPASE 7774 N4.-4 1461 SPASQ, .+1 4040 4040 0001 1 0000 GOODIE. 0 /GET CONVERSION TYPE 1124 TAD OUTSTG ISTRING LOCATIN FOR OUTPUT OPTIONS 199 /FLOURESCENCE PROGRAH 11/19/81 PALB-V1OA NO DATE PAGE 12-1 1015 21465 4534 UMS 1 ENTURP /INTERPRETTER "1. 55466 000’ PDUHE T 1017 21467 bass UMP I CDDDIE 1018 21470 0000 UPSET. O IROUTINE T0 SETUP RSF POINTERS 1019 21471 7240 CLA CMA ‘ 1020 21472 3015 DCA 15 /DATA PICKUP 1021 21473 1031 TAD NOVAL CMA 1023 21475 3063 DCA TEMP5 /-# OF POINTS-1 1024 21476 1022 TAD START 1025 21477 3030 SETUPS. DCA HAVE 1026 21500 1023 TAD START2 1027 21501 7141 CIA CLL 1028 21502 1030 TAD HAVE 1030 21504 5670 JHP I UPSET /RETURN 1031 21505 4543 JMS I LINCOM 1032 21506 7630 SZL CLA 1033 21507 5670 JMP I UPSET 1034 21510 7325 CLA STL RAL IAC 5 1036 21512 3015 DCA 15 1037 21513 2063 182 TEMP5 1039 21515 5473 JHP I MONIT IALREADY OUT OF DATA 1040 21516 1076 TAD INTVL2 1041 21517 4542 JMS I NEGA1 /NOP OR CIA 1042 21520 1030 TAD HAVE /UPDATE HAVE 7 UMP SETUP3 1044 21523 0000 UNDOI 0 /CHANGE PSEUDO HAVE PARAMETER INTO REAL P ARAMETER 1045 21523 7417 LSR 1046 21524 0002 2 1047 21525 1146 Z77. TAD P310 /ADD IN 200 IF HAVELENGTH 1048 21526 4477 JMS I PRTDEC 1049 21527 5722 JMP I UNDO 200 /FLOURESCENCE PROGRAM 11/19/81 PALB—ViOA NO DATE PAGE 13 1050 1600 41600 1051 21600 4000 PMCP. 4000 /START OF PM TABLE (CE? 712.01 COO? GOTCH1. C‘ /-’.—‘E" PSF VALUES. FLOAT 1053 2.602 2063 IS: TEHPE /OUT OF DATA? 1054 21603 7410 SKP 1055 21604 5473 UMP I MONIT IVES 1056 21605 1030 TAD HAVE 1057 21606 7141 CIA CLL 1058 21607 1025 TAD END2 1059 21610 7450 SNA 060 2:611 5215 UMP GUD2 /DK. LAST POINT , .2... L L'. 454:: vJMS I LINCOH It 1062 21613 7620 SNL LA A .; 1063 21614 5473 UMP I MONIT /PAST END, DONE V 1064 21615 6231 GUD2, CDF 30 /MOVE IN DATA 1 1065 21616 1415 TAD I 15 3 1066 21617 3107 DCA R1 1067 21620 1415 TAD I 15 1069 21621 3111 DCA SI 1069 21622 1415 TAD I 15 1070 21623 3110 DCA F1 1071 21624 6221 CDF CODFLD 1072 21625 5226 UMP GOTU /*4.*4 TAD EMFLG2 /CHECK FOR EM OR Ex 1073 21626 1107 GOTU. TAD R1 1074 21627 4540 UMS I NORM /FLOAT ANS SAVE R 1075 21630 3363 RFL 1076 21631 1111 TAD SI 1077 21632 4540 UMS I NORM 1073 21633 3360 SFL 1079 21634 1110 TAD F1 1030 21635 4540 UMS I NORM 1091 21636 3366 FFL 1032 21637 1076 TAD INTVL2 1093 21640 4542 UMS I NEGA1 1054 21641 1030 TAD wAVE 1055 21642 3030 DCA HAVE 1086 21643 5601 UMP I GOTCH1 1037 21644 4040 GOEAC, TEXT " SCALE FACTOR= " 1089 21645 2303 1039 21646 0114 1090 21647 0540 1091 21650 0601 1092 21651 0324 1093 21652 1722 1094 21653 7540 1095 21654 0000 1096 1654 *.—1 1097 21654 0001 1 1093 21655 4040 00MARX, TEXT " MAX=" 1099 21656 1501 1100 21657 3075 1101 21660 0000 1 1102 1660 «.—1 1103 21660 0001 1 1104 21661 0521 0E02. TEXT "EGUIVALENCE wAVELENGTH (HAVENUMBER)=“ 201 /FLDURESCENCE PROGRAM 11/19/91 PALE-V1OA NO DATE PAGE 13-1 1105 21662 2511 1106 21663 2601 1107 21664 1405 1108 21665 1603 1109 21666 0540 1110 21667 2701 1111 21670 2605 1112 21671 1405 1113 21672 1607 1114 21673 2410 1115 21674 4050 1116 21675 2701 1117 21676 2605 1119 21677 1625 1119 21700 1502 /FLOURESCENCE 1125 1126 22200 1127 22201 1128 22202 1129 22203 1130 22204 1131 22205 1132 22206 113? 22207 :134 22210 1135 22211 1136 22212 1137 22213 1138 22214 1139 22215 1140 22216 1141 22217 1142 22220 1143 22221 1144 22222 1145 22223 1146 22224 1147 22225 1148 22226 1149 22227 1150 22230 1151 22231 1152 22232 1153 22233 1154 22234 1155 22235 1156 22236 1157 22237 1159 22240 1159 22241 1160 22242 1161 22243 1162 22244 1163 22245 1164 22246 1165 224 POINT MANUAL 1166 22250 1167 22251 1168 22252 1169 22253 1170 22254 1171 22255 1172 22256 1173 22257 1174 22260 1175 22261 11 22262 EE MANUAL) 1177 22263 1178 22264 1179 22265 202 PROGRAM 11/19/91 FALB-VIOA NO DATE PAGE 14 2200 42200 0000 RR. 0 /OUTPUT RAu R 4407 JHS I FLTPT 5525 FGET I RFLOT oooo FEX 5600 JMP 0000 35. o /DUTPUT s 4407 JMS I FLTPT 5526 FGET I SFLDT 0000 FEXT 5605 JMP 35 0000 FF, 0 /FLUOR 4407 JMS I FLTPT 5527 FGET I FFLOT oooo FEXT 5612 JHP I FF 0000 CO. 0 /CORRECTED FLUOR F/R 0000 A99. 0 /A99OBENCE, —LOG(S/R)=LOG(R/S 4407 JMS 1 FLTPT 5525 FGET I RFLOT 6156 FPUT FTEMPo 3525 FMPY I RFLOT 6525 FPUT I RFLOT 5527 FGET I FFLOT 6161 FPUT FTEMPI 3526 FMPY I 5FLOT 6527 FPUT I FFLOT 5525 FGET I RFLOT 4527 FDIV I FFLOT 0000 FEX 7240 CLA CMA 1044 TAD 44 7500 5MA 5247 JMP .+7 7200 CLA /AB=O R/3<=1 4540 JHS I NORM 0044 44 4540 JMS I NORM 2357 LOGAB 5620 JMP I A39 4540 JMS I NORM‘ /N LOG2 /FOR ALGORITHM SEE FLOATING 0044 .44 , 4407 a0M3 I FLTPT 3321 FMPY LOG2 6530 FPUT I BUFI 5525 FGET I RFLOT 4527 FDIV 1 FFLOT oooo FEXT ' 7201 CLA IAc IHAKE 1<=X€2 3044 DCA 4407 JMS I FLTPT 2531 FSUB I ONEFLT /SUTRACT 1 TO GET ”Y" FOR SERIES (5 6532 FPUT I BUF3 3354 FMPY L3 1351 FADD L7 203 /FLOURESCENCE PROGRAM 3 11/19/61 PALS-VloA NO DATE PAGE 14-1 1180 22266 3532 FHPY I BUF3 1181 22267 1346 FADD L6 1182 22270 3532 FMPY I BUF3 5 3 1138 22276 3532 FHFV I BUF3 1189 22277 1332 FADD L2 1150 22300 3532 FMPY I BUF3 1191 22301 1327 FADD L1 1192 22302 3532 FMPY I BUF3 1193 22303 1530 FADD I BUF1 /THIS IS THE NATURAL LOG 1194 22304 6357 FPUT LOGAB /SA E FOR USE “1TH AB 1195 22305 5156 FGET FTEHPO 1196 22306 6525 FPUT I RFLOT 1197 22307 5161 FGET FTEMP1 1193 22310 6527 FPUT I FFLOT 1199 22311 5357 FGET LOGAB 1200 22312 3362 FMPY GEOFAC /HULTIPLY BY A GEOMETRIC FAC TOR 1201 22313 0000 FEXT 1202 22314 4765 JHS I EXPNT 1203 22315 4407 JMS I FLTPT 1204 22316 3525 FHPY I RFLOT 1206 22320 5620 JMP I AB9 1207 22321 0000 L002: 0 /NATURAL LOG OF 2 1208 22322 2613 2613 1209 22323 4414 4414 1210 22324 0002 COMMON. 2 /LN 10 TO MAKE COMMON LOGS 1211 22325 2232 2232 1212 22326 7307 7307 1213 22327 0000 L1, 0 1214 22330 3777 3777 1224 22342 7211 7211 1225 22343 7776 L5; 7776 1226 22344 2535 2535 1227 22345 3301 3301 1230 22350 0771 0771 1233 22353 4304 4304 1234 22354 7771 LS. 7771 /FLDURESCENCE PROGRAM 11/19/81 PALS-V10A NO DATE PAGE 14-2 1235 22355 4544 4544 1236 22356 1735 1735 1237 22357 0000 LOGAB. 0 1238 22360 0000 O 1239 22361 0000 0 1240 22362 0000 GEOFAC: 0000i 3000; 0000 1241 22363 3000 1242 22364 0000 1243 22365 2415 EXPNT, FEXP 2014 /FLOURESCENCE PROGRAM 11/19/31 PALS—V10A N0 DATE PAGE 15 1244 2400 42400 1245 22400 0000 GCBUFF, 0 1246 22401 0000 o 1247 22402 0000 o 1242 22403 0000 FBF, o 1249 22404 0000 o v 1250 22405 0000 o 1251 22405 2357 LDCABII LOGAB 1252 22407 0000 LOGTT. o 1252 22410 0000 o 1254 22411 0000 o 1255 22412 0000 FAKE. o 1256 22413 0000 o 1257 22414 0000 o 1259 22415 0000 FEXP. 0 € 1259 22416 4407 JMS I FLTPT n 1260 22417 3277 FMPY LG2E “ 1261 22420 6156 FPUT FTEMPO ‘ 1262 22421 0000 FEXT 1263 22422 4537 JMS I YUNORM 1264 22423 3045 DCA 45 1265 22424 1045 TAD 45 1266 22425 3310 DCA FLAG2 1267 22426 4540 JHS I NORM 1268 22427 0045 45 1269 22430 4407 JMS I FLTPT 1270 22431 6161 FPUT FTEHPI 1271 22432 5156 FGET FTEMPO 1272 22433 2161 FSUB FTEMPi 1273 22434 6156 FFUT FTEMPO 1274 22435 3156 FMPY FTEMPO 1275 22436 6161 FPUT FTEMP1 1276 22437 1274 FADD D 1277 22440 6311 FPUT FTEMPZ 1278 22441 5271 FGET C 1279 22442 4311 FDIV FTEHP2 1280 22443 2156 FSUB FTEHPO 1281 22444 1263 ADD A 1282 22445 6311 FPUT FTEMP2 1283 22446 5266 1284 22447 3161 FHPY FTEHP1 1285 22450 1311 FADD FTEMP2 1286 22451 6311 FPUT FTEMP2 1287 22452 5156 FGET FTEMPO 1288 22453 4311 FDIV FTEMP2 1289 22454 3305 FHPY FLOT2 1290 22455 1302 FADD FLDT1 1291 22456 0000 FEXT 1292 22457 1310 TAD FLAG2 1293 22460 1044 TAD 44 1294 22461 3044 DCA 44 1295 22462 5615 JMP I 1296 22463 0004 A. 0004; 2372; 1402 1297 22464 2372 1298 22465 1402 /FLDURESCENCE PROGRAM 1299 1300 1301 22466 22467 22470 22471 22472 22473 22474 22475 22476 22477 22500 22501 22502 22503 22504 22505 22506 22507 22510 22511 22512 22513 22514 22515 22516 B1 LG2E, FLOTL FLOT2: FLAG2, FTEMP2: FLT100. 11/19/81 7774; 0012; 0007; 0001; 0001; 0002; O O; O; 2157; 5454; 2566; 270$ 2000; 2000; 7i 3100; 0 PALS-VIOA N0 DATE 5157 0343 5241 2435 0000 0000 PAGE 15-1 /FLOURESCENCE PROGRAM 11/19/81 PALS-VloA NO DATE PAGE-16 1324 3000 *3000 1325 23000 7000 N1000: -1000 1326 23001 0000 FCNT. 0 1327 23002 0000 RCNT. 0 1328 23003 3344 ONEH. GONEH 132 23004 1124 PLOT: TAD OUTSTG /PLOT THE REQUESTED INFO 1330 23005 4534 JMS I ENTURP IGET REQUEST 1331 23006 0000 PLOPT. O 1332 23007 4547 JMS I DIAG /PRINT DIAGNOSTICS 1333 23010 1364 TAD PLTRET /SETUP RETURN ADDRESS RETPLT 1334 23011 3073 DCA MONIT 1335 23012 4522 JMS I SETUP 1336 23013 4540 JMS I NORM 1337 23014 3161 YCAL /CLEAR YMAX BUFFER 1338 23015 4540 JMS I NORM 1339 23016 3627 XOFF /CLEAR OFFSET BUFFER 1340 23017 4523 GORSH. JMS I GOTCHA /GET RSF 1341 23020 4606 JMS I PLOPT /CALCUALTE Y 1342 23021 4407 JMS I FLTPT 1343 0010 FABS=10 ICOMMAND TO TAKE THE ABSOLUTE VALUE OF THE FAC 1344 23022 0010 FABS 1345 23023 2361 FSUB YCAL 1346 23024 0000 FEXT 1347 23025 1045 TAD 45 1348 23026 7710 SPA CLA 1349 23027 5217 JMP GORSH 1350 23030 4407 JMS I FLTPT 1351 23031 1361 FADD YCAL /BIGGER 1352 23032 6361 FPUT YCAL 1353 23033 0000 FEXT 1354 23034 4506 JHS I TTOINT 1355 23035 7200 CLA 1356 23036 5217 JMP GORSH 1357 23037 1074 RETPLT. TAD MUNSAV /COME HERE AFTER FINDING MAX 1358 23040 3073 DCA MONIT /RESET ABORT 1359 23041 1371 TAD OPLTMX /MAX= 1360 23042 4501 JMS I TYPIST 1361 23043 4407 JMS I FLTPT 1362 23044 5361 FGET YCAL 1363 23045 0000 FEXT 1364 23046 4406 JMS I FOUT /PRINT LARGEST Y VAL 1365 23047 1372 TAD GFAC /ASK FOR " SCALE FACTOR=" 1366 23050 4501 JMS I TYPIST 1367 23051 4405 JHS I FLIN 1368 23052 4407 JMS I FLTPT 1369 23053 6361 FPUT YCAL 1370 23054 0000 FEX T 1371 23055 1057 TAD NLAT /GET LAST CHAR 1372 23056 1135 TAD NASTY /UAS ASTERISK? 1373 23057 7640 SZA CLA 1374 23060 5266 JMP 1+6 1375 23061 7001 IAC /SET PEN UP FOR OVERPLOT 1376 23062 4541 JMS I DRPLOT /MOVE PEN TO ORIGIN 1377 23063 0000 0 1378 23064 0000 O IFLOURESCENCE 1379 23065 1380 23066 1381 23067 1382 23070 1383 23071 1384 23072 1385 23073 1386 23074 1387 23075 1388 23076 1389 23077 1390 23100 1391 23101 1392 23102 1393 23103 1394 23104 1395 23105 1396 23106 1397 23107 1398 23110 1399 23111 1400 23112 1401 23113 1402 23114 1403 23115 1404 23116 1405 23117 1406 23120 1407 23121 1408 23122 1409 23123 1410 23124 1411 23125 1412 23126 1413 23127 1414 23130 1415 23131 1416 23132 1417 23133 1418 23134 1419 23135 1420 23136 1421 23137 1422 23140 1423 23141 1424 23142 1425 23143 1426 23144 1427 23145 1428 23146 1429 23147 1430 23150 1431 23151 1432 23152 PROGRAM 7200 Z 7 7240 P 4523 G 1353 P 207 11/19/81 PALS—V10A NO DATE PAGE 16-1 JMP PLTARE TAD P117 06, CLA INOP TAD START2 MOL DVI 120 CLA MUY 120 CLA MOA DCA PLTEM /START2 ROUNDED TO THE NEXT LOWER 10 LTARE. CLA CMA JMS I DRPLOT /INITIALIZE JMS I SETUP CLA IAC DCA 10 /SET PEN STATUS TAD RET2 /SET RETURN DCA MONIT ORSH2V JMS I GOTCHA JMS I PLOPT /GET Y VALUE JMS I FLTPT FMPY YCAL FADD I XOFFI /ADD IN OFFSET (0 OR 500) XT JMS I YUNORM DCA YREG TAD YREG CIA CLL TAD PP1750 /CHECK FOR OFF SCALE SZL CLA JMP PLOKE TAD PP1750 /TOO BIG DCA YREG TAD BELL JMS I PRINTI /SOUND ALARM LOKE. TAD YREG TAD P12 /OFFSET TO ALLON FOR TICS ON BASELINE DCA YREG TAD NAVE CIA TAD PLTEM JMS I NEGA1 ‘TAD INTVL2 CIA . |JMS I NORM IX COORDINATE JMS I FLTPT FDIV I XSCL3 EXT JMS I VUNORM TAD P12 /ALLOU FOR TICS DCA XREG TAD 10 /PEN STATUS JMS I DRPLOT IFLOURESCENCE PROGRAM » 11/19/81 PALS-V10A NO DATE PAGE 16-2 1434 23154 3010 DCA 10 1435 23155 4506 JMS I TTOINT /LOOK FOR INTERRUPT 1436 23156 7200 1437 23157 5306 JMP GORSH2 1438 23160 3613 XSCL3. XSCAL /PLOT BUFFER XAXIS SCALE FACTOR 1439 23161 0000 YCAL 1440 23162 0000 0 1441 23163 0000 O 1442 23164 3037 PLTRET. RETPLT 1443 23165 1750 PP1750. 1750 1444 23166 0117 P1171 117 1445 23167 0000 PLTEMIO 1446 23170 3200 RET2: R- I 1447 23171 1655 GPLTMX» GOMARX 1448 23172 1644 GFAC. GGFAC 208 /FLOURESCENCE 1449 1450 23200 1451 23201 ASELINE 452 23232 I453 23103 1454 23204 1455 23205 1456 23206 1457 23207 1458 23210 1459 23211 1460 23212 1461 23213 1462 23214 1463 23215 1464 23216 1465 23217 1466 23220 1467 23221 1468 2322 1469 23223 1470 23224 1471 23225 1472 2322 1473 23227 1474 23230 1475 23231 1476 23232 1477 23233 1478 23234 1479 23235 1480 23236 1481 23237 1482 23240 1483 23241 1484 23242 1485 23243 1486 23244 1487 23245 1488 23246 1489 23247 1490 73250 1491 23251 1492 23252 1493 23253 1494 23254 1495 23255 1496 23256 1497 3257 1498 23260 1499 23261 1500 23262 1501 23263 1502 23264 1503 23265 209 PROGRAM 11/19/21 PALS—vIOA N0 DATE PAGE 17 3200 .3200 1057 RET2I. TAD NLAT IRETURN HERE AFTER PLOT 1135 TAD NASTv /IF 4 ON SCALE FACTOR. AVOID DRAwINC B 7650 SNA CLA 5264 JMP LASPLT /wAS OVERPLOT SO DON’T Box 1313 TAD PP117 7000 IPS. NOP /CLA 1025 TAD END2 7427 MGL DVI 0120 120 7605 CLA NOV 0120 120 7701 CLA MGA 3240 DCA PLTEN2 IROUNDOFF END TO NEAREST TEN 1314 TAD RET3 ISET NEH ABORT ADDRESS 3073 DCA MONIT 1240 TAD PLTEM2 , 4276 JMS XXPL /CALCULATE PLOT POSITION 0F END 1 3224 DCA PLTEND Q 7001 IAC 4541 JMS I DRPLOT IMDVE TO END OF PLOT 0000 PLTEND . o 0012 1224 TAD PLTEND 3231 DCA PLUT10 471 JMS I SIDE /PLOT SIDE (10 TICS) 0000 PLUT10, 0 xx COORD DF SIDELINE 001 12 /YCDORD 0F SIDELINE 0012 12 /DIRECTION AND LENGTH OF TIc 0144 144 1716 TAD I PLTEMI /NOH PLOT TOP 3244 DCA PLTEM2+4 471 JMS I BASTOP 0000 PLTEM2. 0 /x STARTING POINT 7660 -120 1762 1762 /v COORD 1774 1774 /Y COORD 0F TIC 0000 0 /x ENDING POINT 4715 JMS I SID DE 0012 12 /x COORD OF SIDELINE 1762 1762 xv CODRD OF SIDELINE 7766 1—12 /DIRECTION AND LENGTH OF TIC 7634 r144 IDIRECTION AND DISTANCE BETuEEN TICS 1240 TAD PLTEMZ 3263 DCA KKR 1244 TAD PLTEN2+4 3257 DCA L 4717 ans I BASTOP r 0000 0 - 0120 12 0012 12 0000 o 0000 MKKR. 0 /ENDINC POINT OF PLOT 1074 LASPLT, TAD MDNSAV 3073 DCA MONIT /RESET ABORT POINTER /FLDURESCENCE 1504 23266 1505 23267 1506 23270 1507 23271 1508 23272 1509 23273 1510 23274 1511 232'. 1512 23276 1513 23277 1514 23300 1515 23301 1516 23302 1517 23303 1518 23304 1519 23305 1520 23306 1521 23307 1522 23310 1523 23311 1524 23312 1525 23313 1526 23314 1527 23315 1528 23316 1529 23317 1530 23320 1531 23321 1532 23322 1533 23323 1534 23324 1535 23325 1536 23326 1537 23327 1538 23330 1539 23331 1540 23332 1541 23333 1542 23334 Sr: :7=~r 1544 23-3; 1545 23337 1546 23340 1547 23341 1548 23342 1549 23343 1550 23344 1551 23345 1552 23346 1553 1554 23346 1555 23347 1556 23350 1557 23351 1558 23352 210 PROGRAM 11/19/31 PALS-V104 NO DATE PAGE 17-1 1224 TAD PLTEND Inc MDPw /WflmEHMFIMH 3273 Dc +3 7001 IAC 4541 OMS I DRPLOT oooo NORMz 0000 0 5473 OMP I MONIT 0000 XXPL. o /CALCULATE x COORD OF PLOT 7041 CIA 1716 TAD I PLTEMI 7041 cIA 4542 OMS I NEGA1 /NOP OR CIA 4540 OMS I NORM 0044 44 . 4407 OMS I FLTPT g 4721 FDIV I XSCALI /SCALE 0000 FEXT 4537 OMS I YUNORM 1136 TAD P12 5676 OMP I XXPL 0117 PP117. 117 3264 RET3. LASPLT 3400 SIDE. PLTSID 3167 PLTEMI. PLTEM 3447 BASTOP. PLTBAS 0062 P62. 3613 XSCALI. XSCAL 0000 NORMAL. o /NORMAL12E NUMBER IN Ac 3046 3045 DCA 45 1337 TAD PP27 3044 DCA 44 IEXPONENT 1722 TAD I NORMAL 3340 DCA SINGLE 2322 SZ NORMAL 4407 OMS I FLTPT 7000 FNDR 6740 FPUT I SINGLE 0000 0027 .P27 :7 0000 SINGLE. 0 /INTEGER INPUT 4405 OMS I 4537 OMS I YUNDRM 5740 OMP I SINGLE 1605 OGNEN. TEXT ”NEH?" 2777 0000 3346 4,—1 0001 1 0015 GGPER. 15 001 12 2516 TEXT "UNITS/INCH? " 1124 /FLDURESCENCE 1559 23353 1560 23354 1561 23355 1562 23356 1563 23357 1564 1565 23357 1566 23360 1567 23361 1568 23362 1569 23363 1570 23364 1571 23365 1572 23366 1573 23367 1574 23370 PROGRAM 2357 1116 0310 7740 0000 3357 0001 *.-1 SFL. O 0 RFL: O O FFL: O 211 11/19/81 212 /FLOURESCENCE PROGRAM 11/19/81 PALB-VlOA N0 DATE PAGE 18 1575 3400 #3400 /LAST 0F PLOT GARBAGE (ROUTINES TO PLOT BASE LINES AND SIDES) 1576 23400 0000 PLTSID. o /PLOT SIDELINE 1577 23401 1136 TAD P12 1573 23402 7040 CMA 1579 23403 3034 DCA TEMP1 710 TICS 1530 23404 4243 JMS GOTP 1531 23405 3223 DCA PLUT xx COORD 1532 23406 4243 JMS GOTP 1533 23407 3224 DCA PLUT+1 xv COORD 1534 23410 4243 JMS GOTP 1535 23411 3017 DCA 17 /DIRECTION OF TIC 1536 23412 4243 JMS GOTP 1537 23413 3016 DCA 16 /DIRECTION OF PLOT 1533 23414 5220 JMP .+4 /ENTER IN MIDDLE 0F LOOP TO PLOT FIRST TI c 1539 23415 1224 PLUT4. TAD PLUT+1 1590 23416 1016 TAD 16 1591 23417 3224 DCA PLUT+1 1592 23420 7240 CLA CMA 1593 23421 3010 DCA 10 § 1594 23422 4541 JMS I DRPLOT I 1595 23423 0000 PLUT.0 1596 23424 0000 o 1597 23425 2010 182 10 1593 23426 5240 JMP PLUT2 1599 23427 1223 TAD PLUT /PLOT TIC 1600 23430 1017 TAD 17 1601 23431 3235 DCA PLUT3 1602 23432 1224 TAD PLUT+1 1603 23433 3236 DCA PLUT3+1 1604 23434 4541 JMS I DRPLOT I 1605 23435 0000 PLOT3, o r 1606 23436 0000 o 1607 23437 5222 JMP PLUT-l /MOVE PEN BACK 1603 23440 2034 PLUT2, ISZ TEMP1 /PLOT AGAIN/7 1609 23441 5215 JMP PLUT4 vaS 1610 23442 5600 JMP I PLTSID /NvET. RETURN 1611 23443 0000 GOTP. o 1612 23444 1600 TAD I PLTSID 1613 23445 2200 132 PLTSID 1614 23446 5643 JMP I GOTP 1615 23447 0000 PLTBAS. 0 /PLOT BASELINE OR TOP 1616 23450 4315 JMS GOTB 1617 23451 3035 DCA TEMP2 1613 23452 4315 JMS GOTB 1619 23453 4542 JMS I NEGA1 1620 23454 3017 DCA 17 1621 23455 4315 JMS GOTB , 1622 23456 3276 DCA PLOO1+1 /YCDDRD 1623 23457 4315 JMS GDTB 1624 23460 3305 DCA PLOO5 /v COORD TIc 1625 23461 4315 JMS GDTB 1626 23462 3016 DCA 16 /END POINT 1627 23463 5267 I JMP .+4 /ENTER LOOP TO PLOT FIRST TIC 1623 23464 1035 PLTBSI. TAD TEMP2 1629 23465 1017 TAD 17 /SET NEw x /FLOURESCENCE 1630 23466 1631 23467 1632 23470 1633 23471 1634 23472 1635 23473 1636 23474 1637 23475 1638 23476 1639 23477 1640 23500 1641 23501 1642 23502 1643 23503 1644 23504 1645 23505 1646 23506 1647 23507 1648 23510 1649 23511 1650 23512 1651 23513 1652 23514 1653 23515 1654 23516 1655 23517 1656 23520 1657 23521 1658 23522 1659 23523 1660 23524 1661 23525 1662 23526 1663 23527 1664 23530 1665 23531 1666 23532 1667 23533 1668 23534 1669 23535 1670 23536 1671 23537 1672 23540 1673 23541 1674 23542 1675 23543 1676 23544 1677 23545 1678 23546 1679 23547 1680 23550 1681 23551 1682 23552 1683 23553 1684 23554 213 PROGRAM 11/19/31 PAL3-V10A NO DATE 3035 DCA TEMP2 1035 TAD TEMP2 4721 JHS I XXPLI 3275 DCA PLOO1 /x COORD 7240 CLA CMA 3010 DCA 10 4541 JMS I DRPLOT oooo PLOOI. o 0000 o 2010 182 10 5307 JMP PLOO2 1275 TAD PLOO1 /PLOT TIC 3304 DCA .+2 4541 JMS I DRPLOT 0000 o 0000 PLOO5. o 5274 JMP PLOO1-1 /RESET PEN POSITION 1035 PLOO2. TAD TEMP2 /DONE? 7041 CIA 1016 TAD 16 7640 SZA CLA 5264 JMP PLTBSI /A0AIN 5647 JMP I PLTBAs /DONE oooo GOTB. o 1647 TAD I PLTBAS 2247 152 PLTBAS 5715 JHP I GOTB 3276 XXPLI. XXPL oooo UNORM.o /UNNORMALIZE NUMBER IN PAC 1045 TAD 45 IRETURN NITH # IN Ac 7750 SPA SNA CLA 5722 0MP I UNORM /NUMBER <=0 4407 JMS I FLTPT 1355 FADD AHALF /ROUND OFF oooo FEXT 1044 TAD 44 7750 SPA SNA CLA 5722 JMP I UNORM /LESS THAN 1 1044 TAD 44 7041 CIA 1354 “TAD P14 7500 *SMA 5343 JMP .+3 _ 7240 CLA CMA /OVERFLON 5722 JMP I UNORM 3350 DCA .+5 1046 TAD 46 7421 MGL 1045 TAD 45 7415 ASR 0000 o 7413 SHL 0001 I 5722 JMP I UNORM 0014 P14,14 PAGE 18-1 ...-45-n1‘u»; ‘ 5 w--<'.~r- .. 214 IFLOURESCENCE PROGRAM 11/19/31 PALS-v1oA NO DATE PAGE 13—2 1635 23555 0000 AHALF, o 1636 23556 2000 2000 1637 23557 0000 o 1633 23560 0000 BUFFI. o 1639 23561 0000 0 1690 23562 0000 o 1691 23563 0000 BUFF2, o 1692 23564 0000 o 1693 23565 0000 o 1694 23566 0000 BUFF3. o 1695 23567 0000 o 1696 23570 0000 o ‘1 '4‘ W 1 IFLOURESCENCE PROGRAM 11/19/31 PALS—v10A NO DATE PAGE 19 1697 6554 #6554 /IN FLOATING POINT INTERPRETTER 1693 26554 3620 FAB /ABSOLUTE VALUE 1699 26555 6000 6000 /NEGATE FAc 1700 3600 *3600 1701 23600 1216 XAx. TAD GPER /AK ”UNIT/INCH?" 1702 23601 4501 OMS I TYPIST 1703 23602 4405 OMS I FLIN 1704 23603 4407 OMS I FLTPT 1705 23604 4617 FDIv I A100 _'Oé 23605 6213 FPUT XSCAL 1707 23606 0000 FEXT 1703 23607 7325 CLA STL IAC RAL /3 1709 23610 1213 TAD XSCAL 1710 23611 3213 DCA XSCAL /MULT BY 3 1711 23612 5473 OMP I MONIT 1712 23613 0001 XSCAL. 1 /SCALE FACTOR DEFAULT uITH 20 UNITS PER I NCH 1713 23614 3146 3146 1714 23615 3146 3146 1715 23616 3347 GPER. GOPER 1716 23617 2514 A100. FLT100 1717 23620 0000 FAB. o /ROUTINE TO TAKE ABSOLUTE VALUE OF FAC 1713 23621 3047 DCA 47 /CLEAR OUT 3RD NORD OF PREC 1719 23622 1045 TAD 45 1720 23623 7710 SPA CLA 1721 23624 4626 OMS I .+2 /NEGATE 1722 23625 5620 OMP I FA 1723 23626 6000 6000 /LOCATION IN FLOATING POINT OF ROUTINE NEGAT ER 1724 23627 0000 XOFF, o 1725 23630 0000 o 1726 23631 0000 o 1727 23632 0000 OFFSET. o 1723 23633 1237 TAD PDDsoo 1729 23634 4540 OMS I NORM /SET OFFSET TO 500 (HALF SCALE) 1730 23635 3627 OFF 1731 23636 5632 OMP I OFFSET 1732 23637 0764 PDDsoo, 764 215 /FLOURESCENCE PROGRAM 11/19/81 PALS-VioA NO DATE PAGE 20 1733 4200 .4200 1734 24200 4540 AVERAGE: JHS I NORM /CLEAR SUMMATIN BUFFER 1735 24201 4237 1736 24202 1124 TAD OUTSTG ICHOOSE OUTPUT OPTION 1737 24203 4534 JHS I ENTURP 1738 24204 0000 AVPARM. O 1739 24205 4547 OMB I DIAG /PRINT DIAGNOSTIC MESSAGES 1740 24206 3544 DCA I TYGH 1741 24207 4522 JMS I SETUP 1742 24210 1236 TAD AVRET 1743 24211 3073 DCA MONIT /SET ABORT RETURN 1744 24212 4523 JMS I GOTCHA 1745 24213 4604 JMS I AVPARH 1746 24214 4407 JHS I FLTPT 1747 24215 1237 FADD AVBUFF 1748 24216 6237 FPUT AVBUFF 1749 24217 0000 FEXT 1750 24220 2544 182 I TYOM 1751 24221 5212 JHP .-7 1752 24222 1074 RETAV. TAD MONSAV 1753 24223 3073 DCA MONIT 1754 24224 1544 TAD I TYGM 1755 24225 4540 JMS I NORM 1756 24226 3560 BUFF1 1757 24227 4407 JHS I FLTPT 1758 24230 5237 FGET AVBUFF 1759 24231 4530 FDIV 1 BUF1 1760 24232 0000 FEXT 1761 24233 4503 JMS I IITYCR 1762 24234 4406 JHS I FOUT 1763 24235 5473 JMP I MONIT 1764 24236 4222 AVRET. RETAV 1765 24237 0000 AVBUFF; O 1766 24240 0000 0 O 1768 24242 3062 LE: DCA TEMP4 1769 24243 1267 TAD INTO /ASK FOR SIZE OF INTVAL 1770 24244 4501 JMS I TYPIST 1771 24245 4500 JMS I NUMIN 1772 24246 3075 DCA INTVAL 1773 24247 1300 TAD SNOOP /START OF STRING OF LE. NU CHANGES 1774 24250 3034 DCA TEMP1 TAD 1 TEMP1 /GET AN ADDRESS 34 1776 94252 7450 SNA /END NITH o 1779 24255 1034 TAD TEMP1 1780 24256 7001 IA 1781 24257 1062 TAD TEMP4 1782 24260 3036 DCA TEMP3 1783 24261 1436 TAD I TEMP3 1784 24262 3435 DCA I TEMP2 1785 24263 2034 152 TEMP1 1786 24264 2034 182 TEMP1 1787 24265 2034 182 TEMP1 216 /FLOURESCENCE PROGRAM 11/19/81 PALB—V10A NO DATE PAGE 20-1 1788 24266 5251 1789 24267 4270 1790 24270 1116 1791 24271 2405 1792 24272 2226 1793 24273 0114 1794 24274 7540 1795 24275 0000 1797 24275 0001 1798 24276 7201 1799 24277 5242 1800 24300 4301 1801 24301 0537 1802 24302 0624 1803 24303 0644 1804 24304 0737 18 24 0 BDA AND HAVENUMBER 1825 24331 70 1826 24332 5730 1827 24333 0000 1828 24334 7000 1829 24335 5733 JMP LE3 INTO, .+1 TEXT "INTERVAL: “ *.-1 1 NU. CLA IAC JMP LE SNOOP..+1 PARM1/ADDRESS LEIN/LENGTHCOMMAND NUIN/NUMBER COMMAND 0 NEGll. 0 /AC NEGATER DUE TO BASIC DIFF BETWEEN LAM NOP /CIA OMP I NEGII. LI'NRER. o /L1_NK COMPLEMENTER FOR SAME REASON NO JMP I LINKER /NOP MAY BE CML /FLOURESCENCE PROGRAM 4400 24400 0301 24401 0400 24402 0515 24403 0503 24404 0530 24405 0530 24406 1725 24407 0671 24410 2303 24411 1000 24412 2022 24413 1400 24414 2417 24415 5106 24416 2315 24417 5025 24420 0411 24421 5003 24422 2014 24423 5000 24424 0126 24425 4200 24426 1405 24427 4242 24430 1625 24431 4276 24432 3001 24433 3600 24434 6161 24435 5010 24436 6167 24437 5014 24440 2217 24441 4512 24442 2301 24443 5100 24444 0705 24445 5103 24446 0000 24447 2222 PUNCH. 24450 2200 24451 2323 24452 2205 24453 0606 24454 2212 24455 0161 24456 2220 24457 0000 24460 0000 USER ERROR 24461 4715 24462 4503 24463 1270 24464 4501 24465 1154 *4400 CMDSTG. STIGMA. RR 2323 SS 0606 FF 0 DINOG. JMS I VISAD 217 11/19/81 PALS-VIOA NO DATE PAGE 21 ISTRINGS OF COMMAND OPTIONS 0301 /CA CALIB 0515 0530 E / 0S3 ‘ /SM FOR AUTO SMOOTH i 11 DISPLA /PLOT ON SCOPE 4 /XA /SET X-AXIS SCALE /11 /17 IRO /SAVE A RUN /GE /GET TABLE FROM DISK O 2222 /STRINGS OF OUTPUT OPTIONS FOR PLOT. /AND AVERAGE /FF IA 0161 1 /PRIMARY CORRECTED ABSORDANCE AB9 O /ROUTINE TO PRINT BRIEF DIAGNOSTICS TO MI /PRINT SOME ADVICE TAD SMFLG IFLOURESCENCE 1885 24466 1886 24467 1887 24470 1888 24471 1889 24472 1890 24473 1891 24474 1892 24475 1893 24476 1894 1895 24476 1896 24477 1897 24500 1898 24501 1899 24502 1900 24503 1901 24504 1902 24505 1903 24506 1904 24507 1905 24510 1906 24511 1907 24512 1908 24513 1909 24514 1910 24515 218 PROGRAM 11/19/31 PALS-v10A NO DATE PAGE 21—1 4277 OMS NOGIN /TELL IF AUTO SMOOTHED 5660 OMP I D N00 4471 ODIG. .+1 V 2315 TEXT “SMOOTH7: " 5 1717 1 2410 r 7772 4040 1! 4000 4476 «.-1 0001 1 0000 NOGIN. o 7650 SNA CLA /PR1NT Y IF AC IS 1 7305 CLL CLA IAC RAL /SET TO 2 1305 TAD Day 4501 OMS I TYPIST 5677 OMP I NOGIN 4506 GOY. .+1 3140 3140 /v 0001 1 1640 1640 /N 0001 6213 ROTAT. CDFCIF 10 5714 OMP I .+1 3000 3000 1404 VISAD. ADVIS /FLOURESCENCE 1911 1912 25000 1913 25001 1914 25002 1915 25003 1916 25004 1917 25005 1918 25006 1919 25007 1920 25010 1921 25011 1922 25012 1923 25013 1924 25014 1925 25015 1926 25016 1927 25017 1928 25020 1929 25021 1930 25022 1931 25023 1932 25024 1933 25025 1934 25026 1935 25027 1936 25030 1937 25031 1938 25032 1939 25033 1940 25034 1941 25035 1942 25036 1943 25037 1944 25040 1945 25041 1946 1947 25041 1948 25042 1949 25043 1950 25044 1951 25045 1952 25046 1953 25047 1954 25050 1955 25051 1956 25052 1957 25053 1958 25054 1959 25055 1960 25056 1961 25057 1962 25060 1963 25061 1964 25062 1965 25063 219 PROGRAM 11/19/31 PALS—VICA NO DATE PAGE 22 5000 45000 1205 PLOTG. TAD XPLOT /PLOT ON PLOTTER 3141 DCA T 5607 OMP I LPLOT 1206 DISPLA. TAD SCPLOT IPLDT ON SCOPE 5201 OMP .—3 5200 XPLDT. PLOTx 4600 SCPLOT. PLOTSC 3004 LPLOT. PLOT I22 SMll. TAD P13 /SET UP FOR 11 POINT CURVE SMOOTH 3112 Dc R2 1220 TAD UPON 5622 OMP I SMOR 1224 SM17. TAD P21 3112 Dc R2 22 TAD HPON+1 5622 OMP I SMOR 5551 HPON. N1 6663 c1 5400 SMOR. SMOOTH 0013 P13.13 0021 P21.21 1234 SMUST. TAD GSMUST /ASK " SMOOTH? " 4501 OMS I TYPIST 4504 OMS I YORN 7410 SKP /NO 7201 CLA IAc _ 3154 DCA SMFLG /SET AUTO SMOOTHING FLAG ACCORDINGLY 5473 OMP I MONIT 5035 GSMUST. .+ 2315 TEXT “SMOOTH? " 1717 2410 7740 4000 5041 4.-1 0001 1 1377 SMUST2. TAD (SMUSTB) /COME HERE AT END OF ALL SCAN 3073 DCA MONIT /RESET MONITR POINTER FOR LOOPING 1154 TAD SMFLG /SHOULD NE SMOOTH? 7650 SNA CLA 5274 OMP SMUST7 /NO 3267 DCA SMUST4 /SETUP R.s.F POINTER (O=R) 7346 CLA CMA CLL RTL /—3 FOR R.S.F 3270 DCA 5MUST5 1224 SMUSTS. TAD P21 /SETUP FOR 17 POINT SMOOTH 3112 D 2 1221 TAD NPON+1 3114 DCA S2 1031 TAD NOVAL 7040 CMA 1112 TAD R2 7500 SMA 5274 OMP SMUST7 /NO POINTS 3034 DCA TEMP1 /FLOUREscENCE 1966 25064 1967 25065 1963 25066 1969 25067 1970 25070’ 1971 25071 RT OF DATA 1972 25072 1973 25073 1974 25074 1975 25075 1976 25076 1977 25077 1973 25100 1979 25101 1930 25102 1931 25103 1932 25104 1933 25105 1934 25106 1935 25107 1936 25110 220 PROGRAM 11/19/81 PALE-V10A NO DATE PAGE 22—1 7240 CLA CMA 1267 TAD SMUST4 /0=R 1=S 2=F 5776’ JMP SMUST6 /HOP INTO SMOOTHING ROUTINE 0000 SMUST4. 0 0000 SMUST5. 0 2267 SMUSTB. ISZ SMUST4 /COME HERE WHEN DONE WITH A PA 2270 182 SMUST5 /DONE ALL 3? 5252 JMP SMUSTO /NO GO DD NEXT SET 7200 SMUST7. CLA /ALL DONE 1074 TAD MONSAV 3073 DCA MONIT IRESET MONITOR POINTER 5473 JMP I MONIT /AND QUIT 6203 SAVE. CDFCIF 0 5702 JMP I .+1 0400 SAVRUN 6203 GET. CDFCIF 0 5705 JMP I +1 0463 GETRUN 6203 058; CDFCIF 0 5710 JMP I +1 7600 7600 Ir- .14;er a» LfiafimwK . .1 /FLOURESCENCE PROGRAM 1987 25176 1988 25177 1989 1990 25400 1991 25401 P8_OSSIBLE 1992 25402 ELSE 1993 25403 1994 25404 1995 25405 1996 25406 1997 25407 1998 25410 1999 25411 2003 25415 2004 25416 2005 25417 2006 25420 2007 25421 2008 25422 2009 25423 2010 25424 2011 25425 2012 25426 2013 25427 2014 25430 2015 25431 2016 25437 2020 25436 2021 25437 2022 25440 2023 25441 2024 25442 2025 25443 2026 25444 2027 25445 2028 25446 2029 25447 2030 25450 2031 25451 2032 25452 NT WHICH IS 2033 25453 NEARBY POINTS 2034 25454 2035 25455 2036 25456 S 2037 25457 2038 25460 2039 25461 2040 25462 2041 25463 5421 5071 5400 3114 1031 7040 5473 0000 1114 3036 7325 3016 1113 7041 3015 3107 3110 '5400 SMOOTH TAD CMA SMIFT. TAD DCA CLA DCA TAD CIA DCA D P4 . DCA 10 D R2 221 11/19/81 PALS-VIDA NO DATE PAGE 23 . DCA S2 NOVAL /SMOOTH FIVE POINTS ASSUMING CUBIC FIT ISEE “METHODS IN NUMERICAL ANALYSIS" BY NI R2 /# OF POINTS IN SMOOTHING INTERVAL TEMP1 TEMP1 CLA SMOO2 SMOOCH I TYPIST I NUMIN N5 /NOT ENOUGH POINTS /”INPUT 0.1.2 FOR R.S.F" .+3 JMS I TYGM I MONIT /INCORRECT ENTRY IPICKUP POINTER RAR F2 /CREATE TRUNCATED N/2 F2 RAL F2 /3*EN/2I 10 11 IPUT-BACK POINTER R2 TEMP2 SMIFT /NOW GET IN FIRST 5 POINTS TEMP2 /NOW GO THRU SMOOTHING LOOP —2 OMS SMIFT /UNORMALIZE NEW MIDDLE VALUE YUNORM 30 I 11 CODFLD 11 11 TEMP1 SMOO3 I MONIT O /SHIFT IN NEW VALUES AND FORM SMOOTHED POI /STORE IN DATA TABLE S2 /ESSENTIALLV A NEIGHTED AVERAGE OF THE 5 TEMP3 /POINTER TO WEIGHT FACTORS IAC STL RAL /3 /FOR SPACING UP POINTER TO WEIGHT FACTOR F2 15 /SPACE UP 5. THEN SPACE DOWN FIVE DCA R1 DCA F1 ICLEAR SUMMATION BUFFER IFLOURESCENCE 2042 25464 2043 25465 2044 25466 2045 25467 2046 25470 2047 25471 2048 25472 2049 25473 2050 25474 2051 25475 2052 25476 2053 25477 2054 25500 2055 25501 2056 25502 2057 25503 2058 25504 2059 25505 2060 25506 2061 25507 2062 25510 2063 25511 2064 25512 2065 25513 2066 25514 LE OF FACTORS 2067 25515 2068 25516 2069 25517 2070 25520 2071 25521 2072 25522 ER 2073 25523 2074 25524 2075 25525 2076 25526 2077 25527 2078 25530 2079 25531 2080 25532 2081 25533 2082 25534 2083 25535 2084 25536 2085 25537 2086 25540 2087 25541 2088 25542 2089 25543 2090 25544 2091 25545 2092 25546 2093 25547 2094 25550 2095 2096 25550 222 PROGRAM 11/19/31 PALS—VIoA NO DATE PAGE 23—1 3111 DCA 51 1327 TAD PUSHER /PUSH—DONN STORAGE BUFFER 3062 DCA TEMP4 1062 TAD TEMP4 3017 DCA 17 /PICKUP POINTER 1112 TAD R2 7041 CIA 3063 DCA TEMP5 1417 SMU. TAD I 17 /GET A POINT 3462 DCA I TEMP4 IPUSH IT DOWN 1462 TAD I TEMP4 2062 182 TEMP4 /GET BACK POINT FOR AVERAGE 4540 OMS I RM . 0044 44 4407 OMS I FLTPT 3436 FMPY I TEMP3 /MULT Bv WEIGHT 1107 FADD R1 . 6107 FPUT R1 0000 FEXT 1036 TAD TEMP3 1016 TAD 16 /MOVE UP OR BACK 3036 DCA TEMP3 2015 182 5 5316 OMP .+3 7346 CLL CMA CLA RTL /—3 FOR SPACING BACK DOWN TAB 3016 DCA 16 /(LAST 5 FACTORS SAME AS FIRST FIVE) 2063 182 TEMP5 5274 OMP SMU 6231 CDF 30 1410 TAD I 10 2010 152 10 /MOVE IN NEW POINT INTO PUSHDDWN BUFF 2010 152 10 6221 CDF CODFLD 3462 DCA I TEMP4 5653 OMP I SMIFT 6717 PUSHER. PUSHY 5531 SMOOCH. . 1 0516 TEXT "ENTER 0.1. OR 2 FOR R.S. OR F1" 2405 . 2240 6054 6154 4017 2240 6240 0617 2240 2254 2354 4017 2240 0672 0000 5550 A.—1 0001 1 223 /FLOURESCENCE PROGRAM 11/19/81 PAL8-V10A NO DATE PAGE 23-2 2097 25551 7775 H117775 /-36/429 ALL SUCCEEDING FACTORS ARE DIVIDED BY 429 2098 25552 5241 5241 2099 25553 0755 0755 2100 25554 7773 H2; 7773 I9 2101 25555 2536 2536 2102 25556 7023 7023 2103 25557 7775 N3. 7775 /44 2104 25560 3220 2 0 2105 25561 3210 210 2106 25562 7776 N4, 7776 / 69 2107 25563 2445 445 2108 25564 4576 576 2109 25565 7776 N5. 7776 /B4 2110 25566 3104 3104 / 2111 25567 0154 154 2112 25570 7776 U6. 7776 /89 2113 25571 3243 43 2114 25572 4020 4020 /FLOURESCENCE PROGRAM 2115 26663 26664 26665 26666 26667 26670 26671 26672 26673 26674 26675 26676 26677 26700 26701 26702 26703 26704 26705 26706 26707 26710 26711 26712 26713 26714 26715 26716 26717 6663 7775 5726 11/19/81 PALS-VloA NO DATE PAGE 24 *6663 C1. 7775 /THESE ARE HEIGHT FACTORS FOR 17 POINT SHO 4430 0 PUSHYIO 224 /FLOURESCENCE PROGRAM 11/19/81 PALS—V10A NO DATE PAGE 25 2145 /FL33 FLUORO 2146 4600 *4600 INEH SCOPE VERSION OF FLOTSC 2147 24600 0000 PLOTSC, 0 /PLOT ON SCOPE 2148 24601 7500 SMA 2149 24602 5221 JMP SCl ElgfioT 24603 7200 CLA IINITIALXZE /BUT DON’T MOVE UNTIL FIRST ACTUA 2151 24604 3244 DCA SCSAVX 2152 24605 3245 DCA SCSAVY 2153 24606 5600 JMP I PLOTSC 2154 24607 0000 PLOLD. 0 /VECTOR PLOT POINTS 2155 24610 1245 TAD SCSAVY /OLD Y COORD MQL 2157 24612 7325 STL CLA IAC RAL /3 2158 24613 4276 JMS PLPT /OUTPUT Y COORD 2159 24614 1244 TAD SCSAVX GL 2161 24616 7305 CLA CLL IAC RAL / 2162 24617 4276 JMS PLPT /OUTPUT X COORD (THE 3 AND THE 2 ARE O 2163 24620 5607 JMP I PLOLD 2164 24621 3246 5C1. DCA SCBRYT /1 MEANS UNURITTEN PLOT 42 TA 081 2166 24623 4250 JMS PRINT3 ISET FOR VECTOR PLOT 2167 24624 4207 JMS PLOLD IPLOT TO OLD COORD 2168 24625 4256 JMS SCSCAL /GET X TIMES .76 2169 24626 3244 DCA SCSAVX 2170 24627 4256 JMS SCSCAL /SAME FOR Y 2171 24630 3245 DCA SCSAVY 2172 24631 1246 TAD SCBRYT 2173 24632 7650 SNA CLA 2174 24633 5236 JMP .+3 /18 BRIGHT PLOT 2175 24634 1242 TAD 081 2176 24635 4250 JMS PRINT3 /IS DARK 2177 24636 4207 JMS PLOLD /OUTPUT NEH COORD. ‘ 2178 24637 1243 TAD U81 2179 24640 425 JMS PRINT3 /SET BACK TO NORML PRINT MODE 2180 24641 5600 JMP I PLOTSC /DONE. RETURN 2181 24642 0235 081; 235 /VECTOR PLOT 2182 24643 0237 U51, 237 /NORMAL TEXT 2183 24644 0000 SCSAVX, 2184 24645 0000 SCSAVY, IX AND Y COORD 2185 24646 0000 SCBRYT: /IS ONE FOR DARK. 0 FOR BRIGHT 2186 24647 0000 SCXN. 0 ISCRATCH 2187 24650 0000 PRINT3 2188 24651 6046 TLS 2189 24652 7200 CLA 2190 24653 6041 TSF 000 JMP 2192 24655 5650 JMP I PRINT3 2193 24656 0000 SCSCAL. 0 /GET COORD TIMES .76 2194 24657 1600 TAD I PLOTSC 2195 24660 2200 182 FLOTSC 2196 24661 7425 MGL MUY 2197 24662 1370 1370 /760 - 2198 24663 7407 DVI 2199 24664 1750 PD1750. 1750 /1000 225 /FLOURESCENCE PROGRAM 11/19/81 PALE-V10A NO DATE PAGE 25-1 2200 24665 7701 CLA MGA 2201 24666 7141 CIA CLL 2202 24667 1264 TAD PDl750 /CHECK TO SEE IF IT IS OVER IOOOD 2203 24670 7620 SNL CLA 2204 24671 5274 JMP ,+3 2205 24672 7701 CLA MGA ILEGITIMATE 2206 24673 5656 JMP I SCSCAL 2207 24674 1264 TAD PDl750 2208 24675 5656 JMP I SCSCAL /TOO B10 2209 24676 0000 PLPT. O /OUTPUT VECTOR COORDINATES 2210 24677 3247 DCA SCXN /CODE 2211 24700 7413 SHL 2212 24701 0006 6 /GET TOP 5 BITS 2213 24702 1311 TAD P0240 IMORE CODES 2214 24703 4250 JMS PRINT3 2215 24704 1247 TAD SCXN 2216 24705 7413 SHL 2217 24706 0004 4 2218 24707 4250 JMS PRINT3 2219 24710 5676 JMP I PLPT 2220 24711 0240 PD240, 240 2221 24712 0000 SCHOME. O 2222 24713 1320 TAD PD33 2223 24714 4250 JMS PRINT3 /CLEAR SCREEN AND HOME 2224 24715 1321 TAD P014 2225 24716 4250 JMS PRINT3 2226 24717 5712 JMP I SCHOME 2227 24720 0033 P033, 33 2228 24721 0014 PD14114 "229 24722 0000 ENPUT. 0 /INPUT AND ECHO A CHAR 2230 24723 7200 CLA 2231 24724 4777’ JMS KBRK /REST OF MONITOR IS AT 200 2232 24725 7450 SNA 2233 24726 5324 JMP .-2 2234 24727 4776’ JMS TTOUT /ECHO IT 2235 24730 1375 TAD (—212 9736 24731 1774’ TAD FCHAR /GET BACK CHAR AND TEST FOR LINE FEED 2237 24732 7650 SNA CLA 2238 24733 5323 3MP ENPUT+1 /IGNORE LINE FEEDS COMPLETELY 2239 24734 1774’ TAD FCHAR / 2240 24735 5722 UJMP I ENPUT /FLOURESCENCE PROGRAM 24774 24775 24776 24777 2522 25227 25230 25231 25232 25233 25234 25235 25236 25237 0306 7566 0317 0271 5200 1362 7141 1600 7420 7041 3364 7004 3367 1600 226 *5200 /DIGITAL 8—12~U /PLOT SUEROUTINE /CALLING SEQUENCE C(AC)=—1) C(AC)= 01 C(AC)= h JMS PLOTX \\\\\\ PLOTX: 0 SP JMP PLOTA TAD PLOTPN CLL RTR SPA CLA JMP PLOT1 SNL CLA P .+4 DCA PLOTPN JMP .+3 ISZ PLOTPN PLPD JMS FLOTHT JMP PLOT1 PLOTA, CLA PL DCA PLOTPN DCA PLOTNX DCA PLOTNY JMS PLOTNT JMP I PLOTX /DIGITAL 8—12-U /PAGE 2 /PICK UP ARGUMENTS PLOTI, TAD PLOTNX CIA CLL TAD I PLOTX SNL CIA DCA PLOTDX RAL DCA PLOTMV TAD I PLOTX 11/19/81 PALS-V10A NO DATE PAGE 26 INITIALIZE PLOT HITH PEN DOWN PLOT NITH PEN UP X CO—ORDINATE (IN STEPS) (RETURN IF AC=-1) Y CO-ORDINATE (IN STEPS) IMOVE THE PEN? /NO: CONTINUE /ADD PEN STATUS /ANY CHANGE? /NO: CONTINUE /LONER THE PEN /RAISE THE PEN /LONER THE PEN /UAIT FOR FLAG ICONTINUE /RAISE THE PEN /0 TO X CO-ORDINATE /0 TO Y CO-ORDINATE /FETCH PREVIOUS X CO-ORDINAT IFORM NX—NPX /L=O: NXCNPX /ABSOLUTE VALUE OF DIFFERENC /SAVE SIGN BIT /SET NEH IFLOURESCENCE PROGRAM 25240 25241 25242 25243 25244 25245 25246 25247 25250 25251 25252 25253 25254 25255 25256 25257 25260 25261 25262 25263 25264 25265 25266 25267 25270 25271 25272 25273 25274 25321 3362 2200 1363 7141 1600 7420 7041 3365 1367 7004 1367 7110 227 11/19/81 DCA PLOTNX ISZ PLOTX TAD PLOTNY CIA CLL TAD I PLOTX SNL CIA DCA PLOTDY TAD PLOTMV RAL DCA PLOTMV TAD I PLOTX DCA PLOTNY ISZ PLOTX TAD PLOTDX CIA CLL TAD PLOTDY SNL CLA JMP PLOT2 TAD PLOTDX DCA PLOTNA TAD PLOTDY DCA PLOTDX TAD PLOTNA DCA PLOTDY AND PLOTMV TAD PLOTT1 JMP /DIGITAL 8-12-U /PAGE 3 TAD PLOTMV CLL RAR TAD PLOTT2 DCA PLOTNA TAD I PLOTNA DCA PLOT4 TAD PLOTMV TAD PLOTT3 DCA PLOTMV TAD I PLOTMV DCA PLOTDB TAD PLOTDX CLL RAR DCA PLOTNA TAD PLOTDX PLOT2. DCA PLOTMV PLOTS. ISZ PLOTMV SKP JMP l PLOTX TAD PLOTNA PALS-V10A NO DATE PAGE 26-1 /PREVIOUS X /INCREMENT POINTER IFETCH PREVIOUS Y CO—ORDINAT /FORM NY-NPY /<=0: NPY