THE MEASUREMENT OF THE EXTINCTION COEFFICIENT OF A FISH LIVER OIL Thesis for the Degrea of M. S. MICHIGAN STATE COLLEGE William Henry Martin I94? wit)? 1 ... ammunbmwr, L . . .I J. $.19 . ‘V t‘. .33. 1+ 9:7... iga .. ..;.:.x C. \ o., u. ._.\ s‘. 12‘ t‘u'r' '. a“, ' {hi a.i 1"; H q "o' I rrpg 3,; I ’ L I" . (I; v’.. . . . ~54.» I, I n r _’ _- ”A " . 0" U; .A 0 .5 " fir‘t‘ '.I'-:\~ ‘v. yup. ' J. v», x u r' . . .. . . . . .,. I. t .1 .1 n i \. 3|O.‘..).J\.nvlitlt. a?! .. Pvt.“ 1‘. H ’7?“ 1...", r" * rm?“ ~' r7“ 3 I "‘T'T’I"I"‘ r“ .5 AA a '. -‘~V‘-A“ -JA'l C: I-J—J aJaa-L .L V-L LI: CLIMLL.’ U ”—44. .L LE A FILE lIJZR CIL by William Henry Pertin ¢ .. . Submitteg to the Grefiuate School 01 Nichican State Cullege of Jie ulture and mpglied Science in partIzl IIlIilment of the reiuireexente ior the degree of IPH (:1- f‘ 1" - ‘3‘} ’3‘- 'A¢~.L.hJJ.s f HU Aus- U454 e arthelt of Cne:.v iEtIy a h"i 1942 ‘ggrecietion The writer wiehes to eagreee hie aggrecietion to A :ful ea~~ :tione and do Dr. D. T. Suing for his hel (D o I guidance during tue ccuree of this inveetigation. H. #195 CO 133 rI: ntroduction: When Esinp, Vandenbelt, Emmett, and Bird (L) evaluated fish liver oil £c€9€ using a sector photometer they reported a deviation of three and one half percent in their results. Zscheile (13), using a photoelectric spectrOgraph on a similar eXperiment re- duced the deviation to two tenths of one percent. The investigation reported in this paper is an attempt to discover a method of using the sector photo- meter but at the Same time reducing the deviation from the three and one Lali' percent reported by Evans 4:31. using the _Sac£or - instrument. One limitation of the sector photometer is the inability to photograph successive absorption spectra closer than one half unit on the adjustable sector scale. A second limitation results from errors introduced by pipetting --- drainage, density changes of the Solvent, and fluctuation of pipette volume. The inaccuracy of visual interpretation as compared to that of the photo- electric cell presents a third limitation. The apparatus used in this study was a Bausch and Lomb ultraviolet sector photometer eduipped with a Bausch and Iomb medium auarta optical system. The con- tinuum is that of a Hilger hydrogen tube Operating at a potential of 3,000 volts. The one centimeter glass cells with cuartz ends are housed in metal cases. A Bausch and lomb density comparator was used for measuring the the densities of the phot05raphic spectra. The materials used were Eastman practical grade of hexane for solvent; Eastman Spectrum Analysis {1, Anti- helation photographic plates; photographic develOper D-lg. The sutstance used was fish oil {LEES supplied by Parke Davis Company of Detroit, richigan. Experimental: From a hydroaen discharge tube two beams of ultra violet radiation are passed through the spectrOphotometer. The t0p beam ---IO--- passes through the solution and then the “100 percent" sector. The two beams are then collisated, passed through a medium quartz cornu prism and are adjacently photographed upon the plate. * A series of eXposures is made differing only in the ratio of the log of 10/1. The uuantity of light U) pas ing through the solvent is kept a constant --- that is the product of the esposure time and the sector setting is equal to a constant. The concentration of the solution is adjusted in such a manner that the maximum extinction occurs in the region of a sector setting of ten. All concentrations espressed are in terms of grams of solute per 100 cubic centimeters of solvent. Laboratory esperience has dem- onstrated that in the region of a sector setting of ten the accuracy of this instrument is at a maximum. * See page 17 ()3 The plate is then developed in D-19 developer for two minutes, fixed in hypo for two minutes, and washed in running water fifteen minutes before drying. The extinction point of an absorption spectrum is the point where the densities of the 10 beam and the I beam are eiual. The maximum extinction point is the extinction point occurring on the band of the lowest sector setting. The relative densities of the I0 and I beams are m asured on a Bausch and lomb density comparator. The ratio of deflections of the IO and I beams on the micro— photometer is prOportional to the ratio of the relative densities of these beams. The ratio of these deflections, 10/1 for a series of bands is plotted against the corre- sponding sector settings.* For bands of lower sector settings than the maximum extinction point, the ratio will be greater than unity; for bands of higher sector 1 =ettizigs than the maximum extinction point, the ratio Tn will be less than one. The point where the curve crosses the unity line is considered the isodensity point and its corresponding sector setting is read on the "Y" axis. In making any absorption determinations on the sector photometer, it is imperative that the instrument be in proper alignment. A....... v. I o i -o 0-. fo—c-o O ., I Q 0 O 0 III I-r1l'0 . u Oooo'ttoo PLATE/7113 . p . T o o . . i . ‘ v 00..--.. I 0 OOQ.’-~—o-- . .’t§1.'0 O . i v I I A o ‘ Iln‘i.’ '00 .~ ‘-fN-‘—'-’ . - -- The first step in aligning the instrument is to Open the slit, remove the plate holder, and illuminate the source. The image of the source, two dark circular lines, may be seen by viewiné the spectrum. By moving the remote end of the sector section of the photoneter laterally, trese images Lay be brought into the center of the spectrum as defined by the diaphraim. The slit is then closed to the proper width and the relative in- tensities of the two beams which make up the band may be investigated. Then the band is photo;raphed throu;h J. ooen sectors, with no cells in the instrument, the ratio (n (O , l p O r. H p of the nicroghotometer deflections from the team be one if the densities are eiual. in the event that this condition is not realized, the ensities oi these 4. L; beams may be changed by adjus irg the aliinment of the ‘ discharge tube. nhen the tube end which is more remote from the slit is ised, the "I" beam will become darker L3 93 and vice versa. If the two beams tend to overlap at the center, thus for ing a dark line through the center of the band, the following adjustment of the instrument may be made to correct this defect in aligruent. ii the distance between the Fresnel biprism and the slit is diminished, the overlapping of the lines lessens. Balsch and icub reconner7 tlut tie biprism be placed as near the slit as possible without damage to either the slit or the bigrism. However on the instrument used, it was iupossible to place the biprism as close as desired. A minimum distance has used. A series of determinations were made using fish oil §o696 previously reported by EWiflé, Vandenbelt, Emmett, and Bird (2). The olates were visually spotted by two individuals who had had previous esperience in spotting absorption spectra. The maximum extinction values were then calculated according to beer's relationship: Eip 2 ice Ir/I lcm C d I is ei*al to the incident liiht (loo percent). I eiuals the percent oi liéht transmitted at the wave length of the absorption maximum. 0 eguals the percent concentration of the solution. d is egual to the length of the cell in centimeters. Table A indicates that the results of visual spot- tinr may deviate as much as 3.72 percent within them- selves. Also it should be noted that when the mean values of one reader are compared with those of another, there is a difference of only .eo percent. 1 1‘ “he volumes used in table A in =eterminin; the percent concentration were oipetted and then careiully & ‘1‘; E l h. V hed. by using the density of hexane at the tenper- ature when the runs were made, its correct volume may be determined. from this the correct values of the.concen- trations say be calculated. Table B shows this concen- tration correction applied to the sector settinps oi table A. A CCKEARISCN CF IAAILJI LAIIICTIVL VALULS DP 7113898 AT u381 7*..U ill-J .LJJAAJ14 11141.1 lililiK—ILT RlJE’III;E- BELTS BY T40 ILDIVIDUALS llate p Conc. ISector 11 Sector I Eigm II Eigm 107 .0L3LO 10.00 10.00 L0.o7 50.57 158 .0L335 10.50 10.00 L0.LL Ll.£0 169 0C0375 lColl 9050 vOovU @1031 170 .0LL10 9.50 10.00 LO.L7 50.31 173A . L330 10.50 10.00 50.40 L1.05 173B .0L2.30 10.35 10.50 50.45 L0.40 17LA .0L370 10.00 10.00 50.55 L0.58 1758 .05370 10.00 10. 00 L0.55 L0.58 174A .0L335 9.25 9.50 L3.09 51.69 174B .0533 10.00 9.5 L1.00 L1.69 I II Range 1. 75 1.50 Deviation .c7 .75 3 n 2. 72 3.55 Kean Value L0.78 50. 93 % Difference of Mean Values .55 TABLE B A CCLEARISCN 0F KALIKLK.EXT1“'11CF IALULS 0F :L598 AT 530 IU Ls DLTLLIINLD s1 VISUAL LbIHCD RD W31GHT’DQNSI‘1Y RLLILLELLTS Plate % Conc. Sector 11 Sector 1 ElW 11 E179 (Corrected) 1cm lcm 167 .05385 10.00 10.00 L0.45 L0.45 168 .0L3L5 10.50 10.00 LC.3 L0.10 169 .CLL10 10.00 9.50 L0.30 L0. 85 170 .0LLE5 9.50 10.00 L0.30 f39.55 173A .05355 10.10.00 L0.05 L0. 70 1723 oULBS‘S 10.: 5 1.0050 {60.40“ [1.10005 175A .0L370 0.C0 10.00 L0.€0 L0.50 17LB .0L370 10.00 10.00 L0.50 L0.50 1741.11. OOLBEO 9.35 9050 (91060 0.1050 174B . L35 10.00 9.50 L0.75 L1.40 I 11 Range 1.55 1.95 Deviation .78 ..98 % " 3.45 5.11 Mean Value LC.5L LO.5L % Difference .39 The sector settings at the isodensity points were by use of the micrOphotometer. The E79 then determined icm values in table 0 show the corrected sector settings which were used with the uncorrected percent concentra- tions from table A. Table D gives detailed information of the combina- tion of the weight-density corrections used in conjunction with the sector settings as determined by the micro- photometer. The accuracy of the determination of the E1; of a 10m substance depends uyon the accuracy of 10/1, 0, and d. The cells used in the absorytion work reported by this gaper were accurately ground, hence this error is negligib e. Two factors determine the accuracy of the percent concentretion of the solution. They are: (l) the accuracy with thich the solute is wei;hed, and (3) the accuracy exercised in determining the volume of the solvent. The semi-hicro balance mhich mus used to determine the weight of the solute is sccurute to glue or minus five thousandths of a milligrum. hxyressed in .‘ gercent deviation this would oe plus or Linus five hundredths of one cercent. This deviation is also negligible. The volum of the solvent nus measured with (I) a pipette. Since the density of the solvent and the volume of the pipette Vary with the tentersture, consid- ! _ FF? eruble devietion is to we expected. hexsne is un able for pinetting because of its hi ‘0 TABLE 0 iiiirvv Li t“mvr*II=o e .54 L ld..u3_ T9” in IIC’JU‘TL‘TLTLTHR ”IT'T-{CUT 34.1“.” DZELITY'CTNEL STICKS c7 Sector 10g Sector % Conc. Eigm ’11 H 90 c+ (D .00225 Ll.&0 1. 033 .05235 51.70 174A 1743 167 9.25 1.034 .03360 01.75 168 9.45 1.030 . 3335 51.60 169 9.05 1.043 .C;375 01.83 170 8.72 1.057 .03510 31.90 172A 9.65 1.015 .05330 51.50 172E 9.b€ 1.014 .03320 ’.",:1.‘: 170A 9.0 1.016 .03370 51.10 1733 9.6 1.0 5 .O;$.0 £1.05 9 4 9 5 H 9 C) {b § Range .85 Deviation .45 e " 1.:5 1 Fe nValue 51.50 10 TABLE D ”IT" 1' CTIC {£133.03- L1 r589 8- AT 530 ICU .13 D3111“ 11' 3D BY THE riTECDS (11132 ‘ kiL" h AT: .3IJ’T 3:12.1TY Plate Temp. 0C. flgt. Hexane Density Volume Herene grams m1. 167 25.0 15.1210 .6616 19.65 168 22. 1L.2127 .6620 19. 95 169 22.5 1L.0979 .6619 19.78 170 22.5 15.1114 .6618 19.66 1721 24.2 15.0746 .6608 19.78 1722 24.2 15.0746 .6608 19.78 1751 24.5 15.0678 .6607 19.78 1752 24.5 15.0676 .6607 19.78 1741 24.7 1L.0625 .6605 19.77 174B 24.7 15.0625 .6605 19.77 . .. _ . 171’; Plate Sector 105 Sector Jgt. 011 % Conc. Elcm me 167 9.25 1.054 ~.52 . .05265 L1 14 168 9.45 1.020 6.45 .052L5 L1.22 169 9.05 1.042 .55 .05L10 L1.45 170 6.72 1.057 \.22 .05385 51.25 172A 9.65 1.015 6.44 .0L255 51.18 1723 9.68 1.014 6.44 .0L255 51.15 175A 9.6L 1.016 6.46 .05270 51.17 1752 9.65 1.015 6.46 .OL27O 51.06 174A 9. 40 1.027 6.45 . 525 51.60 1743 9.50 1.022 6.45 .0525 51.41 91; Values Volumes “10m r Range .55 mean - Pipette 20 Cma DeVietion .27 " Hat. Den. 19.79 % " .86 Difference .2 1eau Value 51.26 fiDifference 1.05 Sector Settings 105 Sector Mean - Photometer 9.40 1.027 " - Visual 9.95 1.002 Difference .025 % Difference 3,5 11 4. EV KNDJifi” the density C1 hexgne Lt the room ten er;ture* heiune used, it is possible to éeterhihe the accurate volune of he::re. This :reph L10 the correspondin: dutu** iron ghich it 415 deterhined ore accurate for these corrections. . 4m- .,. ..,. .... 1,..- .9 - 4m 4“ i .- .. 1 ,, . ii the tehgeihturc-QLhLILJ 111C oi tne ,reon were ‘VA :4. .:'., +~ . ,.,,, L:... '~ ‘.,"+: .. '1 .':,-1 L. '1 . censi y Lion tie ldttJLMULMHLL LJiLIC i tib1€c is .o (“D *TELCrtE 3 iiiference of .L5 percent from th accepted Vslue. This ”ifference huy result :11; the slight inpurities in the high guulity 00hm€Toiu1 hensne used, uni 110m the iuct thyt the comparison Vu11c Les interpoluted, while the value iuotec in the critical tu 1es use actually determined st 36T0 degrees centi- G! :rsde. Thole D shows a one percent diilerence in . volumes betneen the pipetted volume and the volume determined by the weight-density method. By using the microphotometer to measure the ratio of the relative densities of the ID and I, the visual U‘ method of reading the plate is eliminated, and it i‘ possible to interpolate between the sector readings of the two successive absorption ounds. Table 0 shows that * See page 12 ** See Pages 15, 14 i ---.o... a o -o . ;-._-.o—-o - ‘ O n .---+..-_ . o u -.-.¢. ooo . o -..3,.--. O o.-’.... I o I . . 0 o o --.|.... . 0 I 0 -_+. . -o. ._._.- c ' 0 . *_ ._¢_—__-..-_ ....r. t...— l 9 O l --~ot-o-. D I- oo. 0-. I 9.0.. o o 6 o .. n v Q o i V -.--;,....- I 0 I o-..-.. u 0 O ... n 1 o--.y._-- 4....“ J ..-.__ —.—-..-.._.4- c...- I ._... o “-kH-‘o . .vh-O-o-oo—o lb DLKSITY or 331133 Corrections Applied Buoyancy " v 15. I m M = S a Vm Vs 0‘ «+ ( Vm - VS)