WOMEN. MECHANISM OF ERGOSTROL IRRADiAflONu STUDIES OF WAVELENGTH AND SOLVENT EFFECTS ON WTAMW Dz SYNTHESGS Thufis for the Dam of Ph. D. AAW STATE WVERSITY Louis H. Sharp. 1957 is; Wfifi :Ihasxs MI-CHIGAN “ESTATE UNWERSHY EAST AREA .1. Al 't.?G\N ., LIBR/l 1-3 ‘,~' PiliChigim State University MICHIGAN STATE UNIVERSITY UF AGRICULTURE AAD APPLIED SCIENCE DEPARTME' .T OF CHEMISTRY EAST LANSING, MICHIGAN MICHIGAN STATE UNIVIRSIIY us AGRI'.UL‘IURE A;.o APPLIED SCIENCE DEPARTMENT OF CHEMISTRY EAST LANSING, MICHIGAN PHOI‘OCIIEMICAL MECHANISM OF ERGOSTEIOL IRRADILTION-v- STUDIES OF “MOTH AND SOLVENT MEETS ON um D. SIXTEESIS By Louis B. Sharpe ATHESIS Submitted to the College of Advanced Graduate Studies of Elohim sut- erlxernity of Agriculture and Applied Scienc- in path]. fulfillment. of the requirement- tor the degree 01' worm 01" PHEOSOPH! Department of Chemistry 1957 DEDICATION Th1. than 1. dedicated to the memory of Dwight '1'. Mn; undu thou guidance the only part or thin work m momliahed. LCKNOWEDG‘ENT The author viabea to upme hie aincere appreciation to Profeaaor Jane. C. Sternberg for hie guidance and in- valuable oounael during the later atagea of thia art, and particularly for hie guidance in the interpretation of the reeulta. Aclmovledgnent ia aleo due to Parke, Devil and Company of Detroit, Michigan, tor their grant in eupport of thin Iork, and to Dr. Orson D. Bird of that organization for hie intereat and guidance during our new connltationa. W 111 PHUI‘OCKfliICAL KTECHAHISH 0F ”EGCSTIICL HIIiABIATICHo-v STUDIE": OF mmwsm AHB SCLVECHT J‘FE‘IJTS CR VITAEIH I}; snmmsxs By Louie H. SIwrpe “WC? Sub-“ted to the College of Advanced Graduate Studied of Kiohigan state Univeraity of Agriculture and Applied Science in partial fulfillment of the requirement. for the degree of DOCTOR OF PI-IILOSéPH!‘ Departuont of Chemistry rear 195? II MA A w“___ A A ABET-ACT 5 am ha been made of the effeota of 001nm (ethanol, diethyl ether, m, and oyolohenne) and mum melmgth (21.332, 25372. 26513, 23013.. 29673., and 31323) on the couree or the photo- chemical conversion of ergosterol to vitamin D“. a method of calcudctiou, utilizing 133:4: punched cards, hea been mood for epectrophotometrio detormdnation o: the cowoeitiou or the irradiation either”. Compositions have been determined aa a function of time in irradiation! involving various oombbwtione of wavelength and 'aolvent eonditione. rm oalmflationa m based on a :1" component ult- anointing of ergoatarol, lmnioterol, teohyetorol, oaloiterol and toxieterol. Since the owletion or the calculations, however , the We of mother intermediate, preoaloiferol, ha been demtrated in other leboratorioa. Eeglect of thia component deters aw quanti- tative interpretation or the calculated results and form a more quali— tative interpretation. the roenlte abetantiato tho previouely reported favoring o! taohyaterol by aborter wavelengths mad uee o: etlmzol aa a com, and indicator that longer wavelengths favor precoloiferol. An urination at the eolvent effect is given in tame of the viaeoeity and degree of hydrogen bonding of the eolvont. The me- length effect ie explained in term of the relative abaorption ”efficient. of the aeveral irradiation produc to. A mechanical, uhich mu nee a: ionic excited linglat cum aa MW, and is able to account for the formation or all of the irradiation procmote, ie poetulated for the reaction. A novel W for the unwound precalciferol 1a propoaod. TME OF CONTENTS Page Immmonti...0......OOOOOOCOOOOOIOIOOOCOO...00....0.00.00... Interim"...u....o.5........6.u..’u..‘an...and"... (3me Of 13h. RCMheeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee NEWOOOOO00.00.000.00000000000.00....OOOOOC0.0.0.0000... - v '- ' Material. Ind W.tu.eeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee mom.aeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeaeeeeeeeeeeeeeee -~ - -- - v c --v M “D WflMeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee hihm‘hicll MW“ at the mu;;eeeeeeiaeeeeeeaeeeeeiee Prep‘r‘tion Of Experimental Bataan...“unusasfia...” Preparation.of the ApproximationrDecks.....a..z.3.3...3.... m Curve-Fitting HethOdeeeeeeeeaeeeeeeeeeeeeeeeSEeeezeeeee thought. Comm "1. “CM 0: muoneeeeeeeeeeeeee W! OF mrseeeeeeeeeaeeeeeeaeeeeeeeeeeeeeeeeeeeeeeeeeeeee ma W0“.eeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee 1330035on “D MERPRETATION...uuunuuu.................. Rmm $11! and tho Reaction WWeeeeeeeeeeeeeeeeieeee ‘e Btcrooohemical Change. During Rmuoneeeeeeeeeeeeeee B. Photochemistry and Electronic Chcngee During Reaction The Ionifl Excited $11181?“ Suueeeeeeeeeeeeeeeeeee The Non-Ionic “it“ Singlet smeaeeeeeeeeeeeee TheIEUuflet ExdruxiSteteIflmnnlIneeocituhnlldmit Th3 him MP1“ antennshauiusu“as..." co “lvmt Effect on th. Reactian....u..5'.’..a...ua.u. D. wavelength Dependence of the Reaction................ ”MW m mm WOIOCCODOOOOOOCOOOOOOOOO000:0:0000006 um CWOCOOOOOOOOOCOOOIOOOOOOOOOOOOOCCOOCOOOOOOOOOOOOOO vii ‘00 0‘ VIP H 0.0.. INTRODUCTION Historical The photochemical behaviour of ergosterol toward ultraviolet radiation has been a subject for study by many investigators for the past thirty years. Interest in the subject eroee men studiee of the changes occurring in the ultraviolet ebeorption epectra of 'sterols during vitamin D formation directed attention to ergosterol. An excellent sumacry of the work up to 1952 may be found in Sebrell and Harris' book, 1313 am (1). Rosenhein and Webster (2) demonatrated that irradiation of ergosterol with ultraviolet light produced a preparation far more potent in vitamin D than was obtainable by irradiation of cholesterol, the sterol then under utensive investigation as a precureor or vitalin D. This discovery led to the belief, for any years, that the ability of cholesterol to produce anti-rachitio preparation upon ultraviolet irradiation was due, not to activation of the cholesterol itself, but to activation of ergosterol present, as an impurity, in the cholesterol. This belief was shown to be erronecue several years later by Weddell (3). who proved beyond doubt that the vitamin D from the pro- vitaain of cholesterol was not the same as the vitamin D from ergosterol. It as suggested by Caller (h), and proved by Hindeue 9.1., g. (5), who synthesised the oompmmd, that the provitanin present in cholesterol ' us 7-dahydrocholesterol, a sterol very similar to ergosterol. Several other provit‘aleins activatable to vitamins D by ultra- vieletlightareknom, Mttheyarenotomerciallyeignirieantin- CW to ergosterol or 7¥demdrooholssterol. Ergosterol is synthesised by yeast and by a number of fungi and other lulu- organisns) it is obtained oemercially neinly by attraction from you” and to a small extent tron the meliun of aspergillus nicer. a large amount of the earlier work dealing with the conversion or ergosterol to the vitamin entered around the use of spectral absorp-y time changes to provide clues to to the mhanisn of the conversion. “we the process is rather complex, this qlproaoh yielded extremely confusing results, with different sorksre being unable to reach agree. nut even on the matter of the characteristic absorption band of the Vitals. Judicious combination of spectrographic and chancel tech- siws by various vorkers seen shoved, hosever, that the conversion as at a simple process, but one vhich proceeded through several inter-ediate compounds. rheasrk offieerink andvaniijk (6) didmchtoclarifythe situation. These workers Vere also among the first to study the effect or in; (above 21503) and short (below 27502) wavelengths of ultraviolet light on the spectral changes occurring during the reaction. ‘l'hey sored, hm, in believing that upon long uvelength irradiation but one Mt, the vituin, he. formed. About this time it became clear to Window (7), from work in his laboratory, that five or six irradiation products, unprecipitable by digitonin (unlike ergosterol), nay fern during irradiation of ergosterol. He and his coworkers were able to isolate and character- ise luxdsterol (8), techysterol (9), suprasterols I and II (10), and caloii'erol (ll) which is vitamin D... In addition to these products, Hindus and Auhagcn (12) proposed the existence of a 'protachysterol' in order to explain certain changes which occurred in the absorption spectrum of the irradiation products when they were allowed to remain in the dark in a closed system practically tree of oxygen. The evidence, pro and son, for. the existence or protachysterol is about equal, but such a command has never been isolated from the irradiation mixture. Recent mrk by Vellus gt 9... (Delhi-5,16) has demonstrated the existence of a 'precalcii‘erol' uhich changes into calcircrol in solu- tien, especially when unarmed. “there is, evidently, an equilibrium between calciferol and precalciferol, with calciferol always predomi- mting at equilibrium. The transition from precalciferol to calciferol proceeds in the dark and the relation between the two compounds is presumed to be a phenomenon of transitory cycliaation. The toxic compound, toxisterol, has been sham to follow calcio tsrol in the sequence of irradiation products. Its structure is still in doubt, but its absorption spectrum has been published by Hindus (l?) and its preparation is the abject of a patent granted to Linsert (18). Ergosterol and all of its irradiation products are isomers, and their structural farmlas are given in Figure l. ERGOSTEROL FIGURE 1 Ergosterol is known to be dec~orposed photochendcaJJy by ultra- violet light between the mvelengthe 23002 and 31322. Decomposition by light of wavelengths shorter than 23002 has not been investigated, but 31323 seem to be the upper limit for decomposition. It is knoun that both the wavelengths employed for irradiation and the solvent in mich the irradiation is carried out have a marked effect on the relative abundance of the several irradiation products produced and on the speed of decomposition. Thus, for the some sol- vent, the short wavelengths (25003) are known to favor fast reaction and tachysterol formation, while the longer melengths (31002) favor slow reaction and lumisterol formation , each product predominating at the wanes oi' the other. Sindlarly, for irradiations carried out at the some Invelength, the medium obtainable yield of calciferol is reportedly much greater in other than in alcohol, while toxisterol production is much less in other than in alcohol. Althougz a tremendous amount or time and «more has been poured into the study or the photochemical conversion or ergosterol to vitamin 13,, no systematic method has been develOped or applied to detamins the concentration relationships which exist between the several irradiation products at various stages during the irradintion, or men the irradiation is carried out under varying conditions of solvent and irradiating nvelsngth. Furthermore, no atteupt has been made to explain solvent and irradiating wavelength effects or to postulate a mechanism for the reaction (see Discussion and Interpre- tation for sumry or more recent work). Object of the Research ' The object of this research was to study more closely, in a systematic leaner under controlled conditions, the effects of solvent and irradiating sevelmgth upon the photochemcal conversion of ergosterol to vitamin D2, and to develOp a method by means of which it would be possible to identify, and determine the concentrations of, the several irradiation products in an irradiation mixture. EXPERIMENTAL Materials ad Mews Ethanol. Commercial absolute ethanol (Commercial Solvents Corporation) was treated with silver oxide to remove aldehydes and carefully distilled through an efficient fractionating column. No attmnpt was made to obtain the ethanol in an anhydrous state. mm Ether. Anhydrous, c. P., dietlvl ether (Merck) was allowed to stand over anhydrous, C. P., sodium sulfite and sodium hydroxide (both obtained from the J. 1'. Baker Chemical Company) for several hours prior to use. It m then distilled directly from these two reagents immediately prior to use. 8kg]; Solve " . This material, obtained from Commercial Solvents Corporation, as chromatographed on a column of silica gel (activated at 250°C for he hours) 3.75 centimeters in diameter and about 75 centi- meters in length. rh. first 1200 milliliters passing through the column had a transmittance of not less than 99 percent at 23002. This material, since it consists mostly of n-hexane, sill be referred to as lie-hexane throughout the remainder of this lurk. leoheotane. Eastman Kodak's practical grade of cyclohexane was ohromatographed in a manner similar to that used for nehexsne. This aatsrial had a transmittance of not less than 98 percent at 2300!. Erggsterol. A good grade of ergosterol, obtained from the Iontroee Chemical Compaxv, was recrystallised twice from a mixture or 2 volume - of 95 percent ethanol and 1 volume of bensene. The purified material m stored under carbon dioxide at «10°C until use. It exhibited an 21:“. o: 281 at 28202 in ethanol. Monochromatic Light Source. A Banach and Lord: Grating Hone- chromator equipped withanHanoviaAlpine SunBurnerTypeSHuasueed as a source of ultraviolet light for all of the irradiatione carried out. The nonochromator had a focal length of 250 millimeters, an effective aperture of 2/13.}; and a linear dispersion of 663 per milli- aster; nu grating, blazed tor first-order in the range 20004.0002, had a ruled surface of 50 by 50 millimeters and contained 600 lines par nillimetm'. Both entrance and mt slits Vere maintained at 2.5 fillineters, giving the exit been an effective band sidth oat 1652. gpegm hotometgig Measurements . A Beckman Hodel DU Spectroe photonsterequippedsithhydrogendisohsrgstubemused forall asasurenents. The slit width of the instnment was not held constant. W. 'l'he cells deployed vsre of the demuntable 'sandsioh' type and consisted of a thin, circular, glass ring (the spacer), with a center hole diameter .r’ 13 millimeters ”sandwiched“ between two quarts plates, the entire assemly being held tOgethar by means of a specially constructed compressive some type holder. Leakage of solution was eliminated by smearing a small amount of an appropriate grease on either side of the spacer before the cells sire “settled. A silicone vacuum-stowed]: grease was used for ethanol solutions, and a viscous form of the glycerol-dextrin-nsnnitol W AAAAAAAAAAAAA VV "v'vv'vvvvv END PLATE / CELL BODY . SPACER l—-QUAR'rz WINDOWS TEFLON‘WASHERS C 0 ER phosphor-bronze stock 0.02" x 0.5" FIGURE 2. DIAGRAM 0F IRRADIATION CELL AND CELL HOLDER. ALL PARTS ARE ACTUAL SIZE. ALL HACHINED PARTS ARE BRASS. m described by Heloche and Frederick (19) was used for other, n-hmne and cyclohmne solutions . It might be of interest to mention a problem which arose in comection with the greasing oi‘ the cell spacers. is mentioned previously, it was necessary to apply silicone peace to the cell spacer to prevent leakage in the case of alcohol solutions. This grease was, or course, transferred to the quartz cell faces wherever spacer and cell face came into contact. The grease endured the cleaning process even when a variety of organic solvents were used, and us spread over the entire area of the cell face during the final wiping. This tenacious film or silicone changed the transmission characteristics of the quarts faces to the extent that spectral Isasurenuents of identical ergosterol solutions were not reproducible. The method finally used to remove the last trace of silicone use to treat both cell races and spacer with hot Tuning sulfuric acid for several minutes , and follow this treatment with a thorough distilled water sash. No such problem was encountered with the other grease, since it was water soluble. Detailed drawings of a cell, its holder, and the cell mount used to position a pair of cells (reference solvent and solution) for seamrmt in the spectrophotometer, are given in Figure 2. The glass cell spacers were made from microscope slide or cover glass on special order by the Jones Optical Company of Camridge, Massaclmsetts. Qince aicroscope slide or cover glass is or reasonably uniform thick- nus, latching of pairs of spacers presented no special problem. The pair simply was cut from the same region of a given piece of glass. The cell spacers used were or three thicknesses: 2.22, 1.10 and 0.57 millimeters, as measured with a micrometer. Procedure Solution of ergosterol of knot. conontrations sere sade up in the appropriate solvents. The concentrations esployed sore each as toss-rethstallsolutienshadapprodaatelythsssneultrevulet absorbaaoe, regardhss of cell thickness. This absorbance use roughly 0.3 at 27102. A given solution or ergosterol m then placed in the soluuon cell, the appropriate solvent placed in the reference cell, and the spectrum nemred over the region 2350 to 30002. The nono- chsosatos was then'sdjusted to give the melength of light desired ad the solution sell placed at the focus of the exit been from the sonochrsnatos'. After a given tine had elapsed the cell see med tro- ts be. and the spectral ot the solution was reneasured. The «11 us then replaced in the bean for another period of time, then mad and the spectral ot the solution was again measured. This Wmsqsetedagainaluagainuntil the rsactionsas thought to have moaned to the desired extent. The cells sure then dis. W, lashed and dried. Rrpsterol solution use again introduced into the cell, the parties- lar variable under study as changed, and the entire procedure repeated. u this leans dot-ensure obtained for all possible combinations of three 0011 new. four solvents and for the six irradiation moi-um» 21.83, 2537. 2651.. 2801., 2967 and 31322. 10 DATA AHD CALCULATIONS Hsthemticg mtment of the Data The original plan for obtaining the concentrations of the con- patients present in the irradiation mixtures at various times called for application of Beer' a Law to the ultraviolet absorption spectra of the mixtures; this required solving a system of simultaneous linear equations, consisting of one equation for each component suspected to be present. Accurate ultraviolet absorption spectra of ergosterol, lulisterol and calciterol were obtained in this laboratory, and the best «man spectra at tachysterol and toxisterol were obtained from the literature (9, l7). Conpuison spectra of the abovenentioned pure eorlpounds are preeented in Figure 3: and the data in Table 1. The usual method of analysis or the spectral data by simultaneous linear equations see found inapplicable to this particular system beosuse of the close similarities in spectra of the components of the fixture. The absorption naxina of the spectra of several of the possible ooapenents occur at nearly the same wavelength. For example , the spectra of ergosterol and lumisterol are very similar sith respect to both wavelength and intensity of their respective manna. A glance at the absorption spectra of the pure compounds will serve to illustrate this point. 'l'his similarity results in a breakdom of the simltaneous linear equation appreach through the fact that the equations are not sufficiently independent: the so-oallsd characteristic deterunant of 700 l °ERGOSTEBOL -LUHISTEROL OIACHYSTEROL OCALCIFEROL ~.-~ OTOXISTEROL __ 600 500 400 o 5 no u: 300 ~ f 4h :i { ‘ h 200 T 'd — — e; " .1 1oo*——--———- I L 0 .4 W p __ I 230 250* . 270 290 310 wavsLenoTH(uILLInIcaons) of. FIGURE-3. ABSORPTION SPECTRA OF ERGOSTEROL . KND ITS ISOLATED IRRADIATION PRODUCTS. TABLE 1 1% . E1cm. WAV?£§§GTH ERGOSTEROL LUHISTEROL TACHYSTEROL CALCIFEROL TOXISTEROL 230 55* =-* .-u-‘? 258* - 76¢ 235 58 61 -— 289 -- 240 66 70 109 320 140 242 a- on- 76 -- -- -- 245 77 87 -- 356 314 247 -= 96 -- =’ °‘ 250 90 114 178 389 377 252 -- 126 -- -- -- 255 119 144 245 425 314 257 ?¢ 157 -' -- -- 260 174 179 340 450 154 262 190 190 -= -- u‘ 265 194 201 425 463 76 267 -- 211 °- -- " 269 -- ~~ 462 " " 270 261 228 456 445 64 271 269 232 -- “7 " 272 265 232 445 436 58 273 250 229 1- -- “ 274 237 226 -- " " 275 233 221 500 396 53 277 234 220 -600 -- -- 278 -- 220 =- " " 280 267 221 633 329 45 282 281 217 624 299 42 285 222 187 577 251 37 289 145 -- -' '“ " 290 147 133 478 177 30 291 152 -- -- " " 292 157 -- 455 -- -- 293 161 119 -- -- " 294 161 -= -- -- " 295 150 105 419 104 -- 297 -~ 81 -- " " 300 34 41 284 61 -- * Ethyl alcohol Determined in: ¢ Diethyl ether ULTRAVIOLET ABSORPTION SPECTRA OF ERGOSTEROL AND ITS IRRADIATION PRODUCTS the system, which should be large for successful application of the sethod, is nearly equal to zero. It sas evident, then, that some other method for resolution of the irradiation airturss would have to be round. A curve—fitting nethod was chosen, since it was felt that, by latching .. sore wave-v lmgths than there were components, a unique solution might be obtain- able despite linear dependencss asong certain news of the components at certain of the melengths used. the schens, briefly, involved calculation of the absorption . spun-a corresponding to a large muiaer or hypothetical mixtures of the coaponsnts, taken two, three, four and five at a time} the oalcu» lated curve which matched, or most closely approximated, the experi- santally determined spectrum of the irradiation mixture us then selected. in eight-point natch proved to be a convenient curve-fitting device for a five-component determination, and the points selected were melengths or 2500, 2600, 2650, 2700, 2750, 2800, 2850 and 29002. The effects of slight cell variation and solrentr. evaporation prior to placing the solution in the cell were minimized by dividing the absorb- sacs data obtained during a run by the corresponding initial absorbence values for the ergosterol solution in the cell Just before irradiation: the absorbsnoe ratios obtained were used in all calculations. These data are presented in Tables 2 through 31. Became the method required a large amount of repetitive calcu- lation, use was made of an International Business Machines 6024 calculating Punch (lull—capacity machine) and an IBM 6011 Electronic TABLEZ TABLES 2-31 -- TIME DEPENDENCE OF ABSORBANCE RATIOS AT SEVERAL WAVE- 2900 0 AT 2800 2 50 057MM CELL R A T I 2700 27 O ETHYI. ETHER 2600 2483A A a s o R a A N c E O LENGTHS UNDER VARIOUS IRRADIATION CONDITIONS mmmombmmmwmfib vommmhwmmHMRw moammmmmmbbbm dHHHHHHHHHHHH mmvmomomfivmmm mmfimamammmmmm qmoammmfimmmma OOHHHHHHHHHHH HHMFMOHFNO®HV mmomwmmmvwbmm 0000HHHOHHHHO dOHHHHHHHHHHH meOHVMOMb®MH mpvmmmommfimmm qmoammmfifimmao OOHHHHHHHHHHH NNOCDN\O0\OC\2‘¢[\0M \0\O0L00®‘¢0HNNOH 02000000000000 OOOHHHfiOHHHHO vaccooommvmmo ommwmmmwbmmmm OOOHNNNHHHHHO AHHHHHHHHHHHH mbvmmmmammmem ommmooummmmmo OOOHNNHfiHHHOO AHHHHHHHHHHHH 1 2483A A a s o R a A N c E ommvneevooomo mmovmwwmommmb Hfimvmmmmmvvvm AHHHRHHHHHHHH mooomomoooooo oammvohmmmmvo ooooooooaaamn H H H AT 0 057mm CELL R A T ETHYL ALCOHOL E 8 TIM 2500 2600 2650 2700 2750 2800 2850 2900 MIN mmmmvmmmfimvo mmmvrmoammvm mommmovmmmmm dHHHHNNNNNNH FMOMI‘LONHLDVOH \OONF\000\O\OLO0N QOHN‘QWOF-CDCDQWM OHfiHHHHv-IHHHH momamvmmammm bmovamomwvmn mmfimvmbbhhmm dOHHHHHHHHHH mammommaavov mmmmbmvoommm moamvmbwmbmm dHHHHHHHHHHH vmmmmmvmmmmm mombommafimmm mooammvmmemo dHfiHHHHHHHHH wwmmmmmmmmmm HWQOVM©000MH ooamvmmbbwmm HHHfiHHHHHHHH mmmbbavrpmvm mmbmhmnmmvmm qoammvmmmmva fiHHHHHHHHHHH manhmnmmwvmfi mvmfivmavmmmb qmmmwbmmmmbm HHHHHHHHHHHH 0000100000000 0HNM‘Q’WONLOCDOLO 0000000HHHMLO H 0) ._,‘_¢ TABLEZ TABLES 2-31 -- TIME DEPENDENCE OF ABSORBANCE RATIOS AT SEVERAL WAVE- 2 50 2900 2800 2700 27 ETHYL. ETHER 2600 2483A A a s o R a A N C E 0 LENGTHS UNDER VARIOUS IRRADIATION CONDITIONS mmmombmmmwmfin bommmrwmmfimab moammmmmmrbbm dHHHHHHHHHHHH mmvmomomfivmmm mmamamdmmmmmm QO0HNNNHNNONH OOHHHHHHHHHHH HHMbmmemmmHv mmommwmmvmbmm 0000HHHOHHHHO dOHHHHHHHHHHH VCDMOHVMOMFCDMH mpvmmmommfimmm quHmmNHHNNHO OOHHHHHHHHHHH mmommwmmmvuom wmmmmmvmfimmofi qmmoooomoooom OOOHHHHOHHHHO vavwoovmmvmmo ommmmmmmhmmmm 000HNNNHHHHHO HHHHHHHHHHHHH mbvmmwmammmvm ommwooummmmmo OOOHNNHHHHHOO HHHHHHHHHHHHH ( 2483A A a s o R B A N C E ommvuvovooomo mmovmmrmommmb Hfimvmmmmmvvvm HHHHHHHHHHHHH mooomomoooooo 0HNMVWFONW®V0 00000000HHHNW H 2900 A T 2800 2850 0 ETHYL ALCOHOL 057MM CELL R A T 2650 2700. 2750 2600 2500 mmmwvmmmdmvo mmmvrmoammvm mommmovmmmnm daaaammmmmma FMOMPmNHmVOH WONFWO0®W®0N 00HNVOF000©M 0HHHHHHHHHHH momamvmmaomm bmovamomwvmn mmfimemhbhhmm dOHHHHHHHHHH MRmmommfiHvov mmmmbmvoommm moamvmummbmm dHHHHHHHHHHH vwmmmmemmmmw momhommafimmm mooammemmvmo dHHHHHHHHHHH wmmmmmmmmmmm HWQOVMWOO0MH ooanvmmbbomm HHHHHHHHHHHH ,WNWFFHVFPMVN mmbwrmmmmvmm qoammvmmmmva HHHHHHHHHHHH mamumnmmwbma mvmavmavmmmb Rmmmwbmmmmbm HHHHHHHHHHHH 000000000000 0HNMVWONWQOW oooooooaflamm H N TABLE 3 O O 0 N 0 L0 CD 02 JI- .1 m< O U C _ (I) N 20 2 :‘CD 9 L0 ol— F N I O O U 3 I1] 20 < x2 3 [I] \O I< 02 201 . 2: < \0 no 02 CD on mm C O L!) < N mm 22 FE mommmmombomafievn mmmmmmmmbammobmv 00NVMNH00mMNNHHH dHHHHHHOOOOOOOOO MOHLDOMLO\00|>-VML\00\O \OOOWNHFCDNC000VNHO QOHHHOCDWLDMNHHHHH 0HHHHH0000000000 omamfimawbommmarm mbwomVHmmvmmHomm 00OHO0m©VMNHHHOO dOHHHOOOOOOOOOOQ 500NOH000MFFOL‘V0 mmmmmmmrowmmmaoo mCHHfiomwmmmHHHHH, dHHHHHOOOOOOOOOO CCVFNHEmemvmmnm vamommmmmvmmomm mmooombmommfiafioo defiaaooooooooooo OFMWLOWNWKOPLOL‘000CD NOD-NQDUJNNLONMMOFMN 00HNHO0L‘LOVMNHHHH HHHHH0000000000 P'NLD‘Q’LOOHCDNOL000N‘Q'N NFVQVWNVO©00VH®© 00HHH030FLO‘Q’MNNNHH. HHHHHHOOOOOOOOOO mbmwmmmmbhoomfimfi mvofiwmfimowmmvmom Hmvmmmemfimmwmmme HHHHHHHHHOOOOOOO L0000LO0L0000000000 OHNMV.\OE“0NU)COV0\OCDO CCCOCCCCHHHNMMV0 H M H 2900 057mm CELL R T i o A T 2650 2700 2750 2800 2850 CYCLOHEXANE 2600 2483A A'B s o R B A N C E 2500 mmmmovvmmmfivmfi mmmvommvmommmm 0OHMVMO®0mMNHH dHHHHHHOOOOOOO Hmemawmmewmmmm hwmmmwmmmmmmmm 00OHHO®©VMNHHO doaaaaoooooooo Harommvmmmmvmm FmVHMCHHVVHMHF 000HHO®©VMNHHO dOHHHHOOOOOOOO WENOHONQVHmHmN mommrovmovmpmm mooaafimmvmmafio dHHHHHOOOOOOOO hmawbmmmmmvofim wmvmmfimmmnfimmw mmoooohmvmmfifio dOHHHHOOOOOOOO MFHMMFVFVVWWNV HVNQHVOHMMONOM ooaamfimbmvmmafi HHHHHHCCCCCCCC vme©MNVHmmmmb mmmmomevmmmmmv .00HH02H0I‘LO‘Q'MNNH HHHHHHOOOOOOOO mmfimammammwvmv NHOWm00©MHNOHO HmmvaVMHombmm HHHHHHHHHHOOOO L0000L0000000000 0HNMVW0NLOCDVVOWCD 0000000HHH‘NMM‘Q’ H v- »H TABLEQ AT ETHYL ETHER 057mm CELL R A T O 2537A A a s O R a A NxC E TIME MINS 29.00 2650 2700 2750 2800 2850 600 2500 VFPNHQOH>ON mmvmommoamd qonmbhmbbwm OHHHHHHHHHH womanhmmmmc mommmmmmmae COHNNNHNNNH dHHHHHHHHHH 0HNMHOVOHV‘CD 0000-00400000 0.00HNNHHHHO 00HHHHHHHHH 'NH0H00000M0 0~MVMOCOI>0050 00HNNNHNNHH dHHHHHHHHHH NOVOFNHHHO0 FQWHVNNNNQM mmoafiaooomm dOHHHHHHHOO MMOOVQMOOHQ MQOQMHHOOFO OONNMMNNNHH HHHHHHHHHHH ONOOHMOOVOO VO0V050VOH0 OOHNNNHHHHO HHHHHHHHHHH 000140400000? 500F0000000 HMV0000000V HHHHHHHHHHH mOOOmOOOOOO* 0HNM‘VNO002.00‘¢ 0000000HHHN H H vm ETHYL ALCOHOL 057MM CELL R A T ABSORBANCE 2537A A T 2800 2850 2900 0 2600 650 2700 2750 2500 thONthmmbmmO moommmvmmbmomm qmmmmvmmvnaomm HHHHNNNNNNNNHH HOFNVVOMQNNOFH waNHHFMQNQQMH 00M0b®mwFFOVMH dHHHHHHHHHHHHH weamvmmawmfimmv nmvvmammrammvm momkubbmmvnmo dHHHHHHHHHHHHH FHONOFWV®00000 OOHHmmmbovambv OHmebhb>00MNO HHHHHHHHHHHHHH Ohmommvmnwemmfi mvmmmmfibnmbwwm 00HMVVOVVMNH00 dHHHHHHHHHHHHO 0N00000FMMHMVO vvmmmmfibmbmomm oamvwmbmmmvmmo HHHHHHHHHHHHHH 00MOH®NV500050 Ommoavmmmmmmwm OHmvmmmmvvmmam HHHHHHHHHHHHHO [\NHOCDV'0H‘Q'002000 NFCDOHO0CDON£~0OM 0200!.‘0000000-00‘51' HHHHHHHHHHHHHH 00000000000000 ~0HNMV’00N-00V‘0‘00 0000000HHHNMM‘Q’ H m. A ;- .—- " 1 7'4 '. . . TABLE 5 A A E 2600 2650 2700 2750 2800 2850 2900 2500 0PFOV0000HHH00 00VM000VOL‘MH00 QHVLOHL‘VMNHHHHO HHHHH000000000 0000HL‘MHMO0HNL‘ 0VONO0FO0N00>0 00HN00JMNHH0000 0HHH0000000000 [\VNMONL‘.VMMCDV\OM 0HVONHNN005000 00HHCDOMNHH0000 0HHH0000000000 WQPVONFHMVONMO 00HMO‘Q000OOHCOF-00 020020200002HH0000 0HHH0000000000 0HCOb-H\OL\0L\C\200‘¢H [\H000b-0HO0C0000 00005VMNHH0000 dHHHOOOOOOOOOO’ 0002000‘6'02505-1000 0NOF0000NON000 0HNNO0V‘NNHHH00 HHH0000000000 mmovmvomomvmmm vamommmvmomemo OHNNQOVMNNHHHH HHHHOOOOOOOOOO mavmmovmmmommm ombmmmeHmHme RMOOVHmmhmmvmm HHHHHHOOOOOOOO 00000000000000 0HN000N070V’0000 000000HHHNMMV0J H M 2900 A T 2800 2850 0 057mm CELL R A T I 2750 CYCLOHEXANE 2600 2650 2537A A a S O R B A N C E 2700 ommmmammmmmva ommmvammmommm oommMObmvmmRH AHHHRHOOOOOOO 00N00‘Q'0L‘HHV'0N 0OVNOVVONH000 00HNOL‘0MMNHHH 0HHHH00000000 vmvovmfimoamam wommvaabmmvmm moammbmmmaaao dHHHOOOOOOOOO mmwmmmmfimOCVR 000VO0NOHHOMO moammbmnnmaaa dHHHOOOOOOOOO mwaomvmmwmmnh Odomombmmmvmm moaamwvmmaafio dHHHOOOOOOOOO HNO0L‘0‘O‘Q‘0‘Q‘H00 MOH000000l‘020‘d’ 0.0N02000‘Q'MNNHH HHHHH00000000 0FOMNOCQHM‘OF00 MO0E‘0000t‘fi'0’fi'0 0.002020000v0020202 HHHHH00000000 H00050VHL‘L‘ML‘N [‘0CDH‘Q'F-H0‘Q'V'M0‘Q' HNVO0VMH00‘P00 HHHHHHHHH0000 0000000000000 05402000102001.0000 000000HHHNV‘MV H v ~.02 TABLE.6 AT 2800 2850 2900 057MM CELL R A T O 2700 2750 ETHYL ETHER 2650 2600 2654A A a s O R B A N C E 2500 bomvaHmmom bmHHHmmmmmm momwwmmmmvm dHHHHHHHHHH 0NOHO0N000b MO0FVCD‘Q'NOM0 000NMHHH000 00HHHHHHHHO memomvmmmmfi mmwvmmmwvbv mmommfiooomm dOHHHHHHHOO HML‘CDL‘L‘V‘P-NCDW AFOMH00VN0NQ QOHMMHHHHOQ dHHHHHHHHHO wmmmnvmmamb muchbmbmmum macafiommmmb dOHHHHOOOOO WNQNmemOOm mbthMOMHmH OOHMVNHHHOQ HHHHHHHHHHO [‘00H000NV‘0H Mbmm0HMOFNH 00HMMNHH000 HHHHHHHHHHO mommmmovmmm N0000PWHFNO mnmnmhmwmmv HHHHHHHHHHH 00000000000 0HN000NO0V’0 .OOOOOOHHHNM- H H -m ETHYL ALCOHOL 057MM CELL 2654A 2900 AT 2800 2850 0 R A T l 2600 2650 2700 2750 SORBANCE B 00 mmmmovmmowvmu OOvmwmmmmvaO OH0®NMNNHOFVV HHHHNNNNNNHHH mmmmmmommomoa omemrormmmmmm momvmbmmmvmoo dHHHHHHHHHHHH mmvmaammmamrm 0HO>0NmN00000 mommmommvmamm dHHHHHHHHHHOO mmmavmvmfifiuam Obwvvwmhmvmvm momvwowmmvmom dHHHHHHHHHHHO mommmfioaommwm bmmmfimmvmfiemv moamvvmmmmomm dHHHHHHHHHHOO mmmvwomammwwm evawmmmmmmmmfi camcowmmmvmoo HHHHHHHHHHHHH mmvwoomommmvn 0mmmmmm©vmomm qflmmmmvvvmmoo HHHHHHHHHHHHH HHOVObmhmmnwm werromamommmm momemmmmmmwwm HHHHHHHHHHHHH 0000000000000 0HN000NO0VTO0V 000'0'00HHHNM‘Q’0 H (\2 - M TABLE 7 057mm CELL O A T RAT 2600 2650 2700 2750 2800 2 50 2900 2500 00VOQONQFNOVH ommmmammoommm QHVOHO0MMNHHO HHHHHOOOQOOOO mmamamvhwawmm vmmfiaafimmmomm qoammmvmmfiaoo OHHHOOOOOOOOO vwomomvmmmbmm vmmnvmvmmmmmv 00HH®0MNHHOOO dOHHOOOOOOOOO Hwomvommeomv 00MMNHH®NVO®0 mommmwvmmaaoo dHHHOOOOOOOOO FOFON®0HVOMOV waomommmmmmhm 00HO®00NHHOOO dHHHOOOOOOOOO vmnmoammnmvmo ovooammmmmmmm OHMMOOVMNHHHO HHHHHOOOOOOOO 0VMON0000000V 000>MMVMVOHCDM .0H02020l‘0‘Q'MNNHr-l HHHHHOOOOOOOO 0L\*\0.0L\00000\0‘¢CO 0‘00L‘00VH00000 NVOL‘0MHOCDL‘00V“ HHHHHHHHOOOOO 0000000000000 0HN0000200.‘¢0\00 OOOOOOHHHNMMmM H M 00 50 V‘Q’M000MVOMNON 0N‘Q’MNMML‘0HVOV O>HVOVOFOVMNHH 0HHHHH0000000 V0000HOMNN‘Q’50 V000VONONNPMO 00HNO§0VMNHHO 0HHHH00000000 vwmmmommavmwv mowmmmomoomam moamormmnmaao dHHHHOOOOOOOO vooavmmaomram EVOOOVNOHmem moamoumvnmaao dHHHHOOOOOOOO 00NHMM00bVO0N bflmmmmhmmommm 0OHHO0VMNNHHO dHHHOOOOOOOOO Ofimmmamehmmm VNFOVOH®OQVO0 qammamwvvmmma HHHHHOOOOOOOO V0MMV00NF00100 0MP‘Q’CDHOCDO0NWCDM QHNMHCAl‘l-DVMMNN HHHHH00000000 mmuvmumvroanm mommMNOWVOHHm mowhmovaOmmm HHHHHHHHHHOOO 0000000‘000000 0HNM‘O0NO0V000 000‘00.0HHHNMMV‘ fl v TABLE 8 AT 0 ETHYL ETHER 057mm CELL R A T 2804A A a s O R B A N C E T I‘ME M INS 2600 2650 2700 2750 2800 2850 2900 500 PmmHMMbmmmv mmmmhmmvmon mOOvamvnmm dOHHHHHHHHH 000VO0H‘0l‘00 VMOH005000N 0:000HNH0000 000HHHHHH00 ®H®HMNOFFOG mmmmavamvmw mmmmaafiommm dOOOHHHHOOO Mvamommhwm P>HOHOHMVHM qmoommmfioom OOHHHHHHHHO 000000000300 FWL‘HONOM‘Q’0V’ 02000HH0000OOVOOMMOHOO 00NOHOHV®0NVO mmoomaowmvmma dOHHHHHOOOOOO FMVFFH0300HM00 0VO0F000N0000 0000000POV’MNH 000HHH0000000 V000HH‘Q‘0L‘000C0 H‘Q‘MHOMN000L‘0L“ qoammnmommvmm HHHHHHHHOOOOO 0002HNMU10NOH00 ‘0100020000‘6'002020 00HNMMMH0000V 'ARHHHHHHHOOOO M‘OHV’0M0‘Q‘HO0FN 00000FOHO0HOM HMOE‘0HNNOL‘0MH HHHHHNNNNHHHH 0000000000000 0HN000NO0V000 OOOOOOHHHthv’ H 8" -"‘~ 2900 AT 2800 2850 057MM CELL O R A T 2750 2700' CYCLOHEXANE 2650 BANCE 04A O R 2600 NNL‘L‘0H000V‘NOH0 ovammbmmmvmmmm 0:00HVVMNHCDO‘Q’MN 00HHHHHHH00000 HO0VL‘0NL‘0CDL‘000 MOHO0HMHMHO0M0 0000HH0000VMNH 0000HHH0000000 moonoovommvwmm VHMFNNMNNHOVNO mmmmaaommmvmmfi dOOOHHHOOOOOOO HOQVPPVNwmofimb bbmmmmommoomvm 0000HHH0000MNH dOOHHHHOOOOOOO [\FONOMONHV’0NON 00bHHONNMMl‘b-V’0 0000HH00®0VMNH 000HHHH0000000 mvaommwommfimmw Roommvmmmbmwom ooaannmaommmvn AHHHHHHHHOOOOO ‘0MOMVMVH0.0V'0N0 'NWMOVFV’FOHONFO QOHNMMMNN-OCDK‘LOV’ HHHHHHHHHHOOOO mmmovumflmmbvmm 000V000FOH0000 HNVOQOHHHOQNWM HHHHHNNNNNHHHH 00000000000000 0740040003‘00‘0'0‘000 0000'00.HHHNNM~¢U} H v . If TABLElO 2900 AT 2800 2850 0 057MM CELL R A T 2700 2750 ETHYL'ETHER 2650 2600 2967A A a s O R a A N C E 2500 V‘Q’0‘d’00000‘d’fi' V'MVOL‘0L‘VOVCQ 00000000000 0000HHHH000 mmmmwmmmama mammmmmmmvm mmmmmmmmbhb dOOOOOOOOOO VHOHOI>0VI>HN VN000FVNO‘MH 00000000FPF 00000000000 H00,H\00HOL\NM 0005000-VFVN mmmmoommmmm‘ dOOOHHOOOOO [‘0l‘V’0000000 0VMM‘Q’NO0N00 00000000050 00000000000' mmwmmnmnomm OH0000FONWM QOOOHHHHHOO HHHHHHHHHHH bmflmmmommwm anmammwmvmm OOOHHNNNNHH HHHHHHHHHHH MMO000H0000 MMON00‘Q‘0L‘NL‘ HWV‘0-FOOHHHO HHHHHHNNNNN 00000000000 0HNM©0100V00 OOOOOHHHNMMC H H -m 0 o 0 m 0 L0 02 JP J w<° U 0 CD 2:0m 2 ”"0 ° 0 ot-b <02 J 2% O 0 0m“ _| N (0 J20 > m I<£ l- mm 0 00V 000HHHHH00000 VOFVO0H0‘00‘Q’0V 00000000200000 00000000500VM 0000000000000 NVVVVFONHN000 MOF000VONO0N0 0000000FFO0VM 0000000000000 FOF000000000M 0000V000000N0 000000000b000 0000HHH000000 0F05V0000H005 0MMMOVOVO0POF 0000000000000 0000000000000 vemonabmoovmm ON®MHMHONQ00O qooammmaflmmhm HHHHHHHHHOOOO OFwabmbmfimpm NvomVNmQMVMvm QOHHNNNNNHomb HHHHHHHHHHHOO OOOOOOOOOOOOO mmvmmombomomo HNV00OOHNHHO0 Aaaafimmmmmmma 0000000000000 0HNMO0NO0V000 000000HHHNMNV, H M I!) 2900 2850 2700, CYCLOHEXANE 2650 2967A s O R B A N C E 2600 2500 mm 22 F2 0VOMHO0HOVO0H0 VMONFHHFMOFF00 0000HNNHHO0FOV 000HHHHHH00000 hommvmbmmfimmvv mommvmmmmmmmvc qmmwmmmmmbwmvm OOOOOOOOOOOOOO mmmmauamwmmovn momommmomwwmvn mmmmmmmmmbwmvm OOOOOOOOOOOOOO M00HFNV00N00HM mvmmvmvomOOOHm 00000000005000 0000HHHH000000 00500QMEF00000 00V0000VO0NV00 0000000000FO0M 00000H00000000 vmammwmommmmmm ONbHHVVHhmmovm oooammmmaommbw AHHHHHHHHHOOOO vvmmnmmmnnvaoo 00H000000HVOVH qoaammnmmmaomm HHHHHHHHHHHHOO meHVOmhmmmbov vumomvmmnmmmmm ‘HNV00000HHH000 ,HHHHHHNNNNNNHH 00000000000000 Ofimmmmmmwvowmm OOOOOOHHHmvam H v m TABLE 12 2650 2700 2750 2800 2850 2900 ETHYL EITHER 3132A A a s o R a A N c E 2600 2500 TIME MINS 000H [‘00‘?’ 02000 0000 0000 0002? 0000 0000 00H0 00NV‘ 0000 0000 Fbmb bmmm mmmm dooo V000 F0020 02000 0000 00HV 0P~0H 0000 Hooch Mmmm 0P00 qmmm oooo ovam omvv oooo AHHH 0000 H0002 oooav AT 2800 Q57MM CELL R A T o ETHYL. ALCOHOL _ 3132A ABSORBANCE TIME MINS 2900 2850 2600 2650 2700 2750 2500 vmmmmamvma mmhvnmmmmo mmmmmmmmmm dooooooooo maommommmm mmmmvmammm mmmmmmmmmm dooooooooo ovwvmbwvmm mmmmvmomhv mmmmmmmmmm dooooooooo MmNLDLOx—l‘d'pmm 000>h00v0fl mmmmmmmmmm dooooooooo 000NO0H000 00Fb00VNHO mmmmmmmmmm dooooooooo mmommwwmmo 00OOHHHHHO mmoooooooo doHHHHHHHH vmmbbmfifiam ooammvmmmv qooooooooo fifiHHHHHHHfi mwomahmoma Hmovmmbomm OOHHHNN000 AHHHRRHHHH 0000000000 00200020‘d'000 0000x-{H02‘00‘Q' H 02 0 TABLEJJ Q57MM CELL 2900 A T 2800 2850 0 R A T l 2700 2750 2650 2600 2500 000H5050mm muvmmmoamu mmmmwmmhwm dooooooooo 0HO0V5H5V0 55MHON000M mmmmmmbwmm dooooooooo mommmbmmvm 050050550H mmmmmmhwmm dooooooooo mmmvmmmmvo mpmmammvwm mmmmmmmbmm dooooooooo mmmmmmoovm muvmcmamvu 0000000500 dooooooooo 00HOV0Ox0r-GN 0000002005?! 0000000055 6000000000 ooommmmmom 000050N5N5 0000000005 Aaoooooooo 00500VVNNH H050050V05 OOOHHfiHNNN ARHHHHHHHH 0000000000 H000N0‘d’000 ooooaammvofl H 0 0 2850 2900 057mm CELL R A T l o A T 650 2700 2750 2800 CYCLOHEXANE R B A N C E 32A 0 2600 3 0H00v-lx—I50H005‘Q' 005000NHO0V5V 0000000000050 0000000000000 NV50050H50050 mmrmmvaommmmb qmmmmmmmwmbbm ooooooooooooo ommvomovvammv 00500VH000000 mmmmmmmmmmmbm doooooooooooo (\20fim00000‘0005 0005000000‘Q‘5V 0000000000050 0000000000000 00000000020200‘6‘ 005500NH0005V‘ 0000000000050 0000000000000 000000H50000N 0000000500000 0000000000005 0000000000000 vovovvvovvmmm mOmOmmmOmmmvm qOmOmmmOmmmmm oaoaoooaooooo 0000000005500 000000H05HV00 OOOOOOHHHNN00 AHRHHHHHHHHHH 0000000000000 0HNOVO0N00‘Q‘00 0000000HHHN00 H v 0 1h TABLE 0 ETHYL ALCOHOL 111MM CELL A T 2483A A a s o R a A N C E 2800 R A T 2700 2900 2 50 2750 265.0 2600 mmoooavmommm mmbmwmmvmmvo moammammmmmm 0HHHHNNNNNNN 0V0020005H0202x-1 505V05505‘d’00 0:00‘400000000 OfifiHfiHHHfiHHr-i mwomommmmoom havommbmnonw mmoammhmwwhm 00HHHHHHHHHH H000H00200V50‘ 0NOVOHOL~5000 0:00H020000050 OHHHHHHHHHHH vmvmvwommwmm 00VO0Hv5505m mooofivmmmmvm dHHfiHHHHHHHH wmmmwmmmmoom HOH000N55V5H OOHHMm5555wm HHHHHHHHHHHH vmmrvvooommm mmdmmmmafimam qodamvmowmmm HHHfiHHHHHHHH vumowuhmmomm ombwbmnmmmmm HHNOV0000005 HHHHHHHHHHHH 000000000000 0HHN000N00‘Q’0- 0000000Hr-i-H0204; 02‘ m ,4. 2900 o A T 800 2 50 LllMM CELL O R A T 27 E 2700 N HEXANE B A N C 2650 2483A s o R 600 A B 2500 vmvmwmvmmaoa mmmm>mmmmava 0OH00000H000 dHHHHHHHHOOO H5000000NHV5 bmmvmvmmwmbm qmoamvvmmwmfi OOHHHHHHOOOO 0VHONOH000NN 000H00000HVO 000HNO0H50NH dOHHflHHHOOOO 0N050505HVVO wmmvomvovmmm mooanvvmmmmfi dHHHHHHHOOOO .50055HNO0V00 50NO0VOHO0NN 0000HNN050NH 00HHHHHH0000 bummmvvmmmom HOHOHNN00000 QOHNOVVH000H HHHHHHHHOOOO mmfimmmmomomm amammmmvvmmm qoaamnmamwmm HHHHHHHHOOOO muammommmmmw OHOHH005VON0 fiNMV00000H50 AHHHHHHHHHOO 000000000000 0‘HHNO‘Q’0000‘V0 0000000Hx-h-l020 (\2 :0 fl TABLElS 141MM CELL CYCLOHEXANE 2483A a s o R s A N C E 2900 o A T 2800 2 50 R A T I 2700 2750 2650 2600 A 2500 omboommmmovmm omvmmmmavvmmfi QOHNVOVHO0MNH HHHHHHHHOOOOO VH000055000H5 wmmwmbmoowbom mmooamommvmmo dOHHHHHOOOOOO mmmmwmvmmmmma bmmmmmmmmnmmm mmooamovmvmao dOHHHHHOOOOOO mmmmmmmommmma mfimomwmmmvuom QOOfiHNO50VNNO OHHHHHHOOOOOO VOH0000NO0N00 0000N‘Q'0rl00000 0:000v-lr-l050VNv-10 0HHHHH0000000 HOH00000N05N0 N000‘Q’0000HV50 00HHNNO0000NH HHHHHHT-i000000 0500000NNH000 05HON00000V0m qoaammomhwvma HHHHHHHOOOOOO 0005H0000000H mammvmmmammbm fimmnvmmVMfimmm HHHfiflHHHHHOOO 0000000000000 0HHN0000200‘Q’05 0000000HHHN'0V N V‘ H . . _. 2900 AT 2800 2850 111MM CELL o R A T 2750 E 2700 N HEXANE a A N C 2650 A R 2600 2537 s s o 00 A 25 mm 52 F2 50020050050 H000000V0‘Q’ 0H0000HONH HHHHHHHOOO NM50VONO0H 500H0000HH mommvamvma dHHHHHOOOO 0O0HON000N mvwmwmommm momvnomvao 0HHHHfiOOOO 00VHHO0VFN OOVH050500 qammvamvma HHHfiHHOOOO vmomnvmfimm m000H>NObm mommmmbvao dHHHHOOOOO mmoabwmmvo vnmmambmmm oamvvamvmfi HHRHHHCCCO (\20x-l0000v-l55 0VHONHON50 0HOVOHO0NH HHHHHHOOOO 0000550HV5 H000005000 N005500m0v HHHHHHflOOO 0000000000 0HN000NOV0 000000v-h-INO 02 0 N TABLE16 CYCLCHEXANE A.BSORBANCE 2537A LllMM CELL R A T 0 AT 2600 2650 2700 2750 2800 2850 2900 2500 M5HOHQH00000 Humommmomuma NH005500500H AHHHHHHHOCOO mmfimmmmvommr 00NH050Nv005 ooammmmhmvmo Afiaaaaoooooo 000fi00H0005V-l 0VNHOV’V’0r-l005 0.0HNON0000020 0fiHHHH000000 vnmmmmmnbmmo vvmmmvvommvm qoammmmvmmmo HHHHHHOOOOOO 000HO0N00050 N0000HV050N5 0OOHNHm0VONO dHHHHHOOOOOO 5H50HOHOVNON H005050050OH qaamnmmbmvna HHHHHHOOOOOO mmvmmmomommv wmmmvmovaomv 0HHNMNQ500MH dHHHHHHOOOOO ONH000005OMH m000000H0OOH 00V00500HO0V ddHHHHHfiHHOO 000000000000 0rh-h—l00‘00200‘d'0‘ oooooooafiamm,. 02 V ETHYL ALCOHOL A B S O'F? B A N C E 2654A 141MM CELL T A 2800 0 2600 2650 2700 2750 2 50 2900 2500 owmmmmovafldo mommmhomvmmo omvmmmvmmfimm AHHHHNNNNNHH 0050v0000H00 vmmaonmmavmm QOH00550000H OflfiHHHHfiHHHH mvmvvmmmommfi mnmwnvvmmvob qoamvwmmmvmo OHHHHHHHHflHH mhmvvommnmmm mammoommbmma mommmbmmmvna dHHHHHHHHHHH ONHOHO0HOHHO mvmomvmmMfimv moammvvmmmam dHHHfiHfiHHHHO mafimmwwvwvmo mwmbammovpvm qamnmmommvma HHHHHHHHHHHH 050H00500®VH bmmvmvmommmm qamnvmmmvmma HHHHHHHHHHHH mvmmooaamboa mmrmmmmmmnmv qvmormmmmmbw HfiHHHHHHHfiHH 000000000050 0flfi00000200v0 0000000HHHNO (\2 02 0; TABLE17 lJlMM’CéLL o R A T l A T 2750 2800 2850 2900 E E 2700 TIME MINS 0HO0V‘0HOV‘d’ 005002005020 0H000000V‘02 HHHHNHHOOO mmmambmmmw wvmmammmfim qomvmmowma OHHHHHHOOO vmbmmmvvmm mmfimomvmmw mommvammwfi dHHHHHOOOO 00VV00fi5ON 00H000HVHO qomvmmomma HHHHHHHOOO mmmmmmommm Enummmmvmw moammommma dHHHHHOOOO 000502r-l0‘d‘05 0000000000 qamvmmomnmr HHHHHHHOOO 0V0020V’0050‘ mmdaoommam qamvvmovvm HHHHHHHOOO VHO0HV5m0v 005HH00500 qvwmmbmmmb HHHHHHHHOO 0000000000 01-1020000200‘0’ ooooooaanvm 02 0 an.“ - 2900 A T 2800 2850 L11MM CELL o R A T 2750 2650 2700 CYCLOHEXA’NE B A N C E 2654A s o R 2600 a 00 mmmmmaommduwv H005NHNH50000 QvaQQVH05VNH HHHHHHHHOOOOO 500050000HO0V 000V00200020‘U’0H 00H020000000HH OHHHt-lr-lv-IOOOOOO mmvmmmmmbwmao wvvmwmovmhmmo moammmormvmafi dHHHHfiHOOOOOO 050HHO0FNVNNV 00000HNOHOV®H moamnmoumvmaa dHHHHHHOOOOOO meommmbvmmom mvammbamvvobo 0OHHNH000VOHH daaaaaooooooo mmmvammmnmmmb mmmvvmmmmwavw oamnvmomwmvma AHHHHHHOOCOCO Hmamommmnammo hwmaomoomvomm OHNMVMfim5000N Aafiaaafioooooo 02"4'000050‘V‘0H00 .500500HOH002V'0 :02V'000'050‘Q’0H0'0 AHRHRHHHHRHCO 0000000000000 0HH02‘0000200‘Q’00 0000000HHH02‘Q’0 02 v TABLE18 2900 A T 2800 2850 LllMM CELL o R A T 2750 ETHYL ALCOHOL 2650 2700 B A NC'E 2804A s o R 2600 a 00 ovammmmOEmmbm mmmmouvmmmmvm mmoommmmmmbmm dOHHHHHfiHHHHH 00000005H00200 02000005005020V 000000200‘0'00020 0000HHHHHHHHO oumomomvwaomo MOHVVMHMNfimvm mmmmommnmmmfim dOOOHHHHHHHfiO 0Vfi0505HNV5Hm mommmmammommm qmmofimvvvvmmm OOOHHHHHHHHHO ONVVH0505HOH0 mmmmmmnvmmmbv 00000HNNNNHO® dOOOHfiHfiHHHHO Hmmomvvommvmo mmommmmombamm qoafimvvmvvvmo HHHHHHfiHHHHfiH mavmvvumumofin Mbmmmmmvmmmmm QOHHNMVVVVO0H fiHHHHHHHHHHHH 500H0200HH‘Q‘VOV‘ 0V000H5HH0000 .HOV’0500000000 AHHHHHHNNNNNN momoooooooooo' 0Hfi020000200‘d’00 oooooooaaammg 02 02 v. I A T LllMM CELL 0 RA T 2650 2700 2750 2800 2 50 2900 2600 2500. mmvmmmomm vvmvoommm mmOH0550H dOHHHHHHH mmawmammm movmomvao mmmmmmmmm dOOOHHHfiO mvommoawm mommnvmmv 0000HNNHO dOOOHHHHO 00HOHOVH§ 00H0500V0 mmoomnnmm dOHHHHHHO mwmmmmmmm mvmommmvm 0000HHH00 dOOHHHHHO Hmmhwvmmw mmmovmwmm ooammvvno AHHHHHHHR roommommfi 050HOHHOV ooamnvvnfl HHHHRHHHH HHO0HVO00 oamovmmdv mnmwmmmom ARHHHHHNH moooooooo ORmnwmmmo OOOOOOHHM 02 0 v TABLE 19 CYCLOHEXANE 2804A A a s O R a A N C E L11MM CELL AT 2800 2850 2900 0 R A T I 2650 2700 2750 2600 2500 Evamflvombm vmamambmvm maoammmmmh OOHHHHHHHO VOHOH5000H 000V00020002 00000H02H00 OOOOHHHI-IOO H000VHO0N0 vmmumavmoo mmmmommamm dOOOHHHHOO 50VH0000VH wmrmmmmmmv mmmoammmmm dOOHHHHHOO 5000NOVOHV \0\O\OO\\O\OL\C\2L\I—i QQOOOHHHmm OOOOHHHHOO mmvmmmmmmm HV00050V00 QOOHN000O0 HHfiHHfiHHfiO 0000000000 V00050050‘Q' 00HH020VTOH0 HHHHHHHHHO .‘000V0200000 5VO0V‘0002HO r10‘1’0500005 HHHHv-lI-i020202I—l momOOOOOmm OHHNMWQNOV OOOOOOOHNO 02 v .. v 50 2900 2 C 2800 R A 2700 2750 ETHYL ALCOHOL 2650 2967A ABSORBANCE 2600 2500 [ME INS T M omvoomomammm 0V005HOVH5H0 mmmoommmmafiw dOOHHHHHHHHO 00H0000HOV00 mooommmbvmmm mmmmmmmmmmmm OOOOOOOOOOOO V5000x-h-l02000I-4 0000HO0VH00'0 000000000000 000000000000 VH05000000H0 mmwuammmvamv mmmmoooooomu dOOOHHHHHHOO omvmvwmmmvmm 0VVV0005VN55 mmmmmmmmmmmw OOOOOOOOOOOO ONO0V000050m H000VNVNHONQ QOOOHNNNNHHO fiHHHHHfiHfiHHO 00V050V‘00000 0200\050050’Q'Hb- 000Hr-i0202020202020 fiHHHHHHHHHr-IO Ohmvmmmmoavm 000000HOOH00 Hmvvommmoooo ARHHHHHHNNNH 000000000000 0HH020000200V0 0000000HHH020 02 02 U) TABLE 20 2900 AT 2800 2850 O L11MM CELL A T 2750 R H A E 2650 2700 2600 2500 mm 22 k2 V5NV005500 vmmmmmmama qmmoammmom OOOHHHHHHO VOH5H5ON00 voommammbm mommmoommm dOOOOHHOOO mmwomowooo nomovmwmmfi mmmmmmmmmm OOOOOOOOOO VON®5H50V0 00500HN5©N 00000HH005 dOOHHHHHOO mmmmmwommm mvmmmmommm mmmmmmommw dOOOOOHOOO vmmomammva 0NOV0000VH OOOHNNNNHO HHHHHHHHHO mmdbmmmmmm mmamvmmmmo OOHHNNNNHO AHHHHHHRRH 5V000020HHO ~<1'50 00000002 r102? 0000000 Hr-IHHHr-IHHHH 0000000000 0H020000200‘d' 000 000HH00 02 0 0 L11MM CELL T 2750 CYCLOHEXANE 2967A A a s O R B A N C E AT 0 RA TIME MINS 2850 2900 2800 2650 2700 2600 2500 55OHONHOHOH vvrfiomvmvmv qmmoammmmom OOOHHHHHHHO mbmmmmmmvmm mmmmmoaommb mmmmmooombv dOOOOHHHOOO 0000200000‘Q'V‘ 000H‘Q’HOHV55 0000000005‘1‘ 00000HHH000 M00H0000N50 mmwmmomommm 00000HHHO00 dOOOHHHHHOO 5055000N500 mmmwmvvmmoa qmmmmooommm OOOOOHHHOOO hmmmvabvmm HV5H550000N OOOHHNMNNO5 HHHHHHHHHHO 0000NHOHNMH mudvafimmwmp QOHHNMMMNHO HHHHHHHHHfiO mmmvmommmma 0000NO0VOHH HOV05mOOOO0 AHHHHHNNNNH 00000000000 CHI-102000020r-l0 0000000HH00 02 V 0 TABLE21 AT 0 LllMM CELL RA T ETHYL ALCOHOL 3132A A a s O R a A N C E TiME MINS 2600 2650 2700 2750 2800 2850 2900 2500 000000000H0‘Q’0 05555000050002 0000000000000 0000000000000 mambmmommmovm mbmmvmamvvmmm mmmmmmmmwwmbm OOOOOOOOOOOOO VOH50000H5005 0500VH05000VH 0000000000050 0000000000000 0N500000H5000 0055505000H00 mmmmmmmmmmmmw OOOOOOOOOOOOO 000000000HOV’H 000500Vr02r-l051-l0 0000000000000 0000000000000 mowawmoowoomm 0HHNN0000000N 0000000000000 RHHHHHHHHHHHO Hmmammmfivvvmfi Hmmvmmmmmmmva QOOOOOHHHHHHO HHHHHHHHHHHHH mmvomvaaommmo vmfimovwmmmmmo OOHHNMV000500 AHHHHHRHHHHHN 0000000000000 0HH020000200V00. OOOOOOOHHHNMQ5 02 02 0- LllMM CELL I O A T 2800 RA T E 2700 2m 2900 2850 2750 2650 2600 ommmeme 00050VH00 Ommmmmmwb HOOOOOOOO OH0000000 005500000 ommmmmmmh AOOOOOOOO movvmwhwv mmmvmmmvm mmmmmmmmb OOOOOOOOO 0N0V’0VH50 00000000H 000000000 H00000000 OO00O5VNO ommmhvmmo ommmmmmmm AOOOOOOOO 00000002Vf0 00000000H 000000000 HHHHH0000 omoooommm OmOOOOmmv OmOOOOmmm AOHHHHOOO mmmbmmamm HHOVOV000 OOOOOHHNN AHHRHHHHR 000000000 0v-l02000200’d' 00000HI~|00 02 m 0 TABLE 22 C 2550 2700 2750 2800 2850 2900 CYCLOHEXANE 2600 3132A A a s o R‘B A N c E 2500 orfimmfiom ommmvmmh qmmmmmmb Hooooooo 000000020 0050H500 00000050 fi0000000 00005050 0050H000 00000000 00000000 00505055 0050V’H00 0.0000005 H0000000 omwm¢mmo ommmmavm 00000005 Hooooooo ooooowmm ooooomma qoooommm HHHHHOOO ,0000000‘d’ OOONNHOCD 0.0000000 HHHHHHHO ooamvmmm mmrmommm qooammmv HHHfiHHHH 00000000 0HO0N0002 0000H1-l00 (\2 v 0 ETHYL. ALCOHOL 2483A A B s o R a A ZBZMM CELL RA T A T 2800 2850 2900 0 NCE 2650 2700 2750 2600 2500 V000000H5N0050 0000V00000HN00 0:000HV00000V020 OHHHNNNNNNNNNN 0000VOVO0NOHNN 5000V‘NH‘Q‘V‘0NON0 00fiN000000050V‘ OHHflHHHHHHHHv-IH NO0VONHO0H5000 bmovmmmoommmmw 00HN0550®500V0 00HHHHHHHHHHHH MH5H000005500V 0000H5VO0HVO0H 00HN050000500V dHHHHHHHHHHHHH V00005®50HN500 momhmovmvovmvb mooammmmmmvmmfi dHHHHHHHHHHfiHH Humoammmov>vm> mwmabommwavmwm QOHMm55555mmVM HHHHHHHHHHHHHH N000500NOH‘Q‘00H 05050000000000 00HOV00fi0000v002 HHHHHHHHHHHHHH mmvnmvommmmvmm Hm0fiv000000005 ammmbmmmmmmmbw HHHHHHHHHHHHHH 0000-0000000000 0HN000N00-V0000 000000.HHHN“,00V0 m N ‘H TABLE 3- T 2750 2800 2850 2900 E 2700 HEX A N ZQSQ A 600 A B 2500 0ONONVVV05055 0VOVVO0HNO0H0 _00fl000H05VONfi dHHHHHHOOOOOO 0VN05VNV000V0 000VN500VV000 000H000000NHO 00HHHH0000000 MN5H®0005NO®O mbmovmommoomm mmoaammwvmmfio dOHHHOOOOOOOO 05VV000V0000H bomwfimwhmmfimm mooamomwmmmao dHHHHHOOOOOOO vvmmmfifimoammm 50000HO0V0000 00000050VNHHO doaaaoooooooo MFW005VN0000H HVN005500000N 00HH000000NHH fiHHfiHHOOOOOOO 0H0005NO0N05H NONO0V5050000 QOHfiH0050VONH HHHHHHOOOOOOO mmfimvommbvvmm mmmwvomvmmmmm fimmvmmMHomwmv AHHHHHRHRCCOC 0000000000000 0HN000NO0V000 ooooooaaamnnv0 0 0 H 2900 ZBQMM CELL R A T a o A T 2 50 2700 2800 2850 CYCLOHEXANE 2600 2483A A a s o R a A N C E 2500 Hv00v00500000fl mmvmwmvbmfimmmv 00H00500VH05V0 dHHfiHHHHHHOOOO mmmamonmwmmmvr 55NH0550NOVMMM HHNOVVONH0000N JHHHHRHHHCCCCC 05000000V500N5 00H0000HO0N050 0000HH00000VOH 000HHfiHH000000 ommmmmomomuvam 0V0000000V050N oooammmambmvnm AHHHHHHHCCOCOC mmmpmammaoamma Rmmvvvmmmowmmm mmmmoommuwvnma doooaaoooooooo 0VON5NN5®00000 maumaomvnmhmmb mooammaombmvmm dHfiHHHHHOOOOOO owmaomvmmmvvmm mmmooapmmmmomm 55000000000000 ooooafioooooooo OHOV0000v00005 mmammmmrmwmomo oamvwwwmvmoomh ARHHHHHHHHHHCC 00000000000000 0HN000NO0V0000 ooooooaflamnnvw m . v TABLE 21., a22MM CELL I o A T RA T ETHYL. ALCOHOL. 2537A A B s o R B A N C E 2600 2650 2700 2750 2800 2850 2900 2500 CHmvommvmmomHm oommfihmmmmmmam qavrmvvvmmombv HHHHNNNNNNNHHR -‘¢‘¢(\200C\20‘¢000000 5HHH‘Q’VON00‘Q‘000 000V5000500‘Q’NO OHHHHHHHHHHHt-IH 00005H005NHO0V momvvmmmvvmmmm 00H0055000v0fi0 dHHHHHHHHHHHHO mmmmmpommfimmmv 0VN0055V500000 0ONM055500VOHO dHHHHHHHHHHHHH 0055H5000H005v NOHOV050HNHOwm 0OHNVVVVVONHO® 0HHHHHHHHHHHOO mmmmvmmommamfim H505H500mmmmvm oommmwwwmvmmam AHHHRHHHHBHHHQ 50000000000000 000HONNO050N0‘Q‘ 0.0020‘1'000‘Q'0020200 HHHHfiHHfiHHr-IHHO moommmmmawmmmm 05050mmm0000H0 fimvmhmmmwbmmmm HHHHHHHHHHHHHH 00000000000000 0HN000N00V000~00 ooooooaaamnnvp 0 N (\Z azzmm CELL .R A T I o A T 2800 2 SO 2900 2700 A E E 2600 650 2750 2500 H0500000VO0V0 r-l0‘ H00000VOHOV50 OOOHNNNNNH005 HHHHHfiHfiHHHOO mmpmomppaombm mmammummbmwov OOHHNNNNNNHHO HHHHHHHHHHHHO wowomamvwbumm HVV05VHOO00H5 mmmowoafimmmmo AHHHHNNNNNNNN 0000000000000 0H010.000200‘¢000 ~OOOOOOHHHmmmg 0 0 2967A CYCLOHEXANE 222MM CELL A B s O R B A N C E R A T 2500 O A T 2800 mm F2 2900 2850 2650 2700 2750 2600 mmmvmmmmomoumm mmommmmfimmovmm 00OONMMONHRO05 00HHHHHHHHHH00 0H000000.000HO0 0H000r-I000002050 00000000000500 OOOOv-lv-lr-IOOOOOOO 0NNfi000OHONO0O MHHVOH00000000 qmmmooommmmhwm OOOOHHHOOOOOOO mmmmmmmeoaapmm 000H0000V000Nm 0000HH00000050 dOOHHHHHHOOOOO 0000000O0H0051—l 000000HO0H005H 00000000000500 OOOOHHHHOOOOOO mvmammvmwhaamm 00050050H0000H QOHHNNNNNH0000 HHHHHHHHHHHHOO mvmmmfipuhmmmoo 05VO0H000HOH00 QOHNNMNNNNHH00 HHHHHHHHHHHHHO mmmmeOOOOHan mmawmwmmmmvmvv fimmwmmmmmmmamb HHHHHHHHHHHHHH 00000000000000 »‘0H020000100‘¢‘000 0 000000HHH020000 m v 0,0 TABLE:m OO 29 0 AT 800 -2 50 322MM CELL 2259 R A T 00 2 giifl ETHYL ALCOHOL £600 3132A A a s o R a A N C E 0000000000000H 00050000VNH000 00000000000000 00000000000000 00000000000000 00050‘Q’0NH050H0 00000000000005 00000000000000 V000H000‘Q‘NOHOV 00050VONH050H0 00000000000005 00000000000000 AT 0 00VH‘Q‘0000V’HN5H 00005000V0N050C§F 00000000000000 00000000000000 BZMM CELL < K V000000500NO0H 0000500VOH000V 00000000000000 [1] 00000000000000 [1] 20 N0NN0000000000;Z 00000000000050 00000000000000I< 00000HO0H00000 2m oooommmmvvvomm m ooooaaaammmmfia< qooooooooooooomo HHHHHHHHHHHHHHM Hm M vabvumvmamafim m 0HN050H000000H0 000000HHHNNNNO HHHHHHHHHHHHHH mooooooooooooo oammwmmmwvoomm ooooooaaammmvm TIME MINS 0 02, \o. 2600 2650 2700 2750 2800 2850 2900 2500 Hommmamfimm 000H0005NV 0000000550 6000000000 VO005MHONH 5VH05H5N00 qmmmmmbbwm oooooooooo omwommmooo 0VH0000H00 0000005500 dooooooooo 5550500Nfifi mwvmomfimvw 0000000550 6000000000 00H00’Q‘0H0‘d’ 000x—l0000v—l0 0000005550 0000000000 0000050\OH‘¢ 000000205‘6‘5 0000000005 H000000000 0000'fi'550002 00055‘6'020002 0000000000 HH00000000 oamvvmfimmm mwommavmma ooaafimmmmm AHHRHHHHHH 0000000000 H000NOV’000 0-000H1-{0200‘d’ 0 0 0. _7 .7, TABLE31 CYCLOHEXANE . 3132A A B S o R a A N C E a22MM CELL RA T AT 2800 2850 2900 0 2650 2700 2750 2600 omovowvmmom 0050VVHO0V0 ommmmmmmmmp Aoooooooooo omnamvaawmo ommmmfimmmvo 00000000555 40000000000 movovmmmmba ommwmamwomfi ommmmmmmmbv Aoooooooooo 00HV000VO0V 0005VVHO0H5 00000000005 H0000000000 00000000000 0005V’V’x—I0‘fi'00 00000000005 H0000000000 vmommvaavbo oooomommmmo oooomommmmm HHHfiOHOOOOO mmoomoooomv oaamammmopm ooooooooomm AHHHHHHHHCO mmmmmbbmwmm H0005HOH000 COCHHmmmmvv AHHHHHHHHHH 00000000000 0H000020V’000 00000HHNO‘Q’0 0 v 0 . 12 Calculating Punch (ZO-step program machine), in conjunction with other of the attendant IBM machines necessary to the handling of punched cards. All of the equipment which was used is available in the fabulating Department at Michigan State University under the super- vision of Mr. Frank Martin. Since the computational procedure was developed specifically for this problem and is not a standard use of the IBM equipment, it will be described in some detail. The terminology used is that commonly alployed in describing punched-card calculations . {rag gtion of Maximum Data A master oard, X-pnnched in column 80, was prepared for each of the irradiation times at which the spectrum was seamed. The time, in dilutes, use punched in colums 65-67 of the meter card. For each irradiation time, the total mmber of scannings made in the cowlete set of runs as “tinted; trailer cards were then inserted behind each latter, the nnfisr of trailers corresponding to the mmber of sosnnings ends at each time. The trailer cards were then gang-punched sith the tine in columns 65-67 from the meter cards. These new cards were then arranged into a set of S9 decks (one for each run, 59 of inch were ssde at different conditions of wavelength, solvent and cell thickness), the cards in each deck hearing a one-to-one corms- pondence to the irradiation tines observed for the given run, with the cards in chronologcal order, i.e. the 5-minute card first, the 10- ldmte cud next, and so on. The decks were temporarily identified without»punohing in the particular conditions of wavelength, solvent and cell thickness of the corresponding run. .L set or master cards, X-pnnched in column 80, was prepared, with the ergosterol absorbanoe values for a different run.punched into each card as follows: Er sterol absorbence Columns at 2500 R 1.3 2600 - u-e 2650 - 7—9 2700 ~ 10.12 2750 ' 13-15 23% : 16-18 2 , 19-21 2900 ~ 222-21; .An identifiying code number for the conditions of each run (wavelength, solvent and cell thickness) was also punched into each master card as follows: Irradiszp , Code nnnber Solvent ——-v r w ELM” “hm 9: 9°11“ 62 Code number Thickness Code number m 1 ~ m in com; 62 2537 2 ether 1 0.57 l 265k 3 ethanol‘ 2 1.10 2 2801; h n-hexane 3 2 .22 3 gig; g cyclohexane h A three-digit code thus described each run. Each of these prepared master cards was then combined with its corresponding time deck from the set of S9 decks, and all of the internation (columns l~2h, 62-6h) transferred to each of the decks from its corresponding master card. These new cards were then punched with their appropriate absorbance values for the irradiated material as follows: gosorbance W Columns :ngtgr Q. $332? as :333: 33:32 3352: i333 2850 ' 10.16 2900 " hé-hB The result was a set of 59 decks of cards identified with respect to time of irradiation, melongth of irradiation, solvent, cell thick- nose, and the original ergosterol absorhanoe values at each of the eight selected wavelengths in eight 3-coluxm fields of the card, and with the corresponding absorbance values of the irradiated material punched into eight 'other 3-colum fields on the same card. These cards were then all gang-punched with an X in column 80 for calculator control purposes, interspersed with blank trailer cards by means of the IBM Collater, and passed through the 602-1. wired to perform the desired divisions, i.e. columns 25~27 by 1-3, 28-30 by h-6, etc., the quotients being punched as a five-digit result in the trailer cards as follows: Ahsorbance of irradiated mstg£m__ £5522. Absorbance of ergosterol 9225553’ ‘ at 2500 2 6-10 2600 ' 11-15 2650 ' 16.20 2700 ' 21-25 2750 ' 26—30 2800 ' 31-35 2850 ' 36oh0 2900 - Ll-hS JMachine capacity'necessitated that the cards‘be passed twice through the machine, the speed of calculation'being approximately 16 divisions per sinnte. The cards, as they came from the calculator, were passed through the IBMLReproducer and all identifying information (wavelength, solvent, cell thickness, time) transferred from each divisor-dividend card to the corresponding quotient card. The quotient cards were then sorted out and interpreted. These interpreted cards constituted the experimental deck. {reparation of the approximation Deck It was decided that reasonable ranges of concentration over which the various components might be expected to vary would be as follows: Ergostsrol 100 to 0 percent Lumisterol O to 100 ' Tachysterol . 0 to 60 ' Calciferol 0 to ho ” Texisterol O to 15 ' To cover completely all these ranges of concentration for all possible conbinations of these components taken one, two, three, four 16 and five at a time to one-percmt accuracy would require that a deck of sons 700753000 cards be calculated. this seemed to be a rather staggering task. Accordingly, a compromise decision was reached to calculate cards shich would cover the desired concentration ranges and take into account all possible meme, but to lower the desired {accuracy to five percent and use an interpolation method to arrive at closer approximation to the eucperireental data. In «dc to put the approximation deck abeoxtance values at the specified malengths on the same basis as the experimental deck values, the g; on. by the corresponding ergosterol fig" values to give absorbence ratios. values of the various pure comments were divided All calculations were then performed using these absorbance ratios ., The approximtion deck was prepared by beginning with two cards: one hundred percent ergosterol (100E) and five percent lumisterol minus five percent ergosterol [ A (Ia-EM. The absorbence ratio values, to four decide]. places, sore punched in the same fields as for the experimntal deck, and, in addition, the percentages of the various. components present were punched as follows: any; percent Ergoeterol (E; hé-hB " Lundsterol (L -h9-51 ' Tachysterol (T) 52-53 I Calciferol éD; Sh-SS ' Toxisterol X 56-57 These tin cards, 100E X-punched in column 77, and A (L-E) X-punched in columns 77 and 80, were combined with a trailer card and 17 passed through the 602-1 in such a manner that the A(L-E) card was put into storage and 1003 card added algebraically to it, field for field, the result being punched into the trailer card. Negative values in the A(L—E) and were indicated by an X-punch in the highest order column of a field. Three passes through the machine with different control boards were necessary to obtain the complete trailer card, which then contained the absorbence values of a mixture composed of ninety-five percent ergosterol and five percent lumisterol with the percentages punched in the appropriate fields. This 951.“ 51. card, thed in column: 80, was then combined with the [la-E) card - and a trailer card and passed through the machine three times to obtain a new card which contained the absorbence values for a mixture of ninety percent ergosterol and ten percent lumisterol. This process was repeated again and again until the ergosterol value was reduced to sero percent and the lumisterol value raised to one hundred percent. a deck of twenty-one cards was thus obtained which contained the absorbanoe values for all possible combinations of ergosterol and lumisterol for the case in which the percentages of the components were mltiples of five. This deck was then interspersed with blank cards and combined with a five percent tachyeterol minus five percent lumisterol [AG-1.)] card, X-punched in columns 77 and 80, and passed through the machine three tines. The trailer cards, containing all combinations of ergosterol, lnnisterol and five percent tachysterol, were separated m: the other cards, lupunched in column 77, interspersed with blank 18 cards, downed with the A (r-i.) card and passed through the machine three mere times. The resulting trailer cards, containing all possible combinations of ergosterol, lumisterol.and ten percent tachysterol were then separated fron.the cards containing five percent tachystarol, x-punched in column 77, interspersed with blank cards, ceibined.with the (Q,(f€L) card and.psseed through the calculator in the same manner as previously. This process was repeated over and over agsdn until the concentration of tachysterol m raised to sixty percent. ill pmiously generated cards, except those containing zero per- cent lunisterol were then interspersed with blank cards, conbined with a five percent calciferol aims five percent lumicterol I A (D-L)] card and the same procedure as before was followed until the concen- tration of caloiferol was raised to forty peromt. The same procedure was then followed using a five percent tonsterol ninus five percent lumisterol card in combination with the previously generated cards, until a.concentration.o£ fifteen.percant toxieterol was reached. The resulting deck of cards, approximately'fortyefive hundred in nuaber, then contained the absorption epectra over the desired cone centration.ranges for all.pessib1e combinations of the components (the total concentration.adding up to one hundred percent), the percent concentrations of the components being multiples of five. e t Method We considerable research into the problem of finding some method for selecting the five percent approximation card which bore 19 the closest absorbence ratio fit to a given experimental eard, it became apparent that it would.be meaningless to find any one "closest fit" card. Rather, it was necessary to devise a scheme whereby it would be possible to select certain groups of cards such that the absorbanee ratio values on these cards lay'uithin certain limits, these limits being equally'spaced above and below the absorbence values on the experimental card. In.other words, the procedure would have to be such as to select all those five percent approximation cards flhich‘bore curves lying in a certain definite region about the experimental curve. The limits of the region were chosen in the following manner: Absorbance ratio values at each wavelength were calculated for all possible combinations of five components taken two and four at a time at the two percent level, such that the total percentage for a particular conbination was zero, i.e. coMbinations of the type 223 - 2a, 2a. - 2%, 2n - mun”... 2:22 . 2a - 23:) + 211'. 2:5: - 2e. - 2%3 e 2%X,...,..., etc. The absorbence ratio values were then examined, and the largest value (over all the possible conbinations) for each particular wavelength was ascertained. These values, when added to their corresponding absorbence ratios on the experimental card, gnve the upper limit, and when.eubtracted gave the lower limit, of the absotbance ratios of five percent approximation cards which rould be acceptable for further consideration as possible matches to the experi~ mental card. 20 The procedure for selecting acceptable five percent approxi- nation.cards for a given experimental card was to sort the calcu~ lated approximation deck on each absorbance ratio field and to accept only those cards which fell‘between the appropriate limits on all of the sight fields (the number was later reduced to seven, as there seemed to be some doubt as to the correctness of the tachysterol absorbanoe value at 29003). These cards were then reproduced and placed behind the experimental card, and the original five percent approximation cards returned to their file for further use on the next experimental.card. The selected cards where then interspersed with blank trailer cards, and run through the calculator in such a.manner that the absorbence ratios on the experimental card would.be subtracted from the corresponding ratios on the five percent approximation cards, and the result punched in the blank trailer cards. The trailer cards, "difference cards,“ were separated from the other cards, gangvpunched with the identification code and time from the experimental card and then punched.(by reproduction) with the concentration information from the corresponding five percent approximation cards. The result was a set of cards punched with the difference between the absorbence ratios on.the experimental card and on the five percent approximation cards. These difference cards were then passed through the 60h Electronic Calculating Punch to obtain the sum of the squares of the absorbencc ratio differences; the sums of squares for each card were then.punched into columns 71-76 of the same card. 21 Those cards could then be arranged into sets, each set of cards having identical percentages of tachysterol, calciferol and toxisterol but varying percentages of ergosterol and lunisterol. It was then possible by inspection of the sun of the squares values to pick, from each set, that card which had the least sum of squares value. These least squares cards were the cards which bore the closest approxi- nations to the experimental card. Each of these least square cards as then combined with a two percent correction deck (a deck of cards containing all possible combinations of the five components taken two and four at a time at the tan percent level, the total percentage on any one card adding up to am). This deck saa interspersed with blunt we. and passed mg: the calculator, so that the absorption ratio differences on the correction deck cards were added to those on the least squares card, and the resulting values were punched into the blank trailer card. li'he identification code and time were trans- ferred from the experimental card to the trailer cards which were then "corrected" least aqua-es cards, in which the percentages of the minus components in the least squares card had been changed in pairs and in fours in a negative and positive manner by increments of two percent, with corresponding changes in the absorbence ratio difference values. The sum of squares values was obtained for these ”corrected” cards as before and the least squares card selected. These least squares cards rare the final approximation to the experimental card. The data thus obtained are presented in Tables 32 through 52, in which $ n; is the sum of squares value and If: nil the absolute sum of the differences. l2 o1| 21:? (Percent) TABLE 32 Calculated Composition of Mixture .(Mins) Time 00 00 mm . Code 28 30 72 “19 11 00 00 xx 03 00 DD 00 00 T7 00 ‘00 LL p72 ,30 ‘00 :5 35 69 00 5 5 111 84487 14082 00100 22484 94844 12120 00000 00000 xxxxx 00233 00000 DDDDD 00302 00000 TTTTT 23030 00000 LLLLL 00822 56121 00000 552:: 87723 43778 00000 00000 11111 00000 12631445 32391713 00000000 33805149 00688325 42021210 00000000 00000000 xxxxxxxx 02223553 00000000 DDDDDDDD 05305302 00000000 TTTTTTTT 82572585 00000000 LLLLLLLL 03532257 82360033 00000000 EEEEEEEE 28588523 16528856 00000000 00000000 22222222 00000000 907521 532762 000000 701746 941385 433231 000000 000000 xxxxxx 222553 000000 DDDDDD 850502 000000 777777 727385 011111 LLLLLL 533552 047054 000000 asses: 888328 730723 000000 000000 333333 000000 7785653 5242513 0000000 4377446 0249134 9645932 0001000 0000000 xxxxxxx 2250055 0000000 DDDDDDD 0570202 1000000 TTTTTTT 2753030 1112222 LLLLLLL 8305803 0503764 0000000 EEEEEEE 8332020 6273013 0000000 5555555 4444444 0000000 487 703 000 434 884 333 000 000 xxx 785 000 DDD 200 000 TTT 033 222 LLL 375 325 000 EEE 827 340 000 000 666 000 076 881 000 581 509 563 000 000 xxx 707 010 DDD 320 000 777 702 122 LLL 358 205 000 EEE 033 561 000 555 777 090 659 506 000 355 237 567 000 000 xxx 723 011 DDD 000 000 TTT 778 111 LLL 332 500 000 as: 387 256 000 000 999 000 34253 62914 01000 17994 71206 23311 00000 00000 xxxxx 02303 00000 DDDDD 03022 00000 TTTTT 30300 00000 LLLLL 53780 40120 00000 EEEEE 22705 59779 00000 55555 00000 00000 121 0‘3 “1.4.91; (Percent) TABLE 33 Calculated Composition of Mixture Time (Kins) Mm , Code 37030 73517 00010 65893 08277 .12140 00000 00000 Xxxxx 30332 00000 DDDDD -22303 00000 TTTTT .35282 “00000 LLLLL 53228 15041 00000 E EE E E ,70075 7.4947. 00000. 00090 11111 00000 121 1924254 1912767 0000000 8710291 2496789 2411201 0000010 0000000 xxxxxxx 0023382 0000000 D DD DDDD 8285203 0000000 TTTTTTT 7370352 0101111 LLLLLLL 7502723 0500203 0000000 E EE EEEE 803 0550 7388575 0000000 2000000 2222222 0000000 1657 7132 0100 0391 5923 3575 0000 0000 xxxx 2350 0000 DDDD 8525 0000 TTTT 7352 .1222 L L L L 0250 0214 0000 ease 3733 7453 0000 0000 3333 0000 493 552 100 224 777. 302 111 000 xxx 030 000 DDD 077 100 777 733 233 LLL 003 003 000 EEE 377 552 000 ,555 .444 000 98377 64927 00010 38197 32407 79734 11122 00000 xxxxx 00353 00000 DDDDD 07722 10000 77177 72058 34444 L L LL L 03007 02013 00000 EEEEE 38080 52531- 00000 @0000 66666 0.0000 7742 3100 0000 7823 7222 507.9 2332 0000 xxxx 0283 0000 DDDD 7202 0000 TTTT 2808 5565 L L L L 3805 1302 0000 EEEE 8022 2031 .0000 0000 9999 0000 151 025 000 695 222 103 333 000 xxx 032 000 DDD 853 000 TTT 580 556 LLL 025 002 000 EEE 720 331 000 000 222 1.11. 705 330 110 833 872 216 333 000 xxx 233 000 DDD 552 000 TTT 772 556 LLL 803 001 000 EEE 850 232 000 000 555 .111 60T ozn 05x'03003 059 0205 013L 180 64 66 00 14 08 09 2.1 00 xx 78 00 DD 20 00 T7 80 .45 LL 82 00 00 EB 50 34 00 0.0 00 33 14.18 4037 0.110 0975 0393 1232 0000 0000 xxxx 0033 0000 DDDD 0002 0000 TTTT 2330 0000 L L L L 5572 3.410 0000 E E E E 3273 6579 0000 5555 @000 0000 131 12124 2 £01 (Percent) TABLE 3a Calculated Composition of Mixture two (Kins) Mm Code 81322 80363 00010 14940 475424 21050 .00000 00000 xxxxx 103332 00000 DDDDD 05203 00000 TTTTT 50382 ,00000 LLLLL 20023 60242 00000 EEEEE 32270 39747 00000 00000 11111 00000 131 64.957 33502 10010 55551 33883 4.1381 00000 00000 xxxxx 02583 00000 DDDDD 03502 00000 TTTTT 52833 11011 LLLLL 58527 84014 00000 EE E E E 05775 03763 00000 00000 22222 00000 226970 507652 000000 064676 824664. 343652 000000 000000 xxxxxx 225883 000000 DDDDDD 525322 000000 TTTTTT 585588 111111 LLLLLL 882200 472026 000000 E E E EE E 003227 305751 000000 000000 333333 000000 9528 1261 0000 1497 2716 3534 1000 0000 xxxx 2780 0001 DDDD 7502 0000 TTTT 0285 1111 L L LL 3878 5241 0000 55:5 8875 2425 0000 5555 4444 0000 2314 2815 0010 6187 4685 1221 0000 0000 xxxx 0033 0000 DDDD 0002 0000 TTTT 3,330 0000 LLLL 0020 3.410 0000 EEEE 8725 5589 0000 5555 0000 0000, 141 509045 316656 000000 704988 771745 121000 000010 000000 xxxxxx 003382 000000 DDDDDD 203203 000000 TTTTTT 250352 000000 LL. LL L L 872708 350101 000000 EEEEEE 882575 539787 000000 000000 111111 000000 62533953 45054591 00000000 36850259 94921478 12312560 00000000 00000000 xxxxxxxx 22233385 00000000 DDDDDDDD 85272023 00000000 TTTTTTTT 27030388 00101100 LLLL LLL L 03802202 03503601 00000000 EEEEEEEE 83873222 85285887 00000000 00000000 23222222 00000000 58793 06.4.4.0 00000 73974 47599 44120 00000 00000 xxxxx 22553 00000 DDDDD 05527 10000 TTTTT 72350 01111 LL L L L 88557 04130 00000 5555: 33233 73657 00000 00000 33333 @0000 livil 2 £131 (Percent) TABLE 35 Calculated Composition of Mixture Time (Hins) mm Code 0866152 0081650 1000000 6830436 6612268 9241217 0000000 0000000 xxxxxxx 2558872 0000000 DDDDDDD 3825230 0000001 TTTTTTT 7285870 1111111 LLLLLLL 3232780 7050222 0000000 5525555 5320558 0727445 0000000 5555555 4444444 0900900 141 8862542 0309574 0010000 1759109 2442146 7544931 0000100 0000000 xxxxxxx 5778030 0000111 DDDDDDD 2830522 0000000 TTTTTTT 5728035 1011111 LLLLLLL 8852528 7046003 0000000 EEEEEEE 0032005 0731773 0000000 0000000 6666666 0000000 09851 40650 00000 47946 30300 12121 00000 00000 xxxxx 00233 00000 DDDDD 20302 00000 TTTTT 03030. 00000 LLLLL 30827 46120 00000 55555 57728 53778 00000 55555 00000 09009 211 35311635 00574451 00000000 28062993 82299442 13121220 00000000 00000000 xxxxxxxx 00223353 00000000 DDDDDDDD 50305032 00000000 TTTTTTTT 28572835 00000000 LLLLLLLL 57382222 37460403 00000000 EEEEEEEE 85738778 51428485 00000000 00000009 11111111 00000000 317634 906350 000000 720290 631177 322311 000000 000000 xxxxxx 223355 000000 DDDDDD 857032 000000 TTTTTT 720835 011111 LLLLLL 387728 140623 000000 EEEEEE 033270 737154 000000 000000 222222 090000 854891 336461 000000 512861 503848 332565 000000 000000 xxxxxx 235880 000000 DDDDDD 325527 000000 TTTTTT 238837 221121 LLLLLL 855258 662024 000000 555855 577728 004642 000000 00000@ 333333 000000 956368 027620 000000 269637 316467 624433 000000 000000 xxxxxx 257858 000000 DDDDDD 558522 000000 TTTTTT 552588 222222 LLLLLL 850232 630063 000000 EEEEEE 003020 036603 000000 555555 444444 000090 I2 M 5.13% (Percent) TABLE 36 fCalculated Composition of Mixture Time (Kins) Mm Code 5161 5145 0000 6794 0166 5322 0000 0000 xxxx 0778 1000 DDDD 5032 0000 7771 3078 2322 LLLL 0337 0643 0000 EEEE 2005 6022 0000 0000 6666 0000 211 6065 2810 0010 9870 2963 2659 0000 0000 xxxx 0235 1110 DDDD 0302 0000 T771 2730 2122 LLLL 0323 4027 0000 25:3 8520 2640 0000 0000 9999 0000 60 73 00 29 05 87 00 00 xx 72 01 DD 00 00 77 20 22 LL 33 50 00 SE 85 16 00 00 22 11 058E 012L 20T(MN>10x 00746 004 150 22731 40755 00000 88694 28646 22100 00000 00000 xxxxx 00323 00000 DDDDD 20332 00000 77777 25023 00000 LLLLL 88207 35021 00000 EEEEE 87235 53977 00000 55555 00000 00000 221 5184152 3818024 0000000 6365109 8503414 1312211 0000000 0000000 xxxxxxx 2233323 0000000 DDDDDDD 5075032 0000000 7777777 7258300 0100111 LLLLLLL 3807232 3500543 0000000 EEEEEEE 3857223 5287345 0000000 0000009 1111111 0000000 78 06 00 62 52 64 00 00 xx 23 00 DD 37 00 77 73 22 LL 02 51 00 ES 85 15 00 00 22 00 95 42 00 05 44 43 11 00 xx 03 00 DD 05 10 T7 28 33 LL 0? 01 00 ES 87 53 00 00 33 00 0305 013L 4ETCHW’02X 02065 053 045 450794 635402 000000 482633 769492 110110 000000 000000 xxxxxx 003332 000000 DDDDDD 202303 000000 TTTTTT 230030 000000 LLLLLL 802228 461021 000000 555555 873227 438977 000000 555555 000000 000000 231 é Di '2 oil (Percent) TABLE 37 Calculated Composition of Mixture Time (Mins) mm .Code 0860630 4636123 0001000 9543001 9803643 22326600 0000000 0000000 xxxxxxx 2233823 0000000 .DDDDDDD 8070032 0000000 TTTTTTT 2233078 0101100 LLLLLLL 8857232 0706144 0000000 5555555 0827055 8081744 0000000 0000000 1111111 0000000 231 587578 087052 000000 749703 946635 464262 000000 000000 xxxxxx 222585 000000 DDDDDD 055755 100000 TTTTTT 277588 111111 LLLLLL 883075 166103 000000 EEEEEE 883327 501663 000000 000000 222222 000000 427097 245501 000000 071243 893128 625522 000000 000000 xxxxxx 257878 000000 DDDDDD 528732 000000 TTTTTT 037823 221122 LLLLLL 308732 770044 000000 EEEEEE 000055 006622 000000 000000 333333 000900 0555 017L 13T 029 13x 00702 074 0005 O78L 157 con 07x 00542 005 015a 060L 15T 00o 10x 00231 018 045 045 045 92303 50650 00000 66805 01918 22020 00000 00000 xxxxx 00233 00000 DDDDD 00302 00000 TTTTT 23030 00000 LLLLL 55372 45110 00000 EEEEE 32273 54879 00000 55555 00000 00000 241 05866032 33727400 00000000 49522853 35865775 14201120 00000000 00000000 xxxxxxxx 00223333 00000000 DDDDDDDD 50235205 00000000 TTTTTTTT 28553582 00000000 LLLLLLLL 05832222 37430240 00000000 EEEEEEEE 37377878 61458648 00000000 00000000 11111111 00000000 85544 05373 10000 01692 38899 41310 00000 00000 xxxxx 22253 00000 DDDDD 08358 10000 TTTTT 70730 01111 LLLLL 38552 01510 00000 EEEEE 82327 76267 00000 00000 32222 00000 1442 0174 1000 7668 1025 5623 0000 0000 xxxx 2255 0000 DDDD 8585 0000 TTTT 7272 1212 LLLL 0325 3502 0000 EEEE 3883 4164 0000 0000 3333 0000 FmJ 2m? (Percent) TABLE 38 Calculated Composition of Mixture Time (Kins) mm - Code 201557 649232 000000 384049 247896 211272 000000 “000000 xxxxxx .788837 1000010 DDDDDD 7887203 000000 MTTTTTT 558352 111222 LLLLLL .825255 .101525 000000 eases: 2725.73 565131 000000 555555 444444 000000 241 3950 1119 0000 4322 1012 5415. 0000 0000 xxxx 2552 1.111 DDDD 3300 0000 7717 7702 0011 LLLL 8878 3026 0000 EEEE 0788 4640 .0000 0090 5666 0000 804542 184611. 000010 253767 537843 311130 000000 000000 xxxxxx 022353 000000 DDDDDD 0.30032 000000 TTTTTT 302300 000000 LLLLLL 538722 735302 000000 EEEEEE 228703 263597 000000 555555 000000 000000 311 36026 36663 00000 4911.0 99984. 35001 00000 00000 xxxxx 28235 00000 DDDDD 00323 00000 TTTTT 7.5233 00000 L LL L L 35857 824.42. 00000 55555 82572 06446 00000 00000 11111 00000 241079 237921 100000 136558 8485.44 4.4.3221 000000 000000 xxxxxx 225555 000000 DDDDDD 850325 001000 777777 727352 010111 LLLLLL 330750 370453 000000 555555 088238 507324 000000 009990 222222 039090 315 800 000 064 336 864 000 000 xxx 225 000 DDD 507 110 TTT 725 122 LLL 835 053 000 5:5 838 51.2 000 000 333 900 78 37. 00 91 52 19 10 00 xx 07 .10 DD 55 00 77 33 33 LL 73 15 00 as 52 30 00 55 44 99 9413 7743 0000 5.106 1595 777.6 0000 0000 xxxx 7757 0000 DDDD 8875 0000 7777 7702 2233 LLLL 8583 2255 0000 EEEE @303 3300 0000 0909 6666 0909 270822 401565 000000 243215 467539 654446 000000 000000 xxxxxx 700007 011110 DDDDDD 575225 000000 TTTTTT 535885 232222 LLLLLL 887853 512556 000000 EEEEEE 523250 043000 000000 555555 777777 000000 ‘ini‘l 2D? (Percent) TABLE 39 Calculated Composition of lixture Time (Ema mm Code .801533 421884 000000 239735 174572 367443 000000 000000 xxxxxx 233332 111111 DDDDDD 055020 000000 777777 588335 211222 LLLLLL 072273 600436 000000 EEEEEE 372250 056230 000000 000000 999999 000090 311 3322 4332 0000 4351 1225 6586 0000 0000 xxxx 0033 1111 DDDD 0000 0000 TTTT 8888 1111 LLLL 0527 6543 0000 EEEE 2772 1123 0000 0000 2222 1111 12 78 00 47 94. 67 00 00 xx 00 11 DD 00 00 TT 77 11 LL 50 44 00 ES 83 23 00 00 55 11 28205 87774 00000 67462 11552 41182 00000 00000 xxxxx 02385 00000 DDDDD 03203 00000 TTTTT 50330 00000 LLLLL 33222 73300 00000 EEEEE 22070 26689 00000 55555 00090 09090 321 03433244 43580332 00000000 55495256 90310982 22124110 00000000 00000000 xxxxxxxx 22233553 00000000 DDDDDDDD 05550827 10000000 TTTTTTTT .27883305 00001010 LLLLLLLL 88327252 15537042 00000000 EEEEEEEE 88227283 62350836 00000000 00000000 11111111 80090909 864538 286023 000000 440960 110639 353361 000000 000000 xxxxxx 225585 000000 DDDDDD 058557 100000 777777 730330 122222 LLLLLL 887570 250412 000000 EEEEEE 330278 416244 000000 000000 222222 000000 27 22 00 21 78 78 00 00 xx 35. 00 DD 27 10 77 83 23 LL 07 01 00 BE 78 53 00 90 33 00 O38E 008L 42T 10D 02X 01488 037 845 8868 2325 0000 3592 4175 17709 1121 0000 xxxx 3382 0000 DDDD 7757 0000 TTTT 8808 4454 LLLL 2703 2204 0000 EEEE 0578 2130 0000 0000 6666 0000 |£D1| 2 éDi (Percent) TABLE 40 Calculated Composition of Mixture Timo (Kins) mm Code ' 3,21. 24 41 10 96 39 63 22 00 xx 22 00 DD 77 00 77 80 45 LL 03 22 00 SE 38 21 00 00 99 00 591062 045031 000100 257559 648869 130171 000000 000000 xxxxxx 002385 000000 DDDDDD 203203 000000 TTTTTT 250330 000000 LLLLLL 878522 573300 000000 555525 887770 315589 000000 555555 000000 000000 331 192961764 315437468 000000000 866785968 694645779 213317621 000000000 000000000 xxxxxxxxx 033558823 000000000 DDDDDDDDD 520835287 000000000 TTTTTTTTT 703285025 011000100 LLLLLLLLL 572070232 658030222 000000000 555555555 382572853 220848566 000000000 000000000 111111111 000000000 1004749 2361300 0000000 1624727 8514624 3342622 0000000 0000000 xxxxxxx 2255835 0000000 DDDDDDD 8505775 0010000 TTTTTTT 7050880 1212112 LLLLLLL 5322200 4715155 0000000 EEEEEEE 8088520 2051522 0000000 0000000 2222222 0000000 7438 2352 0000 0967 0991 5326 0000 0000 xxxx 5580 0001 Dunn 0552 1000 TTTT 7235 1222 LLLL 5825 3654 0000 EEEE 3028 3011 0000 0000 3333 0000 4827 4031 0000 8125 9365 2523 0000 0000 xxxx 2552 1111 DDDD 0303 0000 TTTT 5252 1111 LLLL 3250 7356 0000 EEEE 0853 0311 0000 5555 4444 0000 65162 02244 00000 35513 42145 05577 10000 00000 xxxxx 35522 11111 DDDDD 30000 00000 TTTTT 25555 00000 LLLLL 08730 55588 00000 EEEEE 22303 32200 00000 00000 66666 00000 450411 568220 000000 361724 934974 241120 000000 000000 xxxxxx 003552 000000 DDDDDD 202303 000000 TTTTTT 230030 000000 LLLLLL 803058 472022 000000 EEEEEE 872277 427966 000000 555555 000000 000000 341 lé D1] :9? (Percent) TABLE #1 Calculated Composition of Hixture Time (Kins) mm Code 3084896 3475302 0000000 8740116 6086552 0251260 0000000 0000000 xxxxxxx 2223383 0000000 0335005 8308227 0000000 7777777 2792883 0010000 LLLLauL 8882257 1570511 0000000 EEEEEEE 0005570 7318367 0000000 0009000 1111111 0009600 341 39075183 05155915 10000000 04071718 68614602 43623551 00000000 00000000 xxxxxxxx 22233855 00000000 Dnnnnnnn 08527570 00010001 77777777 57028373 11211111 LuLLLLLL 08305730 54704111 00000000 EEEEEEEE 55537232 22072555 00000090 00000000 22222222 00000000 959990 501103 001000 96075‘ 823903 774432 900000 000000 xxxxxx 228075 000109 nnnnnn 500770 111001 777777 723552 122222 LLssLL 832530 150032 000000 EEEEEE 837333 ‘15524 090000 090000 333333 900000 55747 27520 00000 41372 27543 ‘1222 00000 00000 xxxxx 70300 01111 DDDDD 80775 01000 77777 20325 22222 kLLLL 32030 51125 00000 EEEEE 08780 14431 00000 55555 4440‘ 00000 3453710821 4321442156 0000000000 0967893095 4070316809 1201112210 0000000000 0000000000 xxxxxxxxxx 5555552255 1111111111 nDDDDDDDDD 5553003322 0000000000 7777777777 2235885577 1111111111 LLBLLL‘LLL 0552523238 2123666654 0000000000 EEEEEEEEEE 8325257838 ‘543000011 0000000000 9900900000 6666656666 0000000000 0452 0530 007 020 00x 00263 078 005 411 ‘590 20% osition of Mixture TABLE #2 (Percent) Calculated Comp Time (Kins) mm Code 41L1 70752 62311 00000 34093 95424 41161 00000 00000 xxxxx 23582 00000 onnnn 02523 00000 77777 53032 00000 LLLLL 87878 85115 00000 seas: 55205 03773 00000 00000 11111 00000 92002587 50131127 01001000 11604206 88970975 24005321 00000000 00000000 xxxxxxxx 22555885 00000000 DDDDDDDD 85082075 00100100 77777777 25038035 00000000 LLLLLLLL 58572023 68248026 00000000 EEEEEEEE 30073202 20530862 00000000 00000000 22222222 00000000 61105059958 42352701673 00000000020 22147422840 33173361609 85630114530 00000000030 00000000000 xxxxxxxxxxx 02255588055 00000000110 DD D D D D D D D D D 25550705820 11111010001 TTTTTTTTTTT 52227073028 00000101110 LLLLLLLLLLL 38300587230 83315725216 00000000000 35555555555 03888377087 04462041551 00000000000 00000000000 33333333333 00000000000 7194.12 926143 300000 558090 202559 714130 200000 000000 xxxxxx 578885 000000 DD D D D D 035270 111101 777777 020385 111111 LLLLLL 232220 530367 000000 EEE E E E 355550 235300 00.0000 555555 444444 000000 6859364 0506032 0000000 5585051 2945543 8403081 0000000 0000000 xxxxxxx 2578837 0000010 DDDDDDD 5837222 1111111 TTTTTTT 5273888 1111111 LLLLLLL 8280753 6241406 0000000 EEEEEEE 0352520 0415150 0000000 0000000 6666666 0000000 3531394 74.15412 0000000 6309704 5185583 2211141 0000000 0000000 xxxxxxx 7000037 0111110 DDDDDDD 5532003 1111111 TTTTTTT 7702330 1122222 LLLLLLL 3578720 4124536 0000000 5552255 8308020 14.30020 0000000 5555555 7777777 0000000 025261 394721 000000 333795 481950 331421 000000 000000 xxxxxx 700330 011111 Dnonnn 585522 111111 TTTTTT 070833 212122 LLLLLL 827255 513035 000000 EEEEEE 038270 04151.0 00000.0 000000 999999 000000 TABLE 43 Calculated Composition of Mixture £1)? |£D1| QPercent) Time (Kins) Mn Code 37385 92645 00000 78377 46604 43221 00000 00000 xxxxx 23332 11111 Dunno 88525 11111 77777 75830 11122 LLLLL 37723 30255 00000 EEEEE 07.700 24.200 00000 00000 22222 11111 411 14 67 00 19 44 11 00 00 xx 77 11 DD 32 11 77 78 11 LL 53 45 00 EE‘ 80 00 00 00 55 11 000E 058L 12T 13D 17X 00257 008 180 05 02 00 39 3.4 03 10 00 xx 57 11 DD 38 10 77 72 01 LL 83 55 00 EE 70 00 00 00 44 22 043E 055L 02T(HM>OOX 00244 041 067E 027L 03T(HM>03X 00492 147 083E 012L 00T(HM>03X 00192 094 005 005 005 4231 07575 45931 00000 77930 114.43 61272 00000 00000 xxxxx 02385 00000 DDDDD .03203 00000 TTTTT 50330 00000 L L L L L 53277 95511 00000 EEEEE 02025 04477 00000 00000 11111 00000 58241867 54731252 00000000 73014562 36133248 23132450 00000000 00000000 xxxxxxxx 22557885 00000000 DDDDDDDD 85528527 00000000 TTTTTTTT 05380382 00000000 L L LL L L L L 88533723 58581154 00000000 E E E E E E E E 20222703 30307634 00000000 00000000 22222222 00000000 55677824 70195315 00000000 87088155 86583345 32124210 00000000 00000000 xxxxxxxx 22558855 00000000 DDDDDDDD 38230750 10111001 TTTTTTTT 27223805 00000010 LL L L L L L L 30872770 48110374 00000000 E E EE E E E E 03337030 40667404 00000000 00000000 33333333 00000000 000E 078L 10T 120 00X 00767 040 0385 038L 07T 15D 02X 0044 045 45 9 8742384 12467023 0 0000000 18361046 1947259 5222400 0000000 0000000 xxxxxxx 2558855 0000000 D DDD DDD 5570500 1101011 7777777 7753823 0011111 LLLL LL L 3007700 3172555 0000000 EEEEEEE 3332232 4604021 0000000 5555555 .4444444 00000000 FM 2 £01 (Percent) TABLE #0 Calculated Composition of Hixture Time Code (Kins) 421 Mm 74991 01305 00000 04480 29910 52212 00000 00000 xxxxx 25855 00000 DDDDD 55000 11111 77777 22887 11111 LLLLL 85200 31365 00000 EEEEE 33278 35301 00000 00000 66666 .00000 671645 419234 000000 903225 985344 035473 100000 000000 xxxxxx 278825 000000 DDDDDD 055052 211111 777777 723825 122222 LLLLLL 832735 000354 000000 EEEEEE 332783 555101 000000 909000 999999 009000 017E 032L 33TIHM>OfiX 00663 030 120 72 51 00 39 32 77 00 00 xx 77 00 DD 50 11 TT 05 33 LL 03 14 00 as 80 30 00 00 55 11 0003 048L 33T£UN>CWWIOOBZS 029 180 2612 8210 0000 5869 0301 2615 1010 0000 xxxx 7370 0101 DDDD 5000 1111 T777 8333 2333 LLLL 2705 1154 0000 5525 8702 3200 0000 0000 ‘444 2222 484 145 000 862 549 453 000 000 xxx 232 111 DDD 378 100 777 732 233 LLL 875 034 000 as: 003 410 000 000 000 333 70437 15820 10000 32061 74357 64325 00000 00000 xxxxx 25550 11111 DDDDD 32872 11001 77777 25805 22232 LLLLL 80203 10345 00000 EEEEE 58780 34100 00000 00000 66666 33333 177 363 000 384 684 116 000 000 xxx 772 111 DDD 850 001 777 250 222 LLL 338 355 000 EEE 000 200 000 000 028 444 951 557 010 489 573 241 000 000 xxx 033 000 DDD 000 000 TTT 230 000 LLL 527 532 000 see 320 467 000 555 000 000 431 967777 049025 000000 465862 600731 342271 000000 000000 xxxxxx 023582 000000 DDDDDD 202303 000000 777777 253030 000000 LLLLLL 830223 796226 000000 535555 802072 103763 000000 000000 111111 000000 lg Dil 2 £01 (Percent) TABLE 45 Calculated Composition of Mixture Time. (Kins) mm Code 431 489872988 252705053 000000000 425491698 319625954 121216340 000000000 000000000 xxxxxxxxx 225558885 000000000 .DDDDDDDDD 850230725 001001000 TTTTTTTTT 250880385 000000000 LLLLLLLLL 887522525 682880266 000000000 EEEEEEEEE 008020700 205008522 000000000 906909900 222222222 000000000 5082723 1604945 0000000 0625593 2975535 7200431 0000000 0000000 xxxxxxx 0255885 0000000 DDDDDDD 2007058 1110100 TTTTTTT 5870830 0001011 LL L L LL L 3708222 8757366 0000909 5555555 0380225 0020411 0000000 0000000 3333333 0000000 510615 206051 000000 237848 434490 627222 000000 000000 xxxxxx 278885 000000 DDDDDD 335270 111101 TTTTTT 220385 111111 LL L LL L 382779 730367 900000 E E E E E E 065900 036300 009.000 555555 444444 090060 0734 3853 0000 0606 1235 1581 0000 0000 xxxx 8037. 0119 D D DD 2503 1111 7777 8507 1121 L LLL 0373 6236 0000 EEEE 2800 0330 0000 0000 6666 0000 75700 30575 00000 86260 11065 38425 00000 00000 xxxxx 03307 11110 DDDDD 55225 11111 77777 55885 11111 LLL LL 37.203 494.66 000.00. eases 70500 15100 00000 55555 77777 00900 49 53 00 56 61 42 00 00 xx 52 11 DD 55 .11 T7 55 11 LL 28 35 00 EE 30 30 00 90 9.9 09 000: 053L 077 15D 15x 00795 016 120 5680 4159 1.110 1724 1932 5262 0000 0000 xxxx 0330 0099 Dunn 0200 0000 TTTT 3030 0000 L L LL 7727 4024 0000 E E EE 0823 5875 0.000 5555 0600 0009 441 |2D1| 2 231 (Percent) TABLE L6 Calculated Composition of nixture Time (Kins) mm Code 441 38807 59410 00000 43492 0745? 53330 00000 00000 xxxxx 02552 00000 DDDDD 02303 00000 77777 30030 00000 LLLLL 03258 96144 00000 55555 73077 03844 00000 00000 11111 00000 216240175 064190420 100001010 203193474 174620456 411177701 000000010 000000000 xxxxxxxxx 235555882 000000000 DDDDDDDDD 578522528 000000000 TTTTTTTTT 200388380 000000000 LLLLLLLLL 372530228 742588155 000600000 EEEEEEEEE 835225202 146300733 000000000 900000000 222222222 000000000 884619553 640404030 000000100 671588935 979946545 232100433 000000000 000000000 xxxxxxxxx 223555882 000000000 DDDDDDDDD 327320278 110111100 TTTTTTTTT 238025387 000000000 LLLLLLLLL 882285203 458124048 000000000 EEEEEEEEE 550035570 320753730 000000000 000000000 333333333 000000000 508710409 045253152 000001000 973464549 165765319 411124131 000000000 000000000 xxxxxxxxx 255588885 000000000 nonnnnnon 553255200 111111111 TTTTTTTTT 778078033 000100111 LLLLLLLLL 307822270 523500247 000000000 EEEEEEEEE 337587822 253165420 000000000 555555555 444444444 000000000 780522053 133731741 000000000 624335420 199325681 831311113 000000000 000000000 xxxxxxxxx 255578888 000000000 DDDDDDDDD 005237520 221111111 177777777 772550358 001111111 LLLLLLLLL 305030237 203640135 000000000 555555555 883825237 463026530 000000000 000000000 666666666 000000000 9902110 1252414 0000000 1418297 2285694 0110151 1000000 0000000 xxxxxxx 2788837 0000010 DDDDDDD 0887223 2111111 T771777 0223887 1111111 LLLLLLL 5827255 4101515 0000000 5555555 3505028 2464140 0000000 5555555 7777??? 0000000 lin1| 2 DE (Percent) TABLE A7 Calculated Composition of Mixture Time (Hins)w Code 441 mm 6499826 0316045 0000000 5757913 0951717 N3102233 «0000000 .0000000 xxxxxxx 7000337 0111110 innnnnnn 8852525 1111111 .TTTTTTT 2258585 fi1111111 L LLL LLL 5208203 ”3035036 .0000000 EEEEEEE .8802570 2530520 20000000 0000000 9999999 0000000 726641 907361 000000 55‘193 395148 514111 000000 000000 xxxxxx 225553 111111 DDDDDD 858532 111111 TTTTTT 727235 010111 LL LL LL 582570 250236 000000 EEEEEE 838320 305320 000000 000.000 222222 111.111 37. 30 00 76 35 22 00 00 xx 77 11 DD 32 11 T7 78 00 LL 03 56 00 EB 30 10 00 00 55 1.1 05 12 00 21 80 31 00 00 xx 04 00 DD 21. 00 T7 00 00 LL 88 62 00 as 07 36 00 00 11 00 511 71 58 00 1.8 07 21 00 00 xx 35 00 DD 25 00 TT 20 00 LL 97 73 00 ES 43 15 00 00 23 90 18 20 00 79 15 11 00 00 xx 56 00 DD .45 00 TT 3.2 00 LL 54 75 00 ES 33 13 0° 00 33 00 42 04 00 96 62 13 00 00 xx 68 00 DD 67. 00 77 54 00 LL 23 85 00 BE 18 02 00 55 1“ 09 28 75 00 67 37 33 00 00 xx 89 00 DD 76 00 TT 78 00 LL 85 7.7 00 SE 02 00 00 00 66 00 094. 267 000 730 6.45 522 000 000 xxx 523 111 DDD 532 111 777 023 000 LLL 332 377 000 EEE 700 300 000 000 555 1.1.1 85 02 00 66 77 32 00 00 xx 55 11 DD 52 11. 77 03 00 LL 30 57. 00 EB 70 10 00 00 88 1.1. ‘éDil ,2 £%_ (Percent) TABLE #8 Calculated Composition of Mixture Time (Mins) mm Code 61 04 00 79 60 11 00 00 xx 14 00 DD 01. 00 TT 1.0 00 LL 78 40 00 BE 17 58 00 005 005 521 88 10 00 49 30 02 00 00 xx 46 00 DD 22 00 77 00 00 LL 63 31 00 EE 89 57 00 00 11 00 7680 3340 001.0 3083 7158 2270 0000 0000 xxxx 3486 0000 Dunn 4374 0000 TTTT 3.402 0000 LLLL 2806 8704 0000 E E E E 8152 0.184 0000 0000 2222 0.000 443 132 000 408 81.4 148 000 000 xxx 587 000 non 563 000 TTT 547 000 LLL 883 7.37 000 £55 740 041 000 000 333 000 003E 072L 107(Hfl>09x 00245 026 045 96 20 00 19 87 74 00 00 xx 09 10 DD 86 00 TT 02 11 LL 93 .47 00 EE 30 20 00 00 65 00 5538243 2424064 0000000 2177429 7.511065 1638540 0000000 0000000 xxxxxxx 7803330 0011111 D D D D D D D 3855270 1111101 TTTTTTT 7255830 0000011 LLLLLL L 8582725 6220266 0000000 EEEEEEE 5725055 0446400 0000000 0000000 9999999 0000000 976997 475446 000000 079839 032258 162330 100000 000000 xxxxxx 772330 001111 DDDDDD 853322 111111 TTTTTT 257780 000001 LLLLLL 830228 57.4336 000000 EEEEEE 508550 102330 000000 000000 222223 111111 591574223 206232375 000000000 073753409 722432294. 897194131 000000000 000000000 xxxxxxxxx 700233352 011111111 DDDDDDDDD 587385270 111111101 TTTTTTTTT 522725830 000000011 L LLLLLLLL 338352258 743512566 000000000 EEEEEEEEE 073525500 023154100 000000000 000000000 555555555 111111111 047256 145473 000000 433571 580191 712131 000000 000000 xxxxxx 023352 111111 DDDDDD 7.33232 111111 777777 27787.8 00000.0 LLL LLL 832058 465636 000000 EEEEEE 355700 201030 000000 000000 888808 111111 £1)? |énil (Percent) TABLE A9 Calculated Composition of Mixture Time (Kins) mm Code 5709 1960 0000 2042 6151 6451 0000 0000 xxxx 2355 1111 DDDD 5350 1111 7777 2527 0000 LL LL 8208 6636 0000 E E EE 37180 0030 0000 0000 .4444 2222 521 904 441 000 505 890 442 000 000 xxx 557 111 DDD 230 111 TTT 225 000 LLL 838 666 000 EEE 370 000 000 000 000 333 OOT 12D 15X 00840 022 0005 073L 360 7.8 20 00 75 3.6 30 00 00 xx 40 00 DD 00 00 77 00 00 LL 30 0.4. 00 SE 30 96 00 55 00 00 531 .43 03 00 94 32 21 00 00 xx 04 00 DD 11 00 77 .10 00 LL 88 73 00 EE 07 25 00 00 11 00 72 23 00 37 06 14 00 00 xx 27 00 DD 42 00 TT 23 00 LL 75 85 00 EE 53 03 00 00 22 00 626 493 000 492 409 150 000 000 xxx 375 000 DDD 7.75 000 TTT 335 ooo LLL 2.10 8.48 000 EEE 525 040 000 000 333 000 78 51 .10 68 63 92 00 00 xx 87 00 DD 07 10 77 57 00 LL 96 37 00 EE 83 30 00 55 44 00 09T 09D 09x 00545 098 0003 073L 060 22416 00164. 01000 76356 86236 65752 00000 00000 xxxxx 80330 01111 DDDDD 85303 11111 TTTTT 02585 00000 LLLLL 25727. 55366 00000 EE E E E 28275 21300 00000 00000 99999 00000 04 06 00 24 52 57 00 00 xx 50 11 DD 37 11 77 50 00 LL 03 67 00 EE 70 00 00 00 22 11 OOOE 068L 02TJKH>12X 00626 029 150 95 56 00 40 86 12 00 00 xx 04 00 DD 01.. 00 77 00 00 LL 74 40 00 EE 31 59 00 55 00 00 541 562 303 000 135 027 313 000 000 xxx 146 000 DDD 122 000 777 100 000 LLL 263 731 000 EEE 589 257 000 000 1.11 000 126 316 000 168 73.4 316 000 000 xxx 258 000 DDD 456 000 TTT 210 000 LLL 818 850 000 EEE .488 037. 000 000 222 000 ZED? ‘2ED1I (Percent) TABLE 50 Calculated Composition of Mixture Time (Mins) mm Code 967 704 000 312 941 735 000 000 xxx 758 000 DDD 857 000 TTT 253 000 LLL 720 284 000 EEE 632 504 000 000 333 000 541 35 51 00 99 98 .47 00 00 xx 89 00 on 78 00 TT 97 00 LL 50 75 00 EE 16 02 00 55 44 00 62.40 .4033 1200 7990 8231 9770 0101 0000 xxxx 0101 1111 DDDD 0277 1100 TTTT 0821 1011 L L L L 3919 5175 0000 EEEE 7002 1501 0000 0000 6666 0000 006E 062L 12TZHH>10X.00954 128 075 6165579490 3270316561 0000000100 2924464701 5929317029 4232155020 1000000100 0000000000 xxxxxxxxxx 2770033300 0001111111 DDDDDDDDDD 0858552023 2111111111 TTTTTTTTTT 0252558308 0000000110 LL L LLLL LL L 3337855287 7471314565 0000000000 EEEEEEEEEE 5003222202 0305352101 0000000000 0000000000 9999999999 0000000000 958068242 664133002 000000011 459442477 497062563 713632473 000000000 000000000 xxxxxxxxx 702333552 011111111 D D D D D D DD D 858872302 111111111 TTTTTTTTT 272230838 000001010 L LLL LLLL L 008273228 662016366 000000000 E E EE E E EE E 380502200 104650300 000000000 000000000 222222222 111111111 0115 8953 0000 0112 8311 .4323 0000 0000 xxxx 0325 1111 Dunn 8752 1111 TTTT 2358 0000 LL LL 0283 6.466 .0000 EEEE 0502 1200 0000 0000 5555 1111 953 113 000 321 370 033 100 000 xxx 055 111 DDD 753 111 TTT 025 000 LLL 307 756 000 EEE 080 010 000 000 888 111 000E 07$_(HN'10D 17x 01425 075 092E OOBL OOT 00D 00X 00154 084 240 010 611 01 53 11 84. 2.4 .43 00 00 xx 20 00 DD 00 00 77 00 00 LL 08 01 00 EE 82 98 00 00 33 00 0675 033L 00T(NM>OOX 01172 255 060 Code 621 631 641 Time (Mins) 005 O N 0 MN HHH HP 30. 99 AA 00mm ”N 0% max €36 660 90 @629 69 0955 0905* o m m mm mm mm 00 C300 00 DO 0 «Ii-Q UIO\0\ Am (MW 4 we tomm \O-Q HUI 03 WNW mm 0905 085E 0555 0835 054E 073E mm 010 001 (’10 FIN mm 00 CO CH (DO 00 \00 083E OBOE 0735 0655 083E OBBE OBOE TABLE 51 (Percent) 005L OOT 00D 010L O 0 N f” I" Hm UQO COCO com on FF? Fl" GO 090 CO OO 0 (DUI ANN A0 LAO 0) I” F O H O r 015L O35L 014L 046L 0 (0 (0 rr rr r 0000 OO 00 FF (0 i4 HP H0 00 O '5] 010 001 (no I” I" O (0 .q r. 035L 014L 007L OSOL rr so 000 oo 00 OCT oo NO MOO NO 00 CO 44 444 44 44 44 , O O .1 007 00D 00D 00D 00D 00D 00D Calculated Composition of Mixture 00x 00x 03x 00x 00X 00X 00x 00x 00x 00x 00x 00x 00x 00x 03x 05x 00x 00 000 00 oo oo so 000 oo oo 00 HH HNN HH H0 H0 2 éDi 00010 00020 mo mHo mo 00 um Hm HQH pm me NH 00017 00056 00281 00893 00649 01465 OO 00 CO CO CO 09 CO CO 00 am AN WA (his men Hm 00083 00082 00147 00139 GOO GOO UNION (DION oomxo 12%| 004 O O Ox 00 OFF 00 CO 00 HP OHO Obi NH 01b! ‘04 «04003 HO 01b! mm D (0 O\ 046 111 214 099 (0 4 0) O O CO CO. CO A A U01 bib! WA A -l (DID 001 UN TABLE 52 2 Run Time Calculated Composition of Mixture éDi |éD1| Code (Kins) (Percent) 641 -120 00T 00D 05x 30593 154 120 OOT 00D 00x 00472 108 150 00T OOD 02x 01023 220 150 00T 00D 05x 01219 251 150 00T 00D 05X 01235 254 150 00T OOD 02x 01074 215 180 00T 00D 02x 00942 216 180 OOT 00D 05x 01012 233 180 00T 00D 02x 00932 211 22 Thong? Gonoerrdg the flatbed of Calculation A limitation of the method of calculation employed was that it led to a manor of pcuible choices as to the “closest fit“ card corresponding to a given experimental card, rather than a single “closest tit" card. This is evident from the tabulation of the data in Table: 32 through 52. This limitation is not too serious for cone aideraticn of. the percent concentration or tacm'terol, calciferol and toxisterol, because the spread of value: for theee components in not too large over the possible choice of cards. It is, however, very eel-ion. in the cue of ergosterol and lunietercl, because the concen- trations of then two components, for the possible choice of cards, vary over a rather large range-v-thie ie, of course, a necessary conse- quence of the similarity between the epectra of ergosterol and ludeterol. Use or a more extensive correction deck would probably narrow the pescible choice of cards, but eubetantial reduction of uncertainty would, undoubtedly, require a correction deck so large that the mmber of calculations necessary could become completely unwieldy} it would prove easier instead to prepare the one percent apprcxzmation deck of seventy to seventy—five thousand cards in the first place. In View of this, it may again be emphasized that the treatment of the data here employed repreeented a compromise between the amount and value of the information obtainable and the lumber of calculations necessary to obtain this information. 23 It my aloe be nontioned here, further to euphaeise the neceseity tor the conpromiee, that the number of individual addition, sub- traction, multiplication and divioion Operations performed in thie work us in the neighborhood of cone five hundred thou-and, and that the total number of carde handled uaa approximtely thirty-five thousand. Due to the fact that the method of cowutation m unable to provide unique solutions to the experimental data, the concentration- time curvee in Figures )4 through 15 each represent only one of the new pouible sets or such curved. The eete selected eoened, to the author at the time, to be the neat reaeonable. PERCENT COIPONENT PERCENT COMPONENT 100 eERGOSTEROL ‘OLUMISTEROL ‘OTACHYSTEROL 80*—' ‘CALCIFEROL - xTOXISTEROL R 411 60 “q .p O 20 2804A DIETHYL ETHER 0°57MM0 CELL ‘ l p o , o 10 20 30 4o 50 6o TIMEGMINUTES) 2654a DIETHYL ETHER 0L57MMO CELL . I eERGOSTEROL oLUMISTEROL OTACHYSTEROL‘ ~CALCIFEROL sq XTOXISTEROL 1 Run 311 C e O 1;; I, ‘ . f 4o-—+———— T“ f i v 20 ' e -— x i ' ‘7“: O . L , T I 1 an o 10 20 30 4o 50 60 TIME(MINUTES) FIGURE 80 ERGOSTEROL AND ITS IRRADIATION PRODUCTSo VARIATION OF CALCULATED CONCENTRATIONS WITH TIME° PERCENT COIPONENT PERCENT COIPONENT 100 I Run 621 OERGOSTEROL OLUIISTEROL 80 " 60 40 20 0 ________i 0 20; 40 60 80 00 120 140 160 . TIIE(HINUTES) 100 .22éza_TTT 80 O 60 , eERGOSTEROL o OLUHISTEROL .oTACHTSTEROL ;!CALCIFEROL xTOXISTEnOL Run 521 l . I If 0" ‘40 0 80 100 120 140 160 ' TIne(uINUTES) 31}2A ETHYL ALCOHOL OoSYMM. CELL FIGURE/'15.. ERGOSTEROL AND ITs IRRAOIATION PRODUCTS. VARIATION OF CALCULATED CONCENTRATIONS WITH TIME. 2h SUMMARY OF RESULTS general Conclusions Before any theoretical treatment of the data is attmpted, some general features of the reaction will be pointed out. Conclusions may be reached as to the effects of the irradiating savelongth on the course of the reaction. In general, regardless of solvent, the short savelengths favor tachysterol formation, the median wavelengths favor calciferol formation and the long melengths favor lumisterol formation. The madame yield of calciferol seems to occur when 28th or 29672 “much is employed, although 29672 seems, in general, to be somewhat less effective than 2801.2 (see mm 16). The maxim yield of tachysterol seems to occur at 26513 with lesser yields at 25372 and 21.832, except where ethanol is employed as the solvent, Ihen 2h833 and 25372 seen to be most effective (see Figure 17). The behaviour of ethanol uith respect to the formation of tachysterol is umual and will be discussed in some detail later. With regard to the solvent effect it may be stated that the best yields of calciferol are Obtained in ether and n-hexane at 280142. although at other wavelengths ether and n-hexane are not necessarily the best solvents (see Figure 18). Tachysterol formation is definitely favored when ethanol is employed as the solvent; by far the largest yields of tachysterol are obtained in ethanol when an irradiating Inve- length of 280$ or below is employed (see Figure 19). In general, l l T T I I 'ZEIE 'ZEIE 5" me: ”I “.963 u: A j a: g "088 j g I 117092 0 . t-‘l t-l 53 , a 1 E lvv59z I Vt598 vzzsz * VAESZ . i—-—- ‘— vEsz vgevz.- l L I I 1 1 1‘3; 9. “5 ° 21? 3 “‘ ° T T l ' T I 'l' ‘ZEIE I VZEIC E I “962 E' I M962 I twee fl [ nose :2 E . c: "592 "S92 VLESZ ’ VASSZ ”2872 I o- V€8t3 1 I 1 7 1 1 if? S “‘ ° ‘3 S “‘ ° (INSOHSH )‘IOHZJI O‘IVO .210 N01 IVHIHEONOO MIX“ MIIUI YIELD OF CALCIFEROL' AS A FUNCTION OF IRRADIATING ILVELENGTH IN VARIOUS SOLVENTSI. FIGURE 16- 1 I 1 r l l 'ZEIE 'ZEIE A A , 3 M96; §L V6963 § "092' a I nose ‘4 - o A .4 E [—— 3 "S93 _ I "593 VAESZ ' VAESZ vista V£873.- I n l l 1 « 13; S \n o 1:: S \n o I I 7 I l j VZEIE ; VaEIE E IVA96'2; . g I H.962 I woe-e ' g I V1708? a « e-a . '. z I ' a . -.-- I} -.-— c: V7592 17992 VLSSa ' VAESZ Vista I . vsevz l 1 1 _ 1 L 23 a m o 1:: a m o (35130836 )‘IOHEJI O‘IVO JO MOI IVHIHSONOO IONIXVR FIGURE 16 IAXIIIUN YIELD OF CALCIFEROL'AS A FUNCTION OF IRRADIAI'ING WAVELENGTH IN VARIOUS SOLVENTS. . dNMHm ea _ _ . 38m ETHYL ALCOHOL domom l"r|l||l|ll||lll|L «ummm DIETHYL ETHER 4 3 co N CYCLOHEXANE O 3 Aazuommmvuommammmo¢a ho onadmazmozoo IDIHNdI N-HEXANE «omom nmmmm «mmomh . , — — _ P a 0 0 0 6 5 4 0 2 L 00 1 :FIGURE 17. llAXIllUM YIELD OF TACHYSTEROL as A FUNCTION or IRRADIATING WAVELENGTH IN VARIOUS SOLVENTS. 2804A M*dxwmouoho mznxmm l Enomoodd .mwmam mmmam — _. . _ *dewmonowo _ madxum — Homoond P ._ _ 2483A' 5 050 1 l _ 5 0 2537A d _ _ MIdNHMOA Ho m2 m How 044 mumam . — - 5 O 5 0 1 1 3132A uzdwwmonowo madxmm Homoond fl mmmam 2654A ¢N¢MOAOHO m m V no OQHd mmmam 15- . — 0 5 0 1 .AazuommmvflomMMHoAdo ho 20Hadmazuuzoo IbiHNdl SOLVENT AT VARIOUS WAVELENGTHS OF IRRADIATION. FIGURE 18. HAXIIIUN YIELD OF CALCIFEROL AS A FUNCTION OF HAXIHUI YIELD OF TACHYSTEROL AS A FUNCTION OF SOLVENT AT VARIOUS WAVELENGTHS OF IRRADIATION. a a 4 _ _ — _ — — _ 4 _ 1 fl — _ a az4uumanumo uz4ummonowo uz4xumoaowo ,1; madman mu Manama _ Ma madman M 6 3 8 9 1 2 . Smog: 2 nomoofl 3.. 8834 manna mumau Fl: mamas I F _ r _ p _ _ _ _ L c _ _ _ w_ _ 0 O 0 0 0 0 0 O 0 0 0. 0 O 0 0 0. O 0 6 5 4 3 2 1 5 4 3 2 1 6 5 4 3 2 1 q a. n ,. .T .- 4 I ._- . a _ _ _ q _ . _ _ _ unexumonowo uzexmmonowo - .uzaxmmonowo _ A; madman .h uzdxum —. . .. .a mzdxum# 3 7 4. 8 no; 1 lllllllll Illli Hm, . a .8884 2 ‘ 3834 2 .8832 mmmau fl. mmmam _ sane» _ _ p h r _ _ — _ b L _ F F ._-. . _. O 0 O O 0 0 0 o O 0 0 0 0 0 0 0 0 0 0 0 6 5 4 3 2 1 5 4 3 2 1 6 5 4 3 2 1 Agony“ Vnommamwmoda ho BOH admafiozou Ina—”Nd: FIGURE19. 25 other end nv-hmne behave alike as solvents, ethanol is extreme in its behaviour, and cyclohexane behaves more like ethanol than like other and n-hexane. The rate of disappearance of ergosterol appears greatest in ether and a-hezans, less in cyclohexane and least in ethanol, except at 31323 there the rate of disappearance of ergosterol is independent of solvent. is night be expected, the rate of appearance of lumieterol is solvent dependent in the same general manner as the rate of ergosterol disappearance. The rate of tachyeterol appearance, on the other hand, is greatest in ethyl alcohol, less in cyclohexane and least in ether and n-hexane which both behave alike. The rate of appearance of caleiferol shove the same solvent dependence as the rate of appearance of tachysterol. Note that although the rate of appearance of calciferol is greater in ethyl alcohol than in ether, the maxim attainable concentration of calcifercl is greater in other than in ethyl alcohol at 28023, Il'he rate of appearance of toxisterol is solvent dependent in a manner similar to W1 and calciferol. ‘ Several attempts were made to treat the rate of disappearance of ergostaol kinetically, in order to get an estimate of the quantum efficiency” of the isomeriaation of lmnisterol. Several different mechanism sore considered, but the results of calculations based on these nechmisns proved to be extremely variable. It is believed that the concatrationdtine curves of ergosterol and lnnisterol must be known with a greater degree of certainty before a kinetic treatment 26 It is indeed unfortunate that the data uhieh should be the most amenable to a kinetic treatment 2. the very data can be successful. which are the nest in doubt. Calculated quantum yields of ergosterol disappearance are given in Table S3 . TABLE 53 WANTUM 3333 OF ERGOSTE‘RG. DISAPPEARANOE Nmeber or Moleoules in Cell- 25 x 10 L2; ‘“ _ Bolvmt wave-6 Tine Number of Number of Wu length (Kins . ) Quanta Holecules Yield 2 Absorbed Decomposed Ether 2&8} 10 6 x 1015 h x 1015 0.7 " 2537 S 12 x " 3 x " 0.2 'l 2651; 3 16 x " 7 x " 0.14 ' 2801; S 11 x " 8 x " 0.7 " 2967 lo 11 x ' 6 x " 0.5 ll-hmne 21183 5 3 x " 2 x " 0.6 ' 2537 5 12 x ' 6 x " 0.5 " 2651; S 16 x " 1h 1 ' 0.8 " 2801; 5 ll 1 " 8 x " 0.7 " 2967 S 6 x ' 10 x ' 1.7 Cycloheaane 21:83 10 6 x ' h x " 0.8 ' 2537 S 12 x ' 1; x ' 0.3 ' 2651; 5 16 x " 8 1 " 0.5 ' 2801; 5 ll 1 " 3 x " 0.3 " 2967 5 6 x " 2 x ' 0.3 Ethanol 21.83 2O 12 x " 5 x " 0.1; "2 2537 10 25 I ' 6 I " 0.2 ' 2651.; 10 33 x " 1.1 x ' 0.1 ' 2801; 10 22 x " 6 x ' 0.3 ' 2967 10 ll 3 " S x " 0.5 1 1 1 1 27 DISCUSSION AND INTERPRETATI ON Recent Work and the Reaction Mechanism The earlier studies discussed in the introduction to this thesis had indicated that the irradiation reaction proceeded irreversibly through the identified intermediates in the order: ergosterol --s- hunisterol --b tacl'wsterol --9 calciferol --e- over-irradiation products. Some recent investigations by three groups of scientists in three different countries-7ellus and his associates (3,h,5,6,20) in France, Havinga and coworkers (21,22,23,2h,25,26) in Belgium, and the group headed by Inhoffen (27,28) in Germany have domnstrated two important features of the reaction which were as yet unknown at the time the emperinental work and calculations described in this thesis were completed. One of these features is the importance of the intermediate, precalciferol (first discovered by the group under Venus), in the reaction scheme: precalciferol, identified tentatively by the Velluz group as a 6,7-cis isomer of calciferol (but postulated by the Havinga group to be a 6,7—cis isomer of tachyeterol), has recently been sheen to be a Injor pflnl'y product of the reaction (29). It isomerizes thermally to caloifercl, slowly at room temperature and more rapidly at eonsIhat elevated tenperatures; it now appears likely that pre- calciferol say be a necessary precursor of calciferol. The structure of 28 precalciferol mains in doubt; its ultraviolet spectrum resembles that of oalciferol in band shape but with considerably lower intensity (very comparable to that of ergosterol and WWI} ihils its infrared spectrum lacks a band (around 900 on“) which characterizes a terminal methylene group (such as that found in celeiferol) . The postulated structures of. precalciferol are shown below along with the currently generally accepted structures for calciferol and tachysterol: PRECALCI FEROL (VELLUZ) Rs: TACHYSTEROL PRECALCIFEROL (HAVINGA) Havinga and his associates (22,23) have shown the occurrence of an iodine catalyzed inter-conversion of precalciferol and tachysteroli this reaction, along with the infra-red evidence, tends to favor the Havinga structure. A cisoid relationship of the double bonds (as shown by both structures) seems necessary to explain the ultraviolet spectrum; this point is discussed in some detail by Brands and Wheeler (30). 29 Mother-dormantdisoevm,aadebythelevin¢am,is tbatthssulieriudisatedirrenrsibilitycfthe reaction sequence doesnot,iatast,esist. wwcmuummt awottheinteraeflatescaagobasktssseeesscstheteraebeliend amatnmouunmmnum,awmomi mmM-Mmamwmnmw iasisatssdireotlen. nelateetpepoeedreastioeseqa-seiaas fellows: Heat macro]. :3 salon-fine ever- “ An" mugs was «natural -—e finalisation: We). be r . a 1 The ceission o: precalciferol in the calculations introduces an “patent source or error, casting a serious shadow over any quanti- tative application of the results . me qualitative inplications of the calculated results raisin essentially valid, homer, if vimd an: a recognition or tn {acts mich are consequences at the relatioflxip of. the paealcifercl spectra to the spectra of the other mad-8 (1) the m1 percentages sill be, in general, too high, since the spectra: of precalciferol consida'ably overlaps that o: Mineral, and 3O (2) the small indicated anmnts of calciferol probably corres- pond to appreciably larger amounts or precalciferol, which has a similar spectrum, but 01‘ lesser intensity. In the long wavelength irradiations, the apparent direct conversion of ergosterol to lumisterol met now be considered to be a conversion to lunristerol plus precalciferol-win better accord with the known (by bio-assay) production of vitamin D, at these wavelengths. In the discussion which follows, a mechanism for the reaction is preposed on the basis of (l) earlier studies, (2) the results presented in this thesis, and (3) those nest recently published investigations which have appeared since the coxupletion of this study. A. StereochendcalfiChjagges Hiring Reaction Consider now the stereoohenical changes which must take place in ring B or the ergosterol nolecule in the formation of the various irradiation products. The formation of lmnisterol appears to require only a simple inversion of the angular methyl group C”. This undoubtedly proceeds through fission of the somewhat strained bond between C; and Cm, after vdxich the methyl group On first becomes . planar with ring A and then moves below the plane as the 094.31,3 bond is reformed to give lmuisterol. A study of Fisher-Hirschfelder models reveals that the motion must be considerably more complicated, however, since the inversion of the angular met‘zwl group is greatly restricted by the hydrOgens on C. and Cu; the inversion actually requires a nearly 360° relative rotation of the two large fragments of the mole- cule about the bond Joining C5 to Us. This rotation is, however, so sterically restricted that it can occur freely only if accompanied by a pair of rotations about the bond joining Co to 6,. The ergosterol molecule is oriented cis with respect to this bend: a trans relation- ship about this bond is required to bring the two large portions of the molecule free of one another to permit the full rotation about the bond C,,-(:‘ to occur, and a second rotation (returning to a cis eon- tiguration with respect to the 0.42, bond) is then ”(paired for the formation of lunieterol. During these rotations the molecule passes through sp- 00mm (in... configurations with ring 3 open) closely approaching the probable configurations of taclvsterol and precalciferol; a stabilisation of the mleeule while in one of these configurations should lead to the formation of the corresponding inter- mediate.. i proton or hydrogen atom transfer from On to Cg, during rotation or after formation of one of the intermediates, is required for the production of calciferol. , The consideration of nolecular models leads to interesting conclu- siens regarding the probable configurations of precalciferol and tachystsrol. ll'he configuration of tachysterol indicated on page 28 is found to be a sterically favorable form shich permits a planar relationship to exist om all three double bonds. in alternative confomtion with all transoid relationships between the double bonds also more reasonable, but suffers a rather close steric interaction of the hydrogen on C. with hydrogens on.Cl‘ and 015} the interaction is closer*then any existing in.the preferred conformation. It is pmflflcuhuiymemmn¢Myin‘mflmuemmmwthnxtmnia«mmpmuflwmflladiso- tachysterol, obtained and characterized by the Inhoffen group (31), has the structure of tachysterol with tho A-8,9 double bond replaced by a. 13-8, 1h double'bonds iso-tachysterol in its most favoreblo storie conformation would then.hsve the all transoid relationship‘beteeen mnmlelnnms ISO-TACHYSTEROL TACHYSTEROL 33 which should lead to a more intense and longer wavelength ultraviolet absorption than the partly traneoid, partly cieoid structure of tachycterol 0 kg 0 ‘ t - transoid Int c — cisoid I4 ISO-TACHYSTEROL TACHYSTEROL The ultraviolet absorption spectra of isotsctqsterol and tachyeterol display the peaks indicated bolas (31) in excellent accord with the postulated structures. m.(x) maze tachysterol 26h0 18,000 2800 23,000 29h0 20,000 isotschysterol 2800 31,000 ‘ 2900 h1,000 3020 31,000 Study of the models also suggests a very plausible structure for precalciferol, vmich seem to explain all. the observed properties of this intermediate compound. The conformation proposed by Hevinga 33 3. cannot possibly be achieved for steric reasons, since a planar arrange- ment of the (10,5), (6,7), and (8,9) double bonds sould require a coalescing of the hydrogens on C4, and 09. It is, in fact, not storically feasible to obtain a simltaneoue planar relationship of all. three 3h double bonds it it be accepted that precalciferol. is a 6,7-ois isomer of either calciferol or tachysterol. Accepting the Rantings conclusion (based, as mentioned earlier, upon infrared spectre and iodine- catalysed isomerization) that precalciferol is a 6,7-cis isomer of tachystecrol, it is found to be sterically highly improbable for the (ta-Cm double bond to lie in a planar relationship to the C.-C., double bond. The sterically most favored conformation for precalciferol, ‘ than, has the c,-c., and 0.4:, double bonds in a oieoid relationship, in a plane perpendicular to the plane of the substituents on the cs-clo double bond. Thus the structure has only two conjugated double bonds capable of resonance, and these are in a constrained cisoid relation- ship not unlike that imposed by the closed ring structures of ergosterol and lumisterol. The ultraviolet spectral similarity between the open ring compound, precalciferol, and the closed ring compounds, ergosterol and lumisterol, is thus to be suspected. The preposed model for precalciferol has, in com, the follow- ing features in its fever: (1) it is consistent with the apparent absence of a terminal methylene group, as indicated by infrared, (2) it is consistent with the ultraviolet spectral similarity between precalciferol, ergosterol, and lumisterol, (3) it is consistent with the iodine-catalyzed isomerisation of precalciferol to techysterol, 35 (h) it 5.: the first sterically favorable conformation which would result immediately upon rupture of the 0,-010 bond during reaction, sith a mum rearrangement of bulky peeps in the molecule, and (5) it retains C. and the Wagons on 01. in close proximity, making the proton or hydrogen atom transfer to form calciferol eterichJy plausible and favorable. (See three dimensional representation of precalciferol structure b.10'e ) 3 / \. a \_ Proposed Structure of Precalciferol 36 B. fihotochemim and Electronic Changes During Reaction It has been shown that the photochemical behaviour of ergosterol and its irradiation products exhibits marked dependence on the irradiating wavelength and on the solvent in which the irradiation is carried out. The quantum yield of ergosterol disappearance as given by the crude calculations based on this work is within an order of magnitude of unity. It is also known that the presence of oxygen during the irradiation dose not affect the yield of calciferol sipifi- cantly, but leads to side reactions producing materials which make the separation of calciferol from the irradiation mixture difficult. A striking feature of the reaction is that all of the changes which occur (hiring the reaction takeplace within the sane ring in all of the compounds involved. Ex'gosterol and all of its irradiation products are, therefore, structurally highly similar. The ring (ring 3) in which all changes occur is conjugated (the only conjugation in the molecule) and is undoubtedly responsible for the strong ultraviolet absorption maxim which occur between 2600 and 28002 in ergosterol, lmnisterol, tachystsrol, precalciferol, and calciferol. Postalations as to the mechanism by which the reaction proceeds met, therefore, explain the changes occurring in this ring. Since the reaction is at least initiated by light, the primary I step met involve the absorption by ergosterol of a quantum of radiant energ. Ergosterol has no Impaired electrons in its normal ground electronic stateuthat is, it is a singlet molecule. The molecule, upon absorption of the quantum, can then find itself in one of four 37 states: an ionic excited singlet state in which there is charge separation in ring 133 a stable excited singlet state not disarm clinically from the nornal ground state) an excited singlet state at a level above the bond dissociation energy of a particular bond} or an excited triplet state in which there are ten unpaired electrons in ring B. The ergosterol molecule in any of the above excited states (except the dissociated singlet) can undergo either of the rollouing deactivating secondary processes (neither of which contributes to chemical change in the molecule): (1) it say give up its energy as heat by stepwise loss of vibrational energy, principally through collision with solvent molecules or other molecules in the system, or (2) it may give up its emery as fluorescent or phosphorescent radiation by spontaneous downinrd electronic transition. Both of these processes would tend to dindniah the quantum yield of the photochedcal reaction. Another possibility is that the excited molecule my collide with another ergosterol molecule in the system, or Iith a solvent molecule, and undergo reaction with it. This possibility is ruled out, sinee only internal changes in the molecule are observed, and since the reacflon has been shown to take place at liquid nitrogen tempera- ture. It renains to be explained, then, how and why the observed changes in ring 3 take place in those excited ergosterol molecules Ihich do not suffer deactivation. The discussion which follows 38 considers the possible rate of the ergosterol molecule in each or the tour excited states which were listed previously (lords singlet, non- polar singlet, singlet above dissociation limit, (ii-radical triplet). The Ionic Excited Singlet State: Due to the large magnitude of the absorption coefficients of ergosterol at the various manna, it is reasonable to suppose that these mine correspond to the different vibrational bands of an I'allowed" transition-obs. , a transition, of high probability, a: the molecule to one of its electronic excited states. Consideration of the region in which the ultraviolet absorp-b ticn occurs, and the large aspimde or the absorption coefficients, leads to the conclusion that the important transitions here involved are most probably of the s-e v type, 1..., transitions from normal singlet ground states (a) to was excited singlet mm (1!). Such transitions new be crudely represented by valence bond structures as m M 115‘ (s) «Cl-5’“) :05 m m (a) who ~ ( m m m m M m (a) 115’ m w Lm m «(15’ (1) (Ni (V) follows: The mited state (V) can be characterized in terns of different percmtago contributions from each of these individual ionic valence band structures, as well as from nonaionic structures: the percentage contributions will differ for different structures, since the indi-v vidual structures themselves possess differing energies. Itispossible, interuofthesestructaree,togiveamali~ .. tative picture of the manor in which certain rotational notions of a part of the ergosterol molecule can lead to the several irradiation products. Once the bond c,-c,, has brokm, the chain cowpoeed of 0,, 6,, 0., and C, can undergo rotational notion. Due to steric inter- action, rotation is probable only about the 0,-0. and 0.41, bonds. Structures (3), (h), (i), (J), (a), and (n) nay rotate about the 0.3-0. single bond, while structures (k), (l), (a), and (n) say rotate about the 0.43., single bond. stracturee (g), (h), (i), and (J) are favorable to the foraatien of precalciferol; this intersediate would result from rotation of the chain 6., c... 0., and C. and its attached groups about the ore. bond (insuchsdireetionastoplace 0,, c,,pc,, endC. inaplaneat right angles to, and behind, the plane of ring A) followed by stabili- sation of this configuration by a rearrsngment of the electrons accospanied by loss of excess energy, either by fluorescence or by collision. In a similar manner, the quinoid-type atruchu-cs (a) and (11) light undergo the same processes to fora precalciferol. It is also possible that during rearrangement of structures (a) and (n) ho Wrmmummwutmc.-c,wwtma nae stable taehrsterol structure when-echoed. Structures (Hand (l)e.lsadtotaeh7steroldireetlybyarotstionabcstflxec.-c, wwwammmcpc.m. StruchreeCh) “(unearth-stabilisadbresntributiaasfrosthewpmuptsd m~(o)m(p),nspeeumn thee-Who structu-es "Malaruammertefmoaflsiforoldirecflr. Wauwmmummfmcuufibetm ordering.rotationthilecnandc.areincloseprorimtr,oritlv umaftcprudcifmlisfcrudsimeouandfithnrsuinin close proximity. It is possible also, in a protonio solvent like ethanol, that before rearrange-en to the stable precalciferol structure Kcaneoeursprotoaisgivenuptothesolvutbyonandaprotonfron thesolvantistakuoubyce; Thelattereaseughtssplainthenre rapidhildupof caleiferelinethanolttnninthe other (non-pretense) solvents. In an event, it appears possible that oalciferol can be produced directly either fro: ergosterol a m- precalciferol. I is mentioned before, the conversion of ergosterol to ludaterol requires, due to stario factors, a nearly 360° relative rotation of thetsolargefrapsutsaboetthec.-O.bcnd,aftertheG.-cmbend isbrokan,toparsit the 01. astlvlg'ouptcinvert. This rotation unlypudbhifitumdbyspdrofmtatiomabout theO.-c.,bond. Thusstruotures(n)and(n)appeartobethoee through shich lasistml for-etion directly fros ergosterol is possible, since both permit, sithout further rearrangement, free rotation about the 65-0. and 0.4}, bonds. They both also possess a formal charge on 0., and m structures uhich would require a minimum of bond roe arrangement to fern lumisterol, after rotation and inversion has taken place. This complete inversion necessarily involves passing through structures closely approximating those of precalciferol and tachyeterol, so that stabilisation to these other intermediates may “intercept“ an excited molecule "bound for“ the lunaterol configur- ation. The Hop-Iggy; Ehgcitgd Singlet State! is mentioned previously, the ergosterol molecule upon absorption of a quantum of energy might find itself, alternatively, in a stable excited singlet state. This stable state is not different chemically from the normal ground state and oennot, of itself, lead to chemical change in the molecule. However, the excited molecule may undergo a radiationlees transition to another low-lying excited state which my be a triplet state. The triplet state is a di-radical and would be capable of producing themed. change. It is entirely possible that the ergosterol molecule wold be able to undergo the same types of rearrangements as postulated for the ionic excited state, with the driving force provided by the tn unpaired electrons of the dioradical (see the following section). There is a serious objection to the use of the di-redical mechan- ism as a total explanation of the observations. The radiationless transition tron “cited singlet to excited triplet state has a very in. probability. It mid, therefore, be expected that the quantum yield would be extranely low, cheese, in reality, the quantum yield is found to be rather near unity. The Shzglet Excited guteime Dissociatign Limit: Another reasonable possibility is that absorption may lead to a singlet * excited state at a level above the dissociation limit of some particu- lar bond in the mlecule. In such a situation, eithin the period of a single vibration (about 10"“ see.) the molecule will dissociate, at that bond, into two fragments. Becmse of the strain present at the 0,-010 bond, this bond till be particularly “sceptible to rupture. when, and if, such rupmre occurs, a rather unique situation exists; because of the ring structure of on molecule, the two fragments cannot fly apart as two independent free radicals, but they must instead remain in proximity through the remaining bond network of ring D. The (ii-radical state produced mat, according to the Spin Conservation Rule, be a singlet state, since we have postulated that the original mited state as a singlet (the triplet state is considered in the next section). in organic free radical would normally soon abstract a hydrogen from a nolecule in the solution or pick up oxygen, or some other free radical substance; the free radical here, however, is hindered by the bulky sterol structure, so its reactions are most probably intre-nolecular, although some ongen sensitivity has been mted. Warmers, energetic considerations strongly favor the participation of the two separate radical ends of the molecule in a coupled process-4.», one in which both ends become sinmltaneously satisfied. A consideration of the “conjugated resonance' structures 143 of the di-radical, structures which can be reached merely by re- arrangement of the existing electronic spin, reveals the following di-radicals: W i ‘ I , . eP ’ . “ : ‘2 Possible Diradical """' f ' Structures . . ‘f Normal Ergosterol m _.._., The last structures (6) and (f) may be referred to as "hypersonju- gative resonance" structures, and suggest the labilization of the. methyl hydrogen atoms, since these hydrogens are allylic to the conju- gated system and are hence reactive. The reunion of the odd electrons into a stable bond is, of course, a likely process, because the odd electron spins are already apposed. The reunion requires physical Juxtaposition of the atoms containing the odd electrons, and the reunited molecule need only lose some excess energy by collision to become a stable molecule. The divradicals pictured above are capable of undergoing a series or rearrange-ants, in a manner entirely analogous to those described in the section on the Ionic Excited Singlet State, to tone the irredi-b etion products of ergosterol: the odd electrons here replace the formal charges or the ionic states. There are ten tests which scold seen in opposition to this explana- tion. First, the appearances of the abeoiption spectra or «panel, lnnisterol, and tachysterol in this region all indicate a selladetined band structure characteristic of transitions to stable vibrational levels in the excited state: a transition above a dissociation limit should shes a continuous, not a banded absorption curve. Second, the bond rnphire theory cannot apply to tachystarol, since the head there is already broken, and the extremely high extinction coefficient there strongly favors the ionic eacited state in at least that one ease. It seems desirable, for the Ike of woposing the silplest possible theory, to reject an: explanation shioh requires use of parts ct tw rather different approaches, if a single approach can explain the same facts. The singlet mu above a dissociation limit picture sand appearcqableeteqlainingnoresultsmcheannotbeasplsinedby the ionic excited state theory alone. The Excited my; State: Since the direct transition from the nor-l ground state to an excited triplet state is a "forbidden“ transition and since the absorption coefficients of the conpounds involved are quite large, this mechanism is ruled out. 16 There is, hossver, at least one piece or evidence which indinates that there may be low-lying triplet states which are in certain cases reached thermally, although opticaJJy inaccessible.‘ consul, it presentinthesystenduringirradiation,1eadstoasnnanonntot side reaction which does not affect the overall yield of calcitercl appreciably (32). Since oxygen is a di-e-edicei it appears likely that some (Ii-radicals are torned thermally iron ergosterol or its irradi- ation products through loin-lying triplet states or by fission of the bond between 0, and 0,0. The dirradicals then undergo reaction with oxygen to produce side precincts. ' i lot-lying thermally accessible triplet state for precalciferol also offers a va'y plausible explanation for the thermal conversion of this compound to caleiferol. In sum, then, it may be stated that the ionic «cited state appears to be the only type of excited state which is able to give a morally acceptable picture or the observations. In addition, postna- lation of low-lying thermlly accessible triplet states provides an emulation of the oxygen sensitivity of the reaction and of the therml conversion of precalciferol to calciterol. The non-ionic excited singlet state, the optically excited triplet state, and the excited singlet state above the dissociation limit may all be ruled out as possible iiqaortant excited states. the first, became it re- quiresaradilationless transition to a triplet state (a 'i‘orbiddm' transition) before chemical change can occur; the second, because it remiree a singlet-excited triplet transition (also a “forbidden“ to transition): the third, because the spectra of a anchor of the com- pounds involved shoe well-defined band structure characteristic of transitions to stable vibrational levels in the excited state, and because it cannot explain either the reactions of tachyeterol, or its extremely high extinction coefficient. 6. solvent Effect on the Rgtion ' It has been sheen that the production or the various intermediates from ergosterol met involve rotations of rather bulky portions of the nolooule about certain bonds. It is, therefore, to be expected that the extent of formation of these intermediates would be dependent upon the my barrier presented by the solvait to movement of the rotat~ ing group. This enery barrier would manifest itseli‘ as a solvent specificity of the reaction-e specificity which say be apected to correlate, at least in part, with the viseuity of the solvent. The use of a bulk property, such as viscosity, in describing proceeseson a molecular scale, is not idthout some shortcomings. For example, in the case of alcohol, uhich shoes anasing specificity for the production or tachysterol, there is another effect, on a molecular scale, Ihich enhances the effect due to viscosity alone. Since alcohol is a hydrogen-bonded solvent, there are present, in the liquid, large aggregates of ethanol molecules. Molecular association within these clusters is much greater than between thm. Viscosity measures the interaction betesm aggregates, not the attractions within each cluster: the interactions in each cluster, not between clusters, probably h? deterlines the resistance to molecular notion of the type required for production of the irradiation products. Since precalciferol and tachysterol are the first irradiation pmchcts of ngosterol, and since further rotations are required to produce the other irradiation products, precalciferol and tachysterol should tend to be stabilised in more viscous solvents, in accord with the experimental results. the more rapid conversion to caloiferol in viscous solmte is due to the feet that precalciferol converts thermlly to caleiferol quite readily at, or slightly above recs tapes-stare; stabilisation of precalciferol facilitates hydrogen transfer and enhances oaloiferol formation. It should be pointed out that a less slew non-mechanistic explanation my given for the observed solvent effects. This explan- ation is based. on the fact that during irradiation the eaterial in the sells was not agitated, so that absorption of light neg have hem restrictedtealsyerof-terialnoxttothe cellvindov. The solvent effect could then be characterized as a viscosity dependent bulk diffusion phmn. The eork of Dealer (33) tends to weaken this explanation, believer, since he found that the specific solvent affect us also operative in solutions which were stirred during irradiation. Visoosities (36) of the solvents employed are given below: Viecoeitz (Centipoise) ' Diethyl Ether 0.222 (25°C. n-heocane 0.291; E2530. Cyclohmne 1.02 17 c. 95% Ethanol 2.35 (25°C. 148 D. Wavelength Dependence of the Reaction From preliminary re-eocamination of the data, in light of the recent work thich has demonstrated the inportance of precalciferol as a member of the irradiation sequence, it appears that precalciferol, rather than lumisterol, is the primary product of ergosterol irradi- ation at 29672 and 31322 (wavelengths at which the absorption of precalciferol is rather small). Since the absorption by precalciferol at these wavelengths is small in comparison to ergosterol and tachy~ sterol it is to be expected that buildup of precalciferol would occur. it shorter wavelengths, the buildup of tachystercl is defthelq‘ favored. The ratio of the absorption coefficients of tachysterol to those of ergosterol and its other irradiation products is much smaller at shorter wavelengths than at longer wavelengths of irradiation. Tachysterol tends , therefore, to build up to greater concentrations at the short uvelengths, since the fraction of the total incident radiation absorbed by tachysterol, and leading to its breakdown, is much smaller at shorter than at longer savelengths. It now appears that the wavelength dependence of the buildup of all the intermediates can be explained in a sixdlar nanner. 149 SUML'STIONS Fm FURTHER WRK The work here presented has suggested optimum conditions for preparation of the intermediates. These intermediates should be prepared and their ultraviolet absorption spectra carefully studied in several solvents, individually, and in mixtures with one another. The method of calculation used in this work should be improved saith the object of obtaining accurate and unique solutions of the data. The heats of combustion of all of the compounds in the irradi- ation chain should be accurately determined in order that information concerning the energy relationships which eccist between the various serpent-sis be obtained. Paramagnetic resonance absorption studies should be carried out - shils the photochendcal reaction is in progees in fluid or glassy media in order to determine whether or not triplet states are produced during the reaction. Study should be made of the fluorescence and phoephoreacence spectra, in rigid media, of all of the compounds involved in the irradiation chain, in an attempt to gain more information on the scary relationships between the stable molecules and their excited states. Model compounds should be studied in respect to their fluorescence, phosphorescence, ultraviolet spectra, and photochemical behaviour. The quan‘hm yields of all of the compounds from their pure procursors and from srgosterol should be determnod under various condition of solvent and irradiating molang’oh. The viscosity effect should be further studied, perhaps by using homologous serio- of solvents, or by varying tho “aperture to obtain s range of visoositios in each of the psrticulsr solvents and. The rosalts of this work suggest that a tub melongth irradiation might yield osloiforol in the irradiation mixture much less commi- nstod with etha- irradiation products than mud be possible from nor-J. irrsdistion nothods. Ergoaterol might be irradiated at 21633 insthmlup to thsuximhuildup of “wastrel; themixburefidzt than M irradiated um; 28021 or 29671 was now-rel tau-orbs "row: and solution]. only Wily. anm CITED 1. Sebrsll, U. 3., Jr., Harris, 3. (editors), Lb: Vitamins, Vol. II, The Academic Press, 1110., Nov York, 1951;. 2. Rossnhoim, 0., Webster, 1. 1., 310011911. J., g, 381 (1927). 3. Weddell, J., J. Biol. Chem, 192, 711 (1931.). h. cm”. a. x., Sci. J. Roy. con. Sci... 14, 1.1 (1931.). s. Hindus, 1., Laura, 11., Schenk, 1., 11:11., 522, 98 (1935). 6. Resrink, E. 3., vsn max, 1., Biochem. J., a, 1291; (1929). 7. mm, 1., Chan. Abs" 25, 132 (1931). 8. mm, 1., mm, x... Psmhols, 3., mm, an, 265 (1932). 90 W. As, Voniflardsr, F" LEW. ‘s’ mo. m, 188 10. Hindus, A" Goods, J... Kosor, J., Stein, 0., Lam, M2, 17 (1930). n. W, 1., 1.1mm, 1., 9013130, 14., Am... ygz, 252 (1931). 12. Hindus, L, suing-m, 3., z. physiol. chem, as, 108 (1931). 13. 70113:, L. Potit, 1., Hichsl, 0., Roussosn, 0., Capt. roads, ggg, 1287 (19148). 11.. Venus L., ram, 1., mm, 0., Bull. soc. chin. home, 1.5, nis (191.8). 15. Vsllus, x... mm, 0., Capt. mm, g_2_§, 692, 853 (191.9). 16. Venus, 1., mind, 0., Pesos, 11., Bull. soc. chin. Franco _1_§_, 501 (19119). 17. Hindus, 1., fitmm, 1., Russo, r., 1mm. Gas. win. Gottingon, hth. physik. Klssso, Fsohmppe EL 150 (1932). 18. Linsort, 0., U.- 8. Pstsnt Ho. 2,030,377 (1936). 19 . 20. 22. 23. 2h. 25. 26. 27. 28. 29. 30. 31. 32. 33. 313. 52 2:010:31)”, 0. 0., Frederick, W. 0., J. Am. Chen. 800., 2Q, 3261; 1932 . Venus, 1., word, 0., Bull. shim. soc. Promo, 11, 205 (1955). 1({a Sh , E., Bots, J. P. L., Roo. trsv. chin. Pays-43“, 11, 393 19 . Kosvost, 1. I... Vorloop, 1., linings, 3., 19g" 11,, 788 (1955). Veriooy, A., Koavoot, A. L., Havingo, 3., 11$... 11.}, 1129 (1955). linings, 1., Koovoot, A. 1... Yea-100p, 1., 11:19... 11,, 1230 (1955). Havings, E., Vuloop, 1., Komot, A. 1.,“ 3319., 12, 371 (1956). You do mono», J. L. 3., Wootorhor, P. Kevorling 8111mm, J. 1., Ratings, 3., 1mm, 35, 1179 (19565. I I figtfiogghgl? Bruckner, L, Model, 11., mm, 0., Bar. Q1 Inhoffen, H., Ramos" jg, 396 (1956). 5113' Ho Ho Co, Jami“, Jo Po, HOIt, 1'. En, Th. W. E 2.8 (1957). . Broads, n. 1., Whooloa‘, o. 3., J. Chou. Soc. tendon, 320 (1955). Inhofton, 3., Bracknar, 1., «31.1.1, 3., Bar. £31, 1 (1951.). Amos, '1'. 0., 333., Prue. Roy. Boo. (London), 3108, 31.0 (1931). Doslor, U., Sumorios o! Dootorsl Dissertations, Univ. Wisconsin 1, 219 (1933). Handbook of Chodstry and Physics, Chemical Rubber Com, ' Clovolsnd, Ohio, 19117. MTCHTGAN STATE Ua‘iiV'RSITY OF AGRICULTURE AM) A1”? .121) SJTENCE DEPARTMEaT OF CHEMISTRY EAST LANSING, MTQHIGAN WTSTM LTBRARY MTCHTGAN STATE UNIVERSTTY UF AGRTCULTURE AND APPLIED SCIENCE DEPARTMENT OF CHEMISTRY EAST LANSING, memos»