465
V O L U M E 20, NO. 5, M A Y 1 9 4 8 method, each asphalt constituent is recovered and may be used for furt,her study. T h e met,hod has the disadvantage whichis common to other proposed mothods, that only arbitrary fract,ions are obt,ained. ACKKOWLEDGMENT
This report represents the rrsulta of work done under a cooperative agreement’ b e t w w i t h r s Bureau of ?\lines, United States Departmentmof t h e Interior, arid the University of TJ-yoming. LITERATURE CITED
(1) dbrahain, Herhert, “.isphalts and Allied Substanies,” 5th ed., Vol. 2. u. 1224. New York. D. Van Xostrand Co.. 1945. (2) Batchelder, -1.H., and \Yellman, H. B., Proc. Am. Pet/olettm Inst., 19,83-6 (1938,. (8) Beckman, 1.0..Badger, 11. M., Gullekaon, E . I-;., and Stevenson, D. P., I n d . Eng.C‘hem., 33,984-90 (1941). (4) Grant. F. H , and Hoibere. A . J., Proc. Assoc. Asphait Paving Trchnol.. 12.87-122 (Derenlher 1940). (5) Hillman, E. S., and Barnett, B., h‘pfinw, 16, 362-6 (1937). (6) Ibid., 18, 533-8 (1939). (7) Hoiberg, A. J., and Gairis, W.E., Jr., ISD.Evb. Crmnr., ANAL ED.,16,294-302 (1944). ( 8 ) Hoiberg, A. J., Hougen, 0. A., and Zapata. Joseph, Univ. Vis., Eng. ExCt. Sta., B u l l . 86, 67 (1939). (9) IIolde, D. ( t r . by E. Mueller), “Examination of Hydrocarbon Oils and Saponifiable Fats and Waxez,” 2nd ed., pp. 106-8, New Yovk, John Wiley & Sons, 1922.
(10) Holmes, A., and Raphael, A. L., Proc. Assoc. Asphalt Paving Technol., 8 , 105-16 (January 1937). (11) Kalichevsky, Y.,and Fulton, S. C., S a t l . Petroleum S e w s , 23, 33-6 (1931). (12) Knowles, E. C., and Levin, H a I i y , IND.ENG.CHEM.,ANAL.ED., 13, 314-17 (1941). (13) Kreitsei, G. D., and Pneva, L. d.,Byi~lZ.Obmen. O p y t . Lakokrasoch Prom., No. 1, 16-17 (1940). (14) Latig, I?. C., and Thomas, T . W., L-niv. Minn. Eng. Expt. Sta., Bull. 15 (1939). (15) Maass, IT,, Proc. World Pet?oleitm Congr., 2, 557-68 (1933). (16) Marcusson, Julius, “Die natarlichen und kanstliehen Asphalte,” 2nd ed., Leipaig, Verlag von Tilhelm Engelmann, 1931. (17) I*;ellenstein. F. J.. and Kuioers. J. P., J. Inst. Petrolezm. 26. 401-6 (1940). (18) Rosumny, M.,and de Rosset, A., Roads and S t r e e t s , 81, 38-40 I193XI.
(19) Stanfield. K. E.. Bui. Mines, K e p t . Iniest. 3435 (1939). (20) Ibad., 3568 (1941). (21) Stanfield, K . E., and Hubhard, H. L., Bur. Mines Tech. Paper, in preparation. (22) Strieter, 0. G., .f. Resrarch S a t l . Bitr. Standards, 26, 415-18 (May 1941). (23) Suida, H., and Mota, F,, P r t i o l e i t m Z . , 35,527-33 (1939). (24) Thuiston, 1%. R., and Knowles, E. C., I n d . Eny. Chem., 33, 320-
4 (1941). RECEIVEDX a y 39, lM47.
I i q i r o r e d for piiti!ication
tiy
tiit.
Direcror,
Bureau of llines.
Vitamin A Acetate as a Vitamin A Standard N. B. GUEIZKAUT, TI. E. CHILCOTE, €€, A , ELLENBEHGEH, AYI)
K.
\. DITC€IER
The Pennsylvania State College, State College, P a .
Hatches of crjstalline bitamin 2 acetate habing uniform extinction coefficients were prepared repeatedlj from a high-potenc? halibut \iscera oil distillate bj the procedure suggested by IIeng. Purification studies with the cr) stalline material revealed that a product haling a constant extinction Talue and a constant melting point could be obtained through two or three recrjstallizations. Stabilitj studies with the undissol\ed cr?stals and with the crjstuls clissolted i n refined deodori~etl cottonseed oil, in c o r n oil. arid in peunut oil hholred this ester of the
UlIEItOl-S scientific articles havc t w r i publishtd dut,ing the past decade which contained espc.rimerita1 eviderict that cod livrr oil i+ not a satisfactory viiariiiii .standard, (w’n though the oil has IXYW srlecttd with great care and ha,s ht,cn handled and st oi,tJtlutitl(1r c.ontlitions favoring vitamin i,ct ciition. y 193:) (8) iriciic.itted that thc Observations rqmi?txl i i . ~c ~ l as U.S.P.rr~frrrncc cod li\er oil S o . 1 had txvn inaccurately standardized against thcx intornational stsndat~tl01’ that its vitamin =\ potency had d(~tc~iioi~ated with time. Studiw i , c p o i , t t d l)y 1IcFai.lan rt ai. (9) i‘urthct~indi(~att‘dt h a t while freshly opened hot tlw of U.S.1’. reference cod liver oil S o . 1 yir.ldd al)sot,ption values in good agreement with those reported by other csperinic~nters,there was a continuous change i n the ahsorption vslur~of the oil during its use in the laboratory. Subsequent studies by (’oy et al. (4jshowed that this decrease in the ultraviolet absorption value of the oil, in the partially filled bottles, took place even n-hen the bottles were flushed with carbon dioxide and stored under refrigeration. Soon after U.S.P. reference cod liver oil S o . 2 became official, data began to appcar in the scientific literature which showed that A\
\ i taniin to be relati\ el) stable when stored in bacuuni at room temperature and under refrigeration and also when stored in nitrogen under refrigeration. Both the crjstalline witamin and its oily solutions were less stable in an atmosphere of nitrogen than in l a c u u m when stored at room temperature. Data thus far obtained indicate that litamin A acetate offers definite adtantages, as a bitamin A standard, o\er L.S.P. reference cod liter oil and over conimerciallj aiailable cr) stalline titamin A alcohol and 8-carotene. Stitdie9 are being continued.
this oil \vas also uiiruital)lc*as a vitaniin -4standard [ llorgareidge ( I I ) , (’oy et al. (j), Oser et a l . ( I c ? ) , Zscheilc et (I[. ( I ? ) , and (‘allison ct a l . ( 3 1 . These roportP indicaated that this particular vitamin -4 stpntlard tx.causrx of its apparent ed by t)iolotical assav aud I)? ultraviolet ahsorption nieasurementa. As H rcsult, 1;.S.P. refercnce cod liver oil S O .3 kwcarncl official during the kltter part of 1944. I A e I-.S.P. rcfetwicr oils I and 2, oil So. 3 also appears unsatisfactory as a vitamin -1standard. Thus a more stablc and reliable standard \vas needed. Gridgeman ( 7 ) has summarized the objections t o c o d liver oils as a vitamin -1standard: (’oil liver oils at’e difficult t o assay Ily because of the presence of much estraneous 11 when the estraneous absorption is removed, the residual speetrosropic characteristics are not necessarily those of normal vitamin -4;and very strict precrutions are necessary if cod liver oils are to be kept without change for any length of time. Because of these seemingly inherent characteristics of cod liver oils it appears that fish liver oils, in general, are unsuited for use as a vitamin A standard; hence one must look elselvhere for
466
ANALYTICAL CHEMISTRY
a more reliable standard. The present report describes some of the studies which have been carried out in search of such a standard. @-CAROTENE
In 1931, the Permanent Commission on Biological Standardization of the League of Sations (Health Section) set up an international unit of vitamin A potency which was based on the biological activity of 1 microgram of crystalline carotene, the pooled product of seven different laboratories. With the advance in the knowledge of the chemistry of the carotenoids, the international unit of vitamin h potency became more specific in 1934 when the above organization defined the unit as the biological mtivity of 0.6 microgram of pure 8-carotene. As p-carotene has been used as the international standard of vitamin A potency from 1934 and Callison and Orent-Keiles ( 3 ) had recommended it as a standard in preference to U.S.P. reference cod liver oil S o . 2, i t appeared to be the logical material for further investigation. This seemed to be true regardless of the fact t,hat 8-carotene is difficult to obtain and to preserve in the pure form a,nd t h a t much remains to bc 1t:arned concerning the conversion of 4carotene to vitamin A in the animal body. Oviing to the difficulty encountered by various investigators in preparing absolutely pure 8-carotene in the crystalline state, the present studies were restricted to a commercially available crystalline product which had been packed under vacuum in small glass ampoules. Because of its supposedly high degree of purity, its commercii1 availability, and its rather general usage in biological laboratories, this material seemed t o offer possibilities as a vitamin A standard. Therefore, samples of this carotene were obtained and subjected to spectrophotometric examination in order to ascert,ain its uniformity and, if possible, its stability. The carotene was purchased directly from the commercial source, usually in quantities ranging from one to six (10-nig.) ampoules. The various shipments of carotene were examined spectrophotometrically rvhen reccived B t the laboratory or soon thereafter. I n the meantime the ampoules of carotene reniainedstorcd in a refrigerator in the absence of light. In carrying out the spectrophotonietric measurenients, the carotene n-as immediately removed from the opened ampoule, weighed on a microbalance, dissolved in petroleum ether (boiling point 35" to 69" C.), and made to volume with that solvent. From the stock solution thus prepared, a series of ten dilutions was made which were calculated to range in concentration from 0.5 t o 3.5 micrograms of carotene per nil., using petroleum ether as the diluent. Caution was taken not' to expose the crystalline caarotene or the carotene solutions to light or to prolonged standing before carrying out the absorption measurements. The absorption a t 450 nip of each of the ten solutions of carotene was measured by means of a Beckman quartz spectrophotometer, using matched corex cells and a slit nidt,h of 0.2 mni. From the absorption data the 450 m p was calculated. The mean computed values for some of the samples of carotene examined :we given in Table I. When it became apparent that the first ampoule of carotene [control 8,ampoule l ( a ) ] under investigation showed a n 450 mp considerGbly 'lower than t h a t attributable to pure 4carotene, spectrophotometric measurements were carried out on a second series of solutions made from another port,ion of crystals from the same ampoule. Because the mean absorption value of t,he second portion of crystalline carotene was in good agreement with that of the first portion, i t was concluded that the experimental technique employed was reasonably reliable, a t least in so far as reproducibility of data was concerned. As the result of examining sixteen different ampoules of this' carotene, over a period of 18 months, the variability of the product became more fully apparent, with t'he carotene from some ampoules showing approximately 267, less absorption than that from other ampoules. Only two ampoules of the carotene, and these bearing the same control number ( B ) ,showed a n absorption value ap-
proaching that ascribed to pure 6-carotene. While thew were‘ variations in the absorption value of carotene from axnpoulee bearing the szme cont'rol number, the greatest variationsappeared to be between the absorption values of the ampoules of carotene bearing different control numbers. Although the d a t r do not explain why the absorption characteristics of the carotene s u m p l e ~ were so variable, it is evident that present supplicr of cai'oteiic will not serve as a reliable vitaniin A st,andarti. VITAiMIIV A ALCOHOL
Hccause the esterified form of vitsmin h as well as thc. vitamin -1 derived from the provitamin (carotene) apparently passes through the alcohol form i n the *process of metabolism in tht, animal body, it would seem that pure vitxniin A alcohol should constitute an ideal vitamin h standard, f r o m the spectrophotiimetric as well as the biological standpoint, provided this form of the vitsniiii possessed thr rcquired characteristics as to purit>and sta.bility. Inasmuch as crystalline vitamin -1alcohol was available from commercial sources, it was clc&kd that a scJries of absorption measurements should be niaticb on different ampoulcs of this material for the purpose of ascertaining the uniforniit.y Of the available product, although biological tests i n the 1aboratoi.y had already indicated that it \vas unst:tblr whtw used under assay conditions eoinparal)l(~tu t how r.c.c.iinnnic,ridrcl h y t h e (-. Is. Pharmaviipwia S I I , Thirteen ampoultss of c line vitaniiii .I alcohol \rere 1 1 ~ 1 ' chastd during a period of nths for the absorption tests. . i t least one ampoule bearing c w h control number was c,saniinetl immediately on being recrivc,d at the laboratory, while othcr anipoul(Ls of the crystalliric~vitamiti were stortd under refrigeratioil antl cxamined sprctri)photc)nic,tricallyat R latei, date. In carryinE out the absorption measurenic~nts,a portion of the cq-stalline vitamin n-as taken from the freshly opentd ampoule, wighed on a microtialanccl, dissolved in isopropaiiol, ant1 made to volume with this solvmt. From this stock s,Jlution, ten diIutions \ v ( ~ wprepared having a calculated concctitrtttion of vitamin A alwho1 ranging from 0.5 to 3.5 micrograms p~ ml.,using isopropa1: uaual prerautiorij \vew taken to aroid line vitamin or tlir preparcd solutioris t o nding before the ahsorption measurements were carried out. The absorption mcwwrcments were iiiatlc on a Beckman quartz spectrophoto~iietci,at a ~vavcslength of 328 mp, while using niptchcd quai,tz cells antl a slit width oi 0.4 mm. The instrument \vas so a d j u ~ t e dthat t l i r solvc~ntgavcx a 100'; transmittance. From thealisorption data thrx 328 nipvalues \v~r(xcalculatcd. Th(x n i ( ~ value i for the ten dilutions prepared lline vitamin is likcn-ixc pnwntcd i i i from each vial of the ( Table I.
Table 1. 1-ariations in Extinction Coefficients or Ampoules of Crystalline P-Carotene and Cr?staIIine \ itamin 4 ilcohol Determined bj Beckrnan Q u a r t 7 Spectrophotometer" 1 itaimn @-Carotene 111 Petroleriin 1:thrr El% conAm1 ctn. trol poule Date of 4.50 So.
KO.
4 B
Control
i l < o h o l in Iwprot,\ I ilcohol _______ B1'f Ain1 cm, ~ioule Date of 328 \
So
assay
iiip
So.
8 / 1/44 8/ 2/44 8/ 9/44 12/30 '44 12/27/43 12/28/44 12/28/44 12/28/44 12/28/41, 12/28/45 12/28/45 12/29/45 12/29 145 12/29/45 12/29/4R 12/29/45 12/29/45
1900 1880 1896 1958 2554 2536 24'73 2330 2403 2347 2366 2407 2381 2458 2170 2223 2390
S
i 7/44
0
?'13/44
0 0 0 0 0 1' 1' P P 1' 1'
Q Q
Q
Q
assay
7'14f44 7 '31 144 7,31144 6 24/47 b '24 I47 IOfI8 45 10 30145 11/8/45 11/14/45 6/ 23/ 47 6/23/47 12/28/44 12/28/44 6/23/47 6 / 2 3 147
m u
1784 1428 1460 1652 1670 1444 1462 1574 1484 1346 1420 1362 1326 1741 1780 1653 166b
AT-erage E;'?,, values for ten dilutions of each asniple ranging in con(.entration from 3.5 t o 0.5 microgram per ml. 0
V O L U M E 20, N O . 5, M A Y 1 9 4 8
467
Effect of Recrystallization of V i t a m i n A A c e t a t e on Extinction Coefficient a n d TIelting P o i n t
T a h l e 11.
E'%
No. of Crmtallization, 1 2 3
?
1 c m . 325 r i i ~ ~ Crvstallized from Ethyl Methyl forinate alcohol 1153 12iS
1,512 1.51ti
..
1385 1.719
1.518 1515
~ ,
Melting Point Range. Cry-talliwd from Ethyl Methyl formate alcohol .jj:lj156 . o 97 7-58,? 57.8-.59.0
..
34:315j . 3 57: 6 L i S .9
37.4-58.8
f fer(' again duplicate dt~tcriniiiittioiison a portion of the tdlinc vitamin f ~ ~ o nthe, i same ampoulc [portions (a) and ( b ) ] yielded absorptioii data which show reasonably good agreenient. ( I f the thirteen ampouIes of vit,aniin .I alcohol examined, only t l i r c ~welt' . found t n h a w absorption values equal to that, ascribed to pure vit,ariiin -\ :tli~trhol(1750); t n o of these ampoules bore one rwntrol 11~unberanti t h c b third bore a swond control nurnt~csr. ficrc~ito;ain thr ahsoi,ption data sho\v a somewhat greater variat ion ht~t\vec~n thr :tlliplJules of the crystalline vitamin bearing ilifferrnt control iiumt~crst,han betn-ren the different, ampoules of t h i s vitamin healing the same control number. Ampoukbs o f t k cryst:~,llinc vitaniiil Irhich had beqi stored at 35" F. for :tlnwst 3 yc'are yic.ldrtf a1)sorption valucs ( q u a l to or only slightly lorver than those of the contents of cwmparable ampoules when ~iurc~hitwti.\Thile t lict orption data do not explain why the alline vitamin vary so xidely in this rlifferont ampowlrs of 1)hysical charactcristica, they do supprst that the purity of the r,rystalliii(~product was not the same \\-hen placed in the various :~inpoiilwor that perhaps deterioration had taken place. It was c~onr~ludt~d that this source of crystalline vitamin A alcohol \voultl iiot constit,utc a iy~lial)lt~ vitamin A standard, owing l o the variof tiiffer(~nt:tmporiles. :3l)ility i ) f the' i~ontc~nt,. \
ITAMIS A . u x : I - A n :
Iiiitriiiucli a i h \ t t . r and Rut)esori (1 I had found vitamin .I ti' to he, ratlitlr c1:tsily preparcd from the vitamin -4' alcohol :ind to be t h e niost stitble crystallinrb wter of vitimin A thus far i,c:portrld, attentioil I)waiiie focused on this ester as a possible vitamin A stand:ii,d. This interest \vas increased when 3Ieng i j O i wported a proct.tiure for preparing the acetic ester of the vita,min directly from high-potchiicy natural ester distillates, thcreby incartiasing thv possibility of conducting detailed collaborativt~studivr rc-lativc. to the physical and biological properties of vitamin .-i itccstatr. I n i)idvr to ci)nfirm tht. hidings o f 13astc.r and Robeson ( 1 ) and to ascwtain thtl suitahility of this ester as a vitamin A standard, -L: supply of natural vitamin AIester concentrate, prepared by the molecular distillation of halibut viscera oil 'supplied through thcl courtesy of Distillation Products, Inc., Rochester, N . T.) \ v a ~obtained a s the stprting material. The potency of the concciitrate a s determined by the Carr-Price reaction and by direct spectrophotometric measurement was found to be approximately 1,000,000 U.S.P. units per gram. Through experimentation it !vas found t h a t the formation and the isolation of crystalline vitamin -4 a,cetate could be most effectively carried out by following a procedure essentially as outlined by Neng (10) and by tipplying some of the techniques described in detail by Baxt,er and 1ti)t)esoii ( 2 ) . The gcneral procedure was as follows: The vitamin *Iconcentrate was saponified, the nonsaponifiable nuttter was recovered and dissolved in methyl alcohol (15% sohtion), and the alcoholic solution was subjected to a two-stage de.*tcrolation. (Ethyl formate, the solvent suggested by Meng, did iiot prove especially useful in dest,erolating this particular vitamin A concentrate, owing to the presence of a dark red oilv fraction which subsequently interfered with the crystallization of
This dark red oily fraction was found to thy1 glcohol.) The first stage of desterolation was effected a t a temperature of -20" C. and the second stage at a temperature of -70" C'. The met>hylalcohol was removed from the desterolated concentrate by vacuum distillation and the resulting concentrate dissolved in pyridine for acetylation. The acetylation was carried out by adding successive small portions of acetic anhydride and cooling so as to minimize destruction of the vitamin. The acetylated concentrate !vas then maintained a t a temperature of 60" to 65' C. for about 30 minutes, then diluted with water, and the acetylated vitamin v a s removed by a series of extractions with diethyl ether. The combined ether extract was purified by washing with water, dilute hydrochloric acid, and sodium bicarbonate solution a s suggested by Meng (10). The ether solution of the concentrate x a s dried,over anhydrous sodium sulfate and the ether subsequently removed by vacuum distillation. The yield a t this stage was usually about 75% of the vitamin -4 originally present in the concentrate. The ether-free concentrate from the acetylation reaction was dissolved in a mixture consisting of equal parts of ethyl forniate and methyl alcohol a t the rate of 3 parts of concentrate to 2 parts of the solvent. The resulting solution was cooled, seeded x i t h crystals of vitamin A acetate, and placed in a freezing unit a t -20" C. until crystallization took plarr. This usually required about, 5 days for completion.
Recrystallization of Vitamin A Acetate. Because vitamin A acetate cf maximal purity was desired, and the initial crystallization produced a yellow colored amorphous precipitate which had a relatively low 325 mp when dissolved in isopropanol, it, was necespary to subject the product to recrystallization. Consequentl?-, several recrystallization experiments ivere carried out', and the purity of the crystals from w c h crystallization !vas tested by their ultraviolet absorption characteristic when dissolved in isopropanol. The niclting point of each crop of crystals was also determined. In the first experiment, a batch of previously crystallized vitamin A acetate (one crystallization) was recrystallized three times from ethj-1 formate (1.5 ml. of solvent per gram of crystals) a t -20" C. In other experiments, a similar batch of previously crystallized vitamin .4 acetate was recrystallized four times from methyl alcohol (6 ml. of solvent per gram of crystals) a t 5' C. The crystals were always removed from tht: mother liquor by filtering through a precooled Biichner funnel, washed with a small amount of the cold solvent, and dried under a moderat,e vacuum, and the desired portions of crystals 11-ere transferred to 9 nim. X .ere sealed onto a glass 15 em. glass ampoules. These ampoul manifold-type vacuum system, and the tem was evacuated t o a pressure of 3 to 5 microns, as measur,ed a McLeod gape. After being held a t this low pressure for at least 0.5 hour, the capsules were senlcd off and storwl at 5' C. i n thC dark.
The data prcscnted iu Table I1 s h i v that in both experiinonts three successive crystallizations oi tlw product brought about thc maxima1 purity attainahltx under thvse conditions, as indicated by the ultraviolet ahsorption and by melting point measurenients. I n both experiments the color of tlie crystals diminished with subsequent recrystallizationup to and inrluding the fourth crystallization. The use of rthyl formate a.q the solvent resulted in this production of more lightly colored (pale lemon yellow) crystals. The final over-all yield after three crj-stallizations amounted ti) approximately 7.5%. of the original vitamin X present in the concentrate. Baxter and Robeson ( 1 ) had previously noted that production of crystalline vitamin acctate from distilled ester concentrates resultcd i n sharply reduced yields as compared to talline vitamin A alcohol. Perhaps this may bo dur, in part, to thv presence of ricv-vitamin A in the concentrate (14). Reproducibility of Product. Close conforniancc to a dcfinite set of physical, chemical, and biological characteristics is a necessary attribute of a satisiactory assay standard. T o test the relative purity of the various batches of c r y s t a l h e vitamin acetate thus prepared, ultraviolet ahsorption measurements were made on portions of five batches of crystals. One of the five batches had been recrystallized from a combination of odd-lot portions of previously (one crystallization) cr)-stallized vitamin
468
ANALYTICAL CHEMISTRY
Table I l l .
Reproducibility of A Acetate
325 nip of Vitamin
325mr Date of Crystallized lcm. Crystallization from in Isopropanol 1 12/17/46 E t h y l formate 1515 2 11 2/47 hIethyl alcohol 1517 3 1/29/47 Ethyl formate 1533 4 1/29/47 E t h y l formate 1514 3 2/14/47 E t h y l formate 1521" hrithmetic mean 1520 Standard deviation 7.7 Coefficient of variation 0.5 Average of twelve values obtained b y testing twelve different samples of crystals from this batch (coefficient of variation = 0.7). Batch No.
Table IV.
Sonie Physical Constants of Vitamin A Acetate fi1%
325 w ~ : ? r n . 6 ~ 0 n l y LIeltiny Ethyl Carr-Price Pzint. Source of D a t a alcohol Reaction'L C. This laboratory, h h 1545 4210 57,6-58.9 This laboratory, B e 1545 4220 57.7-58.8 Baxter and Robesond 1510 4090 57.0-58.0 Oser et al:! 1570 .. , . a Calculated as vitamin A alcohol equivalent of crystalline acetate b Own prelmration. C Supplied for collaborative studies. d Absorption measurements made a t 328 my. Isopropyl alcohol 1520 1510
h acetate from five different batches of concentrate. The resulting data, included in Table 111, show that very close agreement was attained as regards the tested characteristics. D a t a on a typical sample of crystalline vitamin h acetatca prepared in this laboratory, and on the samples of crystalline vitamin -1acetate supplied for the U.S.1'. collaborative study (16) and on crystalline vitamin h acetate as reported b) other investigators, are given in Table IV.
oily solutions, in vacuum and in nitrogen, a t room temperature and under refrigeration, indicated t h a t the acetate ester of the vitamin )vas reasonably stable. This form of the vitamin was found to be stable when stored in vacuum and when stored in nitrogen under refrigeration. However, when stored in nitrogen a t room temperature there \vas marked evidence of deterioration with time, especially when the crystalline acetate was dissolved in peanut oil. ;ilthough efforts were made to purify the nitrogen used in these studies, there is a remote possibility that traces of osygen remained in the gas and thereby contributed to the instability of the vitamin. The peanut oil employed may not have been representative of t h r most satisfactory pea.nut oil obtainable for this particular purposr. It n-as purc*lirtsedthrough a chemical supply source. As a Tvhole, the data thus far obtaintd strongly indicate t h a t vitamin A acrtate possesses sonic of thc most significant characteristics desired in a vit,amin A standard. I t appears to offer distinct advantages in these respects over the P. Y. P. reference cod liver oil, the currently available crystalline 8-carotene, and the crystalline vitamin =\ alcohol.
Table V. Stability of Vitamin A Acetate in Crystalline State and in Solution as Indicated by Extinction Coefficients (E:?~], 323 nip) .rilllein Stored a t Room Yeinperature Storage, Under In Days nitrogen vacuum 0 30
1-20
Stored in Under nitrogen
I n crystalline state 1522 1522a 1538 1528, 1512" *'1466, 1474b 1522 1532" ,1268, 1254" .. 1529, 1522 ,1464, 1462a !1489!~ 1522n
Refrigerator In vacuun~ 1522 I510
.. 1611,1516
I n cottonsred oil
5.31 5.31 0 5.31 5.31 Stability of Vitamin A Acetate. As a satisfactory vitamin -1 5.32 5.29 31 5.18 5.34 standard must withstand storage during the period of essay ,4..53,4.93 5,27 5,16,3.24 .. without deterioration, it seemed desirable to test the stability '4.90 5.19 .5.21 117 4.61,4.82 5.16 c f vitamin A acetate under conditions simulating those actually encountered during biological assay. I n corn oil Portions of the vitamin crystals (4 to 7 mg.) as well as oil solutions (250 mg.) of the crystals !\-ere sealed in glass ampoules and stored in the absence of light a t room temperatures (25" to 30" allineacctate p r e p a i ~ din this lahoratory. This T,i'2,n. 620 mp value of 1210 for the d i d illctl ester conccntratc was calculated from its content 0 1 vitamin .1 as detrrmiued by reference of its EiFl,i,325 nip valuc~to thc correspondiiig vo,lue for pure crystalline vitamin -1ncetittcx. T h e iiir-est i-:ttion is 1)oinp coiitinurd. I t will eventually include, in addition to thc studies already mentioned, stabilit) studicr oii vitamin A acetate when dissolved in those organic solvents most ccninio~ilyuscd in spectrol,hotornetl.ic tests and othrr studies relating tu the acceptability of this form of the vitamin as a methods of assay. stantlard in biological a n d i i i cht:n~ic~oI-,li?-sical
(1941).
I b i d . , 15, 441-3 ( 1 9 4 3 ) . Embree, N. D., Oil and S o a p , 2 3 , 2 7 5 - 6 (1946). Gridgeman, N. T., "Estimation of Vitamin A," pp. 1-T4, Lon-
don, Lever Brothers and Unilerer, 1944. Hume, E. M., S a t u r e , 143, 22-3 (1939). SIcFarlan; It. L., Bates, P. K., and Merrill, E. C., ISD. E ~ G . CHEX.,AR-AL. ED.,12, 645-7 ( 1 9 4 0 ) . Meng, K . H., private communication, I
