5
VOLUME 20, NO. 5, MAY 1948 method, each asphalt constituent is recovered and may be used for furt,her study. The 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 coopera- tive agreement’ betwwi thrs Bureau of ?\lines, United States Departmentm of the 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. 465 (10) Holmes, A., and Raphael, A. L., Proc. Assoc. Asphalt Paving (11) Kalichevsky, Y., and Fulton, S. C., Satl. Petroleum Sews, 23, (12) Knowles, E. C., and Levin, HaIiy, IND. ENG. CHEM., ANAL. ED., (13) Kreitsei, G. D., and Pneva, L. d., Byi~lZ. Obmen. Opyt. Lako- (14) Latig, I?. C., and Thomas, T. W., L-niv. Minn. Eng. Expt. Sta., (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. Technol., 8, 105-16 (January 1937). 33-6 (1931). 13, 314-17 (1941). krasoch Prom., No. 1, 16-17 (1940). Bull. 15 (1939). (2) Batchelder, -1. H., and \Yellman, H. B., Proc. Am. Pet/olettm (8) Beckman, 1. 0.. Badger, 11. M., Gullekaon, E. I-;., and Steven- (4) Grant. F. H , and Hoibere. A. J., Proc. Assoc. Asphait Paving Inst., 19, 83-6 (1938,. son, D. P., Ind. Eng. C‘hem., 33,984-90 (1941). Trchnol.. 12.87-122 (Derenlher 1940). (5) Hillman, E. S., and Barnett, B., h‘pfinw, 16, 362-6 (1937). (7) Hoiberg, A. J., and Gairis, W. E., Jr., ISD. Evb. Crmnr., ANAL (6) Ibid., 18, 533-8 (1939). ED., 16,294-302 (1944). (8) Hoiberg, A. J., Hougen, 0. A., and Zapata. Joseph, Univ. Vis., Eng. ExCt. Sta., Bull. 86, 67 (1939). (9) IIolde, D. (tr. by E. Mueller), “Examination of Hydrocarbon Oils and Saponifiable Fats and Waxez,” 2nd ed., pp. 106-8, New Yovk, John Wiley & Sons, 1922. 401-6 (1940). I193XI. (18) Rosumny, M., and de Rosset, A., Roads and Streets, 81, 38-40 (19) Stanfield. K. E.. Bui. Mines, Kept. Iniest. 3435 (1939). (20) Ibad., 3568 (1941). (21) Stanfield, K. E., and Hubhard, H. L., Bur. Mines Tech. Paper, in preparation. (May 1941). (22) Strieter, 0. G., .f. Resrarch Satl. Bitr. Standards, 26, 415-18 (23) Suida, H., and Mota, F,, PrtioleitmZ., 35,527-33 (1939). (24) Thuiston, 1%. R., and Knowles, E. C., Ind. Eny. Chem., 33, 320- 4 (1941). RECEIVED Xay 39, lM47. Iiqirored 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, Pa. Hatches of crjstalline bitamin 2 acetate habing uni- form 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 in refined deodori~etlcottonseed oil, in corn oil. arid in peunut oil hholred this ester of the UlIEItOl-S scientific articles havc twri publishtd dut,ing the past decade which contained espc.rimerita1 eviderict that cod livrr oil i+ not a satisfactory viiariiiii A\ .standard, (w’n though the oil has IXYW srlecttd with great care and ha,s ht,cn handled and st oi,tJtl utitl(1r c.ontlitions favoring vitamin i,ct ciition. Observations rqmi?txl ii.~ c~ly as 193:) (8) iriciic.itted that thc U.S.P. rr~frrrncc cod li\er oil So. 1 had txvn inaccurately standardized against thcx intornational stsndat~tl 01’ that its vita- min =\ potency had d(~tc~iioi~ated with time. Studiw i,cpoi,ttd l)y 1IcFai.lan rt ai. (9) i‘urthct~ indi(~att‘d that while freshly opened hot tlw of U.S.1’. reference cod liver oil So. 1 yir.ldd al)sot,ption values in good agreement with those reported by other csperinic~nters, there was a continuous change in the ahsorption vslur~ of the oil during its use in the laboratory. Subsequent studies by (’oy et al. (4j showed 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 So. 2 became official, data began to appcar in the scientific literature which showed that \ 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 lacuum 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 coni- merciallj aiailable cr) stalline titamin A alcohol and 8-carotene. Stitdie9 are being continued. this oil \vas also uiiruital)lc* as a vitaniin -4 standard [ llorgareidge (II), (’oy et al. (j), Oser et al. (Ic?), Zscheilc et (I[. (I?), and (‘allison ct al. (31. 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 SO. 3 kwcarncl official during the kltter part of 1944. IAe I-.S.P. rcfetwicr oils I and 2, oil So. 3 also appears un- satisfactory as a vitamin -1 standard. Thus a more stablc and reliable standard \vas needed. Gridgeman (7) has summarized the objections to cod liver oils as a vitamin -1 standard: (’oil liver oils at’e difficult to 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

Vitamin A Acetate as Vitamin A Standard

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V O L U M E 20, NO. 5, M A Y 1948

method, each asphalt constituent is recovered and may be used for furt,her study. The 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 coopera- tive agreement’ be twwi t h r s Bureau of ?\lines, United States Departmentm of the 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.

465

(10) Holmes, A., and Raphael, A. L., Proc. Assoc. Asphal t Paving

(11) Kalichevsky, Y., and Fulton, S. C., S a t l . Petroleum S e w s , 23,

(12) Knowles, E. C., and Levin, HaIiy, IND. ENG. CHEM., ANAL. ED.,

(13) Kreitsei, G. D., and Pneva, L. d., Byi~lZ. Obmen. Opyt . Lako-

(14) Latig, I?. C., and Thomas, T . W., L-niv. Minn. Eng. Expt. Sta.,

(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.

Technol., 8 , 105-16 (January 1937).

33-6 (1931).

13, 314-17 (1941).

krasoch Prom., No. 1, 16-17 (1940).

Bull. 15 (1939).

(2) Batchelder, -1. H., and \Yellman, H. B., Proc. Am. Pet/olettm

(8) Beckman, 1. 0.. Badger, 11. M., Gullekaon, E. I-;., and Steven-

(4) Grant. F. H , and Hoibere. A. J., Proc. Assoc. Asphai t Paving

Inst., 19, 83-6 (1938,.

son, D. P., I n d . Eng. C‘hem., 33,984-90 (1941).

Trchnol.. 12.87-122 (Derenlher 1940). (5) Hillman, E. S., and Barnett, B., h‘pfinw, 16, 362-6 (1937).

(7) Hoiberg, A. J., and Gairis, W. E., Jr., ISD. Evb. Crmnr., ANAL (6) Ibid. , 18, 533-8 (1939).

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.

401-6 (1940).

I193XI. (18) Rosumny, M., and de Rosset, A., Roads and S t ree t s , 81, 38-40

(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.

(May 1941). (22) Strieter, 0. G., .f. Resrarch S a t l . Bitr. Standards, 26, 415-18

(23) Suida, H., and Mota, F,, Prt io le i tmZ. , 35,527-33 (1939). (24) Thuiston, 1%. R., and Knowles, E. C., Ind . Eny. Chem., 33, 320-

4 (1941).

RECEIVED X a y 39, lM47. I i q i r o r e d for piiti!ication tiy t i i t . Direcror, Bureau of l l ines.

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, Pa.

Hatches of crjstalline bitamin 2 acetate habing uni- form 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 A\ .standard, (w’n though the oil has I X Y W srlecttd with great care and ha,s ht,cn handled and st oi,tJtl utitl(1r c.ontlitions favoring vitamin i,ct ciition. Observations rqmi?txl i i . ~ c ~ l y as 193:) (8) iriciic.itted that thc U.S.P. rr~frrrncc cod li\er oil So. 1 had txvn inaccurately standardized against thcx intornational stsndat~tl 01’ that its vita- min =\ potency had d(~tc~iioi~ated with time.

Studiw i , cpo i , t t d l)y 1IcFai.lan rt ai. (9) i‘urthct~ indi(~att‘d tha 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. (4j showed 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

\ 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 lacuum 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 coni- merciallj aiailable cr) stalline titamin A alcohol and 8-carotene. Stitdie9 are being continued.

this oil \vas also uiiruital)lc* as a vitaniin -4 standard [ 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 SO. 3 kwcarncl official during the kl t ter part of 1944. I A e I-.S.P. rcfetwicr oils I and 2, oil So. 3 also appears un- satisfactory as a vitamin -1 standard. 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 -1 standard: (’oil liver oils at’e difficult to 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 A N A L Y T I C A L C H E M I S T R Y

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 Standardiza- tion of the League of Sations (Health Section) set up an interna- tional 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 knowl- edge 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 that much remains to bc 1t:arned concerning the conversion of 4- carotene 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 bio- logical laboratories, this material seemed to 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 spectro- photometrically rvhen reccived B t the laboratory or soon there- after. In 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 to 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 stand- ing before carrying out the absorption measurements. The ab- sorption 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 an 450 mp considerGbly 'lower than that attributable to pure 4- carotene, 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 experi- mental 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 am- poules. Only two ampoules of the carotene, and these bearing the same control number ( B ) , showed an 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 datr do not explain why the absorption characteristics of the carotene sumple~ 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 in the *process of metabolism in tht , animal body, it would seem that pure vitxniin A alcohol should constitute an ideal vitamin h standard, f rom the spectrophotii- metric 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 -1 alcohol 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 in the 1aboratoi.y had already indicated that it \vas unst:tblr whtw used under assay conditions eoinparal)l(~ tu t how r.c .c . i innnic,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 cwh control number was c,saniinetl immediately on being recrivc,d at the laboratory, while othcr ani- poul(Ls of the crystalliric~ vitamiti were stortd under refrigeratioil antl cxamined sprctri)photc)nic,trically at 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 ( ~ w prepared having a calculated concctitrtttion of vitamin A al- who1 ranging from 0.5 to 3.5 micrograms p~ ml., using isopropa-

1: 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 ~vavcs length 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 d that t l i r solvc~nt gavcx a 100'; transmittance. From thealisorption data thrx 328 nipvalues \v~r(x calculatcd. Th(x n i ( ~ i value for the ten dilutions prepared from each vial of the ( lline vitamin is likcn-ixc pnwntcd i i i Table I.

Table 1 . 1-ariations in Extinction Coefficients or Ampoules of Crystalline P-Carotene and Cr? staIIine \ itamin 4 ilcohol Determined bj Beckrnan Quart7

Spectrophotometer" 1 itaimn \ i l < o h o l in Iwprot , \ I

i lcohol _______ - @-Carotene 111 Petroleriin 1:thrr El% B1'f

c o n - Am- 1 ctn. Con- Ain- 1 cm, trol poule Date of 4.50 trol ~ioule Date of 328 S o . KO. assay iiip S o . So assay m u

4 B

8 / 1/44 8/ 2/44

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

8/ 9/44

1900 1880 1896 1958 2554 2536 24'73 2330 2403 2347 2366 2407 2381 2458 2170 2223 2390

S 0 0 0 0 0 0 1' 1' P P 1' 1' Q Q Q Q

i 7 / 4 4 ?'13/44 7 '14 f44 7 '31 144 7,31144 6 24/47 b '24 I47

IOfI8 45 10 30145

11/8/45 11/14/45 6 / 2 3 / 4 7 6/23/47

12/28/44 12/28/44 6/23/47 6/23 147

1784 1428 1460 1652 1670 1444 1462 1574 1484 1346 1420 1362 1326 1741 1780 1653 166b

0 AT-erage E;'?,, values for ten dilutions of each asniple ranging in con- (.entration from 3.5 to 0.5 microgram per ml.

V O L U M E 20, NO. 5, M A Y 1 9 4 8 467

This dark red oily fraction was found to thy1 glcohol.) The first stage of desterola-

tion was effected a t a temperature of -20" C. and the second stage at a temperature of -70" C'. The met>hyl alcohol was re- moved from the desterolated concentrate by vacuum distillation and the resulting concentrate dissolved in pyridine for acetyla- tion. The acetylation was carried out by adding successive small portions of acetic anhydride and cooling so as to minimize destruc- tion of the vitamin. The acetylated concentrate !vas then main- tained 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 as 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 crystalliza- tion 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. Con- sequentl?-, several recrystallization experiments ivere carried out', and the purity of the crystals from wch 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 vita- min 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 fil- tering 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 15 em. glass ampoules. These ampoul .ere sealed onto a glass manifold-type vacuum system, and the tem was evacuated to 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. In both experiments the color of tlie crystals diminished with sub- sequent recrystallizationup to and inrluding the fourth crystalliza- tion. 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 con- centrate. 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

Perhaps this may bo dur, in part, to thv presence of ricv-vitamin A in the con- centrate (14).

Close conforniancc to a dcfinite set of physical, chemical, and biological characteristics is a neces- sary attribute of a satisiactory assay standard. To test the relative purity of the various batches of c rys ta lhe 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

talline vitamin A alcohol.

Reproducibility of Product.

Tahle 11. Effect of Recrystall ization of Vi tamin A Acetate on Extinction Coefficient a n d TIelting Po int

E'% 1 c m . 325 r i i ~ ~ ~ , Melting Point Range.

No. of Crvstallized from Cry-talliwd from Crmtalliza- Ethyl Methyl Ethyl Methyl

tion, forinate alcohol formate alcohol 1 1153

3 1,512 1.719 97 7-58,? 2 1 2 i S 1385 . j j : l j156 . o 34:315j . 3

? 1.5 1 ti 1.518 57.8-.59.0 57: 6 L i S .9 . . 1515 . . 37.4-58.8

f fer(' again duplicate dt~tcriniiiittioiis on a portion of the tdlinc vitamin f ~ ~ o n i the, 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 haw absorption values equal to that, ascribed to pure vit,ariiin -\ :tli~trhol (1750); t n o of these ampoules bore one rwntrol 11~unber anti t h c b third bore a swond control nurnt~csr. ficrc~ ito;ain thr ahsoi,ption data sho\v a somewhat greater varia- t ion ht~t\vec~n thr :tlliplJules of the crystalline vitamin bearing ilifferrnt control iiumt~crs t,han betn-ren the different, ampoules of t h i s vitamin healing the same control number. Ampoukbs of t k cryst:~,llinc vitaniiil Irhich had beqi stored at 35" F. for :tlnwst 3 yc'are yic.ldrtf a1)sorption valucs (qual to or only slightly lorver than those of the contents of cwmparable ampoules when ~iurc~hitwti. \Thile t l i c t orption data do not explain why the rlifferont ampowlrs of alline vitamin vary so xidely in this 1)hysical charactcristica, they do supprst that the purity of the r,rystalliii(~ product was not the same \\-hen placed in the various :~inpoiilw or 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 vari- :3l)ility i ) f the' i~ontc~nt,. of tiiffer(~nt :tmporiles.

\ 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 the 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 col- laborativt~ studivr rc-lativc. to the physical and biological proper- ties of vitamin .-i itccstatr.

In 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 AI ester 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 con- cciitrate as 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 that the formation and the isolation of crystalline vitamin -4 a,cetate could be most effectively carried out by fol- lowing 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 *I concentrate was saponified, the nonsaponifiable nuttter was recovered and dissolved in methyl alcohol (15% soh- tion), 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

468 A N A L Y T I C A L C H E M I S T R Y

Table I l l . Reproducibility of 325 nip of Vitamin A Acetate

3 2 5 m r

No. Crystallization from in Isopropanol Batch Date of Crystallized lcm.

1 12/17/46 Ethyl formate 1515

3 2/14/47 Ethyl formate 1521"

2 11 2/47 hIethyl alcohol 1517 3 1/29/47 Ethyl formate 1533 4 1/29/47 Ethyl formate 1514

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).

Table IV. Sonie Physical Constants of Vitamin A Acetate fi1%

325 w ~ : ? r n . 6 ~ 0 n l y LIeltiny Isopropyl Ethyl Carr-Price Pzint.

Source of Data alcohol alcohol Reaction'L C. This laboratory, h h 1520 1545 4210 57,6-58.9 This laboratory, B e 1510 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.

h acetate from five different batches of concentrate. The result- ing data, included in Table 111, show that very close agreement was attained as regards the tested characteristics.

Data on a typical sample of crystalline vitamin h acetatca prepared in this laboratory, and on the samples of crystalline vitamin -1 acetate 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.

Stability of Vitamin A Acetate. As a satisfactory vitamin -1 standard must withstand storage during the period of essay without deterioration, it seemed desirable to test the stability c f vitamin A acetate under conditions simulating those actually encountered during biological assay.

Portions of the vitamin crystals (4 to 7 mg.) as well as oil solu- tions (250 mg.) of the crystals !\-ere sealed in glass ampoules and stored in the absence of light a t room temperatures (25" to 30" <'.) and a t 5' C. For these studies, solutions of the crystalline vita- min h acetate in purified cottonseed oil (Wesson oil), corn oil (Aiazola oil), and peanut oil were made up so as to contain ap- prosimately 10,000 U.S.P. units of vitamin A per gram. This particular concentration of the vitamin had been reromniendetl by Embree (6). I n each instance, some of the ampoules ueril highly evacuated as previously described, while others were fillrd with nitrogen. T o achieve t,he latter condition the ampoules ivei'e either attached to the previously ment,ioned evacuation inanifold by means of rubber connections or were evacuated and charged by means of a vacuum desiccator. In bothinstances the ampoules m r e evacuated and refilled with nitrogen four succos- sive times to ensure complete removal of osygen. The nitrogt:n used had been previously scrubbed by passing successively through a toxver of alkaline pyrogallol, a tower of concentratcd sulfuric acid, and tubes of anhydrous calcium chloride and indi- cating Drierite (IT. A. Hammond Drierite Co., Yellon- Springs, Ohio). The data obtained to date in the stability studies arc' p r ~ wnted in Table V.

DISCUSSIOK

The results of the foregoing studies show that the current sources of the so-called pure p-carotene and of crystalline vitamin A alcohol cannot be relied upon to yield reproducible data when uskd as vitamin A standards, owing to impurities in the crys- talline product when placed in the ampoule or to degradation products formed after the crystals were packaged. On the other hand, batches of crystalline vitamin A acetate were repeatedly prepared from a fish oil concentrate, and on subsequent recrys- tallization possessed physical characteristics that indicated a high degree of purity. Preliminary stability studies on the prepared vitamin A acrtate, in the crystalline state and in its

oily solutions, in vacuum and in nitrogen, a t room temperature and under refrigeration, indicated that 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 in- stability of the vitamin. The peanut oil employed may not have been representative of th r most satisfactory pea.nut oil obtainable for this particular purposr. It n-as purc*lirtsed through a chemical supply source.

As a Tvhole, the data thus far obtaintd strongly indicate that vitamin A acrtate possesses sonic of thc most significant charac- teristics 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) .rillle in Stored a t Room Yeinperature Stored in Refrigerator Storage, Under I n Under I n

Days nitrogen vacuum nitrogen v a c u u n ~ I n crystalline state

0 1522n 1522 1522a 1522 30 1538 1528, 1512" I510

*'1466, 1474b 1522 1532" . . ,1268, 1254" 1-20 . . 1529, 1522 !1489!~ ,1464, 1462a 1611,1516

I n cottonsred oil 0 5 . 3 1 5 . 3 1 5 . 3 1 5 . 3 1

31 5 . 1 8 5 . 3 2 5 . 2 9 5 .34

117 4 .61 ,4 .82 5.19 .5.21 5.16 ,4 . .53 ,4 .93 5 , 2 7 5 , 1 6 , 3 . 2 4 . . ' 4 . 9 0

I n corn oil

In peanut oil 0 5.06 .5,06 5.06 5 .06

30 4 . 2 4 , 4 . 3 2 4 . 7 4 73 3 . 2 4 , 3 . 6 9 4 . 7 9 , 4 . 6 9 4 : 0 0 , 4 . 8 9 4.94,5.0. ' 90 3 . 0 8 , 3 . 2 0 4 . 7 1 , 4 . 6 9 4 .87 .4 .87 5 .02

k'illed h Filled

a.itti nitrogen n.ith n i t r o ~ e n

1)y nieans of indiridriall~-.

Oscr rt a / . ( I S ) , hoa-ever, a.ppear to favor the use of a distilled cistcr concentrate in preference to crystalline vitamin A acetate as an internal standard in their proposed modification of t,he Carr-Price procedure for the dvtermination of the vitamin A content of hiologic-1 materials. A prime reason for the choice serms to have bwn based on the probability that crystalline vitamin -1 acetate contained oxidized vitamin This deduction was drawn from the results of a prrviously conducted forced osidltion study ( l e ) of both the crystalline vitamin A acetate and the alcohol in solution inethyl laurate. These authors noted, in effcet, that the ratio of the L ~ c ~ l l j , 620 nip to the E;':nl, 325 mp

of the vitamin .1 in the .freshly prepared ethyl laurate solution was lower than the corresponding ratio for the vitamin A in a distilled ester concentrate. This \vas interpreted as indicating partial oxidation of the crystalline vitamin A acetate and alcohol, as both they a,nd Robinson (16) had noted that in the initial stages of forced osidation of vitamin A there was a greater de- crease in chromogenic power in the antimony trichloride reaction than in its rapacity to absorb ultraviolct radiation a t 326 nip.

V O L U M E 20, NO. 5, M A Y 1948

III the authors' studios, lioivever, a distilled ester concentrate (obta.ined from Ilistillat ion Products, Inc.) gave an L:Fn,, 620 m p value that agrcw \vel1 with both the reported value for vitamin .1 acetate (1) arid ivitli the value obtained for the crys- t>alline acctate p r e p a i ~ d in this lahoratory. This T,i'2,n. 620 mp value of 1210 for the d id 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 -1 ncetittcx.

I t will eventually in- clude, in addition to thc studies already mentioned, stabilit) studicr oii vitamin A acetate when dissolved in those organic sol- vents most ccninio~ily uscd in spectrol,hotornetl.ic tests and othrr studies relating tu the acceptability of this form of the vitamin as a stantlard in biological a n d i i i cht:n~ic~oI-,li?-sical methods of assay.

The iiir-est i-:ttion is 1)oinp coiitinurd.

LITERATURE CITED

(1) Baxter, J. G., and Robeson, C. D., J . Am. Chcm. Soc., 64 ,2407-

(2) Ib id . , 64 , 2111-16 (1942) . (3) Callison, E. C., and Orent-Keiles, E., Isn. K x ; . CHEM., -%SAL.

10 (1942).

l . :~. , 17, 378-9 (1945) .

469

COJ., S . H., Sassainan; H. L., and Black, A , , Ib id . , 13, 74-6 (1941) .

Ib id . , 15, 441-3 (1943) . Embree, N. D., Oil and S o a p , 23 ,275-6 (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 (1940) . Meng, K . H., private communication, I<esearch Laboratories,

Distillation Products, Inc., Rochester, N. T., 1946. Morgareidge, Kenneth, ISD. ENG. CHEZI., .\SAL. ED., 14, 700-2

(1942) . Oser, B. L., Mehick, D., and Pnder, XI., Ib id . , 15, 717-24 (1943) . Ib id . , 15, 723-9 (1943) . Robeson, C . D., and Baxter, J. G., J . Am. Chem. Soc., 69, 136-

41 (1947) . Robinson, F. .1., Biochem. J . , 32 , 607-14 (1938). TVilkie, J . B., U.S.P. Vitaniin Advisory Board, Letter 164, pp.

Zscheile. F. P., and Heiwy, R. L., IND. EKG. CHEY., ASAL. ED., 508-20, 1946.

16, 436-8 (1944) .

R E C E I V E D September 5 , 1947. Authorized for publication on riupllst 14 1947, as Paper 1388 in the .Journal Series of the Pennsylvania -4gricultural Experiment Station. Snpijorted in ])art hj. a grant from The Xutrition I.'oundation.

Rapid Colorimetric Determination of Copper in Tin-Base Alloys

GEORGE iYOHW~ITZ,-AMaterial Laboratory, Neu? York iyacal Shipyard, Brooklyn I , S. Y .

i rapid colorimetric method for the determination of copper in tin-base alloys is described. After the sample has been dissolved in hj-drochloric and nitric acids, phosphoric acid is added and the solution is heated. Water and ammonia are added, and the resultant blue copper amine color is measured on a photo- electric colorimeter. The addition of phosphoric acid prevents precipitation of the tin when the solution is made ammoniacal.

H E : author undertook tu develop a rapid colorimetric pro- T cdurcx for thr dt,trxrniination of copper in tin-base alloy.. Two A.Y.T.;\I. nic,thodq ( I , .zj, wither one of which is particu- larly rapid, arc' comnioiily u s d for the analysis of copper in tin- t x w alloys. I n oncs CJF thew procedures (1) the copper is elrc- t,rrilyztd, affcr volatilization of the t in with hydrobromic acid, ijr hytlrobromir :irid antl 1~r~iniine. In the other (2) th r copper is elt~c~trolyz~d in a nitric acid-hydrofluoric acid medium, stripped from ihc' cathodr, and w p l a t d .

Thch rapid colo~imc~tiic inc~thod described in this paper is novel, yebt rsstrcmeIj- simple). .-\iter thc sample has been dissolved in hycli~ocliloric and nitr,ic a(-ids, phosphoric acid is added and the solution is heatrd. 1Yatc.r arid ammonia are added, and the rtwultsnt blue color is nicwured 011 a photbelectric colorimeter. The atidition of thr phosphoric acid prevents the precipitation of the tin whcln the s o l u t i o ~ i is made ammoniacal.

APP4RATUS

hlvtt-Summc1son photoelectric colorimeter. Rectangular 4- c.111. glass absorption cell. Klett-Summerson filter S o . 59 transmittancr mavimuni a t 580 millimicrons).

METHOD

Transfer 1.000 gram of the sample to a 500-ml. Erlenmeyer flask that has a mark indicating the 500-ml. level. Add 10 ml. of concentrated hydiochloric acid and 10 ml. of concen- trated nitric acid in that order, and heat on a hot plate for a minute or two to dissolve the sample. Add 30 nil. of concen- trated phosphoric acid (8.5%) and heat strongly on a hot plate until the solution is a clear grclen (12 to 15 minutes). Remove

the flask from the hot plate and allo\v the solution t o cool some- ivhat,. Xdd about 200 nil. of cold xater, and then add cautiously 120 ml. of concentrated ammonium hydroxide while svirling the flask. Cool to room temperature. Dilute to the 500-ml. mark with n-ater, stoppen the flask, and shake well. Read the blue copper amine color at 580 millimicrons on a colorimeter that has been set to zero Jvith distilled water. Convert the colorimeter wadings to percentaly copper by consulting a curve previously prepared by the use of tin-base samples of knoxn copper content.

The author ran Sational Bureau of Standards samples 51a and 54b in triplicate and obtained the results shown in Table I.

Table I. Determination of Copper Averaye

Cu Present, Cu F;ynd, Cu Found, Ralriiile 70 /O %

24an 3 .75 3 . 7 8 3 . 7 4 i 0 . 0 3 R 7n

54h b 3 .19 3 . 7 3 3 . 2 0 3 . 2 1 + 0.02 3 . 1 9 3 .23

Contains 88.61 Sn, 0.21 Pb, 7.32 Sb, 0.02 Ai, 0.04 Fe, 0.04 As. b Contains 87.48 Sn, 1.81 Pb, 7.39 Sb, 0.029 Bi, 0.028 Fe, 0.052 As.

DISCUSSION

S o mention was f o p d in the literature of t,he use of phosphoric acid to prevent precipitation of tin in ammoniacal solution. The author originally believed that the pyrophosphoric acid formrd by heating the phosphoric acid (4) prevented precipita- tion of the tin. Experiments shon.ed that i f no heat weye