desirable properties in the analysis of formaldehyde. I t afforded a complete separation of each of the components of butanol-formaldehyde solution and formalin. As shown in Figure 3, it separated higher aldehydes, eluting formaldehyde after the time for butyraldehyde. It was of particular interest that Ethofat 60/25 separated formaldehyde by a property other than solubility in the substrate. This was concluded because the formaldehyde peak was wider than the butanol peak which eluted after it, The performance of other substrates supported this conclusion. For example, formaldehyde was eluted from sorbitol after water in a broad peak. At 150" C. the formaldehyde peak was too broad to be used for direct analysis. The long retention was first attributed to the presence of hydroxyl groups present on both sorbitol and Ethofat 60/25. However, Carbowax 6000, which contains only secondary alcohol groups, eluted formaldehyde before methanol. It was then proposed that the unique retention of formaldehyde was due to the formation of a weak hemiacetal with the primary hydroxyl group on the substrate. The proposal was tested by comparing formaldehyde retention on diethylene glycol, hexane-l,Miol, and 2-methyl pentane-2,4-diol. The two latter materials nere not suitable for general use as substrates for formaldehyde analysis because of their high vapor pressures, but were chosen as available sources of primary and secondary alcohols, respectively. Formaldehyde was retnined on diethylene glycol after
of log retention us. 1/T as shown in Table IV.
Elution Order for Several Substrates Elution Per-
Substrate Ethofat Sorbitol
Ordep formance M,W,F,B Good (MB).W,F Poor formaldehyde peak Diethylene glycol M,B,(WF) High vapor vressure Lac 296 F,M,W,B Fair Mannitol hexaF,M,(WB) Anomalolls acetate Sucrose octaace- F,M,W,B G ~ X tate Carbowax 6000 F,M,W,B Fair (FMW),B Poor Apiezon N M = methanol, W = water, B = butanol, F = formaldehyde, ( ) includes peaks not resolved. water and butanol, although the separation from water w m not complete. Formaldehyde eluted from the secondary alcohol before methanol but was retained by the primary diol. It was concluded from this work that the retention of formaldehyde on material containing primary hydroxyl groups was through the formation of an unstable hemiacetal. Influence of Temperature. Column temperature had a marked effect on the elution of formaldehyde from Ethofat 60/25. The separation was studied at seven different temperatures. Chromatograms of butanolformaldehyde solutions obtained at three different column temperatures are shown in Figure 4. From a plot
Figure 5, it was determined that optimum separation was made at 118" c. However, satisfactory separations were made between 110" and 125" C. Solid Supports. The effect of several solid supports on the separation of butanol-formaldehyde solution when using Ethofat 60/25 as the substrate is shown in Figure 6. Columnpak T produced the sharpest peaks for all components. The performance of Teflon 6, not shown on the figure, was between Columnpak T and Fluoropak 80. This work also demonstrated that acetylating the white chromosorb improved the formaldehyde and the alcohol pcaks but did not improve the water peak. When water was present in the sample, Columnpak T was the preferred support. ACKNOWLEDGMENT
The authors thank C. E. Cook for helpful assistance in this work. LITERATURE CITED
(1) Bombaugh, K. J., ANAL. CHEM.33,
29 (1961).
(2) Kelker, Hans von, Z. Anal. Chcm. 176, 3 (1960). (3) McReynolds, W. O., Pittsburgh Con-
ference on Analytical Chemistry and Applied Spectroscopy, 1961. (4) Sandler, S., Strom, R., ANAL.CHEM. 32, 1890 (1960). (5) Walker, J. F., "Formaldehyde," ACS Monograph Series, p. 380, Reinhold, New York, 1953. RECEIVEDfor review April 23, 1962. Accepted July 5, 1962. Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, Pittsburgh, Pa., March 1962.
Purification of Vitamin A by Partition Chromatography in the Analysis of Pharmaceuticals and Margarine T. K. MURRAY Food and Drug laboratories, Department of National Health and Welfare, Ottawa, Canada
b Partition chromatography was applied to the separation of vitamin A from various types of interference, such as oxidized vitamin A and vitamin E, commonly found in pharmaceuticals. Vitamin A was also separated from p-carotene and other interfering material found in margarine. Recovery of the vitamin was almost complete and the absorbance curves of the chromatographed samples resembled that of pure vitamin A. The column was easily prepared, required no standardization, could be used for successive samples, and made possible
the application of the spectrophotometric method to samples which could not otherwise be assayed by this method.
V
A is usually estimated in pharmaceuticals by the spectrophotometric method described in the USPXVI (IO). Lehman etal. (6) pointed out the shortcomings of this method when applied to mixtures of cis and trans isomers, but for most samples it gave an accurate estimate of biological potency (6). Although no ITAMIN
guidance on this matter was given in the USP method, it cannot be applied to samples with grossly distorted absorbance curves. The need for purification in such cases was recognized by the British Pharmacopoeia @), but no procedure was recommended. The International Union of Pure and Applied Chemistry (I.U.P.A.C.), in a report of collaborative experiments ( I ) , recommended that chromatography on alumina be used when the absorbance ratio, 300 m ~ / 3 2 5mp exceeded 0.730, and demonstrated the use of such a procedure in the purification of a VOL. 34, NO. 10, SEPTEMBER 1962
1241
covery
strate that chroniatography improved the accuracy of the results. This paper describes the separation of vitamin A from a variety of interfering substances by partition chromatography and demonstrates the recovery of the vitamin from such samples.
98.5 97.8
PROCEDURE
Table 1. Recovery of Vitamin A from Polyethylene Glycol Column Vitamin A
Recovered after Chromatography, I.U. 89,030 95,290 97.650 96;310 94,570
Vi tamin A Tcst 1 2 3 4
Mean
.4dded, I.U.
90,345 97,435 98,320 96,800 95,725
% Re-
99.3
99.5 98.8
whale-liver oil and a fish-liver oil. The AOAC recently tested (3) a similar system for the separation of vitamin A and carotene from margarine. While alumina sometimes provided a good separation of vitamin A from interfering materials, each batch had to be standardized to ensure uniform retentiveness and, as applied in the I.U.P.A.C. studies ( I ) , required a series of eluting mixtures. Furthermore, this system did not yield a typical vitamin A absorbance curve in the AOAC assay of margarine (3). Tardif’s (8) proposed method involving the destruction of vitamin A with sulfuric acid for the assay of vitamin A in the presence of interfering substances was criticized on theoretical grounds by Gridgeman (4). The use of partition rhromatography for the purification of vitamin D was proposed by Theivagt and Campbell (9). who noted that the same system would probably be useful in the determination of vitamin A. It was used by Wilkie, Jones, and Morris (11) to improve the absorbance curve of several vitamin A preparations, but these authors did not demon-
Table II.
Absorbance Ratios and Recovery of Vitamin A in the Presence of Oxidized Vitamin A
Unchromatographed 310 m p / 334 mu/ 70 325 mp 325 mp Recovery 0 852 0 857
Standard Standard Oxidized “A” 1 0 939 2 0 932 3 0 958 4 0 957
+
Table 111.
A. I.U.
E, I.U.
Standard
... 12.5 6.25
1242
0 0 0 0
852 857
838
849
78 91
89 103
Chromatographed 310mp/ 331m,~/ r0 325 niM 325 m p Recovery 0 852 0 857 0 0 0 0
869 870
882
883
0 0 0 0
U.56
854 861
853
103 103 98
103
Absorbance Ratios and Recovery of Vitamin A in the Presence of Vitamin E
Sample rrjtamin Vitamin
lo00 lo00
Reagents. The reagents for the saponification and extraction of vitamin A are those specified in the USP XVI. For the chromatographic step n-hexane, iso-octane, polyethylene glycol 600 ( P E G 600 Union Carbide Chemicals Co.), and Celite 545 (JohnsManville) are required. Column Preparation. Mix 25 grams of Celite 545 in 125 ml. of iso-octane and add 10 ml. of polyethylene glycol (PEG) 600. Mix for 1 minute with a high-speed blender. Pack firmly into a 15-mm. chromatographic tube t o a height of 150 mm. with the aid of a perforated plunger. Wash the column with n-hexane and keep i t covered with this solvent until the sample is applied. The column may be re-used as long as it gives good results providing it is kept covered with solvent. Method. Saponify and extract pharmaceuticals as described in USP XVI (10) and margarine according to the method of Rice, Primm, and Coombw (7). Evaporate to dryness and dissolve t,he sample in n-hexane so that 2 ml. contains from 400 to 1200 I.U. Apply the sample to the column (preferably in 1to 2 ml. of n-hexane) and elute with the same solvent, a t a flow rate of 40 to 50 drops per minute which may be maintained with gentle suction. Follow the vitamin A band by occasional examination in low intensity ultraviolet light and begin collection when this band is about 5 mm. from the outlet. Collect as little as possible of the eluent following the vitamin A band. 8-Carotene precedes
cn /O
Unchromatographed Chromatographed Re300 mp/ 310mr/ 334mp/ 300mp/ 310mp/ 334 mp/ cov325 mp 325 mp 325mp 325mp 325mp 325 mp ery 0.600 0.852 0.857 0.600 0.852 0.857 .. 1.50 1.14 0.851 0.640 0.872 0.852 95 1.09 1.01 0.847 0.613 0.861 0.854 95
ANALYTICAL CHEMISTRY
vitamin A on the column and may be observed without the aid of the ultraviolet light. The completeness of its collection is judged by visual examination. Complete the determination of vitamin A according to the method of the USP XVI and of carotene as described by Friedman (3). For the purpose of evaluating the method, compare the absorbance ratios, 300 mp/ 325 mp, 310 mc(/325 mp, and 334 mp/325 mp with the ratios found for pure vitamin A. RESULTS AND DISCUSSION
Recovery of Standard. Samples of the USP reference solution of vitamin A acetate were saponified and assayed either directly or after passage through the column. The results of four separate tests, shown in Tablc I, indicate t h a t loss on the column compared favorably with the 2.4% loss on alumina columns reported hy the I.U.P.A.C. (I). Separation of Oxidized Vitamin A. Vitamin A acetate was saponified and then treated with heat, light, and air until i t gave no color with antimony trichloride. A sufficient quantity was then added to samples of the vitamin A standard to alter significantly the absorbance curve and the mixtures assayed (Table 11). The USP method-it., u ithout chromatography -did not accurately estimate the vitamin A because of the interference of the oxidized vitamin. The removal of the interference by chroinatogralihy, as indiraterl by the ahsorhsnw ratios, resulted in a good r c c o i ~ r y of vitamin -2. Separation of Vitamins A and E. Table 111 illustrates the interference of vitamin E in the spectrophotometric determination of vitamin A and the manner in which ’the PEG-Cclite column eliminated this interference and made possible the accurate estimation of vitamin A. Without chromatography it was not possiblr t o estimate the vitamin A content of the mixture by the spectrophotometric method. Assay of Multivitamin Preparations. Multivitamin preparations sometimes contain unknown substances whish interfere with the estimation of vitamin A. When known amounts of the vitamin were added to three such samples, the distortion of the absorbance curve made an accurate estimation of potency impossible. The results obtaiiied by two analysts before and after chromatography (Table IV) illustrate the quantitative recovery of the latter procedure. Vitamin A and Carotene in Margarine. Vitamin A and &carotene were added to two samples of unfortified margarine to the extent usually found in commercial margarine.
~~
Table IV.
~~~~~
Absorbance Ratios and Recovery of Vitamin A Added to Liquid Multivitamin Preparations
Unchromatographed Ya~nple Standard Pharmaceutical No. 1 Pharmaceutical No. 2 Pharniaceutical No. 3
Analyst
3m/ 325
...
0.600
A B A B A
0.788 0.819 0.884 0.988 0.919
Table V illustrates the results obtained by two analysts when these samples were assayed spectrophotometrically after chromatography. The assay of several commercial margarines by this method as well as by the antimony trichloride method (without chromatography) is also shown. The interference usually caused by margarine was almost completely eliminated by chromatography and the spectrophotometric method gave a good estimate of the vitamin A added. The separation and recovery of p-carotene which preceded vitamin A on the column presented no difficulty. In two samples (Nos. 2 and 3) of commercial margarine, the antimony trichloride method gave a greater value than did the spectrophotometric method. The chromatographic system used in these studies offered several advantages over others that have been proposed. It separated vitamin A from a wide variety of interfering substances without modification of the procedure; its components could be used as received and no pretreatment or standardization was necessary; only one eluting solvent was necessary and the column could he used for successive samples. The results were readily rluplicbnted by a second analyst. 1,elinian et al. (6) pointed out the errors that occurred when the USP XVI method was applied t o samples containing cis- isomers of vitamin A. The method described here does not separate these isomers and, in the absence of other interference, provides the same results as the USP method. Its greatest value is its ability t o separate vitamin A from interfering substances commonly encountered such as vitamin E, oxidized vitamin A, and anhydrovitaniin A. For margarine, separation on P E G 600 provided an almost pure sample of vitamin A and made possible the use of the USP spectrophotometric procedure. In this respect, it was much superior to alumina ( 3 ) . Recovery of added vitamin A was good. This method provides a convenient means of separating vitamin .Li and @-carotene in colored margarine and should give a better estimate of potency than the antimony
310/ 325 0.852 0.930 0.944 0.986 1.000 0.990
Table V.
Chromatographed 384/
323 0.857 0.866 0.866 0.855 0.860 0.861
76
Recovery ... 92 88 70 73 94
Absorbance Ratios 334 mM/ 325 mp
310 mr/ Analyst 325 mr
Sample
and carotene and carotene Unfortified margarine S o . 2 and Vitamin A
Commercial margarine S o . 1 KO.2 xo. 3 No. 4
*
310/ 325 0.852 0.862 0.852 0,853 0.846 0.869
331/ 325 0.857 0.854 0.863 0.859 0.852 0.855
%b Recovery 98 101 101 99 100
Purification and Recovery of Vitamin A and Carotene from Margarine
Standard Unfortified margarine S o . 1 and Vitamin A
a
300/ 325 0.600 0.602 0.621 0.600 0.632 0.664
A
A A B B B A A A A A A A A
B
A A A
0 862
O.X.57
0.892 0.886 0.898 0.876 0.862 0.867 0.871
0.850
01856 0.861 0.865 0.865 0.865 0.871 0.861 0.861 0.861 0.861
0.850 0.848 0.829 0.858 0.850 0.852
01842 0.848
0.846 0.853 0.849 0.852 0.851 0.851 0.851 0.870
yo Recovery Vitamin 13A Carotene 9s 98 93 112 94 94 96 92fl loo'
100 1 01)
99 92 98 98 101
107 102b 88b 90* 99*
A Determined by antimony trichloride reaction. Relative t o antimony trichloride reaction on unchromatographed aliquot.
trichloride method when the added vitamin A is a niisture of all-trans and 13-cis. This may be the explanation of the higher values found hy the antimony trichloride method in two samples of commercial margarine. When only all-trans vitamin A is present, the antimony trichloride method should be satisfactory but there is no assurance that this will be the case. It should he noted that Wilkie and Jones (18) did not achieve a good separation of vitamin A from the interference of margarine with the chromatographic system described here. These workers used a larger (25 mm.) column and it may be that the column size is critical. Because chromatography adds considerably to the time spent in completing an assay it is not desirable or necessary to use it for all samples. Purification is obviously necessary when the absorbance peak is displaced and should also be applied in cases of less obvious distortion of the absorbance curve. The I.U.P.A.C. (I) recommends that chromatography be applied when the absorbance ratio 300 mp/ 325 mp exceeds 0.i30,and in our cx-
perience this is a safe criterion. Most of the interference dealt with in this study was much more severe. The analyst must also be on the alert for irrelevent absorbance on the long wavelength side of 325 mp such as occurs when anhydrovitamin A is present. This compound can be detected by an examination of the absorbance curve between 325 mp and 400 mp and can readily he removed by the chromatographic procedure described in this paper. ACKNOWLEDGMENT
The author acknowledges the generosity of Canada Packers Co. Ltd., Lever Brothers, and Frank W. Horner Co. Ltd. for samples of vitamin Afree margarine and pharmaceuticals. H. S. Lakke Gowda, Colombo Plan Fellow from the Public Health Institute, Bangalore, India, and Paula Erdody of this laboratory performed some of the analyses. LITERATURE CITED
(1) Brunius, E., J . Assoc. Of. Aqri. Chemists 42, 657 (1959). VOL. 34, NO. 10, SEPTEMBER 1962
0
1243
(2) "British
J . Biochem. Physiol. 39, 1687 (1961). ( 7 ) Rice, E. E., Primm, E., Coombes, A. I., J. Assoc. Ogic. Agri. Chemists 31,
Chemists 43, 6 (1960). (4) Gridgeman, N. T., J. Pharm. Sci. 50, 449 (1961). ( 5 ) Lehman, R. W., Dieterle, J. M., Fisher, W. T., Ames, S. R., J . A m . Phutm. ASSOC., Sei. Ed. 49,363 (1960). (6) Murray, T. K., Campbell, J. A., Can.
621 (1948). (8) Tardif, R., J. A m . Pharm. ASSOC., Sci. Ed. 49,741 (1960). ( 9 ) Theivagt, J. G., Campbell, D. J., ANAL.CHEM.31, 1375 (1959). (10) "United States Pharmacopeia,'' XVI, p. 938, Mack Publishing Co., Easton, Pa., 1960.
Pharmacopeia," Pharmaceutical Press, London, p. 956, 1958. (3) Friedman, L., J. Assoc. Ogic. Agn'.
( 1 1 ) Wilkie, J. B., Jones, S.
W.,Morris, W. W., J . Assoc. O&. Agri. Chemists,
42, 422 (1959). (12) Wilkie, J. B., Jones, S. W.,Food
and Drug Administration, Waahington
25, D. C., personal communication (1962). RECEIVEDfor review March 10, 1962. Accepted July 8, 1962.
Separation of the 2,4-Dinitrophenylhydrazones of Dicarbonyl and Other Polar Compounds by LiquidLiquid Partition Chromatography EDGAR A. CORBIN Eastern Ufilization Research and Development Division, In order to investigate some highly polar carbonyl compounds found in oxidized whole milk powder, suitable liquid-liquid partition chromatographic systems for separating the mono- and bis(2,4 dinitrophenylhydrazones) of these carbonyl compounds were devised. Three column systems are described, each one suited to handle a different range of compound polarities. Mixtures of acetonitrile and water are used as the stationary phase on Celite columns. Elution is done with methylcyclohexaneor methylcyclohexane-ethyl acetate mixtures. Monocarbonyl, bis(oxoa1dehyde) and diketone bi~(2~4-dinitrophenyIhydrazones) have been studied.
-
D
of the carbonyl compounds associated with the oxidized flavor of whole-milk powder there was obtained a variety of dicarbonyl and highly polar monocarbony1 2,4-dinitrophenylhydrazones(2,4DNPH's). Methods in the literature for separating these derivatives (4, 5) were inapplicable, and a previously published method for monocarbonyl 2,4-DNPH's by the author and others (1) was not suited for these polar derivatives. A liquid-liquid partition chromatographic column was considered the most desirable method of separating these compounds. Three partition columns have been evolved for this purpose since it was found that one partition system could not handle the wide range of polarities encountered. URING A STUDY
EXPERIMENTAL
Reagents and Apparatus. Spectrophotometer to accommodate optically matched test tubes (12 ml. or larger). 1244
ANALYTICAL CHEMISTRY
U. S.
Department of Agriculfure, Washington 25, D.
Chromatographic tube, approximately 25 x 350 mm. with solvent reservoir. Methylcyclohexane. Redistill and collect the 101" t o 103" C. fraction. Acetonitrile. Redistill and collect the 80" to 82" C. fraction. Ethyl Acetate. Redistill and collect the 77" to 78" C. fraction. Dry Column Materials. Analytical grade Celite (Johns-Manville analytical filter aid) and alumina (80 to 200 mesh, for chromatographic analysis, Fisher Scientific Company) were dried in a 140' to 150" C. oven for a t least 24 hours before use. Column Preparation and Use. COLUMN1. An equilibrated eluting solvent is prepared by saturating methylcyclohexane with acetonitrile at room temperature. Fifteen grams of dry Celite are placed in a blending container with 200 ml. of the equilibrated methylcyclohexane and mixed until wetted. I n a graduated cylinder 0.3 ml. of water and 12 ml. of acetonitrile are mixed and then poured as a fine stream into the swirling Celite slurry. When homogeneous, the slurry is poured through a wide-stem funnel into the column, the tip of which has been closed with a pinchcock. Air bubbles are removed with a tamping rod. The pinchcock is opened and the Celite is compacted with 2 to 4 p.s.i. air pressure. The top of the column, which is always kept covered with solvent, is firmed and leveled with a tamping rod. COLUMN2. The eluting solvent in this case is 2% ethylacetate in methylcyclohexane (v./v.). This solvent is then equilibrated by saturating it with acetonitrile a t room temperature. Fifteen grams of dry Celite and 200 ml. of the equilibrated solvent are mixed as before, but only 6 ml. of acetonitrile containing 0.3 ml. of water are added to the swirling Celite slurry. The re-
C.
mainder of the column preparatioii is the same as for column 1. COLUMN3. An eluting solvent is prepared by mixing 6% ethylacetate in methylcyclohexane (v./v.) and then saturating this mixture with acetonitrile a t room temperature. A small layer of Celite deficient in stationary phase is needed at the bottom of column 3. Its function will be explained in the Discussion. Two grams of dry Celite and 60 ml. of equilibrated solvent are mixed by swirling in a tall beaker and then poured into the closed chromatographic tube. After settling slightly, the tube is opened and the Celite is compacted with air pressure. At least 2 to 3 cm. of solvent are allowed to remain above this layer to prevent its being disturbed when the column proper is made on top of it. Fifteen grams of dry Celite and 6 grams of dry alumina are mixed with 200 ml. of the equilibrated eluting solvent. Ten milliliters of acetonitrile and 0.4 ml. of water are mixed and then poured as a fine stream into the swirling Celite-alumina slurry. When homogeneous the slurry is poured carefully into the tube so as not to disturb the lower Celite layer. Air bubbles are removed, air pressure is applied, and the column is finished in the same manner as column 1 or 2. The partition columns are operated in the usual manner taking into account the following suggestions and limitations. The 2,4-DNPH's are dissolved in the proper equilibrated eluting solvent depending upon which column is to be used. A small volume, 10 ml. or less, is most desirable but as much as 20 to 25 ml. can be used if necessary. If any insoluble material is present, it must be filtered out before putting the sample on the column. Flow rate to start should be about 60 drops per minute, When the color starts to move the partition properly, the flow rate can be increased to as much as 150 drops per
