Paper Chromatography of Certain Vitamins in Phenol a nd B uta no I- Propionic Acid- W a te r Solvents EVELYN L. GADSDEN, CECILE H. EDWARDS, and GERALD A. EDWARDS Departments of Home Economics and Chemisfry, The Agricultural and Technical College of North Carolina, Greensboro,
b Separation and detection of vitamins using two-dimensional paper chromatography with phenol and a butanolpropionic acid solvent system have been investigated. The water-soluble vitamins were successfully separated and, depending upon the mixture, can be located following the application of a selected order of detecting agents. Of the fat-soluble vitamins, only a-tocopherol can be identified by the procedures studied.
T
paper chromatographic techniques are now used in studies of amino acids, organic acids, and many other organic compounds, application of this method to vitamins has not been extensive. Many of the procedures now available necessitate the use of specially treated paper (IS, 17, do), or estimation of the vitamin by bioautographic techniques (16, 58, 41). Paper chromatography has been an important analytical tool in this laboratory in the study of the metabolism of the amino acid, methionine, by the adult rat. For this investigation it mas necessary to determine the R, values of pure amino acids using two solvents which were most satisfactory for our purposes (phenol and butanol-propionic acid-water). It was discovered that many of the vitamins migrate to the same general location as the amino acids on paper chromatograms. However, a review of the literature revealed few Rf values in these solvents. The estimation of vitamin A and related compounds on alumina-impregnated paper has been described by Datta, Overell, and Stack-Dunne (13). Rossi (SS) has reported the paper chromatography of vitamin Dz. Brown and Blaxter ( 7 ) have described the separation of a-, p-, and 7-tocopherols by reversed phase paper chromatography on Vaseline-coated paper. Procedures for this vitamin have also been described by others (8, 9, 15, f7, 18). The paper chromatography of thiamine and some of its derivatives has been reported by several workers (X, 22, 28, SO, 5'4,3'6'). Crammer (IO), Hais and Pecakova (IQ), Yagi (4S), and Blair and Graham (4)have described the use of this technique in the separation of riboflavin, its phosphate HOUGH
and nucleotides. Ackermann and Kirby (I), Baddiley and Thain (Z), and Crokaert (11) have studied pantothenic acid. The paper chromatography of nicotinic acid has been reported by several workers (23-26). Procedures for the estimation of pyridoxines (IS, S6), folic acid and its derivatives (16, 81), and choline (36) have also appeared. Bowden and Peterson (6) have investigated the resolution of biotin components in liver by paper chromatography. Winsten and Eigen (S9), Woodmff and Foster (40), and Harrison (2f) have reported studies of vitamin BIZ. Patschky (.%), Mapson and Partridge (Z9), Weygand (S7), and several other investigators have described the paper chromatographic behavior of vitamin C. The present study was to investigate the migration characteristics of certain water- and fat-soluble vitamins in water-saturated phenol and butanolpropionic acid-water on untreated filter paper. EXPERIMENTAL PROCEDURES
Solutions of vitamins were made up in a concentration of 5 mg. per ml., with the exception of p-aminobenzoic acid and a-tocopherol. Because of its lower solubility, p-aminobenzoic acid was made up in a concentration of 2.5 mg. per ml. and a-tocopherol was diluted 1 to 10 with absolute alcohol. Distilled water was used as the solvent for the water-soluble vitamins; absolute alcohol as the solvent for the fat-soluble compounds. The vitamin solutions were individually applied to the lower right-hand corner of 18 X 22 inch Whatman No. 1 filter paper sheets. Buffered watersaturated phenol was used as the first solvent. The aqueous buffer solution contained 6.3% sodium citrate and 3.7% potassium dihydrogen phosphate (100 ml. of phenol per 25 ml. of salt solution), These salts inhibit the diffusion of spots and prevent the migration of a contaminant in the paper. A mixture of 1-butanol, propionic acid, and water was used for the second dimension. Fresh solvent was prepared from equal volumes of two solutions: A (1246 ml. of 1-butanol and 84 ml. of water) and B (620 ml. of propionic acid and 790 ml. of water). The papers were run by the descending technique in paraffin-coated wooden Chromatocabs housed in a constant
N. C.
temperature room maintained a t 24' 0.5' C. A separate cabinet was used for each solvent. A small amount of solvent was kept in a container a t the bottom of the cabinets to bring the atmosphere more quickly to equilibrium with the solvent. After the phenol had migrated down the papers (about 18 to 22 hours), they were removed from the cabinet. The solvent front was outlined with a lead pencil and the papers were dried in a fume hood overnight. They were then turned a t a 90' angle counterclockwise and butanol-propionic acid -water was allowed to descend the papers (14 to 16 hours). The solvent front was outlined again and the papers were dried overnight in a fume hood. Various color reagents were required to detect the different vitamins.
*
RESULTS AND DISCUSSION
Table I presents the method of identification, R f values, and concentrations of the vitamins chromatographed. No attempt was made to determine the minimum quantities which could be detected by paper chromatography. The quantities used were large, as we were primarily interested in determining the R , values of the vitamins. Of the fat-soluble vitamins, only atocopherol was successfully detected by a color reagent after chromatography. Vitamin A and Menadione (vitamin ,K) were yellow in solution and migrated as a yellow spot during the phenol run; therefore, their R/ values in this solvent could be calculated. Their yellow color, however, was lost during butanol-propionic acid migration, and they could not ;be detected with color reagents. We were also unsuccessful in detecting vitamin D and thioctic acid. Antimony trichloride is listed by some investigators as a reagent for vitamins A and D ( 5 ) . Discrete spots were not obtained with this reagent. Many investigators working with the chromatography of fatsoluble vitamins have impregnated their filter papers with alumina [Aln(S04)], coated them with vaseline or paraffin, or treated them in other ways (6, 27). There was no attempt to treat the papers specially, as we wanted to secure R , values for the vitamins using the same procedures as were applied in the two-dimensional chromaVOL 32, NO. 11, OCTOBER 1960
* 1415
tography of other compounds in our experiments (14). Our failure to obtain discrete spots may have been due t o this reason. The water-soluble vitamins were easily separated using phenol and butanol-propionic acid-water. Detection was as follows: The B complex group, riboflavin, pyridoxine, and folic acid fluoresce under ultraviolet light without any special treatment. (A shortwave ultraviolet lamp was used.) Thiamine fluoresces after being sprayed with a n alkaline ferricyanide-nitroprusside reagent (3). Niacin and nicotinamide may be detected by exposure to cyanogen bromide, followed by spraying with 2% p-aminobenzoic acid (5). Vitamin BIZ,which is pink in solution, migrates as a pink spot. Many organic compounds react with iodine vapors to yield a brown spot. Niacin, nicotinamide, choline, and biotin were detected by this method. The R, values shown in the last column were obtained by calculating the ratio of the distance traveled by the compound to the distance traveled by the solvent front. The values listed are for vitamins run as individual compounds. We have, however, also run a mixture of vitamin solutions, separating and identifying the various vitamins by the methods listed above. Because several different methods and color reagents are required to detect the vitamins, it was necessary to determine the order in which these reagents could be used on a single paper chromatogram. Those compounds which retain their color are identified
-.BO ALPHA
TOCOPHEROL
NICOTINAMIDE
Q5
-.70 pAMlNO6ENLOlC ACID PYRIDOXINE
-.60 4
, 0
2 -.so
CHOLINE
II 2
E -.40
fi
PANTOTHENIC ACID
RIBOFLAVIN
a ..
-L---7 dJ INOSITOL
e
ANALYTICAL CHEMISTRY
-30
m
1 -.IO
s Figure 1, Chromatomap of vitamins and related compounds showing migration in phenol and butanol-propionic acid
Paper Chromatographic Behavior of Vitamins and Related Compounds Rf Values hlethod of Identification ButanolQuantity propionic Color Vitamins and Used, Phenol acid-water reagentb Color C V lamp Related Compounds pg.O Vitamin A 250 Yellow ... a-Tocopherol 250 Am-ig Black ... Menadione 250 Yellow ... FCiP None Fluorescent Thiamine 250 Sin Yellow 0.91 0.32 ... Yellow Fluorescent Riboflavin 20 0.83 0.68 I2 Brown ... Niacin 250 DCPI Pink CNBr Orange 0.85 0.69 I. Brown ... Nicotinamide 250 CXBr Yellow FCSP Yellow 0.87 0.60 FeC13 Brown Fluorescent Pyridoxine 250 0.66 0.38 Fin Purple ... Pantothenic acid 250 0.79 0.78 I? Brown ... Biotin 250 0.21 0.12 Amle: Brown Inositol 50 0 . 8 7 0.52 Choline 250 I* Brown .. 0.80 0.69 FeC13 Brown .. p-Aminobenzoic acid 250 PHC Brown 0.34 0.30 ... ... Fluorescent Folic acid 12.5 0.92 0.31 Pink ... Vitamin B12 50 0.34 0.34 AmAg Brown ... Vitamin C 250 a a-Tocopherol in mg. * AmAg = ammonical silver nitrate (6); FCXP = ferricyanide-nitroprusside ( 3 ) ; Nin = ninhydrin; It = iodine vapor; DCPI = 2,6-dichlorophenol-indophenol(3); CNBr = cyanogen bromide (6); FeC13 = ferric chloride reagent (3); PHC = phenolhypochlorite reagent ( 3 ) .
1416
f5
-.20
Positions of spots obtained by plotting Rf valuer given in Table
Table I .
d
ASCORBIC ACID
I
first. Papers are then viewed under ultraviolet light to determine which compounds fluoresce. Ninhydrin, when used as the first color reagent, appears to have little or no effect on the reaction of subsequent color reagents. Papers sprayed with ninhydrin may be exposed to iodine vapors with satisfactory results. After the iodine is allowed to vaporize off the paper, it may be sprayed with ferric chloride or ammonical silver nitrate. Ferric chloride interferes with the subsequent reaction of ammonical silver nitrate and cannot be successfully used before or after this solution. &4map giving the relative positions of the vitamins is shown in Figure 1. Thus, a single procedure which can be applied to several water-soluble vitamins and one fat-soluble vitamin has been developed. It should be helpful in identifying qualitatively the water-soluble vitamins present in a mixture. LITERATURE CITED
( 1 ) ;ickermann, IF'. W., Kirby, H., J . B i d . Chem. 175, 483 (1948).
(2) Baddiley, J., Thain, E. M.,J . Chem. SOC.1951, 2253. (3) Biochemical Institutes Studies, IV, University of Texas, Publ. 5109 (1951). (4) Blair, J. A,, Graham, J., J . Biochem. 56,286 (1954). (5) Block, R. J., Durrum, E. L., Zweig, G.,
"Manual of Paper Chromatography and Paaer Electroahoresis." DD. 398409, Academic Press, New 'Yo& 1958. ( 6 ) Bowden, J. P., Peterson, W. H., J . Bzol. Chem. 178,533 (1949). ( 7 ) Brown, F., Blaxter ,K. L.. Chem. &
Ind. (London) 1951. 633. (8) Brown, J. k,ANAL. CHEM.25, 774 (19.53). ( 9 j B&m, J. A., March, I f . hl., Ibid., 24, 1952 (1952). (10) Crammer, J. L., Xuture 161. 349 . (i948). (11) Crokaert, R., Arch. Intern. Physiol. 56, 189 (1948). (12) Dalgiesh, C. E., J . Biochem. 5 2 , 3 (1952). (13) Datta, S. P., Overell, B. G., StackDunne, AI., Yuture 164, 673 (1949). (14) Edviards, C. H., Gadsden, E. L., Edwards, G. A , , J . Chromatog. 2 , 188 (19.59). \ _ _ _ _
(15) Eggitt, P. W. R., Ward, L. D., Sci. Food Agr. 4, 176 (1953).
(16) Fountain, J. R., Hutchison, D. J.,
Waring, G. B., Burchenal, J. H., Proc.
SOC. Exptl. Biol. Med. 83, 369 (1953). (17) Green, J., Marunkiewicz, S., Kature 176, 1172 (1955). (18) Guerillot, J., Guerillot-Vinet, il., Delmas, L., Compt. rend. 235, 1295 (1952). (19) Hais, I. M.,Pecakova, L., Suture 163, (68 (1949). (20) Hanes, C. S., Isherwood, F. A , Ibzd , 164, 1107 (1949). (21) Harrison, J. S., Analyst 76, 77 11951). (22) Heyndrickx, A,, J . Am. Pharm. Assoc., Sci. Ed. 42, 680 (1953). (23) Holman, W. I. AT,, J . Biochem. 56, 513 (1954). (24) Huebner. H. F.. Nature 167. 119
Lin, P. H., J . Am.
Toc. 75,2971 (1953).
icek, E., Reddi, K. K., Suture 5 (1951).
trer, E., Lederer, >I., "Chromatography," pp. 256-7, Elsevier, Amsterdam. 1953. (28) Malyoth, G., Stein, H. W., Biochem.
Z. 323, 265 (1952). (29) Mapson, L. W., Partridge, S. &I., .\ atu 'e 164, 479 (1949).
. -.
r r
(30) Miyaki, K., Momiyama, H., Hayashi, M., J . Pharm. SOC. J a p a n 72, 688 (19521.
(32) Rossi, C. A., Boll. SOC. ital. sper. 26, 1563 (1950). (34) Siliprandi, D., Siliprandi, N., Biochim. et Biophys. Acta 14,52 (1954). (35) Snyder, J. I., Wender, S. H., Arch. Biochem. Biophys. 46,465 (1953). (36) Viscontini, M., Bonetti, G., Ebnother, C., Karrer, P., Helu. Chim. \ - - ,
19.51\ --, 1 2 R 4 f,----,.
Arfn3A
--""I
( 3'i ) Weygand, F., Arkiv
Kemi 3, 11 (1950). i Si3) Winsten, W.A., Eigen, E., J . Biol. Chem. 17:7, 989 (1949). (39) Ibid., I181, 109 (1949). (40) Woodr,uff, - - - -H. B., Foster, J. C., Ibid., 183,569 (IYSU). (41) Wright, L. D., Cresson, E. L., Driscoll. C. A.. Proc. SOC.Exwtl. Biol. N e d . 86; 480 (1954). (42) Yagi, K., J.Biochem. (Japan)38,161 (1951). RECEIVEDfor review March 1, 1960. hccepted June 21 1960. In,
.
Cou Iometric Determi nation ot t u ropium and Ytterbium at Controlled Potential EDWARD N. WISE and EDWARD J. COKAL Chemistry Deparfment, University of Arizonu, Tucson, Ariz.
b Europium and ytterbium were determined individually b y the one-electron reduction of their trivalent ions in methanolic , tetraethylammonium bromide, with a mean absolute error of less than 0.05 peq. in the sample range o f 0.5 to 20 peq. The reduction of ytterbium was induced b y the reduction of europium, so that only their sum could b e determined b y reduction in samples containing both elements. In such cases, europium was determined in a separate aliquot b y coulometric oxidation in 0.1 M hydrochloric acid, and ytterbium was found b y difference.
T
of controlled POtential coulometry to the determination of the rare earths had led to the development o'f a n excellent method for the determination of europium ( I O ) . This involves reduction of the trivalent europium in dilute HCl, followed by qumtitstive coulometric oxidation of the divalent ion. However, the extension of this method to the determination of the remaining rare earths is not feasible because of the rapid oxidation of their dipositive ions in reactions of the type HE APPLICATION
'13
+ H20
+
XI+'
+
1/2H2
+ OH(1)
+
-
+
~ + 2 H+ 11+3 l / 2 ~ z (2) hlthough reduction of europium(II1) in aqueous ammonium chloride is nearly quantitative when proper background correction is made ( I S ) , the use of a less easily reduced solvent would be expected to yield reductions in which these interfering reactions do not occur. We have found absolute methanol to be a satisfactory solvent for the determination of europium and ytterbium by the one-electron reduction of the trivalent ions. Europium alone in a supporting electrolyte consisting of 0.1M tetraethylammonium bromide (TEABr) in methanol is reduced n-ith very nearly 1 0 0 ~ ccurrent efficiency, with the final electrolysis current approximately that due to the supporting electrolyte. Ytterbium(I1) reacts with the methanol in a manner similar to the reaction with water described above, thus giving less than 1007, current efficiency. However, the rate of this reaction is sufficiently small that a satisfactory correction for its effect can be made. When both europium and ytterbium occur in the same sample, only their sum can be determined by reduction, since the reduction of ytterbium is induced by the reduction of europium. A method of correcting for this induced reduction has been devised (9)$ but
the magnitude of the correction is too great to allow reasonable accuracy in the results for the individual metals. Better results were obtained by determination of the sum of ytterbium and europium at a potential sufficient to cause quantitative reduction of both elements, and determination of the europium in a separate sample by the coulometric oxidation method developed by Shults (10). Suitable control potentials for europium and ytterbium were determined polarographically in the methanolTEABr electrolyte. Half-wave POtentials were approximately 0.0 and -0.78 volt us. the methanolic silversilver bromide electrode. The n-aves appeared to be well formed, but a log i/id-i us. E plot indicated that the reduction of ytterbium in this medium is very irreversible, the reciprocal of the slope being 0.071 rather than the expected 0.059. Preliminary electrolysis and polarographic experiments indicate that the reduction potentials of europium(II1) and ytterbium(II1) in this system do not differ by more than 0.13 volt. A similar effect may be operative in aqueous solutions and could account for the discrepancy between the reported values for the standard POtentials of the Yb-f2-Yb+3electrode VOL. 32, NO. 1 1 , OCTOBER 1960
1417
