Separation of water-soluble vitamins on starch thin layers - Analytical

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DISCUSSION

A variety of organic materials have been decomposed using this technique. Samples, approximately 10 grams (dry weight) in size, of bread, potato chips, clover, rhubarb leaves, strawberry leaves, etc. have been burned with little or no carbon formation. The combustion proceeds smoothly and controllably. Because of the quantity of heat liberated, it is advisable to set the flask in several inches of ice water with a fan directed toward the flask. Also, depending on the analysis, it may be necessary to cool the absorber solution. The combustion should be carried out behind a shield. However, combustions using this size sample have been performed numerous times with no indication of hazard. It has been found in this laboratory that the life expectancy of the ignitor bulb can be prolonged if one uses a Variac to increase the voltage gradually rather than apply the full 110 V with an on-off switch. We have used the described system for fluorine analysis whereby a sample was “spiked” with a known amount of organically bound fluorine. A recovery of 90 & 10% at the 1-ppm level was found. This takes into account the workup of the sample leading to the final analysis. Lisk (I) found a recovery of 80 to 109% at the 1- to 14-ppm level for chlorinated pesticides. However, he first concentrated the chlo-

rinated component in a large sample via a solvent extraction. The chlorinated component was then decomposed. The absorber solution for the above analysis was 30 ml of water placed in a 100-ml graduate. The exit tube from the flask led to the bottom of the absorber solution. Baffles were placed on the portion of the tube immersed in the absorber solution. The purpose of the baffles was to break up the gas bubbles and thus aid in the absorption of the hydrofluoric acid. After the combustion was complete, the oxygen was allowed to flow for another 5 min. The flask and sample holder were rinsed with 0.01N sodium hydroxide to remove any absorbed fluoride. Analysis of the absorber solution for fluoride ion was accomplished via one of the various published colorimetric techniques (2). It is felt that this decomposition technique can be applied to numerous elements, both volatile and nonvolatile. The major limiting factors being the degree of difficulty in converting the element involved to the proper valence state and the choice of the proper absorbing solution required to retain that element. RECEIVEDfor review November 13, 1967. Accepted February 8,1968. (2) R. Belcher and T. S. West, Talanta, 8,853 (1961).

Separation of Water-Soluble Vitamins on Starch Thin Layers S. E. Petrovie, B. E. Belia, and D. B. VukajloviC Department of Chemistry, Unicersity of Novi Sad, Novi Sad, Yugoslavia WATER-SOLUBLE VITAMINS are chemically and physiologically heterogeneous compounds, and most of them are distinguished by high instability. Thin-layer chromatography is a suitable method for analysis of these vitamins, but this method has rarely been used, and few publications have appeared to date (1-6). In published works, the separation of single vitamins from the substances which accompany them in natural materials, as well as the separation of related vitamins and their derivatives is mainly described. Ganshirt and Malzacher (2) separated the group of water-soluble vitamins on silica gel layers containing the fluorescent indicator ZS super using ultraviolet light (254 and 365 mp), sodium iodoplatinate, dichloroquinonchlorimide, and ninhydrin (only for pantothenic acid) for detection. We describe the separation of a group of water-soluble vitamins on thin layers of rice starch using ninhydrin (for all examined vitamins which are not visible in daylight) for detection. EXPERIMENTAL

Procedure. For preparation of the thin layers, which has rice starch (Carlo Erba, Milan, already been described (3, Italy) with addition of gypsum was used. Eight watersoluble vitamins (Pliva, Zagreb, Yugoslavia) (Table I) were examined individually and in the mixtures. Vitamins were dissolved in distilled water and the concentration of each vitamin was examined individually and in the mixture (Table I). Spots of 1 p1 of vitamin solutions were applied by micropipet on the thin layer and chromatoplates were developed by the ascending technique in a glass chamber which contained 50 ml of solvent mixture. The chromatograms

were run in the dark at room temperature without previous saturation of the chamber with solvent. The solvent system was n-butanol-acetic acid-water-pyridine (40 :10 :50: 2), and the upper layer was used for development. The developing time of chromatograms was about 5 hours for a solvent front of about 15 cm. Detection. The ninhydrin reagent, 0.5 gram of ninhydrin dissolved in 100 ml of methanol, was used for detection. The developed and dried chromatograms were heated in an oven for 30 minutes at 160” C. After heating, the cooled chromatograms were sprayed with ninhydrin reagent and heated again for about 10 minutes at 80” C. Colored spots of the vitamins appeared (Figure 1). Ultraviolet light at 254 and 365 mp was used for control. RESULTS AND DISCUSSION

All examined vitamins except Ca-pantothenate and p-aminobenzoic acid were separated by using rice starch as support. When vitamins were chromatographed individually, considerable differences of Rf values of Ca-pantothenate and paminobenzoic acid were obtained (Table I), but when the mixture of vitamins was separated, then Ca-pantothenate and p-aminobenzoic acid moved together. As is evident from (1) S . David and H. Hirshfeld, Bull. Soc. Chim. France, 1963, p.

1011. (2) H. Ganshirt and A. Malzacher, Naturwiss., 47, 279 (1960). (3) E. Niirnberg, Deut. Apotheker-Ztg., 101, 142 (1961). (4) E. Niirnberg, Ibid., p. 268. ( 5 ) K. Randerath, A)?gew. Chem., 73,436 (1961). (6) T. Sasaki, J. Chromatog., 24, 452 (1966). (7) S . M. Petrovib and S . E. Petrovif, ibid., 21,313 (1966). VOL 40, NO. 6 , MAY 1968

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Table I. Rf Values and Color of Spots of Vitamins Concn. of vitamins,

Vitamin Thiamine-HC1 (B,) Riboflavin (B2) Pyridoxin-HC1 (Be) Cyanocobalamin (BIZ) Ascorbic acid (C) Nicotinamide Pantothenic acid (Ca-salt) p-Aminobenzoic acid (PAB)

mg/ml 15.0 1.5 15.0 1.0 5.0 15.0 15.0 2.5

R f X 100 42

UV, 254 mp

UV, 365 mp

...

18 58 79 27 74 69 82

Yellow Blue Dark Dark

Dark Yellow Blue Dark Dark Dark

Yellow

Yellow

...

Figure 1, lower R, values for cyanocobalamin, Ca-pantothenate, nicotinamide, and pyridoxine were obtained when separated in the mixture. Rf values shown in Table I were calculated from the mixture, except for p-aminobenzoic acid. Good separations were obtained with solvent mixture npropanol-pyridine-acetic acid-water (15:10:3:13) when riboflavin was not included in the mixture, because riboflavin in this solvent gives an extended spot. Riboflavin and ascorbic acid gave extended spots when the solvent mixture n-butanolformic acid-water-pyridine (40 : 2:10 : 2 ) was used. We separated some vitamins (thiamine and cyanocobalamin, and riboflavin and ascorbic acid) which Ganshirt and Malzacher (2) could not separate, but we obtained results similar to Bolliger’s (8) on silica gel with water as solvent. Corn starch was unsuitable as a support for the separation of water-soluble vitamins. Our earlier experiences with starch as support in TLC show that bigger particles (20 to 30 p ) of corn starch (the size of rice starch particles is 2 to 10 p ) influenced adversely the diffusion of examined substance molecules, reduced a number of theoretical plates, and made spots diffuse so that they overlapped. It especially accentuated organic and natural substances. Ninhydrin proved to be a suitable reagent for detection and identification of water-soluble vitamins. Ninhydrin has already been used for detection of pantothenic acid (2). Petersen et al. (9) obtained an orange spot when they used ninhydrin for detection of pyridoxamine on paper chromatograms. Ninhydrin has not been used in chromatography for detection of other water-soluble vitamins. Table I shows the colors obtained with ninhydrin reagent. The mechanism of reaction between ninhydrin and pantothenic acid is well known. Ascorbic acid is an active reducing agent and converts ninhydrin to its red-violet reduced form. Color reaction with other vitamins cannot be explained by the mechanism which is known for amino acids, because none of these vitamins has a -COOH group, except p-aminobenzoic acid which has the group, although it is far from the -NH2 group. The amido group in nicotinamide is probably hydrolized, and liberated NH8 reacts with ninhydrin. Thiamine and p-aminobenzoic acid have an amino group and pyridoxine has a nitrogen in the ring, but we have no explanation for the color reaction. Perhaps some degradation products react with ninhydrin. The yellow color of riboflavin,

(8) H. R. Bolliger in “Diinnschicht-Chromatographie,” by Egon Stahl, Springer-Verlag, Berlin, 1962, p. 217. (9) E. A. Petersen, H. A. Sober, and A. Meister, J. Am. Chem. SOC., 74, 570 (1952).

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ANALYTICAL CHEMISTRY

Color Daylight Yellow

...

...

Red

... ... ...

...

...

Ninhydrin reagent Light yellow Dark yellow Reddish

...

Red-violet Light blue Violet Orange

...

a7

2 1

i

1

3

4

5

;

;

;

8

Figure 1. Chromatogram of vitamins 1. Riboflavin Ascorbic acid Thiamine-HCI Pyridoxin-HCI

2. 3. 4.

5. Nicotinamide 6. Ca-pantothenate 7. Cyanocobalamin 8.

The mixture

which is visible in daylight, becomes deep yellow. Cyanocobalamin must be identified before heating. The intensive color of vitamins was also obtained with ninhydrin reagent when silica gel was used for thin layers. We did not determine the lowest concentration limit of detection, because we observed that if concentrations of vitamins, shown in Table I, were lowered, the identification of single vitamins was not reliable. Comparison of the separations obtained on rice starch with results obtained on silica gel as carrier shows that rice starch is inferior only in speed of migration of the solvent. From an analytical point of view, starch is neither inferior nor superior when compared with other supports used in TLC, but it has some advantages which are not of analytical nature, such as inexpensiveness and simplicity in manipulation. For use in TLC, starch is especially suitable in countries which produce it industrially.

RECEIVED for review October 10,1967. Accepted January 8, 1968.