BIOMEDICAL CHROMATOGRAPHY, VOL. 11.59-60 (1997)

SHORT COMMUNICATION TLC Separation of Some Common Sugars on Silica Gel Plates Impregnated with Transition

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Metal Ions

Ravi Bhushan* and Supreet Kaur Department of Chemistry, University of Roorkee, Roorkee--247 667, India

TLC separation of glucose, maltose, lactose, sorbitol and sucrose on silica gel plates impregnated with transition metal ions, Cu(II), NI(II), Zn(I1) or Cd(II), has been achieved. The identification is very distinct by using KMnO, (0.5%) in 0.1 M NaOH as the spray reagent. Two new solvent systems, (A) n-PrOH-H,O (8:4, vlv), and (B) i-PrOH-H,O (8 : 4, vlv), were worked out.

Biomed. Chromatogr: 11,5940 (1997) No. of Figures: 0. No. of Tables: 1. No. of Refs: 14.

INTRODUCTION

Separation and identification of glucose and sorbitol is helpful in following the pathogenesis and progress of diabetes mellitus (Malone et al., 1980; Aida et al., 1990). Recent results (Hadzija et al., 1994) report TLC separation of glucose and sorbitol on Cu(I1) impregnated plates. Impregnation of thin layer plates has been reported to improve the separation of a variety of compounds (Bhushan and Martens, 1996). A search of literature (Sherma, 1980, 1982, 1984, 1986, 1988, 1990, 1992, 1994) indicates a few reports on the separation of mono- and disaccharides on impregnated plates. In view of this, attempts were made for the separation of mono- and disaccharides on TLC Plates impregnated with Cu(II), Ni(II), Zn(I1) and Cd(I1) metal ions.

EXPERIMENTAL

Glucose, lactose, maltose, and sucrose were purchased from SISCO Research Laboratory (Bombay, India) and sorbitol from Sigma Chemical Co. (USA), Silica gel G (10-40 pm) with calcium sulphate (13%) as binder with impurities of chloride, iron and lead (up to 0.02% each) and showing a pH of 7.0 in a 10% aqueous suspension, was from E. Merck (India). The other solvents and reagents used were of A.R. grade and obtained from BDH (England) or SISCO (Bombay, India).

Standard solutions M) of glucose, maltose, lactose, sorbitol and sucrose were prepared in double distilled water. Thin layer plates (20 x 20 cm x 0.5 mm) were prepared by spreading a sluny of silica gel G (50 g in 100 mL distilled water) with a Stahl type applicator. The plates were then dried overnight at 60k2 OC in an oven. The plates were cooled at room temperature and immersed in solution (0.5%) of Cu(II), Ni(II), Zn(II), or Cd(I1) each separately for

Correspondence to: R. Bhushan.

0.5 min. The plates were again dried in an oven as usual. The solutions of mono- and disaccharides were applied on untreated and impregnated silica gel layers at 500 ng level. These were developed at 25k2 OC in paper lined rectan- gular glass chambers using (A): n-PrOH-H,O (8: 4, vlv) and (B): i-ProH-H,O (8 : 4, vlv) as mobile phases for 2 h. The chromatographic chambers had been pre-equilibrated with mobile phase for 15 min. The plates were then dried in an oven at 60+2 OC for about 15 min and cooled to room temperature. The spots were located by spraying potassium perrnanganate (0.5%) in sodium hydroxide (0.1 M) and heating the plates at 60k 2 OC for 10- 15 min.

RESULTS AND DISCUSSION

The hR, value of separated sugars on untreated and impregnated silica gel layers are given in Table 1. The results are an average of at least three identical runs with a standard deviation of k0.40 to k0.50. The resolution was confirmed (Sleclunan and Sherma, 1982) by dividing the distance between two spot centres with the sum of their radii; a value of 1.50 or more showed the clear resolution of saccharides while a value less than 1.50 indicated incom- plete resolution.

In order to optimize the separation conditions, various concentrations of the mobile phase constituents and impreg- nating reagents were used. As a result of extensive experimentation the most successful mobile phases and impregnating reagents were selected and reported herein.

able 1 shows that in general three compounds separated on untreated silica gel layers while all the five compounds resolved on silica gel layers impregnated with metal ions. Although all the saccharides separated on Cu(II), Ni(II), Zn(I1) and Cd(I1) impregnated plates the Cu(I1) impregnated plate was considered to be the best one because the spots on it were very compact in comparison to those on plates impregnated with other metal ions. In the solvent system (A), the impregnation specifically provided a very good separation of maltoselsorbitol and lactose/sucrose, while in

CCC 0269-3879/97/010059-02 O 1997 by John Wiley & Sons, Ltd.

Received 17 June 1996 Accepted 9 August 1996

R. BHUSHAN AND S. KAUR

Table 1. hR, Values of sugars on untreated and impregnated silica gel layers

A B Saccharides a b c d e a b c d e 1. Glucose 36 22 65 83 67 54 40 95 66 40 2. Maltose 62 63 80 55 75 52 50 90 75 50 3. Lactose 84 58 70 79 83 72 78 84 62 78 4.Sorbitol 60 54 84 75 55 64 60 70 70 60 5. Sucrose 86 70 87 41 79 62 87 60 52 87

Mobile phase, (A) n-PrOH-H,O (8:4, w); (B), i-PrOH-H,O (8:4, w); solvent front, 10 cm; developing time, 2 h for A and B separately; detection, 0.5% KMnO, in 0.1 M NaOH; room temperature, 2522 OC. Silica gel plates: a, untreated plates; b, c, d and e, Cu(ll), Cd(ll), Zn(ll) and Ni(ll) impregnated plates at 0.5% concentration, respectively.

the solvent system (B) the impregnation was successful, providing very good separation of glucose/maltose and sorbitol/sucrose; these pairs had very close R, values on plain plates. Furthermore, all the systems reported herein have been successful in separating glucose/sorbitol. Sac- charides are known to form complexes with transition metal ions (Whistler, 1965), and it is known that sugars and polyols are complexing agents for different metals, and that alditols form stronger complexes with Cu(I1) than mono- saccharides (Briggs et al., 1981). Therefore, it can be considered that the sugars under study formed complexes with metal ions, and their separation was governed by the chromatographic behaviour of the complexes so formed. This resulted in complete and clear separation of mono- and disaccharides on silica gel impregnated with metal ions.

The spots of saccharides were detected by spraying potassium permanganate (0.5%) in sodium hydroxide

(0.1 M); it gave a purple background with yellow spots on untreated and Zn(I1) impregnated silica gel layers, a deep violet background with yellow spots on Cu(I1) and Cd(I1) impregnated silica gel layer and a green background with black spots on Ni(I1) impregnated silica gel layers.

Thus, the chromatographic systems reported can be considered as being fast and with simpler methodology of impregnation when compared with TLC methods reported earlier (Hadzija et al., 1994). Being reproducible, they can be recommended for the separation and identification of the reported saccharides from unknown solutions.

Acknowledgements

Thanks are due to the University Grants Commission, New Delhi, and to the University of Roorkee (grant code 106-99194-95) for providing financial assistance.

REFERENCES

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