Chromatography of Urine Sugars

 Chromatography of Urine Sugars-Chromatography is considered a superior method for identification of urine sugars. Thin layer chromatography is quick and sensitive; and therefore it is the method of choice.

Thin layer chromatography 

Reagents:- Solvent mixture

n-butanol     75 ml

Acetic acid    25 ml

Distilled water         6 ml

Aniline-diphenylamine locating reagent

Solution1:

Aniline           1 ml

Diphenylamine        1g

Acetone        99 ml

Solution II:

 85 % phosphoric acid

For use, 10 volumes of solution I are mixed with I volume of solution II. Standards Prepare standard solutions of glucose, lactose, fructose, galactose and xylose in 10% aqueous isopropanol. The concentration should be such that 5 microlitres contain 5 micrograms of the sugar. (1mg/ml)

Silica gel- Use 20 x 10 cm plates with 250 um thickness.

Method:-The optimum amount of urine sample used will vary with the concentration of the sugars present. The reagent strips can be used to roughly estimate the amount of sugars in the urine. Determine the volume of the sample required in such a way that 5 ul will contain 5-10 ug of sugar. Apply the sample and the standards 1 cm apart along the line of application. Place the plate in the chromatography tank. Let the solvent rise to 12 to 15 cm for about 2 to 3 hours. Remove the plate from the tank. Dry it under hot air in a fume cupboard. Spray with the locating agent and heat for 5 minutes in hot air oven at 120°С. above Figure shows position of various sugars, and Table  shows their R, values. 

	Approximate R1 value of urinary
S. NO.	SUGAR	CHARACTERSTIC COLOUR	Approx R1
1.	Lactose	Gray	0.14
2.	GALACTOSE	Gray	0.34
3.	FRUCTOSE	PINK	0.39
4.	GLUCOSE	GREY	0.42
5.	PENTOSES	GRAY BROWN	>0.5