Sciencemadness Discussion Board

TLC tailing problem with monosaccharides

biocat88 - 16-11-2011 at 11:17

I loaded my samples with mixture of glucose and galactose at the first time (developing solvent: ACN/H2O=85:15; color reagent: DAP; upper picture in attachment). It looks that the separation is quite good, and my samples only contain glucose. But when I did the second time (lowerpicture in attachment), I found that my samples have tailing problem, and my samples contain both galactose and glucose. I am really confused.
I am wondering is it possible the composition of developing solvent was changed because of less tight developing chamber, and it will cause the tailing problem?

Many thanks in advance!


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Nicodem - 16-11-2011 at 12:36

It looks normal to me. The peaks for the two compounds are close, so they almost overlap, which might have appeared like tailing to you. Note that the standards behave consistently on both TLC plates.
What is it exactly that you find unusual? Are you not satisfied with the resolution? Try a taller TLC or a different mobile phase. Though, depending on the intended use, the current resolution can be just fine.

Ozone - 16-11-2011 at 17:10

We use either 20:30:50 % nitromethane:water:n-propanol on K5 silica or 39:9:22:30 acetonitrile:ethyl acetate:n-propanol:water on K6F silica. In either case, spot development is done by spraying with an ethanolic solution of 0.3 % (w/v) a-naphthol and 5 % (v/v) H2SO4, air drying, and heating for 10-20 min at 100°C.

These both work well, but it is extremely important to insure that the spots are absolutely dry (without heating them so much that the carbohydrates are damaged...) before developing the plates...failure to do so inevitably results in streaking and poor resolution. Also, don't overload the stationary phase.

Good luck,

O3

biocat88 - 17-11-2011 at 12:41

Quote: Originally posted by Ozone  
We use either 20:30:50 % nitromethane:water:n-propanol on K5 silica or 39:9:22:30 acetonitrile:ethyl acetate:n-propanol:water on K6F silica. In either case, spot development is done by spraying with an ethanolic solution of 0.3 % (w/v) a-naphthol and 5 % (v/v) H2SO4, air drying, and heating for 10-20 min at 100°C.

These both work well, but it is extremely important to insure that the spots are absolutely dry (without heating them so much that the carbohydrates are damaged...) before developing the plates...failure to do so inevitably results in streaking and poor resolution. Also, don't overload the stationary phase.

Good luck,

O3



I wonder that the developing solvent you mentioned is suitable for the seperation of glucose from galactose. BTW, phosphorylated sugar is suitable as well?
Thanks!

Ozone - 17-11-2011 at 16:20

The mechanism involves dehdyration to furfural (pentoses) or 5-hydroxymethylfurfural (HMF, hexoses), http://en.wikipedia.org/wiki/Molisch's_test. Galactose is just as likely to do this a glucose, so, yes. The ketoses, e.g. fructose, react 7-10 x faster wrt. dehydration to HMF.

Although I don't know for sure, but Molisch reagent is quite general and glucose-6-phosphate should also work--the aldehyde group, which is involved with the formation of the chromophore, should still be available. It seems like the sulfuric acid should catalyze the hydrolysis (if there's some water) of that phosphate leading to glucose and then the colored product.

Cheers,

O3

Sedit - 17-11-2011 at 18:47

Slight dilution of your reactants may aid you in gaining a higher resolution as well.

biocat88 - 19-11-2011 at 04:40

Quote: Originally posted by Ozone  
The mechanism involves dehdyration to furfural (pentoses) or 5-hydroxymethylfurfural (HMF, hexoses), http://en.wikipedia.org/wiki/Molisch's_test. Galactose is just as likely to do this a glucose, so, yes. The ketoses, e.g. fructose, react 7-10 x faster wrt. dehydration to HMF.

Although I don't know for sure, but Molisch reagent is quite general and glucose-6-phosphate should also work--the aldehyde group, which is involved with the formation of the chromophore, should still be available. It seems like the sulfuric acid should catalyze the hydrolysis (if there's some water) of that phosphate leading to glucose and then the colored product.

Cheers,

O3



Thank you O3.
I want to know 39:9:22:30 acetonitrile:ethyl acetate:n-propanol:water like you mentioned before is suitable for seperate glucose and galactose well?
Because ACN/H2O (85:15) in my research looks not very satisfactory to seperate these two compounds (the picture shown in the beginning).