biocat88
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TLC tailing problem with monosaccharides
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!
biocat
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Nicodem
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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.
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Ozone
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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
-Anyone who never made a mistake never tried anything new.
--Albert Einstein
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biocat88
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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!
biocat
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Ozone
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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
-Anyone who never made a mistake never tried anything new.
--Albert Einstein
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Sedit
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Slight dilution of your reactants may aid you in gaining a higher resolution as well.
Knowledge is useless to useless people...
"I see a lot of patterns in our behavior as a nation that parallel a lot of other historical processes. The fall of Rome, the fall of Germany — the
fall of the ruling country, the people who think they can do whatever they want without anybody else's consent. I've seen this story
before."~Maynard James Keenan
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biocat88
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| 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).
biocat
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