How Can Chromatography Techniques Be Used to Isolate Specific Biomolecules?

[fuzzy]

Homework Statement

What procedures would you use to obtain gram quantities of each of the following compounds, free of each of the other compounds?
Glycine - monoamino, monocarboxylic amino acid
Glucose - non-ionic monosaccharide
Starch - non-ionic large polysaccharide
Glucose - 5-phosphate - anionic monosaccharide phosphate
mRNA - polyanionic nucleic acid



Compound||||||||||||||||MW
Glycine|||||||||||||||||||75
Glucose||||||||||||||||||180
starch||||||||||||||||||||>>30,000
glucose-5-phosphate||||||258
mRNA|||||||||||||||||||||>>30,000

To answer this question, you must make use of the ionic character of each compound. For example, fructose is non-ionic monomer whereas glycogen is also non-ionic but a polymer. So gel filtration would be useful in this case.

Note that ribose-5-phosphate has three negative charges whereas tRNA has many negative charges and the MW is many folds larger than ribose-5-phosphate.

Alanine is an amino acid with either positive, or neutral, or negative charge depending on the pH of the buffer used.

(when it says ribose I think it means glucose, same for tRNA and mRNA... that's what you get for copy-paste!)

Homework Equations

I think we're only allowed to use three columns, DEAE, CM and Gel.
DEAE=anion exchanger (negative will stick)
CM= cation exchanger (positive will stick)
Gel= filters by size of the molecule...

The Attempt at a Solution

Gel filtration will be good for the non-ionic ones:glucose and starch but if it's used first, it will just give 2 solutions filled with junk, so it has to be used last.
mRNA is negative, glucose-5 is negative, but, mRNA is more negative because of polyanionic.
glycine is... some website said it was non-ionic??

k so nothing is positive?

DEAE-> sticks the mRNA, gluc-5... glucose-5 will come out first followed by mRNA. 2 Solutions down, 3 to go...

solution with: glycine, starch, glucose
CM-> does nothing unless glycine is positive; then you're left with starch and glucose
Gel->starch first, then glucose
done!
But... IS GLYCINE POSITIVE?!? otherwise it wouldn't work...

 

[chemisttree]

What is the pKb of the glycine (amine part) and the pKa of the carboxylic acid part? Can you find a pH where the ammonium will not be protonated? What will happen to the carboxylic acid at that pH? Is there a pH where the ammonium will be protonated and the carboxylic acid remain in the acid form?

 

[Blueshift5]

I would recommend using a combination of techniques to obtain gram quantities of each compound, free of the other compounds. Here is a possible approach:

1. For glycine, which is a non-ionic amino acid, gel filtration can be used to separate it from the other compounds. This will work because glycine has a smaller molecular weight compared to glucose and starch, so it will come out of the gel first. However, if glycine has a net positive charge at the pH of the buffer used, then an anion exchanger like DEAE can be used to separate it from the other compounds.

2. For glucose, which is a non-ionic monosaccharide, gel filtration can be used to separate it from the other compounds, as it has a smaller molecular weight compared to starch. However, if glucose is in the form of glucose-5-phosphate (anionic), then it can be separated using a cation exchanger like CM. This will work because the phosphate group will have a negative charge and will stick to the cation exchanger while glucose will come out in the solution.

3. For starch, which is a large polysaccharide, gel filtration can be used to separate it from the other compounds. This will work because starch has a much larger molecular weight compared to glycine and glucose. However, if there are any smaller impurities in the starch, an anion exchanger like DEAE can be used to remove them.

4. For mRNA, which is a polyanionic nucleic acid, an anion exchanger like DEAE can be used to separate it from the other compounds. This will work because the negative charges on the mRNA will stick to the DEAE while the other compounds will come out in the solution.

Overall, a combination of gel filtration, anion exchange, and cation exchange can be used to obtain gram quantities of each compound, free of the other compounds. The exact order in which these techniques are used will depend on the specific properties of each compound and any potential impurities. It is important to carefully consider the net charge and molecular weight of each compound to determine the most appropriate separation techniques.