How Can Chromatography Techniques Be Used to Isolate Specific Biomolecules?

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SUMMARY

This discussion focuses on the isolation of specific biomolecules using chromatography techniques, specifically DEAE (Diethylaminoethyl) and CM (Carboxymethyl) ion exchange chromatography, alongside gel filtration. The participants conclude that gel filtration is best applied last to separate non-ionic compounds like glucose and starch, while DEAE effectively captures negatively charged molecules such as mRNA and glucose-5-phosphate. The discussion also highlights the importance of understanding the ionic character of glycine, which can vary based on pH, impacting its separation process.

PREREQUISITES
  • Understanding of chromatography techniques, specifically DEAE and CM ion exchange chromatography.
  • Knowledge of gel filtration principles for size-based separation.
  • Familiarity with the ionic character of biomolecules, particularly amino acids and polysaccharides.
  • Basic understanding of pH effects on amino acid charge states.
NEXT STEPS
  • Research the principles of ion exchange chromatography, focusing on DEAE and CM techniques.
  • Learn about gel filtration chromatography and its applications in biomolecule separation.
  • Study the pKa and pKb values of amino acids, particularly glycine, to understand their charge states at different pH levels.
  • Explore the role of ionic character in the separation of biomolecules in chromatography.
USEFUL FOR

This discussion is beneficial for biochemists, laboratory technicians, and students involved in biomolecular research, particularly those focusing on chromatography techniques for isolating amino acids, sugars, and nucleic acids.

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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...
 
Physics news on Phys.org
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?
 

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