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Some questions on TLC, if any one can help? - (Dec/06/2006 )

What are the advantages of TLC?

How can you improve the mobile phase to allow better separation of amino acids?

How can you estimate the sensitivity of the ninhydrin detection system? (As in by calculating?)

Whats an alternative approach to separate amino acids?


TLC is rapid and easy. Detection and resolution can be the problems. To improve resolution of amino acids use a buffer that splits their pKa. You may need a pre-analytical clean up step to get rid of protein fragments, lipids. Use sulfosalicylic acid to precipitate these. You may get a better result if you then desalt. use solvent extraction or ion exchange. You could do a series of extraction washes in different buffers to obtain different classes of amino acids (basic, neutral etc).

You'll get good resolution with cellulose stationary phase. Adding metal ions to ninhydrin helps stabilize the colour complexes. Obviously, compare against a standard set.

Here's a paper that might help, Saifer 1971 "Rapid screening methods for the detection of aminoacidopathies." Adv Clin Chem 14: 145-218.

Tietz Clinical Chemistry also has a decent account of the method.