2. Analysis of amino acids
1. Ion exchange chromatography
Different types of ion exchange resins exist:
– Cation exchange: negatively charged/separation of cations
– Anion exchange: positively changed/separation of anions.
• Obviously, the type of resin to be used will depend on the charge of the
amino acid on interest, itself dependent on the pH of the solution.
+
+
pH >> pI pH << pI
Elution
3. Analysis of amino acids
2. Detection of amino acids: ninhydrin reagent
O
O
OH
OH
Ninhydrin Amino acid
2
O
O
N
O
O
CO2
COO-
NH3
+
R-HC=O
H C
R
Purple!!
While Trp, Phe and Tyr can be detected by their A260-280nm, the other amino acids cannot;
• Ninhydrin reacts with the amine group of amino acids, generating a purple product (yellow in
the case of Pro).
• The ninhydrin reaction allows one to detect and quantify (A570nm) the amino acids contained in
the fractions of the IEX column.
8. • PKU is a hereditary “inborn error
of metabolism” caused by
defective enzyme, phenylalanine
hydroxylase.
• It causes accumulation of
phenylpruvate, which causes
causes mental retardation.
• Sources of phenylalanine such
as aspartame must be limited.
• A substitute for aspartame, known
as alatame, contains alanine rather
than phenylalanine.
Phenylketonuria (PKU)