Ion exchange chromatography and ninhydrin reagent are used to analyze and detect amino acids. Ion exchange chromatography separates amino acids based on their charge using cation or anion exchange resins depending on the pH. Ninhydrin reagent reacts with the amine group of amino acids, producing a purple product that allows for detection and quantification of amino acids in the fractions from ion exchange chromatography. Phenylketonuria is an inborn error of metabolism caused by a defective enzyme, resulting in accumulation of phenylpyruvate and mental retardation. Diet must limit phenylalanine intake through restricting sources like aspartame.

1. ANALYIS AND DETECTION OF
AMINO ACIDS

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.

4. Reaction with Amino Acids

5. Esterification of acid

6. Amide bond formation

7. Metabolic Errors

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)