Organic Chemistry

Section 23.5 Separation of Amino Acids 971

Fractions sequentially collected

>Figure 23.4

Separation of amino acids by

ion-exchange chromatography.

Met

Asp

Thr

Val

Tyr

Lys

SerGlu Ala

Ile

Leu Phe

Arg

40 80 120 160

Gly

Pro

His

NH 3

200 240 280 320

Effluent (mL)

Absorbance

330 370 410 450 490 50 90 130

pH 3.3 buffer pH 4.3 buffer pH 5.3 buffer

>Figure 23.5

A typical chromatogram obtained

from the separation of a mixture of

amino acids using an automated

amino acid analyzer.

determined by the amount of absorption at 570 nm—because the colored compound

formed by the reaction of an amino acid with ninhydrin has a of 570

(Section 8.11). In this way, the identity and the relative amount of each amino acid can

be determined (Figure 23.5).

lmax

PROBLEM 13

Why are buffer solutions of increasingly higher pH used to elute the column that generates

the chromatogram shown in Figure 23.5?

PROBLEM 14

Explain the order of elution (with a buffer of pH 4) of each of the following pairs of amino

acids on a column packed with Dowex 50 (Figure 23.3):

a. aspartate before serine c. valine before leucine

b. glycine before alanine d. tyrosine before phenylalanine

PROBLEM 15

In what order would the following amino acids be eluted with a buffer of pH 4 from a col-

umn containing an anion-exchange resin?

histidine, serine, aspartate, valine

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BRUI23-959-998r2 29-03-2003 1:36 PM Page 971