Amino Acid Analysis Methods

Pre-column derivatization, RP-HPLC

PTC - Phenylthiolcarbamyl
OPA/FMOC - o-phthalaldehyde
FMOC - fluorenylmethyl chloroformate
AQC - 6-aminoquinnoyl-N-hydroxysuccinyl carbamate

Post-column derivatization, ion exchange chromatography

Ninhydrin
OPA
Fluram - fluorescamine

Example: Precolumn Separation

Fig. 1 Typical Amino acid standard (100 pmol) using the Applied Biosystems automated PTC amino acid analysis system (420A derivatizer 130A analyzer). Derivatization and separation of each sample is accomplished in 45 minutes.
Abbreviations: KSU, succinyl cysteine; PEC, pyridylethylcysteine; CMC, carboxymethyl cysteine; Met-O, methionine sulfoxide; AAB, alpha aminobutyric acid; NLE, norleucine; HSer, homoserine
K.A. West and J.W. Crabb (1989) In : Techniques in Protein Chemistry (T.E. Hugli, ed.) Academic Press, pp.295-304.


Example: Post-column Separation


Nihydrin Ion Exchange Amino Acid Analysis

Operating Conditions

Sample:
Column:
Temperature:
Elution Buffers:

Gradient Conditions:

Flow Rate:
Detection:

5 Nanomoles Hydrolysate Standard
Waters Amino Acid Analysis Column
60 C
A) 0.2N Na+, pH 3.15
B) 1.0N Na+, pH 7.4
0-80% B, Curve 8,45 minutes
80-100% B, Curve 8, 15 minutes
0.5 ml/min
546 nm and 436 nm, 0.2 AUFS


Example: Raw Data

Pmol by Area Report

Sample ID: 00901006 (initiated 1/09/96 8:26pm) BASELINE CORRECTED

Turntable Position: 2C Sampling Interval: 1.0 sec
Data Start: 0.00 min Samples In Run: 72
Data Duration: 20.00 min Operator ID: KAW
Peak Ht Threshold: 1000 uAU Int. Std. Amt. : 300 pmol
Calibration File: JAN03DER (initiated 1/09/96 8:47pm)
Reference Time: 0.00 min ISTD Peak ID: Alanine
Reference Offset 1: 0.00 min
Reference Offset 2: 0.0 min

Integration Interval: 0.0 to 20.0 min

PEAK ID
RET. TIME
CAL. TIME
PEAK AREA
PMOL BY
min.
min.
uAU
AREA
Aspartic Acid
6.55
6.49
23850
24.81
Glutamic Acid
6.92
6.86
318955
351.57
Serine
7.62
7.56
243051
321.29
Glycine
8.05
7.99
44476
56.74
Arginine
9.03
9.01
300234
309.56
Threonine
9.28
9.25
8257
9.47
Alanine
9.63
9.6
17428
19.08
Proline
9.87
9.84
146847
158.55
10.28
---
54737
----
11.78
---
528766
----
Tyrosine
12.03
12.03
87542
98.87
Valine
12.93
12.92
4714
4.64
Methionine
13.13
13.14
6043
7.61
14.52
---
30347
----
Leucine
15.02
15.03
170262
184.37
Phenylalanine
15.72
15.57
5647
5.59
15.92
---
6463
----
16.27
---
6063
----
Lysine
16.47
16.48
525632
313.49
17.38
---
321800
----


AAA Data Reduction Overview

An inexpensive integrator will work.

e.g., Shimadzu C-R6A Chromatopac
Beckman System Gold
Hewlett-Packard Chemstation
Perkin Elmer/ABI - Models 920A or 620A
PE Nelson Turbochrom
Rainin Dynamax
Waters Millenium