Amino Acid Analysis Methods

Pre-column derivatization, RP-HPLC

PTC - Phenylthiolcarbamyl

OPA/FMOC - o-phthalaldehyde

FMOC - fluorenylmethyl chloroformate

AQC - 6-aminoquinnoyl-N-hydroxysuccinyl carbamate

• PTC analysis requires salt-free sample.
• Sensitivity is high, usually requiring 0.5-1.0 µg.
• Accuracy can be 90-95% depending on amount analyzed, instrument and operator.
  

Post-column derivatization, ion exchange chromatography

Ninhydrin
OPA
Fluram - fluorescamine

• Sample may contain buffer salts, urea, etc.
• Requires 5-10 µg per analysis.
• Accuracy typically 90-95%.
• Generally less susceptible to performance variations than pre-column methods.

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

• More sophisticated data reduction packages are available such as:

Beckman System Gold

Hewlett-Packard Chemstation

Perkin Elmer/ABI - Models 920A or 620A

PE Nelson Turbochrom

Rainin Dynamax

Waters Millenium

• Important Calculations for AAA
  
  • Calibration
  • Reproducibility
  • Mole %
  • Residues per molecule known and unknown samples
  • Concentration (e.g., pmol/uL or mg/ml)
    

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