Tutorial #1 - Calibration

How to Calibrate an Amino Acid Analyzer

Prerequisites

Amino acid standards, (e.g., Pierce Standard H, 16 AA at 2.5 ľmol/ml + Cys at 1.25 ľmol/ml). Dilute in water for working solution, aliquot and store at -20C.
Peptide and/or protein standard (e.g., BSA from National Bureau of Standards).
Optimize separation and derivatization procedures.

Create a Calibration File

Run 4-6 amino acid standards within linear response range.
Calculate response factor for each amino acid:
Response factor = average peak area/pmol amino acids
or average peak height/pmol amino acids

Compile a calibration file for determining pmol of each amino acid in experimental samples.

Example:

Peak ID

1 Aspartic acid
2 Glutamic acid
3 Serine
4 Glycine
.
.

Ret.time

6.09
6.40
6.65
7.05

Ht/pmol

182.22
175.38
133.94
172.51

Area/pmol

947.31
966.39
855.70
859.65

Amount

300
300
300
300

Calibration file should be updated frequently.
Test quantitation and compositional accuracy with a known protein such as BSA.

How to Evaluate the Linearity Range of Your Analyzer

Analyze in replicate several different amounts of Pierce Standard H spanning the manufacturers specified linearity range.
Plot peak areas vs. amount analyzed.
Amino acid analyzers have an upper and lower limit. It is important to know the limits of your instrument.
Always work in the linear range, below the upper limit. Sensitivity is not an issue for most pre-column systems.

Example: Linearity Determination

Example of linearity determination for the Applied Biosystems Model 420/920/130 System. The data shows a linear response between 1 and 5 nmol. Above 5 nmol, the response of most residues plateaus.

How to Evaluate the Reproducibility

Make several (> 6) injections of Pierce Standard H. (e.g., as for calibration).
Compile retention time and peak area/height data for n = 6 runs.
Calculate the relative standard deviation (RSD) of retention times for each amino acid.
Calculate the RSDs for peak area or height for each amino acid.
Determine the acceptable level of reproducibility for your instrument by repeated analyses over several days, weeks and months by identifying the best (lowest) RSDs.
This not only tests the instrument, but the pipetting ability of the operator.
Remember, this is a quantitative analysis. Test quantitative reproducibility by repeated analysis over time of a peptide or protein of known concentration (e.g., BSA from the National Bureau of Standards).

Example: Reproducibility Statistical Report

Calculate response factor for each amino acid.

Statistical Report

Sample selected:
35501001 (calibration: DEC18DER-12/21/95 5:30 pm)	35501002 (calibration: DEC18DER-12/21/95 6:20 pm)
35501003 (calibration: DEC18DER-12/21/95 7:08 pm)	35501004 (calibration: DEC18DER-12/21/95 7:58 pm)
35501005 (calibration: DEC18DER-12/21/95 8:47 pm)	35501006 (calibration: DEC18DER-12/21/95 9:36 pm)

Peak ID : Alanine

5 peaks found

Sample	rtime	height	pmol	pmol-ht	area	pmol	pmol-ar
ID	min	uAU	by height	correc.	uAU.sec	by area	correc.
35501001	9.52	44914	278.25	300.00	204681	264.24	300.00
35501002	9.55	46359	287.21	300.00	205060	264.73	300.00
35501003	9.55	51353	318.15	300.00	227591	293.82	300.00
35501004	9.55	44834	277.76	300.00	197940	255.54	300.00
35501005	9.57	51409	318.49	300.00	227189	293.3	300.00
35501006	9.57	52410	324.69	300.00	231837	299.3	300.00

Mininum	9.52	44834	277.76	300.00	197940	255.54	300.00
Maxinum	9.57	52410	324.69	300.00	231837	299.3	300.00
Average	9.55	48546	300.76	300.00	215716	278.49	300.00
Std dev	0.02	3543	21.95	0	14723	19.01	0
Rel dev	0.19	7	7.3	0	7	6.83	0

Example: Alanine based on peak area
Response Factor = 215715/300 pmol = 719.05 area/pmol

Example: Alanine based on peak height
Response Factor = 48546/300 pmol = 161.82 ht/pmol

Example: Summary of Reproducibility Data

REPRODUCIBILITY OF AUTOMATIC PTC AMINO ANALYSIS

Amino Acid

Amino Acid Standard

Autohydrolyzed Myoglobin

Retention Time

Peak Area

Percent Relative Standard Deviation (%SD)

ASP	1.36	2.9	10	9
GLX	0.58	3.1	9	11
SER	0.30	2.8	8	10
GLY	0.24	3.0	10	17
HIS	0.36	3.0	9	9
ARG	0.23	3.4	9	10
THR	0.21	2.8	7	8
ALA	0.13	2.5	8	9
PRO	0.16	2.7	32	14
TYR	0.07	2.8	7	5
VAL	0.07	2.7	9	9
MET	0.07	6.7	7	10
ILE	0.06	3.1	7	9
LEU	0.05	3.0	7	8
PHE	0.06	3.6	7	8
LYS	0.07	4.3	7	8


Average %SD	0.25	3.3	9.6	9.6
Amount Analyzed
pmol Protein		-	15	2
pmol Amino Acid	150	150	30-285	4-38

Number of analyses (n)	9	9	6	7

K.A. West and J.W. Crabb (1989) In: Techniques in Protein Chemistry (T.E. Hugli, ed.) Academic Press, pp. 295-304.

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