AminoQuant II

Amino Acid Analyzer

Peter Hunziker
Protein Analysis Unit
Institute of Biochemistry
University of Zurich, Switzerland
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Pre-column Method
Derivatization with
OPA/FMOC
Fluorescence Detection
340/450 nm: primary amines (OPA)
266/305 nm: secondary amines (FMOC)

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Equipment/Procedure
HP 1090 HPLC
HP 1046A Fluorescence Detector
HP Chemstation (Pascal)
Manual Hydrolysis
Derivatization in Autosampler
Manual Data Processing
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Samples
Protein/Peptide Hydrolysate
Free Amino Acids (Culture Media,
Nectar, Lake Water)

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Amino Acids
All standard amino acids
Cys: as CySO3, subtraction method
Trp: not from hydrolysates
OH-Pro, methyl-Arg, -Lys, acetyl-Lys
Some D/L-Amino acids
Glutathione
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 Calibration

Three point Asp Ala

300
calibration
200

(9, 22.5, 90 pmol)

Area
100

Internal standard 0
0 20 40 60 80

(Nval, Sar) Amount on Column (pmol)

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 Dynamic Range

Linear response
Asp Ala

800

up to 150 - 250 600

pmol on column Area

400

(depends on 200

amino acid)
0
0 100 200 300 400 500
Amount on Column (pmol)

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 Accuracy (RSD)

Primary amines: 350

300

1.11 - 1.63 % 250

200

Area
150
100
Secondary amines: 50

5.54 %
0
Asp Gly Pro
Amino Acid

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 Limit of Detection

< 500 fmol on column

Asp Ala

80

60

Depends on
Area
40

fluorescence gain
20

0
0 1 2 3 4 5
Amount on Column (background corrected) (pmol)

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 Limit of Quantitation

Hydrolysate: 40

Not determined

Avarage Error (%)

30

20

10
Routine analysis: 0

>25 ng on column 0 0.02 0.04 0.06

Amount on Column (ug)
0.08 0.1

>0.5 ug for hydrolysis

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 Limit of Quantitation

Free amino acids:

Ala Val Ile

2.5

Amount on column (pmol)

< 500 fmol on 2

column 1.5

0.5

0
0 200 400 600 800 1000
Sample Volume dried

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Run Times
25 min
from
injection to
injection

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Labor Intensity
For up to 18 samples

Sample preparation 0.5 - 1 hour

Data Evaluation 0.5 - 4 hours

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Cost per Sample
(Average of 550 Samples/Year)
(Live Time of Instrument 10 Years)

Consumables 5-6$
Salaries 16 - 19 $
Maintenance 2 - 2.5$
Instrument 13 - 15 $

Total 36 - 42.5 $
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Disadvantages
Sensitive chemistry
Only 1/20 of sample is analyzed
Short column life (approx. 750 runs)
No support for software

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Advantages
No special reagents and chemicals
Turn-on-and-Analyze
No need for long setup
AminoQuant chemistry should also
work on an Agilent 1100

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