1. Determination of amino acids in fodders and raw materials using capillary electrophoresis

2. Methods for analysis of AA contents in fodders
Ion-exchange chromatography (in the amino acids analyzer)
RP-HPLC with gradient elution
Calculation method
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CE - only for free form of AAs (juice, beer)

3. 2 schemes of AAs analysis in fodders by CE
Complete analysis of proteinogenous amino acids (20 AAs)
Express analysis of technologically important amino acids – Lys, Met, Thr, Cys-Cys, Trp.
Pre-capillary derivatization
Direct detection (without derivatization)

4. Direct determination of technologically important AAs
Detection
Optimization of separation, including:
on 190 nm (Lys, Met, Thr, Cys-Cys) acid hydrolysis
on 219 nm (Trp) alkaline hydrolysis
pH and composition of BGE
additives to the BGE (organic solvents, surfactants, macrocycles)

5. CZE separation of the 19 underivatized amino acids
Buffer: 10 mM borate with the addition 10 mM -CD (pH 9.18)
Voltage: +20 kV;
Detection:190 nm.

6. Assay characteristics for technologically important amino acids
Parameters
LOQ
LOD
mg l-1
Range b r %
R.S.D.
% (n=9)
Lys
40 – 200
16.30
0.9982 40
0.40 9 20
Met
10 – 100
2.95
0.9998 10
0.10 4 5
Thr
20 – 100
6.65
0.9997
0.20
Cys-Cys
8.81
0.9988 7
Trp
0.1 – 50
1.45
0.1
0.02
0.05

7. Percentage of the amino acids in samples, (%SD)
The data of the underivatized amino acids determination in the real samples using HPLC, CZE and IEC
Percentage of the amino acids in samples, (%SD)
CZE
RP-HPLC
IEC (arbitral method)
Fish flour
Lys
0.85  0.11
0.94  0.16
0.84  0.09
Met
1.42  0.17
1.65  0.20
1.35  0.12
Thr
1.05  0.13
1.10  0.16
1.02  0.11
Cys-Cys
0.56  0.08
0.65  0.13
0.69  0.12
Mill cake
1.15  0.16
1.18  0.19
1.09  0.14
0.75  0.10
0.69  0.10
0.69  0.09
0.72  0.12
0.80  0.14
0.75  0.16
0.24  0.04
0.36  0.07
0.23  0.04
Mixed fodder
1.52  0.18
1.49  0.13
1.27  0.15
0.45  0.06
0.53  0.06
0.52  0.08
1.11  0.14
0.98  0.15
1.00  0.15
0.33  0.04
0.29  0.04
0.41  0.05

8. Direct determination of Trp
Alkaline hydrolysis
Temperature 40°C
Detection on 219 nm:
increase of detection sensitivity
decrease of interference of concomitant AAs

9. The complete analysis of proteinogenous amino acids
Only for derivative forms
PITC advantages:
simplicity of use
effective separation of the derivatives
sufficient reproducibility of the reaction condition
good solubility of the derivatives in water

10. Derivatization of AAs with PITCCH NH 2
COOH
AAs
pH 10.5 NH C S CH R
COOH
PTC - AAs N C S
PITC + O
PTH - AAs R S NH N

11. CE analysis of PTC-AAs Voltage: 25 kV; BGE: phosphate with ß-CD
Temperature: 20°C
No pressure was applied

12. CE analysis of PTC-AAs Controlled pressure during analysis
Highly precise changeable temperature of a capillary cooling
Voltage: 25 kV; BGE: phosphate with ß-CD
Temperature: 30°C
Pressure 30 mbar was applied after 17 min

13. b-CD as a component of BGE
Chiral activity for Trp, Tyr, Phe !
Protein molecules consist of L-AAs, but mixed fodders’ preparation is followed by insertion of synthetic AAs.
Technology nonobservance – use racemates or D-AAs.
Fodders quality control and falsifications reveal.