Amino Acid Analysis
As RSpak NN-814 is packed with polyhydroxymethacrylate bonded with trace amounts of sulfo groups, the main separation mode is the reversed phase mode. However, due to the sulfo groups, ion interaction also works. Acidic amino acids, such as aspartic acid, are separated by ion exclusion mode and elute faster. Neutral substances are separated by reversed phase and alkaline amino acids are separated by a mixture of reversed phase (in case of hydrophobic structure) and ion exchange mode. Therefore, amino acids basically elute in the following order: first the acidic amino acids, second the neutral amino acids and finally the alkaline amino acids .
Sample : 0.1% each, 20micro-L
1. Aspartic acid
2. Glycine
3. Alanine
4. Valine
5. Methionine
6. Isoleucine
Column : Shodex RSpak NN-814 (8.0mmID*250mm) Eluent : 40mM H3PO4 aq. Flow rate : 1.0mL/min Detector : Shodex RI Column temp. : 40deg-C
Amino acid standards are analyzed using CXpak P-421S. The column pressure is low (3MPa under 0.5mL/min and 63deg-C), because of the 6micro-m of gel is packed into this column. We show the chromatogram of standard amino acids under ninhydrine reaction. 17 kinds of amino acids are separated by baseline. The total time for analysis is within 50 minutes till the last peak of arginine.
Sample :
1. Aspartic acid, Asp, 2. Threonine, Thr
3. Serine, Ser, 4. Glutamic acid, Glu
5. Proline, Pro, 6. Glycine, Gly
7. Alanine, Ala, 8. Cysteine, Cys
9. Valine, Val, 10. Methionine, Met
11. Isoleucine, Ile, 12. Leucine, Leu
13. Tyrosine, Tyr, 14. Phenylalanine, Phe
15. Lysine, Lys, 16. NH3
17. Histidine, His, 18. Arginine, Arg
Column : Shodex CXpak P-421S (4.6mmID*150mm)
Eluent : No.1; 0.12M Sodium citrate buffer(pH3.3)
No.2; 0.13M Sodium citrate buffer(pH3.2)
No.3; 0.11M Sodium citrate buffer(pH4.0)
No.4; 1.02M Sodium citrate buffer(pH4.9)
No.5; 0.2M NaOH + 10% C2H5OH (Rinse solution)
Low pressure gradient:
0min; No.1, 1.2min; No.2, 10.0min; No.3, 21.2min; No.4, 40.0min; No.5
Flow rate : 0.5mL/min
Detector : VIS(570nm) (Ninhydrine reaction; 0.35mL/min, 120deg-C)
Column Temp. : 63deg-C
Amino acids were analyzed using IC YS-50 for cations. YS-50 is packed with polyvinyl alcohol with carboxyl functional group. Amino acids are separated by reversed-phase mode and ionic interaction, as NN-814 column does Alanine and Glycine elute at the same elution time under this condition.
See also Amino Acids (2) (NN-814).
Sample : 0.02% each, 5micro-L
1. Aspartic acid
2. Glycine
3. Valine
4. Methionine
5. Isoleucine
Column : Shodex IC YS-50 (4.6mmID*125mm) Eluent : 2mM Acetic acid aq. Flow rate : 1.0mL/min Detector : Shodex RI Column temp. : 40deg-C
Amino acids were analyzed using anion chromatography column, IC YS-50. YS-50 is packed with polyvinyl alcohol with carboxyl functional group. Amino acids are separated by reversed-phase mode and ionic interaction, as RSpak NN-814 does.
Sample : 5micro-L
1. Aspartic acid 0.8mg/mL
2. Glutamic acid 0.8mg/mL
3. Glycine 0.8mg/mL
4. Valine 1.0mg/mL
5. Methionine 1.0mg/mL
6. Isoleucine 1.0mg/mL
7. GABA 1.0mg/mL
8. Phenylalanine 1.0mg/mL
9. Ornithine 1.0mg/mL
10. Histidine 1.0mg/mL
11. 1-Methylhistidine 1.0mg/mL
12. Arginine 1.0mg/mL
Column : Shodex IC YS-50 (4.6mmID*125mm) Eluent : 6mM H3PO4 aq. Flow rate : 1.0mL/min Detector : Shodex RI Column temp. : 40deg-C
Carcinogenic tryptophan pyrolysis products were analyzed using Asahipak ES-502C 7C (a column for weak cation exchange chromatography).
Column : Shodex Asahipak ES-502C 7C (7.5mmID*100mm) Eluent : 20mM H3PO4(pH2.0)/CH3CN=90/10 Flow rate : 1.0mL/min Detector : Fluorescence detector(Ex. 266nm, Em. 397nm) Column temp. : 40deg-C
S Manabe and O. Wada, Exp. Eye Res., 48 (1989) 351.
Histidine and 1-methylhistidine were analyzed using Asahipak ODP-50 6D (a column for reversed phase chromatography)
Sample : 0.005% each, 5micro-L
1. Histidine
2. 1-Methylhistidine
Column : Shodex Asahipak ODP-50 6D (6.0mmID*150mm) Eluent : 10mM Na2HPO4 aq. Flow rate : 0.5mL/min Detector : UV(210nm) Column temp. : 30deg-C
Dansyl-amino acid was analyzed using ODSpak F-411 (a column for reversed phase chromatography) and a chemiluminescence detectot (CL) and a fluorescence detector detector compared.
Sample :
1.Dansyl-L-Valine
2.Dansyl-L-Proline
Column : Shodex ODSpak F-411 (4.6mmID*150mm) Eluent : 0.1M Imidazole Buffer(pH7.0, adjusted by nitrate)/CH3CN=25/75 Flow rate : 0.5mL/min Reagent : [1mM TCPO in Ethyl acetate}/[0.15M H2O2(30%aq) in CH3CN]=25/75 Detector : CL, Fluorescence detector(Ex. 330nm, Em. 560nm) Column temp. : 30deg-C
High sensitive analysis of metal bonded amino acids was performed using GS320A-M8E (0.8mmID x 250mm) and ICP-MS. GS320A-M8E is a micro-column of Asahipak GS-320 HQ. Using the micro column, It is possible to reduce the injection volume and eluent to 1/200 and 1/100 compared with the case where a conventional column is used, respectively.
Sample :
MeSeCys (Methylselenocysteine)
SeMet (Selenomethionine)
(Left) (Right)
LC : capillary HPLC normal HPLC
(Tubing: 0.03mmID fused-silica capillary tube) (Tubing: 0.25mmID PEEK tube)
Columns : Shodex GS320A-M8E (0.8mmID*250mm) Shodex Asahipak GS-320 HQ (7.5mmID*300mm)
Eluent : 50mM Ammonium acetate(pH6.5) 50mM Ammonium acetate(pH6.5)
Flow rate : 5.5micro-L/min 500micro-L/min
Detector : ICP-MS Se at m/z=82 ICP-MS Se at m/z=82
Courtesy of Dr. Yasumitsu Ogra, Graduate School of Pharmaceutical Sciences, Chiba University
The investigation for metal bonded amino acid is remarkable study for metabolising metals is human body. Selenized amino asid is analyzed using GS320A-M5D and ICP/MS. The sensitivity of the analysis using micro column is higher than that using conventional type column.
Please refer to the page regarding the comparison of micro column and coventional type column.
High Sensitive Analysis of Metal Bonded Amino Acids (1)
Sample : 100nL
1. gamma-Glutamylmethylselenocysteine (gGluMeSeCys)
2. Methylselenocysteine (MeSeCys)
Column : Shodex GS320A-M5D (0.5mmID*150mm) Eluent : 50mM CH3COONH4 buffer(pH6.5) Flow rate : 2.0micro-L/min Detector : ICP-MS (Se at m/z=82)
Courtesy of Dr. Yasumitsu Ogra, Graduate School of Pharmaceutical Sciences, Chiba University