Skip to content

JASCO JASCO

  • News
  • Events
  • E-Store
  • My Account
  • Contact Us
  • Worldwide
Search
Click to view menu
MENUMENU
  • Products
    • Chromatography
      • HPLC
      • RHPLC
      • UHPLC
      • LC-MS
      • Preparative LC
      • Analytical SFC
      • Semi-Preparative SFC
      • Hybrid SFC
      • Fuel Analysis by SFC-FID
      • Preparative SFC
      • Supercritical Fluid Extraction
      • Chromatography Software
    • Molecular Spectroscopy
      • Circular Dichroism
      • High-Throughput CD
      • Vibrational CD
      • Circularly Polarized Luminescence (CPL)
      • Polarimeters
      • FTIR Spectrometers
      • FTIR Microscopy
      • FTIR Portable
      • Raman Microscopy
      • Palmtop Raman Spectrometer
      • Probe Raman
      • UV-Visible/NIR Spectrophotometers
      • UV-Visible/NIR Microscopy
      • Fluorescence Spectrophotometers
      • Film Thickness
      • Spectra Manager™ Suite
    • Refurbished
      • Refurbished HPLC Systems
      • HPLC Switching Valves
      • FTIR Accessories
  • Service
    • Service and Support Plans
    • Service Request Form
  • Applications
  • KnowledgeBase
  • Learning Center
    • Best Practice
      • Circular Dichroism Tips & Tricks for Biological Samples
      • CD Scale Calibration with ACS
      • Fluorescence Tips & Tricks
      • Raman Spectroscopy Tips & Tricks
    • Training Videos
      • ChromNAV
      • SF-NAV
      • Circular Dichroism
      • UV-Visible/NIR
      • Fluorescence
    • Training Seminars
      • Training Registration Form
    • Webinars
    • eBooks
    • Theory
      • Theory of Molecular Spectroscopy
      • Chromatography
  • About Us
    • President’s Message
    • Contact
    • History
    • Careers
  • News
  • Events
  • Worldwide
  • Shop
  • My Account
  • Contact Us

Home / Applications / Analysis of Protein Hydrolysate Amino Acids using OPA Post-column Derivatization by Quaternary Low Pressure Gradient System

  • Industry

  • Technique

Analysis of Protein Hydrolysate Amino Acids using OPA Post-column Derivatization by Quaternary Low Pressure Gradient System

By Satoko Suzuki

PDF IconDownload This Application

January 5, 2024

Introduction

Amino acid analysis has been applied to several categories such as food, medicine, protein science and metabolome study, and is an important measurement technique.

An amino acid analysis system using a low pressure gradient unit with OPA post column derivatization provides excellent repeatability and good separation in a short analysis time.

The analysis results of food analysis and amino acid composition analysis of protein using this amino acid analysis system are reported.

PU-2089 Quarternary Gradient HPLC Pump
PU-2089 Quarternary Gradient HPLC Pump

Experimental

Schematic Diagram

  1. Eluent (Amino Buffer Na-LG(1st~4th))
  2. Degasser
  3. Low pressure gradient unit
  4. Eluent pump
  5. Ammonia filter (AECpak Na-LG)
  6. Autosampler
  7. Column oven
  8. Column (AApak Na-LG)
  9. Reagent 1 (Amino Reagent Na-LG Hypo)
  10. Airtrap 1
  11. Reagent pump 1(Hypo)
  12. Reaction coil 1
  13. Reagent 2 (Amino Reagent Na-LGOPA)
  14. Airtrap 2
  15. Reagent pump 2 (OPA)
  16. Reaction coil 2
  17. Fluorescence detector
  18. Chromatography data system(ChromNAV)

Keywords

430027H

Results

Figure 1 shows the chromatogram of the standard mixture of 20 kinds of amino acids.The sample was well separated within 45 minutes (1 cycle 60 min).

Figure 1. Chromatogram of the amino acid standard mixture (500 pmol each)
1: Cysteic acid, 2: Asparatic acid, 3: Threonine, 4: Serine, 5: Glutamicacid, 6: Proline, 7: Glycine, 8: Alanine, 9: Cystine, 10: Valine, 11: Methionine, 12: Isoleucine, 13: Leucine, 14: Tyrosine, 15: Phenylalanine, 16: GABA, 17: Lysine, 18: Histidine, 19: Tryptophan, 20: Arginine

Figure 2 shows the chromatogram of a sports drink while figure 3 shows the chromatogram of white wine containing GABA.

Figure 3. Chromatogram of white wine containing GABA.
1:Cysteic acid, 2:Asparatic acid, 3:Threonine, 4:Serine, 5: Glutamicacid, 6:Proline, 7: Glycine, 8: Alanine, 9: Cystine,10: Valine,11: Methionine,12: Isoleucine,13: Leucine,14: Tyrosine,15: Phenylalanine, 16:GABA, Lysine,18: Histidine,19: Tryptophan, 20: Arginine

Sample preparation:

Sports drink was diluted 150-fold by 0.2 N citric acid buffer (pH2.2) and then filtrated using 0.45 μm membrane filter.

Figure 2. Chromatogram of a sports drink
2: Asparatic acid, 3: Threonine, 4: Serine, 5: Glutamicacid, 6: Proline, 7: Glycine, 8: Alanine, 10: Valine, 11: Methionine, 12: Isoleucine, 13: Leucine, 14: Tyrosine, 15: Phenylalanine, 17: Lysine, 18:Histidine, 19: Tryptophan, 20: Arginine

Sample preparation: White wineincluding GABA was diluted 10-fold by 0.2N citric acid buffer (pH 2.2) and then filtrated using 0.45 μmmembrane filter.

Figure 4 shows the chromatogram of hydrolyzed myoglobin (horse skeletal muscle). Table 1 shows the results of amino acid composition compared with theoretical value (referred to Japan Bio chemistry DataBook l ,Tokyo Kagaku Dojin). It was confirmed that the amino acid composi tion calculated from this measurement was in good agreement with the theoretical value.

Figure 4 Chromatogram of hydrolyzed myoglobin (horse skeletal muscle).
1: Cysteic acid, 2: Asparatic acid, 3: Threonine, 4: Serine, 5: Glutamicacid, 6: Proline, 7: Glycine, 8: Alanine, 9: Cystine, 10: Valine, 11: Methionine, 12: Isoleucine, 13: Leucine, 14: Tyrosine, 15: Phenylalanine, 16: GABA, 17: Lysine, 18: Histidine, 20: Arginine

Sample preparation:

1. Myoglobin was diluted to 200μg/mL by ultra pure water. 2.The solution filled a 20 μL sample tube and then dried up by centrifugal evaporator. 3. Sample tube was set in vessel for hydrolysis and 0.3 mL of hydrochloric acid was added to thevessel. 4. The sample was heated for 24 hours under vacuumed condition. 5. The sample was hydrolyzed and the residual chlorine was removed using vacuum pump. 6.500μL of 0.2N citric acid buffer (pH2.2) was added to the sample and agitated.

Table 1 Comparison of amino acid composition of myoglobin between the measured and theoretical value.

This document has been prepared based on information available at the time of publication and is subject to revision without notice. Although the contents are checked with the utmost care, we do not guarantee their accuracy or completeness. JASCO Corporation assumes no responsibility or liability for any loss or damage incurred as a result of the use of any information contained in this document. Copyright and other intellectual property rights in this document remain the property of JASCO Corporation. Please do not attempt to copy, modify, redistribute, or sell etc. in whole or in part without prior written permission.

Featured Products:

  • LC-4000 Series RHPLC

  • Column Ovens

  • Tandem LC-MS

About the Author

Satoko Suzuki is a senior manager of the molecular spectroscopy applications laboratory at the JASCO facility in Hachioji Japan.

JASCO Application Note

Analysis of Protein Hydrolysate Amino Acids using OPA Post-column Derivatization by Quaternary Low Pressure Gradient System

Introduction

Amino acid analysis has been applied to several categories such as food, medicine, protein science and metabolome study, and is an important measurement technique.

An amino acid analysis system using a low pressure gradient unit with OPA post column derivatization provides excellent repeatability and good separation in a short analysis time.

The analysis results of food analysis and amino acid composition analysis of protein using this amino acid analysis system are reported.

PU-2089 Quarternary Gradient HPLC Pump
PU-2089 Quarternary Gradient HPLC Pump

Experimental

Schematic Diagram

  1. Eluent (Amino Buffer Na-LG(1st~4th))
  2. Degasser
  3. Low pressure gradient unit
  4. Eluent pump
  5. Ammonia filter (AECpak Na-LG)
  6. Autosampler
  7. Column oven
  8. Column (AApak Na-LG)
  9. Reagent 1 (Amino Reagent Na-LG Hypo)
  10. Airtrap 1
  11. Reagent pump 1(Hypo)
  12. Reaction coil 1
  13. Reagent 2 (Amino Reagent Na-LGOPA)
  14. Airtrap 2
  15. Reagent pump 2 (OPA)
  16. Reaction coil 2
  17. Fluorescence detector
  18. Chromatography data system(ChromNAV)

Results

Figure 1 shows the chromatogram of the standard mixture of 20 kinds of amino acids.The sample was well separated within 45 minutes (1 cycle 60 min).

Figure 1. Chromatogram of the amino acid standard mixture (500 pmol each)
1: Cysteic acid, 2: Asparatic acid, 3: Threonine, 4: Serine, 5: Glutamicacid, 6: Proline, 7: Glycine, 8: Alanine, 9: Cystine, 10: Valine, 11: Methionine, 12: Isoleucine, 13: Leucine, 14: Tyrosine, 15: Phenylalanine, 16: GABA, 17: Lysine, 18: Histidine, 19: Tryptophan, 20: Arginine

Figure 2 shows the chromatogram of a sports drink while figure 3 shows the chromatogram of white wine containing GABA.

Figure 3. Chromatogram of white wine containing GABA.
1:Cysteic acid, 2:Asparatic acid, 3:Threonine, 4:Serine, 5: Glutamicacid, 6:Proline, 7: Glycine, 8: Alanine, 9: Cystine,10: Valine,11: Methionine,12: Isoleucine,13: Leucine,14: Tyrosine,15: Phenylalanine, 16:GABA, Lysine,18: Histidine,19: Tryptophan, 20: Arginine

Sample preparation:

Sports drink was diluted 150-fold by 0.2 N citric acid buffer (pH2.2) and then filtrated using 0.45 μm membrane filter.

Figure 2. Chromatogram of a sports drink
2: Asparatic acid, 3: Threonine, 4: Serine, 5: Glutamicacid, 6: Proline, 7: Glycine, 8: Alanine, 10: Valine, 11: Methionine, 12: Isoleucine, 13: Leucine, 14: Tyrosine, 15: Phenylalanine, 17: Lysine, 18:Histidine, 19: Tryptophan, 20: Arginine

Sample preparation: White wineincluding GABA was diluted 10-fold by 0.2N citric acid buffer (pH 2.2) and then filtrated using 0.45 μmmembrane filter.

Figure 4 shows the chromatogram of hydrolyzed myoglobin (horse skeletal muscle). Table 1 shows the results of amino acid composition compared with theoretical value (referred to Japan Bio chemistry DataBook l ,Tokyo Kagaku Dojin). It was confirmed that the amino acid composi tion calculated from this measurement was in good agreement with the theoretical value.

Figure 4 Chromatogram of hydrolyzed myoglobin (horse skeletal muscle).
1: Cysteic acid, 2: Asparatic acid, 3: Threonine, 4: Serine, 5: Glutamicacid, 6: Proline, 7: Glycine, 8: Alanine, 9: Cystine, 10: Valine, 11: Methionine, 12: Isoleucine, 13: Leucine, 14: Tyrosine, 15: Phenylalanine, 16: GABA, 17: Lysine, 18: Histidine, 20: Arginine

Sample preparation:

1. Myoglobin was diluted to 200μg/mL by ultra pure water. 2.The solution filled a 20 μL sample tube and then dried up by centrifugal evaporator. 3. Sample tube was set in vessel for hydrolysis and 0.3 mL of hydrochloric acid was added to thevessel. 4. The sample was heated for 24 hours under vacuumed condition. 5. The sample was hydrolyzed and the residual chlorine was removed using vacuum pump. 6.500μL of 0.2N citric acid buffer (pH2.2) was added to the sample and agitated.

Table 1 Comparison of amino acid composition of myoglobin between the measured and theoretical value.

Keywords

430027H

This document has been prepared based on information available at the time of publication and is subject to revision without notice. Although the contents are checked with the utmost care, we do not guarantee their accuracy or completeness. JASCO Corporation assumes no responsibility or liability for any loss or damage incurred as a result of the use of any information contained in this document. Copyright and other intellectual property rights in this document remain the property of JASCO Corporation. Please do not attempt to copy, modify, redistribute, or sell etc. in whole or in part without prior written permission.
28600 Mary’s Court, Easton, MD 21601 USA • (800) 333-5272 • Fax: (410) 822-7526 • jascoinc.com/applications

Close

Designed in Tokyo. TRUSTED globally.

View our support plans

Connect with JASCO

  • Facebook
  • Twitter
  • LinkedIn
  • JASCO Sales
  • 800-333-5272

Receive the latest promotions and special offers

  • This field is for validation purposes and should be left unchanged.
  • Careers
  • Press Kit
  • JASCO Privacy Policy
  • Sitemap
  • Environmental Policy

© , JASCO. All Rights Reserved.