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Home / Applications / Rapid Separation of Amino-Acids using UHPLC with OPA Pre-Column Derivatization

  • Industry

  • Technique

Rapid Separation of Amino-Acids using UHPLC with OPA Pre-Column Derivatization

By Miyuki Kanno

PDF IconDownload This Application

November 22, 2023

Introduction

In this application note we report on a method for hiqh-speed automated amino-acid analysis using a UHPLC system with 2.0 mm semi-micro column packed with 2µm C18 stationary phase; this column can dramatically reduce the analysis time (compared with an analytical column) from 38 min down to 7 min; a five-fold improvement in the speed of analysis. The amino-acids were automatically derivatized with ortho-Phthalaldehyde (OPA) using an X-LC 3059AS autosampler, just prior to injection to ensure stable reproducible results.

Amino-acid analysis has been important in many applications ranging from food analysis to protein science. Many different separation and detection techniques have been employed over a long period of time. However, one of the most popular methods remains pre-column derivatization with OPA derivatization, separation using a C18 column and fluorescence detection, offering both simplicity and high sensitivity.

A typical separation normally uses a 4.6 mm I.D x 150 L (or 250 mm) C18 column with 5µm packing material requiring more than 40 mins for a complete analysis.

Experimental

LC-4000 Series
Figure 1. LC-4000 Series HPLC

Automated Pre-column Derivatization System

The derivatization of amino acids was performed automatically using a programmed autosampler, fig. 2 shows the flow diagram of the autosampler. The procedure for derivatizatlon is detailed below.

Figure 2. Flow diagram of the autosampler (X-LC 3059AS)

Procedure for automated pre-column derivatization

  1. Wash needle
  2. Draw reagent and sample with air segmentation
  3. Eject to reaction vial
  4. Mix sample and reagent by air bubbling
  5. Wash needle
  6. Pause to allow reaction to complete
  7. Draw derivatized sample
  8. Injection onto column

(1) Optimization of derivatization conditions

Peak areas of 6 amino-acids were plotted against derivatization reaction time as shown in fig. 3. The derivatives of glycine and lysine showed appreciable decay in fluorescence intensity when the reaction time exceeded 300 seconds. In addition, the lysine derivative has a very low response (as shown by the blue line in fig.3) compared with the derivatives of other amino acids. This phenomenon was also observed by Jones 1). A reaction time of 30 seconds was considered optimal.

Figure 3. Effect of reaction time on peak area

(2) Optimization of Separation Conditions

As shown in fig. 4, pH greatly affected the separation of the histidine and serine derivatives – pH 5.8 was selected. The column temperature also has a significant effect on the separation, shown in orange, green and blue in fig. 5. The column temperature was set to 40°C.

Optimized conditions
Eluent A: 1.0M citrate buffer (pH5.8) 3.5mL in 1L of H20
Eluent B: 1.0M citrate buffer (pH5.8) 3.5mL in 1L of CH3CN/C2H5OH/H2O (30/30/40)
Gradient condition: 1 cycle 10min
A:B = 90:10 – 90:10 – 72:28 – 72:28 – 42:58 – 42:58 – 23:77 – 0:100 – 0:100 – 90:10
0.2min 2.2min 2.5min 4.6min 5.0min 6.1min 6.15mln 7.0min 7.05min
Flow rate: 0.6mL/min
Column: X-PressPak V-C18 (2-µm dia., 2.0mm 1.0. x 5Omm)
Column temperature: 40°C
Detection: Fluorescence; Ex 345nm, Em 455nm; Gain x100

Figure 4. Effect of eluent pH on separation of histidine and serine
Figure 5. Effect of column temperature on separation of several amino acids

Results

This separation was performed under optimized conditions as shown in fig. 6. The blue line represents the gradient profile (%B).

Fig. 7 compares UHPLC with conventional HPLC, extremely fast separation was achieved using UHPLC – the total analysis time was reduced by 80%. and solvent consumption reduced by 90%.

Figure 6. Analysis of standard mixture of 18 amino acids (20pmol/~L each)
Figure 7. Comparison between UHPLC and Conventional HDLC

(1) Precision

Reproducibility of Retention Time and Peak Area

Reproducibility of peak areas  and retention times were calculated from ten replicate injections of 8 amino-acid standards (20pmol each), as shown in Table 1. The relative standard deviations (RSD) were between 0.048 and 0.429% for the retention times, and between 0.546 and 1.90% for the peak areas. Excellent reproducibility of the retention times and peak areas were observed.

Linear Dynamic Range and Detection Limit

Figure 8 shows the linear dynamic range for selected amino acid derivatives. Correlation coefficients (r) were also calculated using the results from the analysis of 7 amino-acid derivatives ranging from 0.2 to 20pmol. Excellent linearity was observed.

Highly sensitive detection was achieved using a fluorescence detector; the detection limit (LOD SN>=3) was between 21 and 49 fmol/µL as shown in Table 2.

(2) Applications in Food Analysis

Figure 9. Analysis of amino-acids in red wine
Figure 10. Analysis of amino-acids in aged rice vinegar.
Aged rice vinegar contains various amino-acids and minerals. Seventeen amino-acids were detected.
Figure 11. Analysis of amino-acids in functional beverage.
This beverage contained 9 essential amino-acids, i.e., His, Thr, Met, Val, Trp, Phe, lie, Leu and Lys.

Analysis of other amino-acids

Figure 12 shows a chromatogram of a standard mixture of 2 amino-acids containing GABA (gamma-aminobutyric acid). GABA is an important inhibitory neurotransmitter in the central nervous system and essential for brain metabolism and function. Asparagine and glutamine were included in this standard in addition to GABA. The analysis conditions were the same as shown in fig. 6.

Figure 13 shows the chromatogram of amino-acids in white wine containing GABA. The label of this wine showed ingredients including lychee juice, which is known to be a good source of GABA. The conditions are the same as shown in fig. 6.

Figure 12. Analysis of standard mixture of 21 amino-acids containing GABA (20pmol/µL each)
Figure 13. Analysis of amino-acids in white wine containing GABA

Analysis of Theanine

Theanine (L-γ-glutamylethylamide) is an analog of glutamine, found almost exclusively in tea plants (and fungi) and can induce a feeling of relaxation. Theanine has a sweet and umami taste and shows a strong correlation with the quality of Japanese green tea. Figure 14 shows a chromatogram of a standard mixture of 18 amino-acids, including theanine. The conditions are different from those used in fig. 6.; the standard contained two additional internal standards – MBA and norvaline. MBA was used as an internal standard for the analysis of green tea, and norvaline for the analysis of black tea; because it is difficult to separate impurity peaks from MBA.

Figure 15 shows the chromatogram of amino-acids of green tea. It should be noted that the green tea contains a large amount of theanine, and is the major component among the range of amino- acids present.

Conditions for the Analysis of Theanine

The separation conditions were selected for the analysis of amino-acids including theanine in tea,  this used binary gradient elution with an ethanol-citrate buffer mobile phase.

Eluent A: 1.0M citrate buffer (pH6.0) 3.5mL in 1L of C2H5OH/H2O (12/88)
Eluent B: 1.0M citrate buffer (pH6.0) 3.5mL in 1L of C2H5OH/H2O (50/50)
Gradient condition: 14 min
A:B = 100:0 – 0:85 – 0:85 – 100:0
9.0 min 0.5 min 0.05 min
Flow rate: 0.4mL/min
Column: X-PressPak V-C18 (2µm dia., 2.0mm I.D. x 50mm)
Column temperature: 40°C
Detection: Fluorescence; Ex 340nm,Em 450nm; Gain x 100

Figure 14. Analysis of standard mixture of 18 amino-acids containing theanine (O.4mg/L each)
Figure 15. Analysis of amino-acids in green tea

Conclusion

  1. High-Speed
    Total analysis time: ca 1/5 (38 min-7 min)
  2. Reduced Solvent Consumption
    Amount ratio: ca 1/10 (60 mL~6 mL)
  3. High Sensitivity Detection
    Detection limit (SN>=3): 21 – 49 fmol/L
  4. Excellent Reproducibility (n=10)
    RSD%: 0.05 – 0.43% (retention time), 0.58 – 1.90% (peak area)
  5. Applicable in a wide variety of Food Analysis
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.

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About the Author

Miyuki Kanno

JASCO Application Note

Rapid Separation of Amino-Acids using UHPLC with OPA Pre-Column Derivatization

Introduction

In this application note we report on a method for hiqh-speed automated amino-acid analysis using a UHPLC system with 2.0 mm semi-micro column packed with 2µm C18 stationary phase; this column can dramatically reduce the analysis time (compared with an analytical column) from 38 min down to 7 min; a five-fold improvement in the speed of analysis. The amino-acids were automatically derivatized with ortho-Phthalaldehyde (OPA) using an X-LC 3059AS autosampler, just prior to injection to ensure stable reproducible results.

Amino-acid analysis has been important in many applications ranging from food analysis to protein science. Many different separation and detection techniques have been employed over a long period of time. However, one of the most popular methods remains pre-column derivatization with OPA derivatization, separation using a C18 column and fluorescence detection, offering both simplicity and high sensitivity.

A typical separation normally uses a 4.6 mm I.D x 150 L (or 250 mm) C18 column with 5µm packing material requiring more than 40 mins for a complete analysis.

Experimental

LC-4000 Series
Figure 1. LC-4000 Series HPLC

Automated Pre-column Derivatization System

The derivatization of amino acids was performed automatically using a programmed autosampler, fig. 2 shows the flow diagram of the autosampler. The procedure for derivatizatlon is detailed below.

Figure 2. Flow diagram of the autosampler (X-LC 3059AS)

Procedure for automated pre-column derivatization

  1. Wash needle
  2. Draw reagent and sample with air segmentation
  3. Eject to reaction vial
  4. Mix sample and reagent by air bubbling
  5. Wash needle
  6. Pause to allow reaction to complete
  7. Draw derivatized sample
  8. Injection onto column

(1) Optimization of derivatization conditions

Peak areas of 6 amino-acids were plotted against derivatization reaction time as shown in fig. 3. The derivatives of glycine and lysine showed appreciable decay in fluorescence intensity when the reaction time exceeded 300 seconds. In addition, the lysine derivative has a very low response (as shown by the blue line in fig.3) compared with the derivatives of other amino acids. This phenomenon was also observed by Jones 1). A reaction time of 30 seconds was considered optimal.

Figure 3. Effect of reaction time on peak area

(2) Optimization of Separation Conditions

As shown in fig. 4, pH greatly affected the separation of the histidine and serine derivatives – pH 5.8 was selected. The column temperature also has a significant effect on the separation, shown in orange, green and blue in fig. 5. The column temperature was set to 40°C.

Optimized conditions
Eluent A: 1.0M citrate buffer (pH5.8) 3.5mL in 1L of H20
Eluent B: 1.0M citrate buffer (pH5.8) 3.5mL in 1L of CH3CN/C2H5OH/H2O (30/30/40)
Gradient condition: 1 cycle 10min
A:B = 90:10 – 90:10 – 72:28 – 72:28 – 42:58 – 42:58 – 23:77 – 0:100 – 0:100 – 90:10
0.2min 2.2min 2.5min 4.6min 5.0min 6.1min 6.15mln 7.0min 7.05min
Flow rate: 0.6mL/min
Column: X-PressPak V-C18 (2-µm dia., 2.0mm 1.0. x 5Omm)
Column temperature: 40°C
Detection: Fluorescence; Ex 345nm, Em 455nm; Gain x100

Figure 4. Effect of eluent pH on separation of histidine and serine
Figure 5. Effect of column temperature on separation of several amino acids

Results

This separation was performed under optimized conditions as shown in fig. 6. The blue line represents the gradient profile (%B).

Fig. 7 compares UHPLC with conventional HPLC, extremely fast separation was achieved using UHPLC – the total analysis time was reduced by 80%. and solvent consumption reduced by 90%.

Figure 6. Analysis of standard mixture of 18 amino acids (20pmol/~L each)
Figure 7. Comparison between UHPLC and Conventional HDLC

(1) Precision

Reproducibility of Retention Time and Peak Area

Reproducibility of peak areas  and retention times were calculated from ten replicate injections of 8 amino-acid standards (20pmol each), as shown in Table 1. The relative standard deviations (RSD) were between 0.048 and 0.429% for the retention times, and between 0.546 and 1.90% for the peak areas. Excellent reproducibility of the retention times and peak areas were observed.

Linear Dynamic Range and Detection Limit

Figure 8 shows the linear dynamic range for selected amino acid derivatives. Correlation coefficients (r) were also calculated using the results from the analysis of 7 amino-acid derivatives ranging from 0.2 to 20pmol. Excellent linearity was observed.

Highly sensitive detection was achieved using a fluorescence detector; the detection limit (LOD SN>=3) was between 21 and 49 fmol/µL as shown in Table 2.

(2) Applications in Food Analysis

Figure 9. Analysis of amino-acids in red wine
Figure 10. Analysis of amino-acids in aged rice vinegar.
Aged rice vinegar contains various amino-acids and minerals. Seventeen amino-acids were detected.
Figure 11. Analysis of amino-acids in functional beverage.
This beverage contained 9 essential amino-acids, i.e., His, Thr, Met, Val, Trp, Phe, lie, Leu and Lys.

Analysis of other amino-acids

Figure 12 shows a chromatogram of a standard mixture of 2 amino-acids containing GABA (gamma-aminobutyric acid). GABA is an important inhibitory neurotransmitter in the central nervous system and essential for brain metabolism and function. Asparagine and glutamine were included in this standard in addition to GABA. The analysis conditions were the same as shown in fig. 6.

Figure 13 shows the chromatogram of amino-acids in white wine containing GABA. The label of this wine showed ingredients including lychee juice, which is known to be a good source of GABA. The conditions are the same as shown in fig. 6.

Figure 12. Analysis of standard mixture of 21 amino-acids containing GABA (20pmol/µL each)
Figure 13. Analysis of amino-acids in white wine containing GABA

Analysis of Theanine

Theanine (L-γ-glutamylethylamide) is an analog of glutamine, found almost exclusively in tea plants (and fungi) and can induce a feeling of relaxation. Theanine has a sweet and umami taste and shows a strong correlation with the quality of Japanese green tea. Figure 14 shows a chromatogram of a standard mixture of 18 amino-acids, including theanine. The conditions are different from those used in fig. 6.; the standard contained two additional internal standards – MBA and norvaline. MBA was used as an internal standard for the analysis of green tea, and norvaline for the analysis of black tea; because it is difficult to separate impurity peaks from MBA.

Figure 15 shows the chromatogram of amino-acids of green tea. It should be noted that the green tea contains a large amount of theanine, and is the major component among the range of amino- acids present.

Conditions for the Analysis of Theanine

The separation conditions were selected for the analysis of amino-acids including theanine in tea,  this used binary gradient elution with an ethanol-citrate buffer mobile phase.

Eluent A: 1.0M citrate buffer (pH6.0) 3.5mL in 1L of C2H5OH/H2O (12/88)
Eluent B: 1.0M citrate buffer (pH6.0) 3.5mL in 1L of C2H5OH/H2O (50/50)
Gradient condition: 14 min
A:B = 100:0 – 0:85 – 0:85 – 100:0
9.0 min 0.5 min 0.05 min
Flow rate: 0.4mL/min
Column: X-PressPak V-C18 (2µm dia., 2.0mm I.D. x 50mm)
Column temperature: 40°C
Detection: Fluorescence; Ex 340nm,Em 450nm; Gain x 100

Figure 14. Analysis of standard mixture of 18 amino-acids containing theanine (O.4mg/L each)
Figure 15. Analysis of amino-acids in green tea

Conclusion

  1. High-Speed
    Total analysis time: ca 1/5 (38 min-7 min)
  2. Reduced Solvent Consumption
    Amount ratio: ca 1/10 (60 mL~6 mL)
  3. High Sensitivity Detection
    Detection limit (SN>=3): 21 – 49 fmol/L
  4. Excellent Reproducibility (n=10)
    RSD%: 0.05 – 0.43% (retention time), 0.58 – 1.90% (peak area)
  5. Applicable in a wide variety of Food Analysis
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.
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