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Home / Applications / CD Spectrum Measurement Using HTCD System

  • Industry

  • Technique

CD Spectrum Measurement Using HTCD System

By Heather Haffner

PDF IconDownload This Application

August 24, 2022

Introduction

Automatic measurement systems have been drawing increasing attention among pharmaceuticals companies due to their ability to analyse multiple samples automatically, saving both time and man power and eliminating any possible human error. Since CD spectroscopy allows the user to easily obtain structural information about proteins and peptides, automated measurement systems can be used to optimize the screening process of a large number of samples.

This application notes introduces the High-Throughput CD system for automatic CD measurements of multiple protein samples.

JASCO J-1500 high-throughput CD system
JASCO J-1500 high-throughput CD system

Experimental

Measurement conditions
Data acquisition interval0.1 nmResponse time2 seconds
Spectral bandwidth1 nmScan speed100 nm/min
Accumulations4 timesPath length1 mm
Emission wavelength350 nmSensitivity850 V

Keywords

J-1500, circular dichroism, Autosampler, ASU-800, FMO- 427, Emission monochromator, excitation, emission, HighThroughput, HTCD, proteins, biochemistry, pharmaceuticals

Results

First the sequence of operation and data acquisition must be defined in advance. The maximum number of samples that can be analyzed automatically is 192. The system will then carry out CD measurements automatically and save the data series. The following sequence is automatically performed by the HTCD system:

1. Loading of measurement condition
2. Baseline correction
3. Sample 1 measurement (cytochrome c)
4. Washing
5. Sample 2 measurement (lysozyme)
6. Washing
7. Sample 3 measurement (human serum albumin)
8. Washing
9. Sample 4 measurement (hemoglobin)
10. Washing

Figure 1. Display of sequence setting

The JFLC-499 CD/emission flow cell was used to obtain both CD and excitation spectra. The results of the automatic measurements of CD, absorbance, and excitation spectra for four 0.1 mg/mL protein samples are shown below. These high speed measurements took a total of 3 minutes per sample (each scan is 45 seconds and there are 4 accumulations).

Figure 2. CD (top), absorbance (middle), and excitation (bottom) spectra of cytochrome c.
Figure 3. CD (top), absorbance (middle), and excitation (bottom) spectra of lysozyme.
Figure 4. CD (top), absorbance (middle), and excitation (bottom) spectra of human serum albumin.
Figure 5. CD (top), absorbance (middle), and excitation (bottom) spectra of hemoglobin.

Conclusion

This application note demonstrates that high quality data can be measured for three different spectroscopy techniques (CD, absorption, and excitation) in a matter of minutes. The J-1500 coupled with the HTCD system allows over 100 samples to be measured automatically without human operation, saving time and eliminating human error.

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

JASCO Application Note

CD Spectrum Measurement Using HTCD System

Introduction

Automatic measurement systems have been drawing increasing attention among pharmaceuticals companies due to their ability to analyse multiple samples automatically, saving both time and man power and eliminating any possible human error. Since CD spectroscopy allows the user to easily obtain structural information about proteins and peptides, automated measurement systems can be used to optimize the screening process of a large number of samples.

This application notes introduces the High-Throughput CD system for automatic CD measurements of multiple protein samples.

JASCO J-1500 high-throughput CD system
JASCO J-1500 high-throughput CD system

Experimental

Measurement conditions
Data acquisition interval0.1 nmResponse time2 seconds
Spectral bandwidth1 nmScan speed100 nm/min
Accumulations4 timesPath length1 mm
Emission wavelength350 nmSensitivity850 V

Results

First the sequence of operation and data acquisition must be defined in advance. The maximum number of samples that can be analyzed automatically is 192. The system will then carry out CD measurements automatically and save the data series. The following sequence is automatically performed by the HTCD system:

1. Loading of measurement condition
2. Baseline correction
3. Sample 1 measurement (cytochrome c)
4. Washing
5. Sample 2 measurement (lysozyme)
6. Washing
7. Sample 3 measurement (human serum albumin)
8. Washing
9. Sample 4 measurement (hemoglobin)
10. Washing

Figure 1. Display of sequence setting

The JFLC-499 CD/emission flow cell was used to obtain both CD and excitation spectra. The results of the automatic measurements of CD, absorbance, and excitation spectra for four 0.1 mg/mL protein samples are shown below. These high speed measurements took a total of 3 minutes per sample (each scan is 45 seconds and there are 4 accumulations).

Figure 2. CD (top), absorbance (middle), and excitation (bottom) spectra of cytochrome c.
Figure 3. CD (top), absorbance (middle), and excitation (bottom) spectra of lysozyme.
Figure 4. CD (top), absorbance (middle), and excitation (bottom) spectra of human serum albumin.
Figure 5. CD (top), absorbance (middle), and excitation (bottom) spectra of hemoglobin.

Conclusion

This application note demonstrates that high quality data can be measured for three different spectroscopy techniques (CD, absorption, and excitation) in a matter of minutes. The J-1500 coupled with the HTCD system allows over 100 samples to be measured automatically without human operation, saving time and eliminating human error.

Keywords

J-1500, circular dichroism, Autosampler, ASU-800, FMO- 427, Emission monochromator, excitation, emission, HighThroughput, HTCD, proteins, biochemistry, pharmaceuticals

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

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