Thermo Scientific ProPac SCX-20 Product Manual
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Thermo Scientific ProPac SCX-20 Product Manual

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Summary of Contents for Thermo Scientific ProPac SCX-20

  • Page 1 ® ProPac SCX-20...
  • Page 2 Product Manual for ProPac SCX-20 Page 1 of 21 PRODUCT MANUAL ® ProPac SCX-20 PROPAC SCX-20 ANALYTICAL COLUMN (4 x 250 mm, P/N 074628) PROPAC SCX-20 GUARD COLUMN (4 x 50 mm, P/N 074643) ® ©2010 Dionex Corporation Document No. 065403...

  • Page 3: Table Of Contents

    Page 2 of 21 TABLE OF CONTENTS SECTION 1 – INTRODUCTION......................3       1.1. Features of the ProPac SCX-20, Strong Cation Exchange Columns ............3     1.2. ProPac SCX-20 Operating Limits and Specifications ................3    ...

  • Page 4: Section 1 - Introduction

    SCX-20 Page 3 of 21 SECTION 1 – INTRODUCTION 1.1. Features of the ProPac SCX-20, Strong Cation Exchange Columns ® The ProPac SCX-20 column is a strong cation exchange (SCX) column designed specifically to provide high-resolution separations of proteins. A grafted cation-exchange surface provides pH-based selectivity control and fast mass transfer for high-efficiency separation and moderate capacity.

  • Page 5: Formats Of The Propac Scx-20 Columns

    ProPac SCX-20 Operating Conditions ProPac SCX-20 strong cation exchange columns are stable between pH 2 and 12, and are compatible with both aqueous mobile phases and those containing solvents, such as acetonitrile. The ProPac SCX-20 strong cation exchange columns can be operated at any flow rate, as long as the backpressure remains below 3,000 psi.

  • Page 6: Section 2 - System Requirement

    2.1. An Inert System is Strongly Recommended The ProPac SCX-20 columns were designed to be used with a standard bore HPLC system having a gradient pump module, injection valve, and a UV detector. A metal-free/inert system is recommended for halide-salt mobile phases which may otherwise cause corrosion of metallic components.

  • Page 7: Section 3 - Operation

    0.1% sodium azide Do not operate the ProPac SCX-20 in the absence of a minimal ionic strength (At least 20 mM). If the ionic strength is too low, the conformation of the stationary phase will be affected, causing a significant increase in backpressure.

  • Page 8: Validating Column Performance

    Validating Column Performance Dionex recommends that you perform an efficiency test on your ProPac SCX-20 strong cation exchange column before you use it. The purpose of column performance validation is to make sure that no damage has been done to the column during shipping.

  • Page 9: Section 4 - Applications

    ProPac SCX-20 is altered. Ribonuclease A elutes first, followed by cytochrome C and lysozyme. Such selectivity differences are quite useful for protein separation and purification applications. Also, the peak capacity for ProPac SCX-20 is superior when compared to ProPac SCX-10 making it the column of choice for protein separation applications.

  • Page 10: Analysis Of Hemoglobin Variants

    F and A. However, two IEF steps, using cellulose acetate electrophoresis with alkaline pH, followed by confirmation using citrate agar electrophoresis at acidic pH are necessary. The ProPac SCX-20 column successfully resolves these hemoglobin species in a single run within 20 minutes.
  • Page 11 Page 10 of 21 Figure 3 shows the separation of hemoglobin A2 using both ProPac SCX-10 and ProPac SCX-20 columns. A faster separation could be achieved on ProPac SCX-20 within 16 minutes with a different selectivity and better resolution of the main peak.
  • Page 12 Product Manual for ProPac SCX-20 Page 11 of 21 Separation of Human hemoglobin and Porcine hemoglobin is achieved with ProPac SCX-20 column (Figure 4). Several closely related variant separations could be achieved in less than 20 minutes. Figure 4 Separation of Human and Porcine Hemoglobins on a ProPac SCX-20 column Document No.

  • Page 13: Monitoring Forced Aspargine Deamidation Of Glycoproteins

    Asn67 were baseline-resolved from each other and from native ribonuclease A in a single chromatographic analysis (9, 10). Figure 5 shows the separation of ribonuclease A and its deamidation products on ProPac SCX-20 at various incubation time points during the course of the forced deamidation. The baseline separation made it possible to quantify the change in amounts of each form within the mixture as a function of time.
  • Page 14 H deamidated RNase A (Peak 2) is present as a small peak. The resolution of ribonuclease A on ProPac SCX-20 is better than ProPac SCX-10 (See Figure 1). The multiple peaks seen as second deamidation variant (shown with an arrow in Figure 5) could be due to the separation deamidated variants of the protein.

  • Page 15: Separation Of Snake Venom Proteins Using Propac Scx-20 Column

    (proteases, polypeptidases), peptidases, and nucleases. Some of these components contribute to the toxicity of the venom. These are separated and used for developing anti-venom therapies. Figure 6 Separation of Snake Venom Proteins on a ProPac SCX-20 column. Document No. 065403-01 ©2010 DIONEX CORPORATION...

  • Page 16: Section 5 - Troubleshooting Guide

    The 4 x 250 mm ProPac SCX-20 should add no more than 2200 psi backpressure at 1 mL/min. The 4 x 50 mm ProPac SCX-20 columns should add no more than 600 psi back pressure at 1 mL/min.

  • Page 17: Peak Efficiency And Resolution Is Poor

    ® Product Manual for ProPac SCX-20 Page 16 of 21 5.1.2. Peak efficiency and resolution is poor. 1. Try to use different eluents (buffer, pH, concentration etc.); to make sure you are using the optimum conditions for your separation problem. 2.

  • Page 18: Appendix A. References

    ® Product Manual for ProPac SCX-20 Page 17 of 21 APPENDIX A. REFERENCES Johnson, A. B., J. M. Shirokawa, W. S. Hancock, M. W. Spellman, L. J. Basa and D. W. Aswad, J. Biol. Chem., 264 (1989) 14262-14271. Paranandi, M. V., Paranandi, A. W. Guzetta, W. S. Hancock and D. W. Aswad, J. Biol. Chem., 269 (1994) 243- 253.

  • Page 19: Appendix B. Propac Scx-20 Column Care

    Usually 5 - 30 min at 1 mL/min is sufficient. Do not exceed 20x the column volume of 0.5 M NaOH (60 mL). The use of high concentrations of base and/or larger volumes of base is not recommended. The ProPac SCX-20 column can be washed at temperatures up to 60 °C.
  • Page 20 ® Product Manual for ProPac SCX-20 Page 19 of 21 Replacing Column Bed Support Assemblies Replace the inlet bed support ONLY if the column is determined to be the cause of high system backpressure, AND cleaning of the column does not solve the problem. NOTE 1.

  • Page 21: Appendix C. Quality Assurance Reports

    ® Product Manual for ProPac SCX-20 Page 20 of 21 APPENDIX C. QUALITY ASSURANCE REPORTS Document No. 065403-01 ©2010 DIONEX CORPORATION September 2010...
  • Page 22 ® Product Manual for ProPac SCX-20 Page 21 of 21 Document No. 065403-01 ©2010 DIONEX CORPORATION September 2010...

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