Basic HPLC and CE of Biomolecules - Softcover

Wehr, Tim; Cunico, Robert L.; Gooding, Karen M.

Softcover

ISBN 10: 0966322908 ISBN 13: 9780966322903

Publisher: Bay Bioanalytical Laboratory, 1998

Synopsis

This text provides explanations and examples of the primary chromatographic techniques for biomolecules (reversed-phase, size exclusion, ion-exchange, hydrophobic interaction and affinity). It also offers practical suggestions for their implementation and optimization. An extensive bibliography furnishes a wealth of background information.

About this title

About the Author

Robert L. Cunico holds degrees in chemistry from Colorado State University and San Francisco State University, where he also did postgraduate work. At Varian Associates, he developed HPLC methods, columns and instrumentation for biomolecules and served as the HPLC training manager. Bob then joined the analytical group at Cetus Corporation (now Chiron) where he developed and validated methods for protein therapeutics. In 1991, he founded Bay Bioanalytical Laboratory, Inc., a consulting and contract laboratory serving the biotechnology and biopharmaceutical industry. As president and principal scientist of BBL, Bob has been a contributor to numerous INDs. Karen M. Gooding began her career in the laboratory of Fred Regnier at Purdue University, participating in the original development of columns and techniques for HPLC of proteins. In 1977, Karen and David Gooding founded SynChrom, Inc. specifically for the purpose of developing and manufacturing HPLC columns for protein analysis. As Analytical Director and President of SynChrom, Karen guided efforts in methods development with the express goal of expediting protein analysis. She has published extensively in the chromatography field and has served as an editor of Journal of Chromatography, Trends in Analytical Chemistry, and, with Fred Regnier, of the book, HPLC of Biological Macromolecules: Methods and Applications. Tim Wehr received his Ph.D. in microbial physiology at Oregon State University and did postdoctoral research in molecular biology at UC Berkeley. He managed the HPLC applications lab at Varian Associates for eight years and worked on development of LC columns and HPLC-based analyzers. For the last eight years he directed the CE chemistry R&D group at Bio-Rad Laboratories, developing CE instrumentation, methods, and application kits. He has published extensively in the separation sciences, and served for nine years on the organizing committee of the International Symposium on HPLC of Proteins, Peptides and Polynucleotides.

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From Chapter 3, HPLC Hardware: Meaningful and valid data can only be obtained if equipment is operating within its specifications. FDA regulated laboratories must have written standard operating procedures (SOPs) for maintenance and calibration procedures, as well as SOPs noting the steps required to check out equipment after maintenance or repair (29). These policies vary from laboratory to laboratory but will always include procedures relevant to installation, instrument qualification, performance qualification, and maintenance. Some HPLC systems have built-in software which assists in implementing procedures and produces documentation for performance qualification (30). System components should always be tested initially, evaluated periodically, and requalified after maintenance and repair. It is often easiest to carry out the performance tests which the equipment manufacturer recommends, but these can be replaced or supplemented by other procedures relevant to the day-to-day use. Some common tests for equipment evaluation are listed in Table 3.6.

Table 3.6 Equipment Validation Procedures
Solvent Delivery
flow accuracy - test flow rate volumetrically with back pressure
flow precision - reproducibility of retention time or peak area for 5 injections

Sampling Device
precision - reproducibility of peak areas for 5 consecutive injections
accuracy - linearity of peak area vs. concentration or volume

Absorbance Detector
linearity - absorbance vs. concentration
wavelength accuracy measurement of wavelengths for maximum and minimum absorbance of a standard compound
noise - measure in mAU

Refractive Index Detector
linearity - RIU vs. concentration
accuracy - calculation from linearity data of dn/dv, a calibration constant relating the change in RIU to that in voltage
noise - measure in RIU
drift - measure change in RIU for at least one hour

Temperature Control
accuracy - measure temperature of eluent
reproducibility - measure temperature of eluent at 5 different times

Gradient Formation and Mixing
accuracy - evaluate linear gradient as outlined in Section IV.C

Edition details

• Publisher: Bay Bioanalytical Laboratory
• Publication date: 1998
• Language: English
• ISBN 10: 0966322908
• ISBN 13: 9780966322903
• Binding: Paperback
• Edition number: 1
• Number of pages: 388