Understanding HPLC Column Retention and Selectivity is designed for users with several years of HPLC experience and provides insight into what happens to cause sample retention and separation inside an HPLC column filled with porous particles. The impact of chromatographic terms (k, á, N and RS) and important physical properties of the packing (particle size, pore size and surface area) on separation performance will be fully explained with examples. Principles of bonding chemistry will also be reviewed and stationary phase chemical structures that have become popular for use under reversed-phase conditions will be shown. The nature of different types of chemical interaction between solute and stationary phase will be described.

The seminar will emphasize small molecule (MW<1000) method development with the reversed-phase (RP) mode. Techniques for evaluating whether column pairs have different selectivity (orthogonality) will be shown. The most popular and important RP phases will be covered including C18, C8, Phenyl and Amide phases. Separation will be compared for different column and sample types to learn which samples might be preferentially retained and separated on the various phase options. The important steps in developing a robust HPLC method will be reviewed. Some guidelines will be presented for intelligent selection and screening of columns to get the method off on the right foot and arrive quickly at the phase which best separates your sample type.

• Review of chromatographic terms and why they are important.
• Using the Fundamental Resolution Equation as a tool.
• Review of particle substrates found inside HPLC columns.
• Description of some common particle platforms.
• Advantages and disadvantages of silica; why it is so popular.
• Importance of substrate physical properties.
• Influence of pore size, surface area and particle size on separation performance.
• Importance of substrate chemical properties and surface modification.
• Review of bonding chemistry and reversed-phase (RP) mode; you should know what is inside your column.
• Practical ranges for column and mobile phase operating conditions.
• Description of solute-stationary phase interactions in RP HPLC.
• Guidelines for developing a successful method.
• All C18 and C8 columns are beginning to look alike.
• Polar RP phases can show better selectivity than C18 and C8.
• Gradient recommendations for fast column screening.

OUR EXPERT PRESENTER:

Dr. Richard A Henry

Dr. Richard A. Henry received his B.S. degree in Chemistry from Juniata College in 1963 and Ph.D. in Analytical Chemistry from The Pennsylvania State University in 1966. After a postdoctoral year in separations at Purdue University with Professor L. B. Rogers, he joined DuPont at the Experimental Station in Wilmington, DE and became one of the first employees of the Analytical Instrument Products Division in their chromatography group. In 1985, he joined the Penn State University chemistry faculty as Director of Analytical Laboratories at University Park, PA where he taught Instrumental Analysis to chemistry majors. That same year, Dick also founded Keystone Scientific, Inc. where he developed and manufactured separation technology products. He retired from both Penn State University and Keystone Scientific in 2002 and remains active as a consultant. He served two terms as Chairman of the ACS Subdivision on Separations (1998-2002) and currently serves on its Executive Committee. Dick lives in State College, PA and can be reached at rhenry@psualum.com.