Conducted by Richard A. Henry, Ph.D.

The initial step in HPLC method development is typically a screening experiment with nonpolar column and polar reversed-phase solvents. Column candidates are usually C18 or C8 because solute retention based on hydrophobic attraction has proven to be selective enough in a large percentage of cases. Although interaction between solutes and the stationary phase surface is highly complex and so far has been difficult to predict accurately, we do know that certain classes of solutes interact preferentially with nonpolar stationary phases that have polar groups embedded within them.

It is becoming possible to examine sample molecular structure and select columns having functional groups that should be more attractive and selective than straight hydrocarbon chains. When C18 or C8 columns cannot be optimized into a rugged enough separation, columns with more polar stationary phase chemistry should be evaluated.

Sample classes that are selectively attracted to different stationary phase types are shown in the table below.

Sample Classes

• Stationary Phase Type
• Interactive Forces
• Wide range of samples C18/C8 Dispersion only
• Acids, phenols, anilines PE (amide, carbamate, urea) H-bond and other dipole
• Hetero and other aromatics Phenyl p donor-p acceptor (p -p )
• Bases, all electron donors Perfluorophenyl p -p, Lewis acid-base dipole
• Range of polar samples Cyano Dipole only

This audio seminar CD will complement previous LCGC seminars on reversed-phase separation and will focus on identifying sample types that separate better on polar embedded (PE) reversed phase columns. Possible separation mechanisms for this special class of column will be described. Performance advantages and disadvantages will be compared to straight alkyl phases. Practical tips for optimizing separations and maintaining PE columns will be included.

This audio seminar CD is presented by Dr. Richard A. Henry, a renowned expert on HPLC.

A detailed outline and Dr. Henry's qualifications follow below.

Digging Deeper into HPLC Column Selection:

When to Choose Polar-Embedded Reversed-Phase

Part I Definition of polar-embedded reversed-phase columns

• Polar-Embedded (PE) or Embedded-Polar (EP) terms used interchangeably
• Functional groups that should be grouped in this RP phase type include amide, carbamate, urea, sulfonamide, and maybe ethers
• Functional groups that should not be grouped in this RP phase type include phenyl, pentafluorophenyl and cyano; these tend to be amphoteric phases that are useful in both RP and NP (and ANP) modes
• Description of molecular interaction forces between samples and phases

Part II Types of samples that should interact preferentially with PE phases

• H-bonding and other dipole attraction as an important force in PE phases
• Types of samples that show strong H-bonding tendencies
• Acids, phenols and certain nitrogen and sulfur compounds

Part III PE phases may show improved peak symmetry for bases over nonpolar alkyl phases

• Description of a possible mechanism for internal base deactivation where polar RP phase strongly shields samples from silanol interaction

Part IV Performance characteristics of PE phases

• Argument for selecting PE phases early during method development
• Rules for optimizing mobile phase and other HPLC variables
• Possible LC-UV and LC-MS limitations
• Stability and storage

Part V Separation examples where PE phase columns show advantages

• Phenols and related natural products
• Carboxylic acids
• Peptides
• Pharmaceuticals

Part VI Summary and trends in new PE phases for HPLC

Richard A. Henry, Ph.D.

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. Dick worked closely with Dr. Jack Kirkland and others in the development of HPLC columns and packing materials. He joined the Penn State University chemistry faculty in 1985 as Director of Analytical Laboratories where he taught Instrumental Analysis to chemistry majors. That same year, he also founded Keystone Scientific, Inc. where he developed and marketed HPLC columns and related separation technology. He retired from both Penn State University and Keystone Scientific in 2002 and remains active as a consultant. Dick has research interests in mechanisms of separation and applications of reduced-bore column technology. He is also interested in new hyphenated analytical techniques, especially the rapidly growing field of LC-MS, and all multidimensional separation methods. He served two terms as Chairman of the ACS Subdivision on Separations (1998-2002) and currently serves on its Executive Committee.