The State of the Art and Future Trends of Size-Exclusion Chromatography Packings and Columns - - Chromatography Online
The State of the Art and Future Trends of Size-Exclusion Chromatography Packings and Columns

LCGC North America
Volume 7, Issue 30, pp. 544-563

Column Purchasing Caveats

SEC users should also keep in mind that some SEC packing material will slowly degrade with time. In the case of polymeric packings stored in nonstabilized tetrahydrofuran, peroxide buildup will degrade the packing over time. Even if stabilized solvents are used for column storage (that is, solvents that contain an antioxidant such as BHT), oxidized products might adsorb onto the packing, changing its adsorptive properties. From extensive studies involving silica packings, long-term storage in aqueous solutions, especially if neutral or slightly basic, can degrade silica, thereby lowering column efficiency and generating fines that will eventually interfere with on-line light scattering detection. In view of these potential problems, buyers should insist on purchasing newly packed columns. Although some manufacturers supply a dated test chromatogram with each column sold, it would be more desirable for all column producers to list the manufacturing date, or specifically the date the column was packed. This information can sometimes be traced if the column serial number is provided.

Our last caveat involving column selection is about the risk of purchasing packings that may contain inadvertently adsorbed components on the surface. Depending on how the packing is polymerized, packed into columns, and washed, it may contain any number of adsorbed components that could possibly change the adsorptive properties of the polymer packing. Packings can also adsorb impurities from mobile phases or from injected samples overtime. In light of this potential problem, we recommend that SEC columns in question be evaluated by injecting polymer samples of interest and measuring column recovery, which should be greater than 95%. Columns also should be tested for unwanted adsorption by injecting the monomer or dimer forms of the sample in question, and measuring elution volume or SEC distribution coefficient, K SEC (equation 2). The K SEC values should be close to unity to ensure the absence of adsorption. If adsorption is suspected, higher-molecular-weight samples will probably be retained on the column, that is, K SEC > 1, invalidating the SEC process. In the likelihood of adsorption, columns should be cleaned of adsorbed components by following the manufacturer's recommendations, or another column brand should be tested.

Anticipated and Recommended Future Trends

SEC is a well-established technique for routine polymer and biopolymer separations, common to most laboratories that deal with macromolecules. As such, developments in column technology have matured and slowed in recent years because many of the parameters that affect the performance of a size-exclusion separation have been researched and optimized.

SEC separations rely heavily on the physical properties of packings, as summarized earlier (1). Thus, in this section we focus on these inherent properties that can only be adjusted and controlled through manufacturing.

Pore Volume

The most important packing properties are the pore size, which define the molecular weight separation range, and the pore volume, V i , or the ratio V i /V o , which helps to establish the resolution of a separation. It appears that we are close to the maximum limit of V i/V o of commercially available cross-linked polymer packings; to obtain a significantly higher V i /V o ratio, a paradigm shift in column technology is required. An open network of structurally sound pores needs to be developed; perhaps a new type of silicate needs to be realized, as with Waters BEH column technology (9).

To substantially increase V i , large-diameter columns can be used, a course taken by Polymer Standards Service's asymmetric column. An expensive proposition is to use preparative SEC columns for analytical work. Experimentally, V i can be increased by simply adding more columns in series. In both of these cases, however, analysis time is compromised unless higher flow rates are used.


blog comments powered by Disqus
LCGC E-mail Newsletters
Global E-newsletters subscribe here:



Column Watch: Ron Majors, established authority on new column technologies, keeps readers up-to-date with new sample preparation trends in all branches of chromatography and reviews developments. LATEST: When Bad Things Happen to Good Food: Applications of HPLC to Detect Food Adulteration

Perspectives in Modern HPLC: Michael W. Dong is a senior scientist in Small Molecule Drug Discovery at Genentech in South San Francisco, California. He is responsible for new technologies, automation, and supporting late-stage research projects in small molecule analytical chemistry and QC of small molecule pharmaceutical sciences. LATEST: HPLC for Characterization and Quality Control of Therapeutic Monoclonal Antibodies

MS — The Practical Art: Kate Yu brings her expertise in the field of mass spectrometry and hyphenated techniques to the pages of LCGC. In this column she examines the mass spectrometric side of coupled liquid and gas-phase systems. Troubleshooting-style articles provide readers with invaluable advice for getting the most from their mass spectrometers. LATEST: Radical Mass Spectrometry as a New Frontier for Bioanalysis

LC Troubleshooting: LC Troubleshooting sets about making HPLC methods easier to master. By covering the basics of liquid chromatography separations and instrumentation, John Dolan is able to highlight common problems and provide remedies for them. LATEST: How Much Can I Inject? Part I: Injecting in Mobile Phase

More LCGC Columnists>>

LCGC North America Editorial Advisory Board>>

LCGC Europe Editorial Advisory Board>>

LCGC Editorial Team Contacts>>

Source: LCGC North America,
Click here