Choosing the Right HPLC Column

1. Set Your Separation Goals. Determine if your application requires high resolution, short analysis time, maximum sensitivity, long column life, low operating cost, or other important factors. 2. Choose Your Packing Material. Read below for an explanation of different specifications and how they affect your separation. 3. Choose Your Column Format. Make sure the column format that you choose has the appropriate affect on your separation.

Packing Materials

Understanding the specifications of HPLC packing materials and how they affect a separation will help you narrow your column choices. You will find all of these specifications for Alltech’s HPLC packings on pages 129-130.

Base Material

Silica-based packings are physically strong and will not shrink or swell. They are compatible with a broad range of polar and non-polar solvents. Most silica phases are stable from pH 2-8, but newer phases, such as Alltima™ HP, are stable from pH 1-10. Silica provides sharp peaks with small molecules. Polymer-based packings, such as methacrylate, are compressible and may shrink or swell with certain solvents. Many polymers are stable from pH 1-14. Because of this expanded pH range, most polymeric columns can be thoroughly cleaned with strong acids or bases to extend column life.

Particle Shape

Most modern HPLC packings have spherical particles, but some are irregular in shape. Irregular particles have larger surface areas, and are relatively inexpensive. Spherical particles offer lower backpressure, and higher performance, stability, and reproducibility than irregular particles.

Particle Size

Smaller particle sizes give higher efficiency and higher resolution than larger particle sizes. However, larger particle sizes offer faster flow rates and lower back-pressure. Typical particle sizes range from 3μm to 20μm, and new 1.5μm particle sizes are available to maximize resolution on short columns. A 5μm particle size represents the best compromise between efficiency and back-pressure.

Carbon Load

For silica-based reversed-phase packings, carbon load indicates the amount of functional bonded phase attached to the base material. Phases with lower carbon loads are more weakly hydrophobic, which may significantly reduce retention times over phases with higher carbon loads. However, a higher carbon load will give higher capacity and often greater resolution, especially for compounds of similar hydrophobicity.

Pore Size

Choose a pore large enough to completely enclose your target molecule. If your molecule is larger than the pore, it will be difficult or impossible to retain. In general, packing materials with a smaller pore size have higher surface areas and higher capacities than packing materials with larger pore sizes. Larger pores are better for interaction with large compounds, such as proteins.

Surface Area

This is the available surface, most of which is within the pores, for interaction with the sample. A larger surface area typically indicates a greater number of pores, and therefore a higher overall capacity. Smaller surface areas equilibrate faster, which is important for gradient elution analyses.

Phase Type

Polymerically bonded phases have functional chains bound to the base silica particle at multiple attachment points and can involve cross-linking between chains, while monomerically bonded phases have a single attachment point. Historically, polymeric packings have resulted in better column stability under pH extremes. However, new high-purity silica phases are very stable whether monomerically or polymerically bonded. Monomerically bonded phases generally offer rapid mass transfer and high column efficiency to better resolve chemically similar analytes.

Endcapping

Silica-based reversed-phase packings have free silanol groups that will interact with polar compounds. Endcapping the bonded phase minimizes these secondary interactions. Choose endcapped phases if you do not want interactions with polar compounds. Choose nonendcapped phases if you want enhanced polar selectivity, for stronger retention of polar organic compounds.