When selecting an HPLC column, it is important to understand the different attributes and how they will affect your chromatography. Chemical properties, such as the type of surface of the stationary phase and pore size, affect sensitivity and retention. Physical properties of the column dimension, including particle size, column length, and inner diameter, affect efficiency and speed.
What Is Pore Size?
Pore size is the average size of a pore in a porous packing. Its value is typically expressed in angstroms. The pore size determines whether a molecule can diffuse into and out of the packing. Therefore, the pore size of the packing material in your HPLC column is important, since the molecules must 'fit' into the porous structure in order to interact with the stationary phase. Smaller pore size packings (pore size 80 to 120Å) are best for small molecules with molecular weights up to a molecular weight of 2000. For larger molecules with MW over 2000, wider pore packings are required; for example, a popular pore size for proteins is 300Å. For polypeptides and many proteins, choose 200-450 Å, and choose 1,000Å and 4,000Å for very high molecular weight proteins and vaccines. For GPC/SEC separations, the molecular weight range for separations is typically given with the pore size information, so the right column can be selected.
What Is Particle Size?
Particle size is the average particle size of the packing in the HPLC column. A 5 µm column would be packed with particles with a definite particle-size distribution because packings are never monodisperse. Particle size distribution is the measure of the distribution of the particles used to pack the LC column. In HPLC, a narrow particle size distribution is desirable. A particle size distribution of dp ± 10% would mean that 90% of the particles fall between 9 and 11 µm for an average 10 µm dp packing.
The standard particle size for HPLC columns was 5 µm for a long time, until the mid-1990s, when 3.5 µm became popular for method development. More recently, as higher speed and/or higher resolution is required, chromatographers have turned to packings with sub-2-3 µm, including 1.8 µm. Shorter columns with these particles can produce faster high-resolution separations. The 3.5 µm particle size operates at a routine operating pressure and may be used on all LCs, including those with a 400-bar operating limit. Short (50 mm and shorter) 1.8 µm columns may be employed on optimized standard LCs, while longer columns may require a higher-pressure LC or UHPLC, operating at pressures from 600 to 1300 bar. Superficially, porous particles have been developed that enable performance similar to sub-2 µm columns but generate lower backpressure, so they can be used with conventional HPLC instruments. If the particle size of a column is reduced by half, the plate number doubles (assuming column length remains the same). However, if particle size halves, column backpressure increases four times.
What About Column Length?
If column length doubles, the plate number and analysis time also double. As column length increases, backpressure increases linearly. For example, a 2.1 x 100 mm column packed with 3.5 µm particles generates about 12,000-14,000 theoretical plates, an efficiency that can provide adequate separation for many samples. By reducing the particle size from 3.5 µm to 1.8 µm, the efficiency of the same 2.1 x 100 mm column is doubled to about 24,000 theoretical plates. However, this column generates a backpressure that is four times greater than the pressure of the same size column filled with 3.5 µm particles. Very often, an efficiency of 24,000 plates is not required, so the column length can be halved to 50 mm, with an expected efficiency of 12,000 plates. The analysis time will be cut in half with this shorter column, and the backpressure is only twice as great as the 100 mm column with 3.5 µm particles.
During method development, choose the column id (for example, 2.1 or 3.0 mm) to accommodate additional application objectives (such as sensitivity, solvent usage) or compatibility with certain instrument types (capillary, nano, or prep columns).
What Column Inner Diameter Should I Select?
When you want to establish a routine method, consider reducing the column dimensions to the smallest available size for your analysis and instrument; smaller columns are often less expensive to buy and use less solvent. In some cases, if column diameter is reduced by half, sensitivity increases by four to five times (assuming the injection mass is kept constant). For example, when a sample is injected onto a 2.1 mm id column, the peaks are about three to five times higher than on an optimized LC than when the same amount of sample is injected onto a 4.6 mm id column. If your instrument is optimized for low-volume columns, as long as linear velocity is maintained, column efficiency, theoretical plates, backpressure, and analysis time are not significantly affected by reducing the column’s diameter.
There are multiple parameters to consider when evaluating a column stationary phase and column dimensions. To perform high throughput analysis, a short column with small particles (e.g., sub-2 µm) may be the best choice. If you have a complex separation involving many sample components, then a long column packed with small particles could be chosen, keeping in mind that the operating pressure of such a column may increase dramatically. If you are performing mass spectrometry, a small internal diameter column (e.g., 2.1 mm id) may be the best choice, due to the lower flow rates used with an MS detector. For preparative chromatography, larger particles (5 or 10 µm) packed into larger diameter columns are often used. For such columns, it is preferable to have a higher flow rate pump to match the flow requirements of a preparative column.
For help in selecting an HPLC column for your method, please reach out to Chrom Tech, and we will be happy to assist. Chrom Tech is proud to have the best customer service in the industry. We look forward to earning the opportunity to be your supplier of HPLC columns and other HPLC supplies.