What Is The Disadvantage To Smaller Particle Size Of The Stationary Phase?

Pore Size vs. Particle Size in HPLC Columns

When selecting an HPLC cavalcade, it is important to understand the different attributes and how they will impact your chromatography. Chemical backdrop, such equally the type of surface of the stationary phase and pore size, impact sensitivity and retention. Physical backdrop of the column dimension, including particle size, column length, and inner diameter, affect efficiency and speed.

HPLC Column

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 tin can lengthened 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 collaborate with the stationary phase. Smaller pore size packings (pore size 80 to 120Å) are best for small molecules with molecular weights upwardly 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 exist 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 cavalcade. In HPLC, a narrow particle size distribution is desirable. A particle size distribution of dp ± 10% would hateful that ninety% of the particles autumn between 9 and 11 µm for an average 10 µm dp packing.

The standard particle size for HPLC columns was five µm for a long time, until the mid-1990s, when three.5 µm became popular for method development. More recently, as higher speed and/or higher resolution is required, chromatographers take turned to packings with sub-2-3 µm, including 1.8 µm. Shorter columns with these particles can produce faster high-resolution separations. The three.5 µm particle size operates at a routine operating force per unit area and may be used on all LCs, including those with a 400-bar operating limit. Short (50 mm and shorter) 1.eight µm columns may exist 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 like to sub-ii µm columns just generate lower backpressure, so they tin can exist used with conventional HPLC instruments. If the particle size of a column is reduced by half, the plate number doubles (bold cavalcade length remains the same). Still, if particle size halves, cavalcade backpressure increases 4 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.one x 100 mm column packed with iii.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.v µm to 1.8 µm, the efficiency of the same 2.1 x 100 mm column is doubled to about 24,000 theoretical plates. Nevertheless, this cavalcade generates a backpressure that is four times greater than the force per unit area of the same size column filled with 3.5 µm particles. Very oftentimes, an efficiency of 24,000 plates is non required, so the column length can be halved to 50 mm, with an expected efficiency of 12,000 plates. The analysis fourth dimension will be cut in half with this shorter column, and the backpressure is just twice equally groovy as the 100 mm column with 3.5 µm particles.

During method evolution, choose the column id (for example, 2.1 or 3.0 mm) to conform boosted application objectives (such as sensitivity, solvent usage) or compatibility with sure 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 bachelor size for your analysis and instrument; smaller columns are often less expensive to purchase and use less solvent. In some cases, if column diameter is reduced by half, sensitivity increases by four to v times (bold the injection mass is kept abiding). For example, when a sample is injected onto a two.ane mm id cavalcade, the peaks are about 3 to five times higher than on an optimized LC than when the same amount of sample is injected onto a four.six mm id column. If your instrument is optimized for depression-volume columns, every bit long as linear velocity is maintained, column efficiency, theoretical plates, backpressure, and analysis time are not significantly affected past reducing the column'due south diameter.

Conclusion:

At that place are multiple parameters to consider when evaluating a cavalcade stationary phase and column dimensions. To perform loftier throughput assay, a brusque cavalcade with small particles (e.grand., sub-2 µm) may be the best choice. If yous have a complex separation involving many sample components, then a long column packed with pocket-sized particles could be chosen, keeping in mind that the operating pressure of such a column may increase dramatically. If you lot are performing mass spectrometry, a small internal diameter cavalcade (east.g., 2.ane 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 ten µm) packed into larger diameter columns are often used. For such columns, it is preferable to accept a higher menses rate pump to match the period requirements of a preparative cavalcade.

For help in selecting an HPLC cavalcade for your method, please attain out to Chrom Tech, and nosotros will exist happy to assist. Chrom Tech is proud to have the all-time client service in the industry. We look forwards to earning the opportunity to exist your supplier of HPLC columns and other HPLC supplies.