An High Performance Liquid Chromatography (HPLC) column is considered to be the most important part of any instrument as this is where the separations occur. In many cases columns are at the cutting edge of separation technology, but are all basically an improved form of standard column chromatography. The main difference is that instead of a solvent being allowed to pass through a column under gravity, it is forced through under high pressure which makes separations much faster.

The higher pressure allows for the use of a smaller particle size for the column packing material. Columns with smaller particles generate sharper peaks with increased resolution due to better packing and reduced diffusion distances for analytes. This allows for faster separations versus low pressure columns.

Ultra High Performance Liquid Chromatography (UHPLC) uses an even smaller particle size than HPLC for the packing material. This has the advantages of producing increased resolution and sensitivity. UHPLC requires even higher pressures than HPLC which means systems capable of handling this increased pressure can be relatively expensive. It is worth noting that methods developed for HPLC do not readily transfer to UHPLC and vice versa.

In selecting the most appropriate column, a number of considerations must be taken into account. These are summarized in Figure 1. Further details on each consideration will be given in the coming sections.

In order to select the right column, the first thing to consider is your application. To match your application to a column there are a few simple steps to work through, the first of which is to calculate the molecular weight of your analyte. In general, compounds are divided into two groups: low molecular weight, <5000 daltons, and high molecular weight, >5000 daltons molecular weight. Assigning an analyte to one of these should be relatively easy. Secondly, you’ll need to find out if the analyte is soluble in water (polar) or an organic (non-polar) solvent. Lastly, the mode of separation can be determined to narrow column choice.

The process is summarized in Figure 2 and works as follows: if your analyte is <5000 g/mol then you should start at the low molecular weight cell, if it is water soluble move on to that cell. This results in a number of separation modes that may be used. Further details on this are given in the coming section

Figure 2: Determination of Separation Mode.

HPLC and UHPLC are both routinely used within analytical research from pure chromatographic studies to life science research and drug discovery. When used in drug discovery, for example, it has been shown to significantly improve the success of the process. It can prevent poor drug candidates from progressing through the discovery process by monitoring factors such as metabolic stability and toxic metabolite production. The technique has also been successfully applied to the separation and analysis of challenging samples such as soil, environmental waters and explosives which are of importance to environmental and forensic laboratories. The usefulness of HPLC and UHPLC can be further enhanced by linking to other systems such as mass spectrometers.

There are many different columns on the market that have been designed for specific applications, such as: Enantiomers; Vitamins; Pesticides; Carbohydrates. It is worth investigating if there are already columns available that have been optimized for your application.

Once the application has been identified, choosing the best column for your application is based on two considerations. The first and most important of these is the type of stationary phase. The factors to be considered in this regards are the column chemistry, the separation mode, the particle size and the retention capacity.

In general columns are filled with porous particles coated with a material that interacts with the injected sample. HPLC uses what is called a true stationary phase: column “chemistries” are bonded tightly to the packing material and do not bleed off.

There are various ways to categorize the types of stationary phase available but the most widely accepted of these is the USP “L” system. This is a list of over 60 column classifications ordered according to the type of bonded phase (C18, C8…), packing material and particle size. Figure 3 shows some of the most common types.

Figure 3: Common USP ‘L’ Classifications.

The column chemistry selected is also dependent upon the separation mode which is going to be used. In general, three primary characteristics of chemical compounds can be used to create HPLC separations. They are:

The next consideration is the size and shape of the particles which make up the column packing material. The majority of new HPLC methods are performed on spherical shaped or spheroidal (almost spherical) particles. Spherical particles provide higher efficiency, better column stability and lower back-pressures compared to irregularly shaped particles.

If all other factors are constant, the smaller the particles are the more efficient the separation. The major problem with a decrease in particle size, from 10-, 7-, 5-, and 3-micron diameters, is that the backpressure increases exponentially. This roughly means a column using 3-micron particles is about twice as efficient as a 5-micron column, but pressures are three times as high.

Although separation efficiencies can be increased by reducing particle size even more, to below 2 microns, the hardware required becomes more expensive as it needs to handle the extremely high pressures. UHPLC systems have been designed to overcome these limitations and UHPLC columns can deliver shorter run times, cleaner separations, sharper/taller peaks, and improved detection limits.

Some applications also benefit from end-capped HPLC columns. A reversed-phase HPLC column that is end-capped has gone through a secondary bonding step to cover unreacted silanols on the silica surface. End-capped packing materials can eliminate unpredictable secondary interactions.

The retention capacity is the time samples spend in the column. It is of importance to both the quality and speed of separations. A column which takes a long time to elute will not be of use to a high-throughput application.

The retention capacity is influenced by surface area and carbon load (the percentage of carbon in the packing material). The pore size and volume of the packing material is also important in determining retention capacity. This is because surface area is inversely proportional to pore size. A larger pore size results in lower retention. The particle pore size is measured in angstroms and generally ranges between 100-1000 angstroms. 300 angstroms is the most popular pore size for proteins and peptides and 100 angstroms is the most common for small molecules.

Depending on your requirements HPLC columns can be quite expensive and you will therefore want to ensure they last as long as possible. Guard columns can be used to protect your analytical column and prolong their lifetime. HPLC guard columns (or cartridges) are installed in front of an analytical column and protect it from strongly retained impurities. An alternative to using a guard system is the use of an in-line filter. Filters are a simple cost-effective means to providing a quick efficient clean up for occasional 'dirty' samples.

Once a packing material has been selected, the physical dimensions of the HPLC column hardware should also be optimized for the desired separation. Larger columns are useful in the scale up process, but smaller columns generally offer greater sensitivity and are therefore more useful in analytical applications.

The column length is determined by how many, and what type, of compounds you have to separate. Long columns provide better resolution and sensitivity but require longer retention times and higher pressure. It is best to choose the shortest column possible for your application without affecting resolution.

The internal diameter (ID) of an HPLC column influences both detection sensitivity and separation selectivity (resolution). The most commonly used ID for HPLC columns is 4.6mm. Small column diameters provide higher sensitivity than larger column diameters for the same injected mass because the concentration of the analyte in the mobile phase is greater. Smaller diameter columns also use less mobile phase per analysis because a slower flow rate is required to achieve the same linear velocity through the column. The major disadvantage associated with smaller diameter columns is that the sample loading capacity is reduced.

In summary:

• Larger ID columns (over 10 mm) are mainly used to purify large scale amounts of material because of their relatively large loading capacity.
• Analytical scale columns (4.6 mm) are the most common type. They are most often used in quantitative analysis.
• Narrow-bore columns (1–2 mm) are used for applications where more sensitivity is required.
• Capillary columns (under 0.3 mm) are used almost exclusively with alternative types of detection such as mass spectrometry.

Choosing the physical dimensions of your column will therefore depend on your application and throughput requirements.

The most recent development in HPLC is the use of fused-core column technology. It is claimed to provide the performance of sub-2-micron particles, higher separating efficiencies similar to UHPLC, but at normal pressures. This technology uses a solid silica particle covered with a layer of porous silica, which is then infused with the bonded phase. A big advantage of fused-core columns over UHPLC is that while methods developed on UHPLC or conventional HPLC cannot be transferred unless the two labs have the same instrument, with fused-core particle columns different labs only need to have the same type of column.

With the advent of a second generation of silica monolith columns, liquid chromatographers now have an alternative to fused-core packings for high-throughput, high-efficiency separations. The more favorable correlation of column back pressure and separation efficiency, shown by fused-core particles as compared to fully porous particles, can now also be achieved with a new generation of silica-based monolithic HPLC columns. While monolith columns have recently emerged as a viable alternative for liquid phase separations they are still being developed for certain applications.

In the next few years, as HILIC and HIC continue to become more widely used, we are likely to see an increase in the number of phases that are available for these techniques. HILIC is especially favored by mass spectroscopists since ionization efficiency is often enhanced in organic solvents and the presence of low or no buffer salt compared to reversed-phase chromatography.

Traditionally, separations are performed on straight phases where one main type of chromatographic interaction governs the separation. To achieve different selectivity, column researchers are now synthesizing phases that have two or three different functionalities. Mixed-mode phases are becoming more widely accepted and may play a future role in enabling scientists to achieve separations that were not previously possible.

Although silica gel and silica hybrids with chemically bonded phases still dominate the applications of HPLC, newer packing materials continue to be developed. Whether the particle size reduction trend will continue is a matter of speculation. Smaller particles will require even higher pressure and place additional constraints on the packing materials, column hardware and instrument consumables. It remains to be seen whether fused-core column technology and second generation monoliths will take over from traditional packing materials.

There are many different types of HPLC column on the market and finding the correct one for your application may seem daunting but, by applying a few simple considerations, selecting the correct column can be made an easier task.

HPLC columns will continue to develop and new technologies will emerge. Visit the SelectScience product library to find out about the latest HPLC columns from leading manufactures and read user reviews. Use the SelectScience application note library to keep up to date with the latest HPLC methods.