This essential guide provides you with all the information you need to know about HPLC columns. Learn about key factors and application considerations that come into effect when choosing the right column.
A High Performance Liquid Chromatography (HPLC) column is considered to be the most important part of any liquid chromatography instrument, as this is where the separations occur. In many cases, columns are at the cutting edge of separations technology, but are all essentially an improved form of standard LC column chromatography. The main difference is that instead of a solvent being allowed to pass through a HPLC column under gravity, it is forced through under high pressure, which makes separations much faster and in turn reduces solvent consumption.
The higher pressure allows for the use of a smaller particle size for the HPLC 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 compared to low pressure columns.
Ultra High Performance Liquid Chromatography (UHPLC) uses an even smaller particle size than HPLC for the packing material. This helps to produce 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 HPLC column, a number of considerations must be taken into account. These are summarized in Table 1. Further details on each consideration will be given in the coming sections.
In order to select the correct HPLC column, you will have to consider your application. There are a few simple steps to work through to match your application to a column, 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. 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 Table 2 and works as follows: if your analyte is <5000 g/mol then you should start at the low molecular weight section of the Table; if it is water soluble, move on and select your required separation mode. Further details on this are given later in this guide.
HPLC and UHPLC are both routinely used within analytical research, from pure chromatographic studies to life science research and drug discovery. When the technique is 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: chirality; enantiomers; vitamins; pesticides; carbohydrates. It is worth investigating if there are already HPLC 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.
Columns are usually 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, a list of more than 60 HPLC column classifications ordered according to the type of bonded phase (C18, C8…), packing material and particle size. Table 3 shows some of the most common types.
Monolith columns contain a single rod of high purity monolith silica for separation with porous channels, instead of the traditional beaded columns. Monolith columns can be utilized with existing HPLC systems with minimal method development and enable fast passage of eluent, while efficiently separating compounds ready for detection.
The column chemistry selected is also dependent upon the separation mode that you are going to
used. In general, three primary characteristics of chemical compounds can be used to create HPLC
separations. They are:
Two primary separation modes are commonly used to exploit the polarity of a chemical compound. These are Normal Phase and Reversed Phase. Both are dependent on the relative polarity of the solvent used and the stationary phase. Reversed phase HPLC is the most commonly used form. In recent years, Hydrophilic Interaction Chromatography (HILIC) and Hydrophobic Interaction Chromatography (HIC) have become more widely used, particularly for the separation of polar compounds and large biomolecules, respectively.
Normal Phase HPLC: Normal-phase chromatography, or NP, is the classic form of liquid chromatography using polar stationary phases and non-polar mobile phases. The analyte is retained by the interaction of its polar functional groups, with the polar groups on the surface of the packing. Analytes elute from the HPLC column, starting with the least polar compound, followed by other compounds in order of their increasing polarity. Normal-phase chromatography is useful in the separation of analytes with low to intermediate polarity and high solubility in low-polarity solvents. Water-soluble analytes are usually not good candidates for normal-phase chromatography.
Reversed Phase HPLC: In reversed phase chromatography, or RP, the stationary phase is modified to make it non-polar by attaching long hydrocarbons to its surface. Typically, these hydrocarbons will be hydrophobic alkyl chains of 4, 8 or 18 carbon atoms in length that interact with the analyte. Manufacturers commonly name their columns depending on the number of carbon atoms i.e. C4, C8, C18. Of these chain lengths, C4 is generally used for separating proteins, while C8 and C18 chemistries are used for peptides or small molecules.
Hydrophilic Interaction Chromatography (HILIC): HILIC is a relatively recently adopted HPLC phase, which is used for the retention of water soluble (polar) compounds. Liquid-liquid partitioning between the polar mobile phase and the water layer at the stationary phase surface is considered to be the main mechanism of action. In this type, polar stationary (e.g. silica and amine) and mobile (e.g. water and methanol) phases are used and an increase in mobile phase polarity results in a decrease in analyte retention. This makes it very useful in separating highly polar compounds, which would be unretained by reversed phase chromatography. Among the most typical analytes for HILIC are amino acids, polar pharmaceuticals, nucleobases, nucleosides, alkaloids and carbohydrates.
Hydrophobic Interaction Chromatography (HIC): HIC, the most recent addition to the different modes of chromatography, is a type of reversed-phase chromatography that is used to separate large biomolecules, such as proteins. It is usually desirable to maintain these molecules intact in an aqueous solution, avoiding contact with organic solvents or surfaces that might denature them. The promotion of the hydrophobic effect (by addition of lyotropic salts) drives the adsorption of hydrophobic areas on a protein to the hydrophobic areas on the solid support. This is thermodynamically favorable, in that it reduces the number and volume of individual hydrophobic cavities. Reducing hydrophobic interaction, by decreasing the concentration of lyotropic salts, results in desorption from the solid support.
HIC is unique in that proteins bind at high salt concentration and delute at low salt concentrations. This is manifested in a reverse salt gradient, which is an immediate indication that HIC is being employed.
Ion-Exchange Chromatography (IEC or IEX) separates analytes based on differences in their surface charge. It is typically used for the analysis of biological samples such as proteins, peptides, amino acids, nucleic acids and glycoproteins. The basic principle governing IEC separations is that analytes with an electrical charge are attracted to ligands on the stationary-phase particles, which bear the opposite charge. The mobile phase typically consists of a pair of aqueous buffers, with one buffer containing a low concentration of salt and the other buffer containing much more salt.
The stationary phases for ion-exchange separations are characterized by the nature and strength of the acidic or basic functions on their surfaces and the types of ions that they attract and retain. Two types of ion-exchange chromatography exist. Cation exchange is used to retain and separate positively charged ions on a negative surface. Conversely, anion exchange is used to retain and separate negatively charged ions on a positive surface.
Anion and cation exchangers are further classified as strong or weak, depending on how much the ionization state of the functional groups vary with pH. A strong ion exchanger has the same charge density on its surface over a broad pH range, whereas the charge density of a weak ion exchanger changes with pH.
The choice of whether to use a strong verses weak cation or anion exchanger depends on the charge of the biomolecule of interest. For example, a weakly acidic molecule, which requires very low or very high pH for ionization, will require the use of a strong ion exchanger because it functions at extremes of pH. You will also need to consider the stability of your molecule. If the substance is stable to extremes of pH (e.g. amino acids), strongly acidic or basic ion exchangers can be used. If substance is labile (e.g. proteins), a weaker exchanger of low charge density is advisable because the molecules can be eluted by more gentle conditions of pH and ionic strength. When choosing an appropriate HPLC column for IEC, it is advantageous to have knowledge of the isoelectric point (pI) of your molecule of interest.
Size-Exclusion HPLC (SEC) separates analytes on the basis of a combination of their hydrodynamic size, diffusion coefficient, and surface properties. It is usually applied to large molecules or macromolecular complexes such as proteins and industrial polymers.
In SEC, the stationary phase is composed of inert particles packed into a dense three-dimensional matrix within a glass or steel column. The stationary-phase particles have small pores and/or channels that will only allow species below a certain size to enter. Smaller molecules will penetrate more of the pores on their passage through the bed and will take longer to elute. Larger molecules can only penetrate pores above a certain size so they will spend less time in the bed. The biggest molecules may be totally excluded from pores and pass only between the particles, eluting very quickly in a small volume. The mobile phase can be pure water, an aqueous buffer, an organic solvent, or a mixture of these.
There are two basic types of size exclusion chromatography. One is gel permeation chromatography (GPC), which uses a hydrophobic column packing material and a non-aqueous mobile phase (organic solvent) to measure the molecular weight distribution of synthetic polymers. The other is gel filtration chromatography (GFC), which uses a hydrophilic packing material and an aqueous mobile phase to separate, fractionate, or measure the molecular weight distribution of molecules soluble in water, such as polysaccharides and proteins.
There are many different commercially available SEC columns, which have different packing material pore sizes (different exclusion limits). When choosing a column, you should compare the calibration curves and select a HPLC column that is best suited to the range of molecule weights to be measured. If your sample is distributed over a wide range of molecular weights, you may want to consider using several columns connected in a series. Many SEC columns have limitations on the eluents that can be used. Using an eluent that is not specifically permitted could irreversibly expand the packing material or cause physical deterioration. You should therefore investigate which solvents readily dissolve the sample in advance and then choose a column compatible with one of those solvents.
The next considerations are size, shape and chirality of the particles that make up the HPLC 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.
Phenyl columns (USP L11) are an alternative to C18 columns and provide a unique selectivity for aromatic compounds through pi-pi interactions. Biphenyl column, are the next generation of phenyl columns. They offer both aromatic selectivity and an increase in hydrophobic retention. These phenyl phases are of particular interest when analyzing drug compounds and residues.
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 back-pressure 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 separation of enantiomers or chiral compounds for analysis can be difficult. Most stationary phases of HPLC columns are achiral; however, some can contain a single enantiomer, which enables such enantiomeric separations. Although enantiomers exhibit identical chemical and physical properties, when passed through a column containing a single enantiomer of the chiral compound forming a chiral stationary phase (CSP), the enantiomers differ in affinity and pass through the column at different times, thus enabling the separation and analysis of enantiomers. As you might expect, these highly specified columns are more expensive than a traditional column.
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 that takes a long time to elute will not be of use to a high through-put 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 its 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.
Choosing the physical dimensions of your column will therefore depend on your application and
The most recent development in HPLC is the use of fused-core columns. 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.
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 there is a low amount 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.
Two dimensional high performance liquid chromatography (2D-HPLC) is an up-and-coming technique, and many leading manufacturers are currently investing money into the development of such systems. 2D-HPLC has the ability to combine two separate columns with differing separation modes. Comprehensive 2D chromatography is the most common approach, whereby the sample is injected into the first column (first dimension – generally low flow rate), and all the eluent is passed into a second column (second dimension – fast flow rate) via a switching valve, which comprises of two loops.
The first loop contains the eluent from the first HPLC column; the second loop content is analyzed on the second column. Conversely, heart-cutting 2D chromatography occurs when only a selected portion of the eluent from the first column (fast flow rate) is ‘cut’ from the ‘heart’ of the initial chromatography and passes through the second column (slow flow rate). Although these are different approaches to this technique, both are significant improvements to the traditional 1D chromatography, as they afford greater selectivity, separation and analysis of complex mixtures, which may otherwise be inseparable.
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 columns 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. Check out our recent article on HPLC vials if you would like further reading!