HPLC columns

If the length of the column increases, the retention time of the analytes also increases. At the same time, a longer column also shows higher soil numbers. The loadability of the column also increases as more material is present. In general, it can be said that the longer a column is...

• the longer the retention time/running time.
• the higher the soil number.
• the higher the dissolution.
• the higher the loadability.
• the more tolerant the column is to dirty samples
• the higher the stability (number of injections).
• the higher the back pressure.

The inner diameter of the column has no direct effect on the separation, provided that the flow rate is adjusted as the inner diameter decreases so that the linear flow rate remains constant. A smaller inner diameter has a different optimum flow rate for the highest efficiency compared to thicker columns. A smaller inner diameter does not increase the plate number but leads to narrower peaks. This can increase the resolution and detection limit. In general, it can be said that the narrower (smaller inner diameter) a column is...

• the narrower the peaks (higher resolution).
• the higher the detection limits (smaller sample quantities possible).
• the lower the solvent consumption.
• the lower the optimum flow rate.
• the lower the loadability.
• the higher the back pressure.
• the lower the stability.

The pore size is primarily dependent on the size of the analytes. In HPLC, the pores should be significantly larger than the analytes, as no exclusion from the pores is desired here. In size exclusion chromatography, the pores are one of the most important factors and separation takes place exclusively via the rejection of the analytes from the pores. In most cases, different relationships can be found between pore size and molecular weight.

• 60 Å pores for small molecules (up to approx. 1,000 Da)
• 120 Å pores for slightly larger molecules (up to approx. 10,000 Da)
• 200 Å pores for larger molecules (approx. 5,000 - 40,000 Da)
• 300 Å and larger pores for large molecules (larger than 20,000 Da)

The molecular weight range also depends on the shape of the analytes and can influence the values given here.

How does the pore size change the separation? In general, the smaller the pores, the higher the specific surface area (e.g. PerfectSil 120 Å has 300m²/g and PerfectSil 300 Å has 100m²/g). The larger surface area then results in longer retention. The diffusion of the analytes is also more difficult with smaller pores, which in turn can also contribute to an increase in the analysis time.

The shape of the particles can have a significant influence on separation. Firstly, a distinction can be made between irregular and spherical particles. Irregular particles have a higher back pressure than spherical particles and a lower number of bases. Modern HPLC phases generally always consist of spherical particles. Another form is also based on spherical particles but has a non-porous core and a porous shell. These so-called core-shell particles (superficially porous particles, SPP) have lower back pressures than spherical particles and higher plate numbers. However, the loadability is lower here compared to fully porous particles. Another point is that core-shell particles have a weaker increasing Van Deemter plot and therefore higher flow rates are possible with a high number of bottoms. As a result, the analysis time can be reduced without losing efficiency.

• Back pressure: irregular particles > spherical particles > core-shell particles
• Soil number: core-shell particles > spherical particles > irregular particles
• Loadability: irregular particles ≈ spherical particles > core-shell particles
• Analysis time: core-shell particles < irregular particles ≈ spherical particles

The specific surface area is partly dependent on the pore size. In general, the smaller the pores, the larger the surface area. However, some materials only have a small surface area even with small pores (e.g. Hypersil 120 Å and 170m²/g, PerfectSil 120 Å and 300m²/g). A larger specific surface area provides more space for the analytes to diffuse and therefore the retention times for larger surface areas are also longer. In addition, a larger surface area also provides more space for functional groups, which leads to stronger interactions and, in turn, longer retention. In general, the larger the surface area ...

• the longer the retention time (longer diffusion and more interactions)

The smaller the particles, the higher the plate number but the higher the back pressure. In addition to the disadvantage of the higher back pressure, it must also be considered that the space between the particles becomes smaller and the frit that holds the particles in the column also has smaller pores. As a result, columns with smaller particles are also clogged more frequently compared to larger particles. As a result, the loadability for smaller particles tends to be lower. In addition, a higher back pressure may also be associated with lower stability (fewer injections). In general, the smaller the particles ...

• the higher the base number
• the higher the back pressure
• the lower the loadability
• the lower the stability can be under certain circumstances
• the faster the column tends to clog

The type of functionalisation determines the type of separation technique used. This is therefore the most important property of an HPLC column. If a C18 group is selected as the modification, the reversed phase is the separation method used. If a separation technique has already been chosen, there are various options for functionalisation. The choice then depends on the type of analytes or the ability of the analytes to interact with the modification. Not sure which separation technique you would like to use? On our page Separation techniques you will find some selection aids to make the choice easier.

A higher carbon content indicates a higher occupancy of the stationary phase with the modification. This also tends to increase the interaction and thus the retention. In some cases, however, the retention can also decrease. A C18 phase serves as an example of this. If a C18 phase with a high carbon load is selected, the interaction with hydrophobic analytes increases, but more polar analytes can be eluted earlier, as access to the polar surface is made more difficult. Therefore, together with the carbon content, the type of modification must always be taken into account. If it is a polar modified C18 phase, more polar analytes are also retarded for longer at a higher carbon content.

Endcapping is used to reduce free silanol groups on the surface. These silanol groups can lead to undesirable secondary interactions, which can result in tailing, particularly with bases. Endcapping can also be used to specifically change the properties of the stationary phase. For example, polar endcapping in a C18 phase enables intended polar interactions. If a sterically demanding endcapping is used, the pH stability of the phase can be increased. Non-endcapped phases also have polar groups in the form of silanols, which can also be advantageous for some analytes.

The support material is the basic structure of the stationary phase. In most cases, this is silica. However, other materials can also be used. For example, polymers can be used for a variety of different separation techniques. The use of metal oxides such as zirconium oxide is also possible. Pure carbon in the form of graphite is also used as a stationary phase, which leads to special retention mechanisms. The advantage of silica is that it is relatively cheap and very stable under pressure. One disadvantage, however, is its limited pH stability at around 2-8. For some analytes, it may be advantageous to use a high pH value (retention of bases). In this case, polymers would be suitable as stationary phases. However, the disadvantage here is that the polymers are not as pressure-stable and exhibit different swelling properties in different solvents. Metal oxide and carbon columns enable special interactions. The manufacturer's applications should be taken into account to determine whether these phases are suitable for a particular separation.

The purity of the support material only plays a major role for silica materials. Older phases in particular (e.g. LiChrospher, Nucleosil, etc.) are produced with old silica gel, so-called type A silica. These silica gels have significant amounts of transition metals in the base structure. These transition metals cause a strongly acidic character in the free silanols. This leads to very strong polar interactions of these silanols with the analytes. Therefore, such type-A silica materials are less suitable for bases. Modern phases have other silica sources, which significantly reduce the transition metals. These are referred to as type B silica materials. These materials have fewer acidic silanol groups on the surface, making these phases more suitable for bases. However, it can be difficult to switch from type A to type B silica materials, as the strongly polar interactions of type A silica phases no longer apply. Here it can be useful to use polar modified or non-endcapped phases.

The pH stability can have an influence if the analytes are ionisable (acids/bases). While bases are neutral in the high pH range, acids are uncharged at low pH values. This can have a considerable effect on the interactions. As silica columns are usually stable in a pH range of 2-8, pH values outside this range are a challenge. Various methods can be used to increase the pH range of a column. Steric ligands on the modifications can shield the surface and make it more stable. Steric endcapping can also contribute to stability. Another method is the encapsulation of the silica surface with a polymer or the synthesis of hybrid materials, which significantly increase stability.

The degree of polymerisation describes the number of bonds that connect the modification to the surface. Mono-, di- and trifunctionalisation is possible. Monofunctionalisation is the most common functionalisation. Here, the modification is connected to the silica framework via a bond. In the case of di- and trifunctionalisation, binding to the surface is possible via two or three bonds. A distinction can be made between horizontal polymerisation and vertical polymerisation. In horizontal polymerisation, the modification should be bound to the surface via all available bonds (this is done using anhydrous solvents and high temperatures). In vertical polymerisation, water is added repeatedly over several steps, which also leads to the reaction of already bound modifications. This results in a thick layer over the silica surface. Vertical polymerisation produces a lower carbon content but fewer free silanols. Horizontal polymerisation shows a higher carbon content but more free silanols.