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The role of pore size in Reversed Phase HPLC

SGE is very excited to launch a new HPLC product line under the ProteCol™ brand.

ProteCol™ is fundamental to the new line of columns which give a continued focus on inert column design which was first created with the ProteCol™ PEEKsil™ offerings (polymer – sheathed fused silica tubing). SGE is also excited to offer a whole new range of ultrapure reversed phase silica which comes in both the unique GLT™ (glass lined tubing) column format as well as with the new PEEK™ lined stainless steel format. The benefit which you get is the most comprehensive inert HPLC reversed phase column which offers from the 150 micron ID PEEKsil™ format through to the new 4.6mm ID PEEK™ lined stainless steel columns.

Why is Inert HPLC Column Design Important?

The non-specific interactions which happen between the target analyte and the silica particles in the HPLC column are very well-controlled with the availability of the ultrapure silica. Today, many of the chromatographers expect that the silica which is sent by the manufacturers is of the highest purity and SGE confirms that we have rigorously researched the quality of silica using the standard reference material (SRM) which is provided by the National Institute of Standards and Technology. When you test the silica using SRM 870 (NIST), it identifies the non-specific interactions which are associated with the metal contamination and also with the non-end capped silanols. (See Figure 1).

The thing which is not considered often is that the role column hardware may play in non-specific interactions – the frit and the internal column hardware can both influence the behavior or analytes with a known metal chelating activity. Many pharmaceutically active compounds and natural products have the potential to interact with the metals. The co-ordination between the metal ion and the analyte is facilitated by the only electron pair on the analyte molecule.

If two electron donor groups (either oxygen or nitrogen) are located in a favorable position, a chelate can easily be formed and while the enthalpy of the complex formation for two monodentate lingands and a bidentate lingand is similar, the chelate is entropically favored and then leads to a stronger interaction. For this reason molecules like quinizarin.

tetracycline or ciclopirox from tailing peaks in the presence of metal in the column or system (See figure 2).

It is fundamental to the new ProteCol™ line. To address this potential risk, SGE’s ProteCol™ column development has focused exclusively on the most inert C8 and C18 phases in a variety of pore and different particle sizes in the most inert column hardware – glass lined PEEK™ lined stainless steel (See figure 3) and PEEKsil™.

Why focus on Reversed Phase and Pore Size?

The most commonly used form of liquid chromatography is reversed phase chromatography and many of the chromatographers try to remain in the reversed phase environment than moving to other less conventional buffer systems.

The thing which is not considered often is the role of Alkane modified silicas which were developed in the 1970s and as they had a better resolution and higher reproducibility, they quickly became the most popular technique in the separation of liquid chromatography. Since the elution conditions and elution order are opposite to what earlier was “normal” chromatography, the term “reversed” phase was coined and remained the general term which was used for describing a hydrophobic bonded stationary phase. When you have to consider the optimal reversed phase column for the separation of a target analyte, the majority of liquid chromatographers made their selection on the basis of the type of bonded phase (C18, C8, C4, Phenyl, etc.), whether the column is end capped and the overall carbon loading.

Don’t forget to choose the appropriate pore size of your reversed phase column!

The thing which is often ignored by many chromatographers is the choice of optimal pore size of the silica for their appropriate application. In liquid chromatography, virtually all the interactions (and therefore retention) take place inside the pore system. The exterior surface of the common porous silica covers less than 1% of the total surface area. To use the available interactive surface, the analyte molecule requires unrestricted access to the particle interior. In many chromatographic applications, pore diffusion is the time limiting step (the slowest step which therefore governs the overall kinetics). After the solute overcomes the film mass transfer resistance, it has to diffuse into the pore system in order to bind itself to the surface as most of the surface is inside a porous particle (>99%). Pore diffusion for larger molecules like proteins, the process of pore diffusion becomes an important factor.

Many molecules have been derived to describe the effect of the pore diameter on the diffusion constant of a solute molecule. These models range from the Fickian diffusion rate is driven purely by concentration (large pores – small solute molecules) – the mean free path of Brownian motion is equal or larger than the pore diameter (collisions with the wall play a major role in the determination of the diffusion rate). One of the extreme cases is the single file diffusion, where the diameter of the solute molecule is larger than the radius of the pore. During this, the molecules are unable to pass each other. Also, an estimation for the steric hindrance at the pore entrance and the frictional resistance within the pore system was successfully provided by Renkin.

A graphical representation of the Renkin model and its implications are shown below in Figure 4.

The main drawback of the large pore sizes in HPLC columns is the reduction in specific surface area. As the size of the pore increases, the accessible surface area which is required for the solute to bind decreases and with it the capacity of the column decreases. Figure 5 shows the specific surface area of commercially available packing material versus the diameter of the pore.

The choice of the pore size which is for a given separation is a compromise between resolution on the high pore diameter and the load-ability (capacity) on the low pore diameter end. A rough guide for a suitable pore diameter ranges (where the pore diffusion coefficient is between 50 and 80% of the diffusion rate in free liquid) is shown in Figure 6.

As figure 7 suggests, the use of a 300 Å  pore size stationary phases does have a practical limit for large molecules and you should also be mindful of the sample and its complexity before considering the separation of large macromolecules on this type of phase. Proteins are macro molecules which are large and diffuse slowly (small diffusion rate constant). Also, there is a greater chance of proteins to cause a steric hindrance once they bind to the pore surface.

In conclusion, SGE recommends you the following guide to select the appropriate pore size which is based on your target analytes molecular weight range.

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