July 1, 2012 (Vol. 32, No. 13)

Although small particle size, nonporous particles for ion exchange such as Agilent’s Bio IEX range offer significant advantages for increased throughput in ion-exchange chromatography, column back pressure can place limitations on the maximum performance that can ultimately be achieved. It is therefore important to understand and appreciate how column pressure can be influenced by factors other than simply the flow rate and column dimensions for these high technology particles.

A grafted hydrophilic polymer layer, containing the ion-exchange functionality, covers the rigid polystyrene (crosslinked with divinylbenzene) core. This helps eliminate nonspecific binding, however the resultant particle is not entirely incompressible or without a slight degree of swell. Consequently it is possible to observe notable changes in column back pressure simply from the buffer composition alone that may not have been noticed when using columns packed with larger particle sizes.

It is important to recognize that, although using a smaller particle size will lead to sharper peaks due to increased column efficiency, column back pressure may begin to become a limiting factor. Furthermore it should also be noted that the choice of buffer salt and the ionic strength of the buffer can also affect the column back pressure as well as separation performance.


Agilent Bio WCX NP3, 4.6 x 50 mm SS

Mobile phase
A: 20 mM or 40 mM sodium phosphate, pH 6.0, or 20 mM or 40 mM MES, pH 6.0
B: A + 1.0 M NaCl

0–20% B, 0–20 minutes; 20–100% B, 20–25 minutes


Injection volume
20 µL

Monoclonal antibody (IgG)

1.0 mg/mL

UV, 280 nm

Agilent 1260 Infinity Bio-inert Quaternary LC system

Results and Discussion

Figure 1 indicates the effect of different buffer salts: MES or 2-(N-morpholino)ethanesulfonic acid and sodium phosphate, 20 mM and 40 mM ionic strength; pH 6.0. Note, although 200 nm tends to give the best peak response for proteins due to the number of amide bonds, the detector wavelength used was 280 nm due to the background absorbance of MES at 220 nm.

The profile of the monoclonal antibody separation is typical for this kind of molecule, with a major peak with very closely eluting impurities, both before and after, resulting from charge isoforms.

Figure 1. Monoclonal separation Bio WCX NP3 4.6 x 50 mm (1.0 mL/min) using different buffer salts and concentrations

The separation at pH 6.0 appears slightly better using MES instead of phosphate buffer, although it may be possible to further optimize the separation at higher pH. As expected, using a higher ionic strength buffer causes the antibody to elute earlier, however the effect was more pronounced with phosphate buffer.

Apart from the high background absorbance at 220 nm when using MES, it was also observed that the column pressure was around 10 bar greater with 40 mM MES compared to the other buffer conditions (Figure 2). In all cases, the column pressure rose during the latter part of the gradient as the salt concentration (and hence the eluent viscosity) increases.

Figure 2. Column pressure using different buffer salts and concentrations

Agilent Technologies

Andrew Coffey

Applications Chemist

[email protected]


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