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by Robert L. Stevenson


Transitioning From HPLC to UHPLC of Proteins

Since the introduction of ACQUITY UPLC (Ultra Performance Liquid Chromatography) by Waters (Milford, Mass.) over a decade ago, there has been increased interest in the effects of high pressure on HPLC samples. Prior to the UHPLC era, the effect of pressure on HPLC was studied, but generally for small molecules and pressures lower than 400 bar. Increasing pressure did affect retention behavior, but not by much. The effect of tem- perature was studied more thoroughly, since it had a large effect and was useful in elucidating the mechanism of retention by Van’t Hoff plots, etc. Early UHPLC systems had a maximum pressure (Pmax

) of 1000 bar, which

has since been extended to over 1500 bar and has the potential to go even higher, especially if submicron column packings prove useful. Since proteins potentially can react to changes in the temperature and pressure encountered during UHPLC conditions, what should the analyst be aware of as the separation proceeds?

Effects of high pressure on proteins The following examples illustrate the effects of pressure, temperature and

heating on proteins.

Professors Szabolcs Fekete and Davy Guilllarme (University of Lausanne, Geneva, Switzerland) studied four proteins—lysozyme, myoglobin, filgrastim and interferon alpha-2A—using a C4 RPLC 1.7-μm Waters BEH 300, 50 × 2.1 mm packed column (see Figure 1).1

This short butyl surface is often used to improve column efficiency for proteins under

reversed-phase liquid chromatographic (RPLC) conditions. Earlier studies showed a 3000% increase in retention with a 1000–bar increase in pres- sure for myoglobin, which was much larger than the increase for a range of small–molecule probes.2

C4 stationary phases are less commonly used

in small–molecule separations, but longer hydrophobic phases, such as C18, are often too retentive for proteins.

Bridgman studied the effect of high pressure on egg whites, which are rich in lysozyme,3

and found that proteins in egg whites are completely denatured at a pressure of 7 kbar.

High pressure and elevated temperature may not always be denaturing or deadly, as we see in extremophiles. Unusual fish in particular inhabit ocean depths where pressure can be in the kilobar range.4

They may have

proteins that function only at high pressure. Acre–size mats of bacteria thrive in hot water at Yellowstone National Park (Figure 2).

BaroFold Inc. (Boulder, Col.), makers of PreEMT, or Pressure Enabled Protein Manufacturing, seeks to improve the tolerability, efficacy and safety of a variety of protein therapeutics. The company denatures proteins by increasing and then decreasing pressure, which can lead to refolding.5

overlap. This raises the question: Are protein structure and function dependent on ambient pressure?

The pressure ranges of UHPLC and the PreEMT process

Figure 1 – Waters Ethylene Bridged Hybrid (BEH) particle technology, available in numerous particle sizes and bonded phases for HPLC and UPLC, offers excellent peak shape and efficiency for basic analytes, a rational array of chromatographic selectivity and improvements in chemical stability at mobile phase extremes, particularly at elevated pH.


Figure 2 – View of a steaming lake in Yellowstone National Park. The lake and exiting streams cover mats of colored living bacteria that are respon- sible for the blue and other colors.

MARCH 2016

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