UHPLC OF PROTEINS continued
changes would be due to resistive heating. The retention of lysozyme and myoglobin was particularly sensitive to changes in temperature.
The column terminus is most interesting— this is where the effects of pressure and temperature can be seen.
The researchers measured changes in retention factors as a function of column temperature. Van’t Hoff plots demonstrated a peculiar curved trajectory. At low temperatures (25 °C), pressure fluctuates, but as the temperature reaches about 50 °C, the plots show an apex followed by a decrease. This indicates that the proteins have undergone a significant change in conformation. For lysozyme, the pressure-induced changes dominate at low temperature (<30 °C). However, the temperature has a stronger influence on interferon alpha, even in the conventional HPLC range.
Conclusion
the pressure is increased from 100 to 1100 bar.2 mode, the increase was smaller still.
In the gradient elution
Frictional heating at the column inlet At the top of the column, frictional heating should be minimal, since the generation of heat from resistance to flow from passing from the injector to the column inlet is minimal.
Column midpoint At the column midpoint, the pressure is reduced by half. The dissipation of pressure with length has been confirmed by coupling two identical columns in series, which doubles the pressure. For a pressure-sensitive protein analyte, the reduced pressure may pass through the pressure point, which favors refolding and renaturation. When this happens, the protein may start to refold. This can be very rapid.7
By column midpoint, the temperature would be higher than at the inlet due to resistive heating. The heating is not adiabatic, since narrow- column-diameter (2.1-mm) columns packed with sub-twos often show resolving power superior to fatter columns. This has been attributed to improved dissipation of heat to the column walls, which provides a more uniform radial and axial temperature profile.
Column terminus The column terminus is most interesting—this is where the effects of pres- sure and temperature can be seen. Because it has been exposed to the most frictional heating, the temperature is highest here and the pressure is lowest, about equal to atmospheric pressure. If the pressure-induced denaturation is reversible, then the original protein could be refolded into its native form. Homologous multimeric proteins can also reassemble and renature. However, the retention times of the component part’s hetero- geneous multimeric proteins is expected to be different, and thus would not be in the same volume for regeneration.
Temperature-induced denaturation is now a possibility. This may not be reversible. Power is dissipated via frictional heating as the liquid races through the packed bed. Fekete and Guillarme studied this by adding a variable-pressure restrictor after the column terminus. This allowed the pressure to be kept constant at 750 bar while the flow rate was varied from 100 to 1400 μL/min. Since the pressure drop was constant, any retention
AMERICAN LABORATORY 50
Proteins that have undergone a structural change during chromatogra- phy may not be representative of the starting material. If the identity of the original analyte is of interest, as it would be in discovery proteomics and preparative applications, the identity of the peaks should be confirmed post–chromatography. This can be done with a quick scan of the starting sample to see if post-column materials are present in the starting sample. Circular dichroism can often reveal if the purified proteins are similar to the reference standards. Running an electrophoresis gel, especially a native gel, with the pre-chromatography sample and collected fractions from a UHPLC should show if any unique bands appear in the collected UHPLC fraction. This would indicate they came from the chromatography.
In conclusion, transitioning a protein assay from HPLC to UHPLC may not be as simple as it is with small molecules. The effects of pressure on proteins separated by ion exchange and steric exclusion chromatography might not be as pronounced as those separated by RPLC. The added pressure raises new possibilities and hence questions that should be addressed as part of a method validation.
References 1. Fekete, S.; Guillarme, D. Estimation of pressure-, temperature- and fric-
tional heating-related effects on proteins’ retention under ultra-high- pressure liquid chromatographic conditions. J. Chromatogr. A 2015, 1293, 73–80.
2. Fekete, S.; Veuthy, J.-L. et al. The effect of pressure and mobile phase velocity on the retention properties of small analytes and large bio- molecules in ultra-high pressure liquid chromatography. J. Chro- matogr. A 2012, 1270, 127–38.
3. Bridgman, P.W. The coagulation of albumin by pressure. J. Biol. Chem. 1914, 19, 511–12.
4.
http://list25.com/25-most-terrifying-deep-sea-creatures/ 5.
http://www.barofold.com/ 6. Broom, A.; Gosavi, S. et al. Protein unfolding rates correlate as strongly as folding rates with native structure. Prot. Sci. 2015, 24, 580–7.
7. Liu, Y.; Prigozhin, M.B. et al. Observation of complete pressure-jump protein refolding in molecular dynamics simulation and experiment. J. Am. Chem. Soc. 2014, 136, 4265–72.
Robert L. Stevenson, Ph.D., is Editor Emeritus, American Laboratory/ Labcompare; e-mail:
rlsteven@yahoo.com
MARCH 2016
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