Gradient chromatofocusing: A new chromatographic technique for protein purification and separation

Gradient chromatofocusing is a new chromatographic technique developed by our research group which overcomes the limitations of the chromatofocusing technique. The design of gradient chromatofocusing is similar to the chromatofocusing technique but has two significant differences with the chromatofocusing technique. One is that a gradient pump system is used to mix the high and low pH mobile phases in gradient chromatofocusing by gradually increasing the percentage of the low pH mobile phase to the inlet of the column, producing a linear pH gradient. The second difference is that common buffer components are used in the mobile phase in gradient chromatofocusing. The use of a gradient pump system makes the control of the pH gradients much easier and allows the use of high mobile phase buffer concentration in gradient chromatofocusing, which cannot be done by the chromatofocusing technique. The use of common buffer species addresses all the limitations associated with the polymeric ampholyte buffer.; In this dissertation, several studies are made to characterize the gradient chromatofocusing technique. In the first chapter, the basic aspects of the traditionally used ion-exchange HPLC techniques for protein separation are described. In the second chapter, the mobile phase buffer concentration effect on protein separation in the gradient chromatofocusing technique using a DEAF column is studied. The results show that the mobile phase buffer concentration has a significant effect on protein separation. With the increase of the mobile phase buffer concentration, the resolution of proteins gradually increases up to a limit, showing the capability of gradient chromatofocusing to manipulate the mobile phase pH and mobile phase buffer concentration to optimize protein separation, which cannot be achieved by the chromatofocusing technique. In the third chapter, the gradient chromatofocusing technique is further characterized by employing a popularly used chromatofocusing column, a Mono P column. The results show that in terms of column efficiency, gradient chromatofocusing is comparable with the chromatofocusing technique but the optimized resolution of protein samples is much better than that obtained by the gradient chromatofocusing technique. In addition, the results also show that gradient chromatofocusing is much more flexible in adjusting pH gradients for better protein separation. In the fourth chapter, the anion concentration effect in gradient chromatofocusing is studied. The results show that the mobile phase buffer concentration effect is actually an anion concentration effect. The results also show the existence of a “magic pH”, at which the proteins can be dramatically resolved. Different behaviors for different salts do exist in gradient chromatofocusing although the general trend is the same for all the salts. In chapter five, a new interfacing technique GCF-ESI-MS which couples gradient chromatofocusing with electrospray ionization—mass spectrometry is studied. This technique is generally the first LC-MS technique which couples ion-exchange HPLC with electrospray ionization—mass spectrometry for protein separation. This technique not only can be used for qualitative study of the protein samples such as determining the molecular weight, but also can be used to quantify the protein samples. Due to these qualitative and quantitative capabilities, this technique shows a great promise being widely used in proteomics.