Applicability of analytical and preparative monolithic columns to the separation and isolation of major whey proteins

The separation and isolation of major whey proteins is already extensively covered in the literature although no study has been published in which monolithic columns were used. In our research we present, for the first time, the use of short convective interaction media (CIM) monolithic columns for the separation of all major whey proteins and isolation of β-lactoglobulin variant A and B (β-LgA and β-LgB) from a commercial product whey isolate (WI). Although our primary interest was directed towards finding a proper monolithic column and chromatographic conditions for the purification and isolation of β-LgA and β-LgB, three additional analytical LC methods, each having its own potential application target, were also developed in the course of our research. On the monolithic diethylaminoethyl convective interaction media analytical column (CIMac DEAE), the separation of major whey proteins was achieved by gradually lowering the pH of the mobile phase. The ever-so-hard obtainable linear external pH gradient was very linear in the range of pH 5.5–3 and the developed ion-exchange (IE) high-performance liquid chromatographic (HPLC) method was amenable to mass spectrometry (MS). A very fast baseline separation, with UV detection, of all major whey proteins was achieved on a prototype CIMac reversed-phase styrene-divinylbenzene (RP-SDVB) monolithic column in only 4 min and the performance of this column proved superior in comparison with the packed particle POROS perfusion column. The developed RP-HPLC–MS method is fast and, due to the MS detector, can offer low limits of detection and quantitation. Finally, in order to fulfill our primary interest, a scale-up method was developed, using a prototype 8 mL analogue of the CIMac RP-SDVB column, for the isolation of native and chemically unmodified β-LgA and β-LgB from WI with purities higher than 90% and 81%, respectively. The proteins were to be used in further protein–ligand binding studies. The developed methods excel in speed of the analysis, sensitivity, resolution, and simplicity. Thus, it is shown for the first time that short monolithic columns are applicable to the separation and isolation of major whey proteins and that their use has some obvious benefits.