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 theseparation 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 courseof our research. On the monolithic diethylaminoethyl convective interaction media analytical column (CIMac DEAE), the separation of major wheyproteins 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). Avery 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.
COBISS.SI-ID: 4909850
The main problem in the densitometric determination of carotenoids is their rapid degradation during and immediately after chromatography, respectively. In this study, we show that 15 ng of lutein, lycopene and β-carotene standardsapplied on C18 RP high-performance thin-layer chromatography (HPTLC) plates pre-developed with dichloromethane-methanol 1:1 (v/v) remained stable for 1 h after the development of chromatogram using methanol-acetone 1:1 (v/v) with 0.1% of 2-tert-butylhydroquinone (TBHQ), which is a substantial improvement of their stability. An HPTLC quantification procedure for free lutein, with densitometry at 450 nm based on the developed method described above, was established and validated. Repeatabilities of the chromatography expressed by the relative standard deviation (RSD) from 6 applications of lutein standard at 5, 15 and 25 ng were 3.41, 1.33 and 1.65%, respectively. The best fit calibration curve from 5 ng to 30 ng of lutein was polynomial. Limit of detection (1.5 ng) and limit of quantification (5 ng) were the best achieved so far. With these chromatographic conditions dietary carotenoids lutein esters, lycopene, free lutein and β-carotene from food supplements werealso well separated and were identified by visible absorption spectra scanned in situ and by mass spectra. Some additional developing solvents with the same type of chromatographic layer are proposed for the fast separation oflutein esters from free lutein in food supplements.
COBISS.SI-ID: 4915226
Shikonin and its ester derivatives belong to a group of secondary metabolites with a wide array of beneficial effects on human health. However, shikonin is principally used in oil-based preparations due to the low solubility of the pigment in aqueous media, and the positive properties of shikonin are not exploited to their full potential. Such low aqueous solubility often results in poor bioavailability, makes shikonin undesirable for oral administration, and restricts its broadened use in the food and pharmaceutical industries. The purpose of this study was to enhance the aqueous solubility of shikonin by the addition of β- lactoglobulin and to characterize the macromolecule-ligand binding interaction by means of spectrophotometry, spectrofluorometry, high-performance liquid chromatography, and mass spectrometry. In the presence of β-lactoglobulin the solubility of shikonin is increased up to 181-fold. One shikonin molecule binds covalently to β-lactoglobulin via Cys121, whereas the remaining pigment molecules most probably bind to the protein via noncovalent interactions.
COBISS.SI-ID: 5114394