Publication details
A CONTINUOUS MODE OF OPERATION FOR LARGE VOLUME DOSING IN ANALYTICAL CARRIER AMPHOLYTE–FREE ISOELECTRIC FOCUSING OF PROTEINS APPLIED TO OFF–LINE DETECTION OF FRACTIONS
| Authors | |
|---|---|
| Year of publication | 2006 |
| Type | Article in Periodical |
| Magazine / Source | Journal of Separation Science |
| MU Faculty or unit | |
| Citation | |
| Field | Analytic chemistry |
| Keywords | capillary isoelectric focusing;neutralization reaction boundary;proteins;fractionation;injected large volume sample |
| Description | Mass spectrometry (MS) is being increasingly used for analysis of proteome complex samples. To remove matrix interferences and to concentrate analytes prior to a MS measurement, sample preparation is often necessary. A useful method for this purpose is Carrier Ampholyte Free-Isoelectric Focusing (CAF-IEF). In this paper CAF-IEF of ampholytes was performed on a commercial apparatus EA101 (Villa Labeco, Slovakia) equipped with a specially made column for samples of large volumes (up to 0.5 ml). A new continuous mode without voltage interruption or electrolyte replacement was developed. In this mode, a low molecular pI marker (PIM 7.4), low concentrations of myoglobin and insulin (16 mg/l), respectively, were concentrated, and then 5-ul fractions collected for off-line analyses. The total time of focusing was 66 minutes. Concentration of PIM 7.4 in the fractions was increased up to 75 times (determined by UV-VIS spectrometry). Concentration in fractions was increased up to 30 times for myoglobin and 10 times for insulin, respectively. |