Biomedical Engineering Reference
In-Depth Information
Fig. 21.1. Ion exchange/reversed-phase chromatographs of complex mixtures. Proteins
from an immunodepleted plasma sample were separated by two dimensions of chro-
matography; IEX (
A
) separates proteins on the basis of charge. Fractions from the first
separation were further subjected to RP separation on the basis of hydrophobicity (
B
;
chromatographs normalised to a scale of 1 and stacked).
4. Eluted proteins are collected into clean microcentrifuge
tubes. Depending upon peak profile, between 8 and 20 first-
dimension samples of 30-90 s can be collected. Fractions
from the first dimension can be injected directly onto the
second, reversed-phase column.
5. IEX fractions are injected onto the reversed-phase column
using a switching valve. The column is washed for 5 min
with low organic solvent (2% (v/v) acetonitrile, 0.1% (v/v)
TFA or formic acid in water) to remove salt and urea.
6. Gradient elution of proteins is performed at 100
l/min
using a linear gradient from 20 to 100% B (90% (v/v)
μ
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