Amino acids were eluted using an elution gradient.
Subsequently, the protein solution was loaded onto a HiTrap Q FF prepacked with Q Sepharose (GE Healthcare Life Sciences) equilibrated with Buffer A and the bound proteins eluted using a buffer elution gradient from 0 to 1000 mM of NaCl.
Peptides were chromatographed using a borate buffer system, namely 10 mM sodium borate, pH 9.3 (Buffer A) and 10 mM sodium borate, pH 9.3 + 0.5 M sodium chloride (Buffer B) and eluted using an exponential elution gradient into 12 × 750 μl fractions.
The crude extract, a dark green oil (1.40 g), was separated by means of column chromatography using silica gel 60 eluted with n-hexane/EtOAc as the elution gradient, yielding 8 fractions.
They were then transferred to the column and eluted as above except the stepwise elution gradient was comprised of 500 ml of 0 1, 1 1 and 1 0 (v/v) CH2Cl2: hexane and finally MeOH, and 2.5 ml fractions were collected.
Using an optimized elution gradient, the main components were separately eluted within 11 min. The typical RRLC-QQQ MS/MS chromatograms of the marker chemicals in Shen-Fu decoction are shown in Figure 2.
Drugs have been separated on a C18 column using an elution gradient based on an increase in the acetonitrile percentage.
While the effect of temperature on the separation was not apparent, effect of initial organic strength was stronger than that of the elution gradient.
In one of several case studies, sequential and simultaneous optimization of elution gradient shape and cut times are compared for a three component separation.
In the optimization step, the loading volume and the elution gradient are optimized with regard to the most important costs: the fixed costs and the feed cost.
The principal factors influencing the separation (acetonitrile and SHS concentration) have been considered for the optimization of the elution gradient through factorial design and multicriteria decision-making.
