A reversed-phase ODS column (TSKgel ODS-80Ts, Tosoh Corporation, Tokyo, Japan) was used for the separation and quantification of glutamate and glutamine, and the gradient elution of the mobile phase was kept at a constant flow rate of 0.8 mL/min.
Separation and elution were achieved using acetonitrile: 0.1% formic acid (90 10, v/v) as the mobile phase at a flow-rate of 0.25 mL/min for a run time of 2 min.
In this case, the run time was longer (50 min) to allow for the elution and separation of these polyglutmatyl folates (Table 2 ).
Gradient elution in nonlinear chromatography is a very powerful tool for the separation of components having a wide range of retentivity.
It was established that the multistep gradient elution developed for RP-TLC separation of pigments can be successfully used as a pilot method for the rational design of gradient elution in RP-HPLC for the separation of the same pigments.
Alignment was performed to correct for the variable elution of peptides during chromatographic separation.
For the separation of redox species strong cation exchange chromatography (SCX) with isocratic elution was employed.
CSPs prepared were tested for the separation of four chiral β-blockers (atenolol, metoprolol, pindolol and propranolol) in normal phase (NP) and polar organic phase (PO) elution modes.
A gradient method was used for the separation of the extracted samples: 0-25 min, 15%-40% A; 25-40 min, 40%-100% A. The elution flow rate and detection wavelength were set at 1.0 mL/min and 254 nm, respectively.
