Separation Techniques
Isocratic versus Gradient Elution
Elution techniques are methods of pumping mobile phase
through a column. In the isocratic method, the composition of
the mobile phase remains constant, whereas in the gradient
method the composition changes during the separation
process. The isocratic method is the simplest technique and
should be the first choice when developing a separation. Eluent
gradients are usually generated by combining the pressurized
flows from two pumps and changing their individual flow
rates with an electronic controller or data system while maintaining
the overall flow rate constant.
Derivatization
Derivatization of samples involves a chemical reaction that
alters the molecular structure of the analyte of interest to improve
detection. In HPLC, derivatization of a drug is usually
unnecessary to achieve satisfactory chromatography. Derivatization
is used to enhance the sensitivity and selectivity of detection
when available detectors are not satisfactory for the
underivatized compounds