Separation isocratic versus gradient

When performing HPLC (see Basic Protocol 2) the time of analysis will depend on the conditions used—i.e., isocratic versus gradient. Isocratic separation of the individual betacyanins, as shown in Figure F3.1.2, requires 20 min, compared to 9 min using a gradient elution system. 

Two analytical methods for priority pollutants specified by the USEPA (38) use HPLC separation and fluorescence or electrochemical detection. Method 605, 40 CFR Part 136, determines benzidine and 3,3-dichlorobenzidine by amperometric detection at +0.80 V, versus a silver/silver chloride reference electrode, at a glassy carbon electrode. Separation is achieved with a 1 1 (v/v) mixture of acetonitrile and a pH 4.7 acetate buffer (1 M) under isocratic conditions on an ethyl-bonded reversed-phase column. Lower limits of detection are reported to be 0.05 /xg/L for benzidine and 0.1 /xg/L for 3,3-dichlorobenzidine. Method 610, 40 CFR Part 136, determines 16 PAHs by either GC or HPLC. The HPLC method is required when all 16 PAHs need to be individually determined. The GC method, which uses a packed column, cannot adequately individually resolve all 16 PAHs. The method specifies gradient elution of the PAHs from a reversed-phase analytical column and fluorescence detection with an excitation wavelength of 280 nm and an emission wavelength of 389 nm for all but three PAHs naphthalene, acenaphthylene, and acenaphthene. As a result of weak fluorescence, these three PAHs are detected with greater sensitivity by UV-absorption detection at 254 nm. Thus, the method requires that fluores-... 

Gradient elution versus isocratic elution — effects of the gradient profile on separation... 

If for each peak pair no differing isocratic conditions in which adequate separation results exist, then a gradient cannot be successful either. This can be seen by exactly overlying lines in the In (A ) versus % -graph. This is a rare but possible case. 

---