Gradient isocratic separations

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. 

When we talk about optimization of serial LC/MS operations, we consider the genuinely serial sequences of actions necessary to perform such analyses including equilibration of columns, sample aspiration, sample injection, isocratic or solvent gradient sample separation, detection, and column washout. 

Some LC/MS users adhere to isocratic separation because of the myths around gradient elution (it is complex to develop and transfer between instruments and laboratories, it is inherently slower than isocratic methods because of re-equilibration, and other reasons summarized by Carr and Schelling6). A researcher may have a very good reason to use an isocratic method, for example, for a well defined mixture containing only a few compounds. The isocratic method would certainly not be useful in an open access LC/MS system processing varying samples from injection to injection. 

The selectivity of different stationary phase materials can be applied using columns in sequence to provide high-speed isocratic separations instead of gradient elution. An example for amino acids analysis is shown later in Figure 4.15, where the same eluent was used for all of the separations and the fraction containing the sample components of interest was switched from one column to another. 

White recently illustrated the use of fast supercritical fluid and EFLC for drug discovery and purification [46]. The optimized isocratic separations used to scale up to preparative-scale separations were often EFL mixtures. For example, Figure 9.13 shows the optimized conditions for the separation of a drug candidate included 30% methanol (with 0.2% isopropyl amine)/C02 on a Chiralcel OJ-H column at 5 mL/min [46]. His work also illustrates by using gradients that start in supercritical conditions and then move into EFL mixture conditions provides efficient and fast separations. 

Refractive-index detection is seldom used for many of the same reasons just mentioned for the spectroscopic detection of amino acids in their native forms. In fact, these problems are even more severe. Refractive-index detection has almost no selectivity whatsoever. Nearly every sample component passing the detector will register a signal. Also, this makes refractive index entirely incompatible with gradient elution. Even for isocratic separation, and detection of only a select few amino acids, refractive index can be very troublesome because of the detector s tendency to drift due to temperature changes in the laboratory (perhaps newer models have fixed this problem ). Finally, detection limits tend to be very poor for refractive-index detection. 

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