Elution separation defined

Reiterating the conditions for a chromatographic separation once again, for two solutes to be resolved their peaks must be moved apart in the column and maintained sufficiently narrow for them to be eluted as discrete peaks. However, the criterion for two peaks to be resolved (usually defined as the resolution) is somewhat arbitrary and is usually defined as the ratio of the distance between the peak maxima to half the peak width (a) at the points of inflection. To illustrate the various degrees of resolution that can be obtained, the separation of a pair of solutes 2o, 3o, 4o, 5o and 6o apart are shown in Figure 12. Although, for baseline resolution, it is clear that the peak maxima should be separated by at least 6o for most quantitative analyses. 

Equation (22) allows the maximum sample volume that can be used without seriously denigrating the performance of the column to be calculated from the retention volume of the solute and the column efficiency. In any separation, there will be one pair of solutes that are eluted closest together (which, as will be seen in Part 3 of this book, is defined as the critical pair) and it is the retention volume of the first of these that is usually employed in equation (22) to calculate the maximum acceptable sample volume. 

The second and third peaks will be the pair of peaks in the mixture that are eluted closest together and, thus, the most difficult to separate (usually given the term the critical pair as they define the severity of the separation). Finally, the fourth peak will be that which is eluted last from the mixture and will determine when the analysis is complete and establishes the total analysis time. The chromatographic system must be designed to separate the critical pair and, as this is the pair that is eluted closest together, all other peaks should also be resolved... 

The choice of variables remaining with the operator, as stated before, is restricted and is usually confined to the selection of the phase system. Preliminary experiments must be carried out to identify the best phase system to be used for the particular analysis under consideration. The best phase system will be that which provides the greatest separation ratio for the critical pair of solutes and, at the same time, ensures a minimum value for the capacity factor of the last eluted solute. Unfortunately, at this time, theories that predict the optimum solvent system that will effect a particular separation are largely empirical and those that are available can be very approximate, to say the least. Nevertheless, there are commercially available experimental routines that help in the selection of the best phase system for LC analyses, the results from which can be evaluated by supporting computer software. The program may then suggest further routines based on the initial results and, by an iterative procedure, eventually provides an optimum phase system as defined by the computer software. 

Column design involves the application of a number of specific equations (most of which have been previously derived and/or discussed) to determine the column parameters and operating conditions that will provide the analytical specifications necessary to achieve a specific separation. The characteristics of the separation will be defined by the reduced chromatogram of the particular sample of interest. First, it is necessary to calculate the efficiency required to separate the critical pair of the reduced chromatogram of the sample. This requires a knowledge of the capacity ratio of the first eluted peak of the critical pair and their separation ratio. Employing the Purnell equation (chapter 6, equation (16)). 

The ability of a GC column to theoretically separate a multitude of components is normally defined by the capacity of the column. Component boiling point will be an initial property that determines relative component retention. Superimposed on this primary consideration is then the phase selectivity, which allows solutes of similar boiling point or volatility to be differentiated. In GC X GC, capacity is now defined in terms of the separation space available (11). As shown below, this space is an area determined by (a) the time of the modulation period (defined further below), which corresponds to an elution property on the second column, and (b) the elution time on the first column. In the normal experiment, the fast elution on the second column is conducted almost instantaneously, so will be essentially carried out under isothermal conditions, although the oven is temperature programmed. Thus, compounds will have an approximately constant peak width in the first dimension, but their widths in the second dimension will depend on how long they take to elute on the second column (isothermal conditions mean that later-eluting peaks on 2D are broader). In addition, peaks will have a variance (distribution) in each dimension depending on... 

Finally, ion chromatography can be used to determine the a-sulfo fatty acid esters. The chromatographic column is a nonpolar poly sty rene/divinylbenzene column and the ion pair reagent is 0.005 M ammonia. In order to reduce the elution time, acetonitrile is added as a modifier with increasing concentration. This gradient technique makes it possible to separate simultaneously ester sulfonates and disalts by chain length. Determination is achieved by standards with defined chain length [107]. 

How then are these ions/decompositions chosen Before considering this we must define, very carefully, the requirements of the analysis to be carried out. Is a single compound to be determined or are a number of compounds of interest If a single compound is involved, its mass spectrum and MS-MS spectra can be obtained and scrutinized for any appropriate ions or decompositions. If the requirement is to determine a number of analytes, their chromatographic properties need to be considered. If they are well separated, different ions/decompositions can be monitored for discrete time-periods as each compound elutes, thus obtaining the maximum sensitivity for each analyte. If the analytes are not well separated, this approach may not be possible and it may then be necessary to monitor a number of ions/decompositions for the complete duration of the analysis. If this is the case, the analyst should attempt to find the smallest number of ions/decompositions that give adequate performance for all of the analytes (remember the more ions/decompositions monitored, then the lower the overall sensitivity will be). 

The capacity factor tells us where the peaks elute relative to VQ. The separation factor (a) tells us where the peaks elute relative to each other. It is defined, for two peaks, as the ratio of the capacity factors, with the larger one in the numerator. 

The chiral recognition ability of a CSP is quantitatively evaluated from the results of chromatographic separation of enantiomers. Figure 3.4 shows a chromatogram of the resolution of benzoin (19) on cellulose tris(3,5-dimethylphenylcarbamate). The (+)-isomer elutes first followed by the (—)-isomer complete baseline separation is achieved. The results of the separation can be expressed by three parameters—capacity factors (k1), separation factor (a), and resolution factor (Rs)—defined as follows ... 

A reduced peak capacity in one domain may be counterbalanced by an increased peak capacity in another domain. If we know the average peak width of a chromatographic separation and the gradient duration, we can calculate the maximum number of peaks that can be separated. (Note peak capacity does not mean that this number of compounds in a sample will be separated they may still co-elute). That means we can operate between two limits (1) a peak capacity of zero representing a flow injection analysis and (2) a minimal required peak capacity that defines the peak capacity to separate all compounds in a given mixture. Unfortunately, especially in the early stages of drug... 

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. 

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