Overlapping separation methods

A six-port valve was first used to interface the SEC microcolumn to the CZE capillary in a valve-loop design. UV-VIS detection was employed in this experiment. The overall run time was 2 h, with the CZE runs requiring 9 min. As in the reverse phase HPLC-CZE technique, runs were overlapped in the second dimension to reduce the apparent run time. The main disadvantage of this yu-SEC-CZE method was the valve that was used for interfacing. The six-port valve contributed a substantial extracolumn volume, and required a fixed volume of 900 nL of effluent from the chromatographic column for each CZE run. The large fixed volume imposed restrictions on the operating conditions of both of the separation methods. Specifically, to fill the 900 nL volume, the SEC flow rate had to be far above the optimum level and therefore the SEC efficiency was decreased (22). 

This equation shows that N increases with Xlcr, the ratio of the migration distance in which separation can occur to the zone width parameter zone overlap. The overall goodness of a separation obviously increases with this ratio, so N is clearly a valid index of separation power, as substantiated more fully elsewhere [8,15]. In chromatography, N is often taken as a measure of column efficiency, a definition that can be extended to other zonal separation methods as well. 

Reality, therefore, forces separation scientists to deal with various aspects of peak overlap. The subject is a complicated one. We present in this section a brief introduction to the nature, implications, and unexpectedly high frequency of overlap. Some theoretical methods for dealing with overlap are outlined in the following section. While this treatment is based on studies of peak overlap in chromatography [33, 34], the concepts are equally valid for electrophoresis and other zonal separation methods. 

Randomly overlapping component peaks in chromatography have been studied by Rosenthal [36], by Davis and this author [33,34], and by Guiochon and co-workers [37,38]. The results may be assumed to apply to other separation methods as well. 

In terms of organization, the text has two main parts. The first six chapters constitute generic background material applicable to a wide range of separation methods. This part includes the theoretical foundations of separations, which are rooted in transport, flow, and equilibrium phenomena. It incorporates concepts that are broadly relevant to separations diffusion, capillary and packed bed flow, viscous phenomena, Gaussian zone formation, random walk processes, criteria of band broadening and resolution, steady-state zones, the statistics of overlapping peaks, two-dimensional separations, and so on. 

Analytical instruments based on either physicochemical separation methods or relying on (bio)chemical reactions require a finite time to run these instruments are usually operated in repetitive, non-overlapping batch mode and deliver results with a certain non-negligible dead time. Generally, data density is low, for instance, in the order of 1 min 1 for FIA or 2 h 1 for HPLC. 

As samples become more complex, the ability of a particular separation method to resolve all components decreases. A statistical study of component overlap has shown that a chromatogram must be approximately 95% vacant to provide a 90% probability that a given component of interest will appear as an isolated peak (9). This is shown graphically in Fig. 10, where the probability of separation is plotted as a function of the system peak capacity for cases where the number... 

Separation methods which can be used to achieve constancy of analysis will now be considered. No attempt will be made to survey these methods exhaustively, but merely to indicate the essential features of the various techniques. (It is to be noted that some of the techniques will necessarily overlap with those used for assessing homogeneity.)... 

A simple compartment type of apparatus may be used to separate a peptide mixture into basic, neutral and acidic fractions (Gordon et al., 1941, 1943 Sanger and Tuppy, 1951a). Since the simplification of the mixture is usually more important than the yield of peptides, it is often advisable to repeat the ionophoresis on each fraction, and in this way clear cut fractionations may be obtained with very little overlapping. This method is especially useful for separating the basic peptides. By... 

In contrast to the calculations described above, nonlinear separation methods try to allocate the area of overlapping peaks by taking into account the true peak shape as far as possible. 

Pitch-scale modification is performed 1 recombining the frames on epochs that are set at different distances apart from the analysis epochs, as shown in Figure 14.3. All other things being equal, if we take for example a section of speech with an average pitch of 100 Hz, the epochs will lie 10 ms apart. From these epochs we perform the analysis and separate the speech into the pitch-synchronous frames. We can now create a new set of epochs that are closer together, say 9 ms apart. If we now recombine the frames by the overlap-add method, we find that we have created a signal that now has a pitch of 1.0/0.009 = 111 Hz. Conversely, if we create a synthetic set of epochs that... 

Since spectra of different orders may coincide or overlap, some method of separating orders is required. Separation of orders is achieved by use of a second dispersing element, either a prism or conventional grating, mounted so its dispersion is in a direction perpendicular to that of the echelle. This combination of dispersing elements produces a two-dimensional, spectral pattern. An example of such a pattern is shown in Figure 3-18, which was... 

Several approaches are possible to minimize the effects of spectral band-spectral line overlap. One method is to use a high resolution spectrometer with narrow slit widths. This method frequently resolves the band into its separate components, thus permitting better separation of spectral line and spectral band components. Another method is to determine if another line is available for use in a different spectral region. For example, there is less OH band interference with the copper 3274.0 A line than with the copper line at 3247.0 A. Cobalt at 2873.1 A is in a region of strong CH band interference, while the cobalt line at 3453.5 A is not. 

Having overlapping peaks in a chromatogram, the analyst has to solve the following question Which separation method is the best for separating these overlapping peaks ... 

To evaluate and optimize separation methods for overlapping peaks, there is a software tool called hilchromet. With hi chromet, the analyst has a tool for determining how well the chosen separation method in his chromatography data system really is it deals with symmetric as well as a symmetric peaks. 

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