Resolution peak capacity

This book is organized into five sections (1) Theory, (2) Columns, Instrumentation, and Methods, (3) Life Science Applications, (4) Multidimensional Separations Using Capillary Electrophoresis, and (5) Industrial Applications. The first section covers theoretical topics including a theory overview chapter (Chapter 2), which deals with peak capacity, resolution, sampling, peak overlap, and other issues that have evolved the present level of understanding of multidimensional separation science. Two issues, however, are presented in more detail, and these are the effects of correlation on peak capacity (Chapter 3) and the use of sophisticated Fourier analysis methods for component estimation (Chapter 4). Chapter 11 also discusses a new approach to evaluating correlation and peak capacity. 

Open capillaries of Spin i.d. and with 10 theoretical plates are rather easy to prepare. Theory shows that a capillary of i.d. <10 pm scores over a packed column as regards peak capacity, resolution and analysis time. The equipment poses a problem that is not insoluble (injection volume 50 pi, flow rate 2nlmin ). 

Detection Limit, Peak Capacity, Resolution Possibilities for Gradient Optimization... 

The analytical capability of a SEC column is sometimes judged by the peak capacity, which is the number of unique species that can be resolved on any given SEC column. This number will increase with decreased particle size, increased column length, and increased pore volume. Because small particlesized medium generally has a lower pore volume and a shorter column length, peak capacities of ca. 13 for fully resolved peaks can be expected for high-resolution modern media as well as traditional media, (see Eig. 2.5). It was found that SEC columns differ widely in pore volume, which affects the effective peak capacity (Hagel, 1992). 

Table 1.4 Peak capacities in modem high-resolution cliromatography ...

Giddings pointed out (32) that separated compounds must remain resolved throughout the whole process. This situation is illustrated in Figure 1.5, where two secondary columns are coupled to a primary column, and each secondary column is fed a fraction of duration Ar from the eluent from the first column. The peak capacity of the coupled system then depends on the plate number of each individual separation and on At. The primary column eliminates sample components that would otherwise interfere with the resolution of the components of interest in the secondary columns. An efficient primary separation may be wasted, however, if At is greater than the average peak width produced by the primary column, because of the recombination of resolved peaks after transfer into a secondary column. As At increases, the system approaches that of a tandem arrangement, and the resolution gained in one column may be nullified by the elution order in a subsequent column. 

The above theoretical analysis of the total number of resolvable components in a complex mixture has shown that in LC, relative to the maximum peak content or peak capacity for closely spaced peaks, a random chromatogram will never contain more than about 37% of its potential peaks and furthermore that only 18% of such components will emerge as single-component peaks having a minimum specified resolution with respect to the neighbouring peaks. 

A practical method for enhancing the peak capacity, and thus the resolution of analytes in multicomponent complex mixtures, can be achieved by changing the mode of the separation during the chromatographic analysis, employing a column switching system in order to optimize a separation. 

The resolution of these columns for protein mixtures, however, was comparably poor. The peak capacity for human serum albumin was near 3 during 20 min gradient elution. Improvement has been reached by covalent binding of PEI (M = 400-600) onto a 330 A silica of 5 pm particle size [38], The peak capacities of ovalbumin and 2a -arid glycoprotein were 30-40 (tgradienl = 20 min). Enhanced peak capacity and resolution probably were due to the more diffuse structure of PEI coupled to silane moieties than that of strictly adsorbed on silica and cross-linked (see Sect, 2.2). Other applications of covalently adsorbed PEI are discussed in Sect. 4.1. 

It is clear that the chromatogram in figure 8 does not have a peak capacity of 29 although a perfectly satisfactory resolution is obtained. 

Another approach to defining the separation capacity of a column is by its peak capacity (the number of peaks than can be resolved at any specific resolution, usually R, i, in a given separation time). For SEC the pe2dc capacity, PCgc, is given approximately by... 

The separating power of a column can Ise expressed as its peak capacity defined as the number of peaks that can be resolved, at any specified resolution level, in a given separation time. For the general case it can be calculated using equation (1.49)... 

Therefore, a 4a separation (R = 1), in which peak retention times differ by four times the width at half-height, corresponds to a 2% area overlap between peaks.1 The maximum number of peaks that could be separated in a given time period assuming a given value of R, is defined as the peak capacity.1 The peak capacity must be greater — usually much greater — than the number of components in the mixture for a separation to succeed. The resolution of two compounds can also be written in terms of the number of plates of a column, N, the selectivity, a, and the capacity factors, k, and k j, as12... 

It is difficult to compare separation techniques in any general way. Comparison may be based on the traditional figures of merit, such as resolution Rs (including column efficiency N, selectivity, retention, and peak capacity), chromatographic speed, sample capacity, sensitivity, detection and column impedance, as well as breadth of application. Usually a tradeoff between these attributes is found. Berger [26] has compared GC, pSFC, cSFC, LC and CE on the basis... 

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