Peak capacity defined

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)... 

It has been argued that in a typical 2DLC proteomic experiment, with only a limited number of fractions submitted for analysis in the second LC dimension, chromatographic peak capacity is less than 1000. This value is considerably lower than the expected sample complexity. Additional resolution is offered by MS, which represents another separation dimension. With the peak capacity defined as the number of MS/MS scans (peptide identifications) accomplished within the LC analysis time, the MS-derived peak capacity was estimated to be in an order of tens of thousands. While the MS peak capacity is virtually independent of LC separation performance, the complexity of the sample entering the MS instrument still defines the quality of MS/MS data acquisition. The primary goal of 2DLC separation is to reduce the complexity of the sample (and concentrate it, if possible) to a level acceptable for MS/MS analysis. What is the acceptable level of complexity to maintain the reliability and the repeatability of DDA experiments remains to be seen. 

In the separation of complex mixtures the total number of observed peaks is as important or more so than the resolution of specific peak pairs. Many of these peaks may not be singlets (see section 1.6.4), but systems that separate the mixture into the largest number of observable peaks are desirable unless only a few components are of interest. The separating power of a column can be characterized by its peak capacity, defined as the maximum number of peaks that can be separated with a specified resolution in a given time interval. Figure 1.16. For the general case it can be calculated using Eq. (1.48)... 

The peak capacity of a column has been defined as the number of peaks that can be fitted into a chromatogram between the dead point and the last peak, with each peak being separated from its neighbor by 4a. The last peak of chromatogram is rather a... 

One final quality parameter that is sometimes specified in test methods is the peak capacity of the column set. In the literature of the OECD, this peak capacity is required to have a value of 6.0 or greater. This peak capacity is defined by Eq. (2) ... 

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... 

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... 

The loss is comparable to that found in 1D gas chromatography of complex mixtures, in which only 2 times or so peak capacity (as defined for maxima) is available than is... 

In their seminal work from 1983, Davis and Giddings used a statistical theory to define the number of peaks observable in 1DLC separation upon the injection of a sample of different complexity on a column of a given peak capacity (Davis and Giddings, 1983). The theory was later extended into 2D separation space (Davis, 2005 Shi and Davis, 1993), also discussed in Chapter 2 of this book. The theory implies that when the 1D or 2D separation space is randomly covered with the number of peaks equal to the separation space peak capacity (area), the normalized surface coverage is... 

While chromatographic peak capacity is not adequate to resolve hundreds of thousands of components, many researchers argue that MS itself is an additional separation dimension with an orthogonal selectivity (separation is based on mass-to-charge ratio). Therefore, the combined resolution of LC and MS is greater than the chromatographically defined peak capacity. The question therefore stands What is the achievable peak capacity of the 2DLC-MS/MS system ... 

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... 

In order to talk about fast gradient separations, we need to review the principles involved in measuring the performance of a gradient separation. We can use the concept of peak capacity to measure the separation power of a particular gradient on a given column. The peak capacity (P) is defined as follows ... 

A convenient measure of the performance of a chromatographic system for the separation of complex samples is the peak capacity P, which is defined as the upper limit of the number of fully... 

The overall separation potential of an electromigration technique can be expressed by the peak capacity ( ), which is defined as the maximum number of peaks that can be separated within a given separation time, usually coincident with the time interval between the first and last detected peak in the electropherogram, while retaining unit resolution for all adjacent peak pairs ... 

Figure 5.11. Peak capacity is defined as the number ne of peaks or zones that can be separated (at a specified Rs) over path length L (or elution volume range Vmmx -Knin) provided by a separation system.

From the view point of the assessment, the quality of an HPLC separation in response to changes in different system variables, such as the stationary phase particle diameter, the column configuration, the flow rate, or mobile phase composition, or alternatively, changes in a solute variable such as the molecular size, net charge, charge anisotropy, or hydrophobic cluster distribution of a protein, can be based on evaluation of the system peak capacity (PC) in the analytical modes of HPLC separations and the system productivity (Peff) parameters in terms of bioactive mass recovered throughput per unit time at a specified purity level and operational cost structure. The system peak capacity PC depends on the relative selectivity and the bandwidth, and can be defined as... 

Since there is no selectivity (a) involved in GPC, the ability to separate a pair of compounds depends upon the calibration curve and the efficiency of the column(s). One measure of the ability of the GPC column(s) to separate is the peak capacity of the column, which is defined as the number of resolvable peaks per chromatogram, n. The peak capacity (16) for a GPC column used in the analysis of small molecules is related to the number of theoretical plates (N) according to the equation... 

Ideally, components that are not separated in the first separation step are resolved in the second. Peak capacity is the number of individual components that can be resolved by a separation method. A mathematical model shows that if the MD separations are orthogonal, then the total peak capacity is the product of the individual peak capacities of each dimension [14], Load capacity is defined as the maximum amount of material that can be run in a separation while maintaining chromatographic resolution. MD separations can be designed to significantly increase the load capacity in a first dimension to achieve enrichment of low-abundance or trace components in a peptide mixture, while the necessary peak capacity may be obtained in the second separation dimension [15]. 

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