Peak capacity value

In general, 2DLC is expected to provide a greater peak capacity (P2o) than single-dimensional LC. The P2d can be calculated theoretically by multiplying the peak capacity values of the first (Pi) and second (P2) LC dimensions (Eq. 1) (Giddings, 1987). 

A comparison of theoretical and practical peak capacity values, summarized in Table 12.2, leads to a conclusion that even the most promising 2DLC setups do not provide for the peak capacity needed to fully resolve a complex proteomic sample. As a result, the eluent entering the MS source typically contains multiple coeluting peptides. 

But Equation (7) defines a maximum theoretical peak capacity. "Practical" peak capacity values, related to the experimental separation of components in a mixture, are lower for several reasons. 

The curves show that the peak capacity increases with the column efficiency, which is much as one would expect, however the major factor that influences peak capacity is clearly the capacity ratio of the last eluted peak. It follows that any aspect of the chromatographic system that might limit the value of (k ) for the last peak will also limit the peak capacity. Davis and Giddings [15] have pointed out that the theoretical peak capacity is an exaggerated value of the true peak capacity. They claim that the individual (k ) values for each solute in a realistic multi-component mixture will have a statistically irregular distribution. As they very adroitly point out, the solutes in a real sample do not array themselves conveniently along the chromatogram four standard deviations apart to provide the maximum peak capacity. 

However, with practical samples the way the (k ) values of the individual components for any given complex solute mixture are distributed is not predictable, and will vary very significantly from mixture to mixture, depending on the nature of the sample. Nevertheless, although the values for the theoretical peak capacity of a column given by equation (26) can be used as a reasonable practical guide for comparing different columns, the theoretical values that are obtained will always be in excess of the peak capacities that are actually realized in practice. 

It is also apparent from Figure 20 that any property of the chromatographic system that places a limit on the maximum value of (k ) must also limit the maximum peak capacity that is attainable. One property of the system that limits the maximum value... 

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) ... 

The dimensionless ratio P/ corresponds to the ratio between the number of visible peaks, under the proposed chromatographic conditions, with the chromatographic column having a peak capacity . Differentiation of equation 5.6 with respect to a gives the maximum possible value of the ratio P/ and shows this to occur at a = 1 then, the maximum ratio P/ can be estimated by the following equation ... 

Thus, an approximate value for the peak capacity will be given by... 

This value appears to be in reasonable agreement with the peak capacity demonstrated by the chromatogram shown in figure 2. It is seen that the micro-reticulated gel gives separations as good as, if not better than, those obtained on silica gel and all the solutes are adequately separated for quantitative assessment. 

Thus, If two identical coluwis with a peak capacity of 25 are coupled in series, then the resultant peak capacity would be about 35, conpared to a value of 625 if the same columns were used in the multidimensional mode. In many Instances formidable technical problems must be solved to take full advantage of the potential of multidimensional systems (section 8.7). 

An automated procedure to measure peak widths for peak capacity measurements has been proposed.35 Since peak width varies through the separation, the peak capacity as conventionally measured depends on the sampling procedure. The integral of reciprocal base peak width vs. retention time provides a peak capacity independent of retention time, but requires an accurate calculation of peak width. Peak overlap complicates automation of calculation. Use of the second derivative in the magnitude-concavity method gives an accurate value of the standard deviation of the peak, from which the base peak width can be calculated. 

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... 

General applications While capillary separation methods produce peak capacities, n, numbered in hundreds, many real-world mixtures (e.g. in the petroleum industry) require values of 104. This can only... 

There has been an attempt to measure the peak capacity in 1DLC and 2DLC by assigning a range of useful retention time between the unretained marker that elutes at ti and some stated value of the retention factor k leading to a zone at tf and plugging in a value for the peak width W. This number is useful but will never be equal to the number... 

As discussed above, in a typical proteomic experiment with a limited number of fractions collected, the practical peak capacity of 2DLC is well below 1000. This resolution is dramatically lower than the values considered in literature. 

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. 

What will happen if we select a short 5 cm 5-pm column We expect that this column will not perform well for the long gradients. Indeed, the best we can do is a peak capacity of about 160 for the 6-h gradient at 0.4mL/min. While this is low, it is only about half of the peak capacity of the 25-cm column. For shorter gradients, the peak capacity barely changes for the 5-cm column it is stiU around 150 for a 2-h gradient. For the 45-min run time used as an example for the other 5- J,m columns, the maximum peak capacity is 135 at a flow rate of just under 1 mL/min. For the 8-min gradient, the 5-cm column starts to become competitive the peak capacity of 96 does not quite reach the value of the 15-cm column, which was 120, but the pressure is much lower. There are two reasons for this. The column is shorter, and the best flow rate is lower (2 mL/min) than for the 15-cm column (4mL/min). We are also far away from the pressure limit this short column can be used for up to about 15 mL/min. 

Figure 2. Comparison of DSC thermograms for native and glutaraldehyde (GA)-crosslinked / -D-glucosidase at pH 5.0 in lOmM NaCl. In order to facilitate comparison of the peaks, the differential heat capacity values plotted for the native enzyme are the actual values multiplied by a fector of 0.571.

Under isocratic development, if the early peaks of the mixture are adequately separated, then the late peaks are often broad, and consequently, at concentrations so low that they are hardly detectable. Conversely, if the late peaks are eluted at a sufficiently low k values to improve detection limits, the early peaks become bunched together and are not resolved. This problem is obviated considerably by employing gradient elution, but if there are a large number of individual solutes present in the sample,then the same problems will arise. These difficulties are caused by the column having a limited peak capacity and it is, therefore, important to determine how to calculate peak capacity and how to control it. From the Plate Theory, the peak width at the base is given by -... 

It is seen that the maximum value of the capacity factor Is inversely proportional to the detector sensitivity or, the minimum detectable concentration. It follows, that the detector sensitivity also sets an ultimate limit on the peak capacity that can be realized from a column. This limit however is fairly high as can be seen from the data given in Table (1) The capacity ratios and peak capacities were calculated for a column having an efficiency of 10,000 theoretical plates, a dead volume of 6.7 ml and a sample concentration of % v/v. 

It is seen from table (1) that by increasing the minimum detectable quantity by an order, from 10-7 g/mi to 10 8 g/ml, the maximum (k ) value is also increased by an order but the peak capacity is only increased by about 0%. Furthermore, this increase in peak capacity is achieved at an increase in retention time from twelve hours to about five days. It follows that, attainment of increased peak capacity by increased detector sensitivity, is extremely costly in time and higher peak capacities are best achieved by the use of columns of intrinsic high efficiency. ... 

The analytical specifications must prescribe the ultimate performance of the total chromatographic system, in appropriate numerical values, to demonstrate the performance that has been achieved. The separation of the critical pair would require a minimum column efficiency and the number of theoretical plated produced by the column should be reported. The second most important requisite is that the analysis should be achieved in the minimum time and thus the analysis time should also be given. The analyst will also want to know the maximum volume of charge that can be placed on the column, the solvent consumption per analysis, the mass sensitivity and finally the total peak capacity of the chromatogram. The analytical specifications can be summarized as follows. 

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