Retention capacity factor

The objective of all chromatographic separation is resolution. This experiment illustrates resolution and the factors that affect it. As discussed in Chapters 1 and 3 resolution cannot occur if the components are not partially retained or slowed down (retarded) by the column. Therefore, before calculating resolution, it is important to use the results of the experiment to calculate the fundamental chromatographic parameters of retention, capacity factor, selectivity, and efficiency. 

The retention (capacity) factor. If t has been determined in order to calculate the adjusted retention time, it is more common to use it to calculate and quote the retention factor (k ) according to the following equation ... 

Metal ions sorption by 2-pyridine carboxaldehyde phe-nylhydrazone supported by chemical binding on a silica surface were confirmed to the Langmuir isotherm. The modified phase was used and applied as a metal-ion extractant for determination of trace amounts of iron, cobalt, nickel, and copper. The relative orders of the Langmuir constants K and the column retention capacity factors K for the four transition metal ions are the same as the natural order of the stabihty constants for their metal chelates Fe(II) < Co(II) < Ni(II) < Cu(II). The structure is given in Scheme 5. 

A solute s capacity factor can be determined from a chromatogram by measuring the column s void time, f, and the solute s retention time, (see Figure 12.7). The mobile phase s average linear velocity, m, is equal to the length of the column, L, divided by the time required to elute a nonretained solute. 

In a chromatographic analysis of low-molecular-weight acids, butyric acid elutes with a retention time of 7.63 min. The column s void time is 0.31 min. Calculate the capacity factor for butyric acid. 

Now that we have defined capacity factor, selectivity, and column efficiency we consider their relationship to chromatographic resolution. Since we are only interested in the resolution between solutes eluting with similar retention times, it is safe to assume that the peak widths for the two solutes are approximately the same. Equation 12.1, therefore, is written as... 

The retention times for solutes A and B are replaced with their respective capacity factors by rearranging equation 12.10... 

Any improvement in resolution obtained by increasing ki generally comes at the expense of a longer analysis time. This is also indicated in Figure 12.11, which shows the relative change in retention time as a function of the new capacity factor. Note that a minimum in the retention time curve occurs when b is equal to 2, and that retention time increases in either direction. Increasing b from 2 to 10, for example, approximately doubles solute B s retention time. 

Adjusting the capacity factor to improve resolution between one pair of solutes may lead to an unacceptably long retention time for other solutes. For example, improving resolution for solutes with short retention times by increasing... 

Generally it was found that resolution R is practically the same for isoeluotropic mixtures methanol and acetonitrile with water. The dependencies were obtained between capacity factors for derivatives of 3-chloro-l,4-naphtoquinone at their retention with methanol and acetonitrile. Previous prediction of RP-HPLC behaviour of the compounds was made by ChromDream softwai e. Some complications ai e observed at weak acetonitrile eluent with 40 % w content when for some substances the existence of peak bifurcation. 

Analytical information taken from a chromatogram has almost exclusively involved either retention data (retention times, capacity factors, etc.) for peak identification or peak heights and peak areas for quantitative assessment. The width of the peak has been rarely used for analytical purposes, except occasionally to obtain approximate values for peak areas. Nevertheless, as seen from the Rate Theory, the peak width is inversely proportional to the solute diffusivity which, in turn, is a function of the solute molecular weight. It follows that for high molecular weight materials, particularly those that cannot be volatalized in the ionization source of a mass spectrometer, peak width measurement offers an approximate source of molecular weight data for very intractable solutes. 

As described above, resolution can be improved by variations in plate number, selectivity or capacity factor. However, when considering the separation of a mixture which contains several components of different retention rates, the adjustment of the capacity factors has a limited influence on resolution. The retention times for the last eluted peaks can be excessive, and in some cases strongly retained sample components would not be eluted at all. 

The time taken for an analyte to elute from a chromatographic column with a particular mobile phase is termed its retention time, fan- Since this will vary with column length and mobile phase flow rate, it is more useful to use the capacity factor, k. This relates the retention time of an analyte to the time taken by an unretained compound, i.e. one which passes through the column without interacting with the stationary phase, to elute from the column under identical conditions (to). This is represented mathematically by the following equation ... 

Often, the retention time is used but, as discussed above in Section 2.3, this absolute parameter changes with column length and flow rate and this precludes the use of reference data obtained in other laboratories. To make use of these reference data, the capacity factor (k ), which removes such variability, must be employed. 

A general approach to the problem of identification, should more definitive detectors not be available, is to change the chromatographic system , which in the case of HPLC is usually the mobile phase, and redetermine the retention parameter. The change obtained is often more characteristic of a single analyte than is the capacity factor with either of the mobile phases. 

Capacity factor The parameter used in HPLC to measure the retention of an analyte. 

Temperature has an influence on the retention and consequently on the capacity factors of carotenoids in HPLC columns. Usually, as the column temperature increases, the retention decreases however, in a polymeric C30 column, after an initial decrease of the t values of cis isomers of carotenoids, the retention of cis isomers actually increases at temperatures above 35°C. This different behavior can be explained by the increased order and rigidity of the C30 stationary phase at lower temperatures that in turn induce preferential retention of long, narrow solutes as the trans isomer and partial exclusion of bent and bulky cis isomers. The greater chain mobihty and less rigid conformation of the C30 at higher temperatures may increase the contact area available for interaction with the cis isomers and also may lower... 

Having chosen the test mixture and mobile diase composition, the chromatogram is run, usually at a fairly fast chart speed to reduce errors associated with the measurement of peak widths, etc.. Figure 4.10. The parameters calculated from the chromatogram are the retention volume and capacity factor of each component, the plate count for the unretained peak and at least one of the retained peaks, the peak asymmetry factor for each component, and the separation factor for at least one pair of solutes. The pressure drop for the column at the optimum test flow rate should also be noted. This data is then used to determine two types of performance criteria. These are kinetic parameters, which indicate how well the column is physically packed, and thermodynamic parameters, which indicate whether the column packing material meets the manufacturer s specifications. Examples of such thermodynamic parameters are whether the percentage oi bonded... 

Retention in HIC can be described in terms of the solvophobic theory, in which the change in free energy on protein binding to the stationary phase with the salt concentration in the mobile phase is determined mainly by the contact surface area between the protein and stationary phase and the nature of the salt as measured by its propensity to increase the surface tension of aqueous solutions [331,333-338]. In simple terms the solvopbobic theory predicts that the log u ithn of the capacity factor should be linearly dependent on the surface tension of the mobile phase, which in turn, is a llne2u function of the salt concentration. At sufficiently high salt concentration the electrostatic contribution to retention can be considered constant, and in the absence of specific salt-protein interactions, log k should depend linearly on salt concentration as described by equation (4.21)... 

Figure 8.25 Schematic arrangement ot a two-column system for separation of a sample comprised of co xinents spanning a wide rage of capacity factors (A) and heartcutting of a group of analytes of similar retention to an analytical column for separation (B).

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