Relative-retention parameters

Although the retention volume is independent of flow rate, relative retention parameters are preferred because they utilize dimensionless parameters as well as providing additional information about the chromatographic process. The relative chromatographic mobility (Re) in liquid chromatography is defined by 

1 Capacity factor (k J. The fundamental dimensionless measure of retention in liquid chromatography is the capacity ratio (or capacity factor) (k ), which is defined as the ratio of the number of molecules of solute in the stationary phase, N, to the number of molecules in the mobile phase, 

By taking into account the volumes of the mobile phase (Km) and stationary phase (F ) in the column, the capacity ratio can be related to the partition coefficient (A d) of the solute between the mobile phase and stationary phase 

The ratio, Vs/Vm, is often referred to as the phase volume ratio. Combining equations (2.16)-(2.18) allows the development of equations (2.20)-(2.22), which relate the capacity ratio of a solute to its retention time and the elution time, of an unretained compound  

Thus defining retention in terms of fe allows calibration of the time scale of a chromatogram in units of t (equation (2.22)) as well as allowing the retention of the solute to be quantified in terms of the extent of its interaction with the stationary phase (equation (2.19)). 

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It has been shown that gas-Hquid chromatographic methods are particularly suitable for a quantitative characterization of the polarity of solvents. In gas-liquid chromatography it is possible to determine the solvent power of the stationary liquid phase very accurately for a large number of substances [98, 99, 259, 260]. Many groups of substances exhibit a certain dependence of their relative retention parameters on the solvation characteristics of the stationary phase or of the separable components. In determining universal gas-chromatographic characteristics, the so-called retention index, I, introduced by Kovats [100], is frequently used. The elution maxima of individual members of the homologous series of n-alkanes (C H2 +2) form the fixed points of the system of retention indices. The retention index is defined by means of Eq. (7-41),... 

Because errors are inherent in the determination of F, substances are chromatographically characterized by relative retention parameters. There are two classes of such parameters some are defined in terms of the content of a component in the stationary and mobile phase, and others in terms of a standard component existing on the same chromatogram with the component in question. 

Starting up from the linear dependence between the logarithm of retention parameters of the members of a homologous series and the number, n, of carbon atoms in the molecule, at constant column temperature, Kovats [16—18] has defined another relative retention parameter — the retention index, I — which is very often employed in the qualitative characterization of a component. The retention time, displayed on the same cliroma-togram, or net retention volume, of the substance i to be identified lies between those of normal hydrocarbons with G and 0 -f-1 carbon atoms in the molecule. Thus, from the standpoint of retention characteristics, this substance may be regarded as a hypothetical normal hydro-... 

Parameter setup Example 6.5. Relative retention of two solutes... 

A parameter defined as the relative retention, or selectivity, is often reported for a given instrumental chromatography system as a number that ought to be able to be reproduced from instrument to instrument and laboratory to laboratory regardless of slight differences that might exist in the systems (column lengths, temperature, etc.). This parameter compares the retention of one component (1) with another... 

GC-Computer System Nowadays, a large number of data-processing-computer-aided instruments for the automatic calculation of various peak parameters, for instance relative retention, composition, peak areas etc., can be conveniently coupled with GC-systems. A commercially available fairly sophisticated computer system of such type are available abundantly that may be capable of undertaking load upto 100 gas-chromatographs with ample data-storage facilities. In fact, the installation such as multi GC-systems in the routine analysis in oil-refineries and bulk pharmaceutical industries, and chemical based industries have tremendously cut-down their operating cost of analysis to a bare minimum. 

The relative retention a=k/ki is a measure of the separation selectivity for two compounds i and j with retention factors ki and kj, respectively, differing by one repeat structural unit, An=l.p in Equation 5.16 is the end-group contribution to the retention factor. The conventional theory describes adequately the retention of oligomers and lower homopolymers and copolymers up to the molar masses 10,000-30,000Da for higher polymers the accuracy of the determination of retention model parameters is too low [95]. 

To compare different chromatographic systems, however, it is more useful to use the relative retention time (also called the capacity factor, k-). This parameter is defined as the retention of the compound relative to a nonretained chemical species, such as a very polar organic compound or an inorganic species such as nitrate ... 

MS/MS, are often not sufficiently accurate that they can be faithfully relied on [33]. In these cases, a statistical analysis should be introduced that would include more parameters that could assist in protein and peptide identification. For example, the observed and calculated relative retention times from reversed-phase columns could be correlated to provide further validation [34, 35]. 

We have shown that fractions collected from broad particle distributions can be reinjected into the sedimentation FFF device for a second run (18). The emerging peaks are relatively narrow, reflecting their small particle size range. Quite obviously, a reinjected fraction run in a carrier of a different density will emerge at a different volume because of the effect of Ap on retention parameter X (see Equation 3). The shift in retention volume (see next section) can be used to calculate the density of the particulate material exactly as outlined for monodisperse populations. 

For better reproducibility of the retention parameters, relative retention times referenced to a standard chemical have been used in several GC applications. The added reference chemical should be chosen so that it elutes in the middle of the chromatogram. There is always some degree of error in the retention time measurement, and the relative retention times of chemicals having short or long retention times might not be very accurate. To obtain better reproducibility, it is recommended that several different reference chemicals are used for the calculation of relative retention times. However, in this case the use of RIs becomes more attractive. It is a much more reliable to use RIs than absolute or relative retention times because in retention indices (RI) measurements, the retention is measured relative to a homologue series. 

We have seen that the selectivity in chromatography can be related to the relative retention of two solutes. However, this parameter does not describe the actual separation between two chromatographic peaks. There are two factors which determine whether or not two peaks are completely resolved, as is illustrated in figure 1.3. The relevant parameters are the distance between the peaks and their width. The distance can be expressed as the difference in retention times (AtR), while the peak width at the peak base (usually determined by drawing tangent lines along the slopes of the peaks) can be denoted by w. 

Figure 1.5 Influence of (a) the relative retention (o), (b) the (average) capacity factor (k) and (c) the number of theoretical plates (A/) on the resolution (Rv) according to eqn.(1.22). In each case the two other parameters are kept constant, k and N are assumed not to equal zero, and a not to equal one.

By definition, the relative retention is larger than (or equal to) 1. Thus, for a normal phase system, where 8S > 5m, it follows from eqn.(3.31) that 5- > Sp and hence the more polar solute will elute last. Again, the reverse is true for a reversed phase system. Because the signs of the two factors in eqn.(3.31) which involve solubility parameters will always be thesame, we may state that it is the absolute difference between the polarities of the two phases that should be maximized. Therefore, the selectivity of a phase system (V) may be defined as... 

Reduced parameters, 66-69 Refractive index (RI) detector, 206-207 Regular solution, 49 Relative retention, 20-21, 22, 77 Repeatability, see Precision Reproducibility, see Precision Resolution, 17-19, 55 Response factors (detector), 104, 125 Response time, 94 Retardation factor, Rf, 71 Retention index of Kovats, 78 Retention ratio, 11, 12, 71 Retention time, 6, 9 Retention volume, 9, 75 adjusted, 10, 75 corrected, 62-63, 75 net, 63, 75 specific, 110 Reverse phase LC, 158 Rohrschneider/McReynolds constants, 137-140... 

Equations (105) and (106) provide an important linkage between the three essential parameters that dictate the overall quality of the chromatographic resolution, namely, the relative retention, expressed in terms of the capacity factor k the relative selectivity a, and the extent of peak dispersion Nt or he i. Higher system performances and thus larger values of Rs, per unit time... 

Assuming LEER, one can determine the relative inputs of individual structural groups, fragments or features to a property measured for a series of compounds in various chemical, physical, and biological experiments. Such obtained structural parameters (descriptors) can then be related to retention parameters. 

The first exploitation of this relationship in a biological context was by Boyce and Milbarrow [28], who showed a relationship between the molluscicidal activity of some A -alkyltritylamines and their Rm values on TLC plates. Many publications have reported the application of TLC to determine the relative lipophilicity of compounds. The first chapter of the book Chromatographic Determination of Molecular Interactions Applications in Biochemistry, Chemistry, and Biology [29[ summarises the theory and presents the major application fields of the TLC method. The advantage of the method for the determination of lipophilicity is that the layers can be easily covered by octanol and by using an aqueous buffer the retention parameter would be directly proportional to the octanol-water partition coefficients. The drawbacks of this method are the limited reproducibility and precision. 

It is interesting to note that by analogy with chromatographic retention parameters, the values of some other properties of organic compounds may be pre-2 sented in the linear interpolated form relative to the set of reference compounds. These equivalent to indices forms are known for boiling points [6], molecular I weights [7], and molar refractions, MR , = (MW/d)(n -1 l)/( + 2), where MW is the molecular weight, is the... 

As shown by Eq. (1), the resolution of components in a liquid chromatographic separation is dependent on (1) their relative retention on a particular chromatographic system and (2) their peak widths. To optimize these parameters for maximum resolution, a clear understanding of their nature and the factors that affect them is necessary. Although the retention time of a component adequately describes the amount of time a particular solute takes to elute from a chromatographic system, a more useful parameter describ-... 

The identification of steroids in an unknown sample can be based on GC or GC-MS parameters, such as relative retention times, retention indices, steroid number, mass spectra, and/or important ion fragments. 

The decisive parameter for the separation of two components is their relative retention, which is called selectivity a. The selectivity is defined as the ratio of the solute retention times of two different signals ... 

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