Chromatographic processes retention volume

As known, SEC separates molecules and particles according to their hydro-dynamic volume in solution. In an ideal case, the SEC separation is based solely on entropy changes and is not accompanied with any enthalpic processes. In real systems, however, enthalpic interactions among components of the chromatographic system often play a nonnegligible role and affect the corresponding retention volumes (Vr) of samples. This is clearly evident from the elution behavior of small molecules, which depends rather strongly on their chemical nature and on the properties of eluent used. This is the case even for... 

Every chromatographic process is controlled by the equilibrium distribution of the solute between the mobile and stationary phases. The retention volume V describing the volume of mobile phase that is required to elute the analyte from the column, is given by Equation 17.4 ... 

The uncertainty in the measurement of elution time / or elution volume of an unretained tracer is another potential source of error in the evaluation of thermodynamic quantities for the chromatographic process. It can be shown that a small relative error in the determination of r , will give rise to a commensurate relative error in both the retention factor and the related Gibbs free energy. Thus, a 5% error in leads to errors of nearly 5% in both k and AG. An analysis of error propagation showed that if the... 

So far the Plate Theory has been used to determine the equation for the retention volume of a solute, calculate the capacity factor of a solute and identify the dead volume of the column and how it should be calculated. However, the equation for the elution curve of a solute that arises directly from the Plate Theory can do far more than that to explain the characteristics of a chromatogram. The equation will now be used in a variety of ways to expand our knowledge of the chromatographic process. 

Any chromatographic process is associated with the distribution of the analyzed substance between the mobile and the stationary phase. In liquid chromatography the solvent, with the volume V0 in the interparticle space, moving along the column at a certain speed, is the mobile phase, and a porous adsorbent, with an overall pore volume of V, is the stationary phase. The distribution coefficient, Kd, equal to the ratio between the concentration of the substance in the stationary and the mobile phase, determines the retention volume, VR, of a given substance in the column according to the basic chromatographic equation... 

Reproducibility of peak height is also quite dependent on the reproducibility of the sample injection. This is especially important on early and thus normally quite sharp, narrow peaks. On such early peaks the width of the peak is controlled more by the injection time rather than the chromatographic process. A fraction of a second increase in injection time can double the width of these peaks and reduce peak height 50%. The peaks most subject to error in peak height measurement from injection problems are those with retention volumes between one and two times the hold-up volume of the column. Peaks beyond five to ten times the hold-up volume are negligibly affected by injection technique. 

Thin layer chromatography and gas-liquid chromatography have been widely applied for the separation and for the identification of thiazoles in reaction mixtures. From systematic studies it appears that thiazole, alkyl- and aryl-thiazoles and benzothiazoles are best separated on stationary phase of low polarity in GLC and with eluents of low polarity in TLC. It has been possible to correlate, for these series of compounds, the RF of TLC or the specific volume of retention in GLC with the number of carbon atoms in the aliphatic side chain, and also with the rate constants of quaternization of the cyclic nitrogen atom. This last observation indicates a significant participation of the nitrogen atom in the chromatographic processes (67BSF846). 

K. S. Yun, C. Zhu, and J. F. Parcher, Theoretical relationships between the void volume, mobile phase volume, retention volume, adsorption, and Gibbs free energy in chromatographic processes, AnaZ. Chem. 67 (1995), 613-619. 

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... 

As the chromatographic process in CCC is based on the partition of a solute between the mobile and stationary phases, the value is the most important parameter in CCC. A Ad value of around 1.0 is most desirable in CCC, wherein a solute with Ad =1.0 elutes with its retention volume equivalent to the total column capacity. In the above two-phase solvent systems, the Ad values of monomers (catechin and/or epicatechin) were greater than 1.0, and those of the ACTs are always smaller than 1.0, suggesting that monomers are more hydrophobic than their oligomers present in ACTs. Among these four solvent systems, we selected a simple binary system of methyl acetate/water for the separation of procyanidin oligomers from ACTs by CCC. 

Let the distance between the injection point and the peak maximum (the retention distance on the chromatogram) be y cm and the peak width at the points of inflexion be x cm. If the chromatographic data are computer processed, then the equivalent retention times can be used. Then, as the retention volume is n Vyn + KVs) and twice the peak standard deviation at the points of inflexion is 2Vn v + Kv ), then... 

In the preceding section, we briefly discussed the separation mechanisms that are exclusively and directly connected with the dimensions of the separated species. Let us designate these mechanisms primary. In the real gel chromatographic systems, however, several other processes are operative, affecting both the retention volumes and the widths of chromatographic zones. These secondary processes can be classified into ... 

The dependence of chromatographic separation on sample size is similar for all elution or bed development processes. Figure 4-1, serves as a general example, whether we are concerned with gas-liquid, partition, paper, ion exchange, or adsorption chromatography. For small amounts of sample, as in Fig. 4-1 (a), sample retention volumes (or Rf values) are constant for variation in sample size (linear isotherm separation). As the sample size is increased, however, a point is at last reached [Fig. 

The fundamental equation for any chromatographic process, relating the retention volume Fr to other quantities, is... 

Unlike other chromatographic methods the mobile phase acts just as a solvent. Its physical properties do not influence the separation process. Solvent gradients do not alter the retention volume. As all compounds leave the column between Vq and Vo + Vi, no sample is lost on the stationary phase. The mobile phase usually consists of an aqueous buffer with an ionic strength of 50 to 100 mM. Typical flow rates are in the order of 0.1 to 1 mL min. ... 

In order to design the appropriate liquid chromatography separation system, it is necessary to nnderstand on molecular level some basic principles and tendencies of the processes taking place in the chromatographic column. Above processes resnlt in differences in retention of sample constituents to allow their mutual separation. Extent of retention of macromolecules within colutim reflects the volume of mobile phase needed for their elution, their abovementioned retention volume, V. For the sake of simplicity, let us consider constant overall experimental conditions that is the elnent flow rate, temperature and pressure drop. The latter two parameters are dictated not only by the inherent hydrodynamic resistance of colunm that is inflnenced by the eluent viscosity, size and shape of packing particles but also by the sample viscosity, which may be rather high in polymer HPLC. Further, only one variable molecular characteristic of separated macromolecules will be... 

The thermodynamics of the chromatographic process is responsible for the solute retention. The retention volume, Vr, is related to the solute affinity constant for the stationary phase, K, by... 

For given values of the retention volumes above, the shape of chromatographic peaks depends on the value of the kinetic parameter k jF. If the only slow process is equilibration of injected substance vapour in the polymer bulk, the simplest theories of chromatographic rate processes [113] give for 7c, ... 

Fig. 5-32 [76]. The chromatograms in the figure present the results for one acetone pressure. The effect of acetone dopant on peak shape and retention volumes persisted even at the lowest pressures investigated [76]. Therefore, the volatile modifier blocking process can be used to mimic (to simulate) the deactivation and to improve the chromatographic efficiency produced by low loading of non-volatile liquid on solid adsorbents.

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