Retention in HPLC

Retention in HPLC is sometimes measured in volume units (mL), rather than in time units (s or m). Thus, the retention volume Vr is the total volume of mobile phase required to elute the center of a given bandX, i.e., the total solvent flow in time between sample injection and... 

Since reversed-phase HPLC is the most commonly used technique in HPLC, it would be a good idea to develop a better understanding of separation mechanisms. The reader would benefit from the basic review on physicochemical basis of retention in HPLC (see Chapter 2, reference 5). 

Discussed below is the impact of various other physicochemical parameters excluding solubility (see Section 15.8.4) on retention in HPLC. 

Retention in HPLC depends on the strength of the solute s interaction with both the mobile and stationary phases as opposed to GC, where the mobile phase does not contribute to the selectivity. An intelligent selection of the type of stationary phase for the separation is made and... 

The factors which influence the reproducibility of retention in HPLC have been studied [55]. The conclusion is that the relative method of re-... 

To derive the relationship of the analyte retention with the thermodynamic properties of chromatographic system, the mechanism of the analyte behavior in the column should be determined. The mechanism and the theoretical description of the analyte retention in HPLC has been the subject of many publications, and different research groups are still in disagreement on what is the most reahstic retention mechanism and what is the best theory to describe the analyte retention and if possible predict its behavior [8,9]. 

Instrumental methods in chemistry have dramatically increased the availability of measurable properties. Any molecule can be characterized by many different kinds of data. Examples are provided by Physical measures, e.g. melting point, boiling point, dipole moment, refractive index structural data, e.g. bond lengths, bond angles, van der Waals radii thermodynamic data, e.g. heat of formation, heat of vaporization, ioniziation energy, standard entropy chemical properties, e.g. pK, lipophilicity (log P), proton affinity, relative rate measurements chromatographic data, e.g. retention in HPLC, GLC, TLC spectroscopic data, e.g. UV, IR, NMR, ESCA. 

Y.L. Xie, J.J. Baeza Baeza, J.R. Torres Lapasio, M.C. Garcia Alvarez-Coque and G. Ramis Ramos, Modeling and Prediction of Retention in HPLC by using Neural Networks, Chromatographia, 41 435 (1995). 

Gas Chromatography (gc). A principal advantage of gas chromatography has been the faciUty with which it can be combined with mass spectrometry for amino acid identification and confirmation of purity. The gc-mass spectrometry combination offers the advantage of obtaining stmctural information rather than the identification by retention time in hplc. 

With eluent MeOH H O = 40 60% w (flow rate -1 ml/min) full sepai ation was achieved within acetylated and non-acetylated on amino group of eight thioesters of 4-aminobenzenthiosulfinic acid with retention in next order for R - = -CH3, -C,H3,-CH,-CH=CH, -C3H3by RP-HPLC on Spherisorb-ODS-2 (250x4,6 mm). The chromatograms were obtained at 254 and 289 nm. Retention was generalized by In = In - S-(p (cp - MeOH volume pai t in range near 0.4-0.6) as shown in Fig. 

Obtained P values for the description of benzodiazepine s retention in reversed-phase HPLC are used. The equation... 

RRT - Relative retention time for elution in HPLC TGA - Thermogravimetric analysis... 

Unlike gas chromatography, in which the mobile phase, i.e. the carrier gas, plays no part in the separation mechanism, in HPLC it is the relative interaction of an analyte with both the mobile and stationary phases that determines its retention characteristics. Hence, it is the varying degrees of interaction of different analytes with the mobile and stationary phases which determines whether or not they will be separated by a particular HPLC system. 

A number of different retention mechanisms operate in HPLC and interested readers may find further details elsewhere [2-4]. It is sufficient to say here that the interaction may be considered in terms of the relative polarities of the species involved. As indicated in Section 2.1 above, there are two extremes of interaction, neither of which is desirable if separation is to be achieved. 

Buffers are used in HPLC to control the degree of ionization of the analyte and thus the tailing of responses and the reproducibility of retention. A range of buffers is available but those most widely used are inorganic, and thus involatile, materials, such as potassium or sodium phosphate. 

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... 

Selection of columns and mobile phases is determined after consideration of the chemistry of the analytes. In HPLC, the mobile phase is a liquid, while the stationary phase can be a solid or a liquid immobilised on a solid. A stationary phase may have chemical functional groups or compounds physically or chemically bonded to its surface. Resolution and efficiency of HPLC are closely associated with the active surface area of the materials used as stationary phase. Generally, the efficiency of a column increases with decreasing particle size, but back-pressure and mobile phase viscosity increase simultaneously. Selection of the stationary phase material is generally not difficult when the retention mechanism of the intended separation is understood. The fundamental behaviour of stationary phase materials is related to their solubility-interaction... 

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