Isocratic composition

Figure 5.13 Curves relating the isocratic composition (p ) to the net retention time under gradient conditions for various values of the isocratic capacity factor. Curves calculated on the basis of eqns.(3.45) and (3.46). Linear gradient 0 — 100% methanol in water. /0= 125 s. Figure taken from ref. [536]. Reprinted with permission. 

Figure 8.9. HPLC chromatogram of an isocratic composite method for a neutral drug substance.

There is an inherent proportioning error in the mobile phase delivered to the column, both during gradient and final isocratic composition. This error may be as high as 0.5%. However, this error can be reduced to less than 0.15 with hi -predsion HPLC instruments of the latest generatioa... 

Two variations of the technique exists isocratic elution, when the mobile phase composition is kept constant, and gradient elution, when the mobile phase composition is varied during the separation. Isocratic elution is often the method of choice for analysis and in process apphcations when the retention characteristics of the solutes to be separated are similar and not dramaticallv sensitive to vei y small changes in operating conditions. Isocratic elution is also generally practical for systems where the equilibrium isotherm is linear or nearly hnear. In all cases, isocratic elution results in a dilution of the separated produces. 

In most situations the eluent composition is chosen to minimize the effects of hydrophobic interaction, but these secondary effects can be used to advantage. By careful selection of a salt and its concentration, specific selectivities for analytes can be achieved without the use of organic solvents. Therefore, many separations usually run by solvent gradient reversed-phase methods can be completed with a purely aqueous isocratic eluent (13,14). 

A separation involving a mobile phase of constant composition (irrespective of the number of components it contains) is termed isocratic elution, while that in which the composition of the mobile phase is changed is termed gradient elution. In the latter, a mobile phase is chosen which provides adequate separation of the early eluting analytes and a solvent which is known to elute the longer-retained compounds is added over a period of time. The rate at which the composition is changed may be determined by trial and error , or more formal optimization techniques may be used [5-7]. 

Isocratic elution The use of a mobile phase of constant composition during the course of an analysis. 

Chromatography is essentially a method of separation based on two phases, one stationary and one mobile. If the composition of the mobile phase is not changed during the separation, the term isocratic elution is used. For separation of complex mixtures with wide ranges of polarities, the composition of the mobile phase can be changed during separation, a process known as gradient elution. 

When a limited range of organic modifier proportions within the mobile phase is considered, the retention changes in RPLC with mobile phase composition are generally adequately described by the linear Soczewinski-Snyder model (Eq. 4) adapted to both isocratic and gradient mode [5]. 

The practices of isocratic and gradient sorptive chromatography are very different. Isocratic chromatography tends to be very sensitive to the details of mobile phase preparation, temperature, pump speed, and sample composition. Gradient chromatography is usually more tolerant of small variations in these factors but may be extremely sensitive to column history, equilibration time, and gradient preparation. 

The conceptual basis for understanding the connection between isocratic and gradient elution is well established and is called "linear solvent strength theory".22 27 Linear solvent strength theory proposes that, for a given solute, mobile phase, and column, if one measures the retention time of an analyte at two organic component concentrations, it will be possible to predict the retention time with any other mobile phase composition. The k value that would be observed in pure water, kw, is related to the actual k by the relationship... 

Jandera, P. and Churacek, J., Gradient elution in liquid chromatography. I. The influence of the composition of the mobile phase on the capacity ratio (retention volume, band width, and resolution) in isocratic elution — theoretical considerations, /. Chromatogr., 91, 207, 1974. 

Liquid chromatography has a number of different configurations with regard to technical (instrumental) as well as separation modes. The HPLC system can be operated in either isocratic mode, i.e. the same mobile phase composition throughout the chromatographic ran, or by gradient elution (GE), i.e. the mobile phase composition varies with run time. The choice of operation... 

Normal-phase (NP) and reversed-phase (RP) liquid chromatography are simple divisions of the LC techniques based on the relative polarities of the mobile and stationary phases (Figure 4.10). Both NPLC and RPLC analysis make use of either the isocratic or gradient elution modes of separation (i.e. constant or variable composition of the mobile phase, respectively). Selection from these four available separation techniques depends on many variables but basically on the number and chemical structure of the compounds to be separated and on the scope of the analysis. 

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