Elution, isocratic

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. 

Isocratic Elution In the simplest case, feed with concentration cf is apphed to the column for a time tp followed by the pure carrier fluid. Under trace conditions, for a hnear isotherm with external mass-transfer control, the linear driving force approximation or reaction kinetics (see Table 16-12), solution of Eq. (16-146) gives the following expression for the dimensionless solute concentration at the column outlet ... 

Table 16-14 gives exphcit expressions for chromatographic peak properties in isocratic elution and huear gradient elution for two cases. 

Band broadening is also affected by the gradient steepness. This effect is expressed in Table 16-14 by a band compression factor C, which is a fnuctiou of the gradient steepness and of equilibrium parameters. Since C < 1, gradient elution yields peaks that are sharper than those that would be obtained in isocratic elution at

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The acceptable separation of Am(III) and Cm(III) by countercurrent chromatography (CCC) was achieved using both isocratic elution, and a new approach to the creation of the reagent concentration gradient in the stationary phase [1]. This way allows reduce the experiment length. 

A liquid chromatography-mass spectrometry (LC-MS) method that can quantitatively analyze urinar y normal and modified nucleosides in less than 30 min with a good resolution and sufficient sensitivity has been developed. Nineteen kinds of normal and modified nucleosides were determined in urine samples from 10 healthy persons and 18 breast cancer patients. Compounds were separ ated on a reverse phase Kromasil C18 column (2.1 mm I.D.) by isocratic elution mode using 20 mg/1 ammonium acetate - acetonitrile (97 3 % v/v) at 200 p.l/min. A higher sensitivity was obtained in positive atmospheric pressure chemical ionization mode APCI(-i-). 

For preparative or semipreparative-scale enantiomer separations, the enantiose-lectivity and column saturation capacity are the critical factors determining the throughput of pure enantiomer that can be achieved. The above-described MICSPs are stable, they can be reproducibly synthesized, and they exhibit high selectivities - all of which are attractive features for such applications. However, most MICSPs have only moderate saturation capacities, and isocratic elution leads to excessive peak tailing which precludes many preparative applications. Nevertheless, with the L-PA MICSP described above, mobile phases can be chosen leading to acceptable resolution, saturation capacities and relatively short elution times also in the isocratic mode (Fig. 6-6). 

On the other hand, the HIP value for ribonuclease was practically independent of the length of the polyether ligate. These stationary phases were also employed for the separation of oligophenylalanines containing up to 4 residues by isocratic elution with 0.5 mol/1 phosphate buffer, pH 6.3. The retention increments of the Phe residues did not depend on the ligate length, too, and were 0.82 and 0.89 for the stationary phases composed of PEOs (1500 and 4000, respectively). 

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. 

WANG H, HELLiwELL K and YOU X (2000a) Isocratic elution system for the determination of catechins, caffeine and gallic acid in green tea using HPLC ,Pood Chem, 68 (1), 115-21. 

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. 

FIGURE 11.27 Densitogram of Azulan extract at 410 nm chromatographed on silica (a) isocratic elution with AcOEt + CHCI3 (1 5) (b) stepwise gradient of 10 to 40% AcOEt in CHCI3. (Eor details, see Matysik, G., Soczewiriski, E., and Polak, B., Chromatographia, 39, 497-504, 1994.)... 

The plate number in equation (4.56) corresponds to the value when the effective value of the capacity factor (equal to k when the band is at the column midpoint) is equal to the capacity factor in isocratic elution for the same column. The effective value of the capacity factor, k, is simply 1/1.15b. In most cases k, will be large and equation (4.57) is simplified by equating l/k, to zero. The resolution between two adjacent bands in a gradient program, again analogous to isocratic elution, is e q>ressed by equation (4.58)... 

Figur 4.31 Sequential, isocratic elution using a stepwise reduction in solvent strength to identify a binary solvent of acceptable strength for elution of a five ca x>nent mixture. In this example the column holdup time was 1 min.

Sample components having a wide range of capacity factor values are not conveniently separated by isocratic elution. 

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. 

In a sample containing many different solutes, with isocratic elution it is sometimes impossible to choose a suitable mobile phase that will result in all k values being within the optimum range. If this is the case, the chromatogram may appear as in Fig. 4.3a. 

With isocratic elution and a sample having solutes with a wide range of polarity it is sometimes not possible to achieve the desired resolution in an acceptably short time. It may be possible to improve the chromatogram using gradient elution. A practical example of the development of a gradient is discussed. 

In order to reduce the time requirements for this separation, gradient instead of isocratic elution was used. As revealed in Fig. 17.9, using this approach the total separation time was decreased to about 50 min. 

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