Isocratic elution disadvantages

If a more accurate prediction of the isocratic elution behaviour is required, then the use of two [529] or more [531,532] different gradients may provide a possibility. The disadvantage of this method, besides the need to perform additional experiments, is that instrumental factors can give rise to quite considerable errors [529,530], so that extreme precautions may be required. 

The packed column suppressor, originally introduced by Small et al., suffers from a number of drawbacks, such as time shifts due to Donnan exclusion effects, band broadening (due to a large dead volume and high dispersion), and oxidation of nitrite, which is easily oxidized to nityrate, due to the formation of nitrous acid in the suppressor. Because of these limitations, they were only practical for isocratic elution. However, the main disadvantage of the method is the necessity for periodical regeneration of the suppressor (also called stripper) to restore its ion-exchange capacity. 

More recently, a less sensitive detection technique, refractive index (RI) detection, has been used. The disadvantage of RI is that one is limited to isocratic elution, because RI detection is affected by changes in the pressure and temperature of the mobile phase. On the other hand, infrared (IR) detection can be used when the solvents in the mobile phase do not absorb infrared light, which can create interference. Because of these limitations, RI and IR have not been used as widely as UV detection. 

Because many fat molecules (for example, fatty acids) do not contain a chromophore, detection by UV—Vis cannot be used. In these cases, a derivatization step is necessary to allow their detection. Other usual LC detection methods include refractive index (RI) and evaporative light scattering (ELS). RI is affected by changes in the pressure and temperature of the mobile phases, so that only isocratic elution can be performed, limiting the separative possibilities. ELS has proven useful for the NPLC analysis of lipids for quantitative application, where information on molecular diversity within a lipid class is not desired. The main disadvantage of both detection methods is that sensitivity is not satisfactory [13]. 

Traditionally, most pharmaceutical assays are isocratic analysis employing the same mobile phase throughout the elution of the sample. Isocratic analyses are particnlarly common in quality control applications since they nse simpler HPLC eqnipment and premixed mobile phases. Notable disadvantages of isocratic analysis are limited peak capacity (the maximnm nnmber of peaks that can be accommodated in the chromatogram), and problems with samples containing analytes of diverse polarities. Also, late eluters (such as dimers) are particularly difficult to quantitate in isocratic analysis due to excessive band broadening with long retention times. 

In isocratic development, a solvent of constant composition is used to effect separation for example, 40% ethyl acetate m hexane. This may be extended to continuous development, in which the TLC plate is left in an isocratic system after the solvent front has reached the top of the TLC plate. This has the advantage that closely eluting bands may be resolved through the use of a nonpolar solvent over several hours. A major disadvantage of this technique is that unstable compounds may degenerate on the adsorbent during this lengthy time penod. 

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