Isocratic analysis

FIGURE 2 A chromatogram showing the calculation for capacity factor (k ) which is equal to t /tg. k is an important parameter defining the retention of the analyte.Typical k values for isocratic analyses are I to 20. 

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. 

Fig. 4. Chromatograms obtained from the analysis of 2 ml of unfiltered Baltic Sea water, by direct injection into two isocratic analysers acidic-neutral (A) basic (B). The glutamic acid peak GLU) represents a concentration of ca. 100 nmole 1". Peak identity ALA = alanine GI,y = glycine GL17 = glutamic acid S i = serine = threonine ASP =...

For isocratic analysis, flow rate has no impact on k or a, since flow has the same effect on % and tg- Both operating pressnre and analysis time are inversely proportional to flow rate. Flow rate also has a significant effect on N, as efficiency is reduced (with H increasing) at higher flow rate dne the higher resistance to mass transfer (the van Deemter C term). 

Use premixed mobile phases for isocratic analysis to reduce mobile phase variations. 

Due to small particle size, the columns packed with micropellicular stationary phases have low permeability (27) and therefore, can not be operated at very high flow rates due to pressure limitations of commercial HPLC instruments. In comparison to porous particles, the surface area of stationary phases per unit column volume is low, and hence, their loading capacity is correspondingly smaller. This is particularly evident in the isocratic analysis of small molecules where the column can be easily overloaded. Therefore, micropellicular sorbents do not appear to offer advantages in the HPLC of small molecules. 

In isocratic analysis, the general motivation is that the larger the supply of a particular kind of sample, the more optimization effort is warranted. In programmed analysis this is not true. In that case, the larger the supply of samples, the larger the urge to look for alternative methods. Therefore, gradient optimization procedures are only relevant if they represent a limited effort. It yet remains to be established just how far the word limited will reach. 

The 0.25 AtG Rule When Is Isocratic Analysis More Appropriate ... 

Most HPLC separations are performed under isocratic conditions in which the same mobile phase is used throughout the elution of the entire sample. Although isocratic analysis is good for simple mixtures, gradient analysis, in which the strength of the mobile phase is increased with time during sample elution, is preferred for more complex samples containing analytes of diverse polarities.1-5... 

Reduce the sample injector volumes <20pL for isocratic analysis... 

The principles of green chemistry by adopting the most efficient and environmentally friendly processes should be practiced whenever possible in the HPLC laboratory. One obvious approach is reduction of solvent consumption by using solvent recycling for isocratic analysis and narrowbore LC columns. Another area is to find ways to reduce sample size and the number of sample preparation steps without sacrificing method performance.2 A case study to illustrate this principle for a tablet assay is shown in Table 11.1. Another example is an environmental analysis of soil/sediment sample3 is shown in Table 7.7. 

Kowalski, J. Simple HPLC analysis for Sudan dyes monitoring Sudan I, II, III, and IV in a single, isocratic analysis. Restek Chromatography Products. Available online. 

Corresponds to 30-40 data points within the integration limits Is valid for isocratic analysis and should... 

The slight hydrophobic character of organic size exclusion bonded phases may be used in a reversed-phase mode to retain proteins [44]. In a study where the concentration of trifluoroacetic acid (TEA) in the isocratic analysis of a peptide mixture on an SEC column was varied, the retention and the peak shapes of the components were affected differentially, yielding good resolution that was not by size [49]. 

Figure 30. Simultaneous isocratic analysis of alkali and alkaline-earth cations with ion-exchange chromatography Column Polyencap WCX eluent 4 mmolA, citric acid ...

Figure 3.72 Isocratic analysis of nonpolariz-able and polarizable inorganic anions on lonPac AS25. Separator column lonPac AS25 column dimensions 250 mm x 4 mm i.d. column temperature 30 °C eluent 37 mmol/L KOH (EG) flow rate 1 mlVmin detection suppressed conductivity injection volume 25 pL ...

Figure 10.203 Isocratic analysis of monofluor-ophosphate in toothpaste. Separator column lonPac ASH eluent 2.5mmol/L NaHC03 + 2 mmol/L Na2C03 flow rate 1 mL/min detection suppressed conductivity injection volume lOpL peaks in the anion standard ...

Macroporous support materials with pore sizes of 200 nm, which have been mentioned above in the discussion of the lonPac AS9-HC, were originally developed for the isocratic analysis of fluoride and short-chain fatty acids together with other inorganic anions. In the past, the reliable determination of fluoride... 

---