Versus gradient elution

Hodges, R. S., Burke, T. W. L., and Mant, C. T. (1988). Preparative purification of peptides by reversed phase chromatography—sample displacement versus gradient elution modes. ]. Chromatogr. 444, 349-362. 

Isocratic versus Gradient Elution. The principles of chromatography just described hold true for isocratic elution schemes, in which the nature of the mobile phase remains constant throughout the de-... 

Fig-1 Fractional migration X L) of solute bands along the column and resulting chromatograms for isocratic versus gradient elution. (Reprinted with permission from L. R. Snyder, M. A. Stadalius, and M. A. Quarry, Analytical Chemistry,... 

Considerations in the Choice of Isocratic versus Gradient Elution... 

Favaro and Fiorani [34] used an electrode, prepared by doping conductive C cement with 5% cobalt phthalocyanine, in LC systems to detect the pharmaceutical thiols, captopril, thiopronine, and penicillamine. FIA determinations were performed with pH 2 phosphate buffer as the carrier stream (1 mL/min), an injection volume of 20 pL, and an applied potential of 0.6 V versus Ag/AgCl (stainless steel counter electrode). Calibration curves were developed for 5-100 pM of each analyte, and the dynamic linear range was up to approximately 20 pM. The detection limits were 76, 73, and 88 nM for captopril, thiopronine, and penicillamine, respectively. LC determinations were performed using a 5-pm Bio-Sil C18 HL 90-5S column (15 cm x 4.6 mm i.d.) with 1 mM sodium 1-octanesulfonate in 0.01 M phosphate buffer/acetonitrile as the mobile phase (1 mL/min) and gradient elution from 9 1 (held for 5 min) to 7 3 (held for 10 min) in 5 min. The working electrode was maintained at 0.6 V versus Ag/AgCl, and the injection volume was 20 pL. For thiopronine, penicillamine, and captopril, the retention times were 3.1, 5.0, and 11.3 min, and the detection limits were 0.71, 1.0, and 2.5 pM, respectively. 

Since other secondary interactive effects, e.g. electrostatic effects, can also contribute with a particular type of RPC sorbent to the retention of peptides as the mobile-phase composition is changed, the shape of these In k versus ip plots then over the range 0.0 < ip < 1.0 takes on the characteristic U-shaped dependencies.112,211 As a consequence of these dependencies of In k on ip, for mixtures of peptides elution usually requires the use of gradient elution conditions, even when closely related or homologous peptides are being separated. 

Two analytical methods for priority pollutants specified by the USEPA (38) use HPLC separation and fluorescence or electrochemical detection. Method 605, 40 CFR Part 136, determines benzidine and 3,3-dichlorobenzidine by amperometric detection at +0.80 V, versus a silver/silver chloride reference electrode, at a glassy carbon electrode. Separation is achieved with a 1 1 (v/v) mixture of acetonitrile and a pH 4.7 acetate buffer (1 M) under isocratic conditions on an ethyl-bonded reversed-phase column. Lower limits of detection are reported to be 0.05 /xg/L for benzidine and 0.1 /xg/L for 3,3-dichlorobenzidine. Method 610, 40 CFR Part 136, determines 16 PAHs by either GC or HPLC. The HPLC method is required when all 16 PAHs need to be individually determined. The GC method, which uses a packed column, cannot adequately individually resolve all 16 PAHs. The method specifies gradient elution of the PAHs from a reversed-phase analytical column and fluorescence detection with an excitation wavelength of 280 nm and an emission wavelength of 389 nm for all but three PAHs naphthalene, acenaphthylene, and acenaphthene. As a result of weak fluorescence, these three PAHs are detected with greater sensitivity by UV-absorption detection at 254 nm. Thus, the method requires that fluores-... 

When performing HPLC (see Basic Protocol 2) the time of analysis will depend on the conditions used—i.e., isocratic versus gradient. Isocratic separation of the individual betacyanins, as shown in Figure F3.1.2, requires 20 min, compared to 9 min using a gradient elution system. 

Gradient-elution versus other HPLC programming techniques... 

Gradient elution versus isocratic elution — effects of the gradient profile on separation... 

Abbott et al. [4] devised a method designed to predict the retention times in gradient elution under the assumption that the retention factor as determined under isocratic conditions is a log-linear function of solvent composition according to Eq. (5), where k is the retention factor obtained in water, ipo refers to the volume fraction of the organic component, and S refers to the solvent strength for which the values can be obtained as the negative slope of plots of log k versus volume fraction ... 

A mapping of the dependence of analyte retention (expressed as the natural logarithm of the retention factor, k) on the mobile phase composition (expressed as the volume fraction of solvent in the mobile phase, (p) in isocratic elution (or as k versus ip in gradient elution) with a minimum of two initial experiments can be used to define the useful range of mobile phase conditions, and can indicate the mobile phase composition at which the band spacing is optimal (see Figure 10). 

Figure 5.8 illustrates various elution modes based on different compositions of the mobile phase. In the diagrams, A represents the composition of the eluent while B indicates the modifier concentration versus time. The simplest gradient elution is the integration of a washing step (Figure 5.8b). It is mostly used if the component of interest can be adsorbed onto the stationary phase under noneluting conditions. After washing off all the impurities, the target component is eluted at increased modifier concentration.

Flo. 24. Peak capacity versus gradient time and sample molecular weight (JF 6). Calculations of model of Table for teveise(H>hase gradient elution of peptides. Conditions as in... 

An example of a separation where both sorption and precipitation-redissolution can be seen for the same sample is shown in Fig. 43 the separation of a SO,000-Da polystyrene sample by gradient elution (water/ tetrahydrofuran gradient, Cig column). The heavy, dashed curve and solid data points represent the solubility curve for this sample, showing the saturation-concentration (Cnn, x axis) plotted versus mobile-phase composition 0. The light curve and open dides are retention data, plotted as mobUo-phase composition at elation (4> versus sample mass (equivalent to... 

Fig. 4. Plots of log Ic versus based on gradient experiments for /3-endorphin-related peptides 7, 8, 10, 11,14-15. The plots were derived from best-fit analysis to the data points obtained from gradient elution experiments, where tQ = 20, 30, 40, 60 and 120 min and / = ml/min. Column, developmental octadecylsilica, dp= 6/im, Pd = 13 nm, 25 cm x 4.6 mm ID. Solvent A, 0.1 % trifluoroacetic add (TFA) in water solvent B, 0.1% TFA in water-acetonitrile (50 50). See Table 2 for the code to polypeptide structure and for the calculated slope parameter S and log k values. Note the changes in band spacing for peptides 7, 8, 10 and 11 which illustrate the potential for selectivity manipulation through changes in the gradient steepness parameter, b. From [4].

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