EXPERIMENT 10 GRADIENT ELUTION

The approach of Jandera and Chura ek allows the optimization of the resolution of one given (arbitrary) pair of sample components and the minimization of the retention volume of another (arbitrary) solute. It requires knowledge of the isocratic retention vs. composition relationships of these three solutes. The information needed may be acquired from gradient elution experiments performed as part of the optimization procedure, or from separate isocratic experiments. The selection of the three arbitrary solutes considered during the optimization process appears to have a large effect on the result and the resolution cannot be optimized throughout the chromatogram. 

Cj(cp) can be obtained e3q)erimentally from isocratic elution experiments at different Cp values, or from linear gradient elution experiments where the ratio G = pL/v is varied. In the latter case, the retention factor is obtained by differentiation of Eq. (16-190) from... 

Because of the very high polarity of water, even trace amounts of moisture in the mobile phase can affect significantly the retention of sample compounds in NPC. Hence, it is very important to control the water content in the chromatographic system. As the water content usually differs in various organic. solvents u.sed as the components of the gradient, the easiest way to control this is to use carefully dried solvents. Finally, it is very important to control the temperature of the column to get reproducible results in repeated gradient-elution experiments, especially in NPC. 

Examples Include binary, ternary, quaternary, linear and multiple-linear solvent gradients as well as for cases Involving stationary phase programming (coupled column gradient elution experiments). 

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].

A major disadvantage of gradient elution in terms of fast analysis remains the time to adequately equilibrate the chromatographic column between two experiments. However, Carr et al. recently demonstrated an excellent repeatability ( 0.002 min in retention time) obtained with two column volumes of re-equilibration instead of the usual 10 column volumes when a small amount of ancillary solvent (1-3% of 1-butanol or 1-propanol) is added to the mobile phase [45, 58]. 

The gradient elution method for HPLC is the method in which the mobile phase composition is changed in some preprogrammed way in the middle of the run. The device that accomplishes this is called the gradient programmer and is placed between the mobile phase reservoir and the pump. It is useful in experiments in which altering the mobile phase composition assists with the resolution of the mixture. 

In isocratic elution, a capacity factor k 5 provides separation from the solvent front and does not require excessive time. For gradient elution, k = 5 is a reasonable starting condition. Let s calculate a sensible gradient time for the experiment in Figure 25-31a, in which we chose a gradient from 10% to 90% B (A = 0.8) in a 0.46 X 25 cm column eluted at 1.0 mL/min. From Equation 25-4, Vm Ld2/2 = (25 cm) (0.46 cm)2/2 = 2.65 mL. We calculate the required gradient time by rearranging Equation 25-6 ... 

This study evaluated the impurity profile of untreated water from a textile plant in Portugal [35]. The organic material was concentrated by extraction from 11 of water into dichloromethane and HPLC-NMR and HPLC-MS experiments were carried out using a reverse-phase separation with an acetonitrile/ D2O gradient elution with H NMR spectroscopic observation at 600 MHz. For the HPLC-NMR studies, the samples were further fractionated into two pools according to their HPLC retention times. The HPLC-NMR studies were carried out in the stop-flow mode and the combination of NMR and MS results yielded the identification or tentative identification of 14 compounds, comprising mainly surfactants, anthraquinone dyes and nonylphenol-related molecules. 

Figure 5.2.3 depicts the HPLC chromatogram of a tomato peel extract monitored by UV absorbance at 469 nm. The separation was performed on a 150 x 4.6 mm C30 column (ProntoSil, 3 xm, 200 A, Bischoff, Germany) at room temperature and a flow rate of 1 ml/min with a binary mixture of acetone/ water, developed for LC-NMR experiments. The 50-min gradient elution was performed in four steps, i.e. (1) an initial 3 min with 75/25 (v/v) acetone/water, (2) a 24-min gradient to 100% acetone, (3) an isocratic step from 27 45 min with 100% acetone, and (4) a 2-min gradient back to the initial conditions. 

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