Gradient slope

Separation selectivity in LC depends on various factors. The most important is the choice of the stationary and mobile phases (Chen et al., 2004 Guo et al., 1987). In addition, the separation temperature (Hancock et al., 1994) and gradient slope (Chloupek et al., 1994) have also been shown to have a moderate impact on LC selectivity. 

The theoretical considerations discussed briefly above have been further enlarged and the enhanced calculation of optimal gradient programmes was achieved involving three factors gradient slope, initial eluent composition and gradient curvature. In the case of an ACN organic modifier the retention of an analyte can be described by... 

An unknown mixture can be screened on a set of orthogonal systems as a first step in the method development procedure. The chromatographic and/or electrophoretic system, on which the best separation was achieved, can then be retained for further method optimization. Sequentially, the pH and the organic modifier composition of the mobile phase can be adjusted to improve the separation on the CS. If necessary, also the temperature can be modified, while for gradient methods the gradient slope can be considered. For CE methods, the optimization steps will be different from RP chromatography methods. Other factors will be optimized depending on the type of CE method, e.g., CZE and MEKC. However, for the development of CE methods, we would like to refer to Chapter 4 of this book. 

Aa and Am are the differences between the constants a and m of Equation 5.7 for the compounds differing by An repeat structural units m is the average constant of Equation 5.7 for the sample solutes A is the initial organic solvent concentrations at the start of the gradient B is the gradient slope (steepness parameter). Equation 5.4... 

The throughput of this eight parallel LC/UV/MS system is 3200 compounds per day for a 3.5-min cycle time per injection of eight samples under current optimized conditions. It could be further increased by increasing the gradient slope and flow rate. We have also determined that five compounds in the standard mixture gave a linear response from 0.01 to 0.4 mg/ml.16... 

If the earliest peaks are jammed together at the void volume, we would want to drop the initial percentage of acetonitrile to 20% to allow these early peaks to interact with the column if later peaks are taking too much time to come off, you would change the gradient slope so that we reached 100 acetonitrile faster to push the late peaks off earlier. 

S = overall energy gradient slope from point 1 to point 2 Sw = hydraulic gradient slope from point 1 to point 2 V = velocity of fluid flow downslope... 

Fig. 8.17. Effect of gradient slope on the separation of PTH amino acids. Column, 150 x 0.075 mm i.d. packed with 3.5 pm/80 A Zorbax ODS eluents, (A) 2 mmol/1 ammonium acetate, pH 7.0, (B) 2 mmol/1 ammonium acetate, pH 7.0, 90% acetonitrile gradient elution with (a) 30-80% B and (b) 30-60% B in 5 min flow rate of mobile phase through inlet reservoir, 100 pl/min applied voltage, 20 kV detection, ESI-MS, 0.5 s/spectrum integration time sheath liquid, 0.2 mmol/1 ammonium acetate, pH 7.0, 90% methanol, 3 pl/min injection, electrokinetic, 2 kV, 2 s sample, PTH-asparagine, PTH-glutamine, PTH-threonine, PTH-glycine, PTH-alanine, PTH-tyrosine (in order of elution). (Reproduced from ref. [82] with permission of Elsevier Sciences B. V.).

Figure 3.1 Graphs illustrating (a) the gradients (slopes) = m of lines having m > 0 (positive), m= 0 (parallel to x-axis) and m < 0 (negative) and (b) the relationship of the gradient, m, to tan 9, where 6 is the angle between the line and the horizontal.

FIGURE 7-24. Separation of an 11-component herbicide sample as a function of the initial organic in the mobile phase, (a) Gradient 15-100% acetonitrile/water over 70 min, R = 2.1. (b) Gradient 25-100% acetonitrile/water over 60 min, R = 1.9. (c) Gradient 35-100% acetonitrile/water over 50 min, R = 1.5. Gradient slope and other conditions the same as in Figure 7-23. (Reproduced with permission from reference 3.)... 

Fig. 12. The separation of a mixture of C-apolipoproteins (VLDL) on a Waters fi-Bondapak alkylphenyl column with a mobile phase of 1% triethylammcmium phosphate, pH 3.2, and acetonitrile as the organic modifier using several gradient slopes. The flow rate was 1.5 ml/min. The different proteins were identified by amino acid analysis and pure standards as follows 1, olipoprotein C-I 2-4, apolipoprotein C-111 with 2,1, and 0 mol of sialic acid in the carbohydrate side chains, respectively 5, apolipoprotein C-II. Adapted from Hancock and Sparrow (1981c).

In Figure 8-18, a mixture of acids and bases was analyzed on three types of columns phenyl, polar embedded, and C18 column. Significant differences in selectivity were obtained. The separation could be further optimized by modifying the gradient slope and employing off-line method development tools such as Drylab for further optimization and resolution of the critical pairs. 

Moreover, once a particular column or columns that have provided the best selectivity are chosen, an automated method optimization may be performed. This would include employment of an integrated HPLC method development system such as AMDS/Drylab such that the gradient slope/temperature... 

Figure 8-19. Effect of gradient slope on the chromatographic separation. Symmetry CIS, 4.6 X 100 mm, 3.5 gm.

Figure 8-20. Effect of gradient slope on the separation selectivity of basic and acidic analytes on Waters BEH C18 colnmn, snb-2-(xm particles.

Gradient slope x (Elution time from probe gradient run - Dwell time) = Isocratic % organic composition + 10% organic composition... 

Alternatively, the results from the gradient runs for each sample can be inputted into Drylab, ACD, or Chromsword for further optimization (see Sections 8.5.6.11). For the predicted experimental conditions (i.e., gradient slope, temperature, flow rate), if desired selectivity and resolution can be obtained, an experiment can be run for verification. The peak purity for the main analyte (MS and DAD detection) should be checked in the verification run. If the desired selectivity and/or the target analyte are not spectrally homogeneous, go to Step 6, Figure 8-37. 

For FIPLC methods, the parameters that can be deliberately modihed are mobile-phase composition, pFl of the mobile phase (if applicable), ionic strength of the aqueous portion of the mobile phase, concentration of mobile-phase additive (chaotropic, ion-pairing), gradient slope (if applicable), initial hold time for gradient (if apphcable), flow rate, column (different lots and suppliers),column temperature, injection volume, autosampler temperature (if apphcable), and wavelength. After changing these parameters, it must be assessed whether the system suitability requirements can be met for the particular FIPLC method. Consider the following two examples shown below. 

---