Gradient retention

Due to instrumental or laboratory variability, the information given by the experimental gradient retention times (tr) is not sufficient to obtain accurate and repeat-able logP values [38]. As described by Eqs. (4), (6) and (7) the lipophilicity measurement in gradient mode is therefore based on the determination of logfeo and S because both terms are necessary to obtain log few. 

In case of fast gradient (below 15 min), S could be considered constant for all the investigated molecules and wiU only have a small influence on the retention time of the compounds. Thus, the gradient retention times, of a calibration set of compounds are linearly related to the ( )o values [39]. Moreover, Valko et al. also demonstrated that the faster the gradient was, the better the correlation between t, and < )o [40]. Once the regression model was established for the calibration standards, Eq. 8 allowed the conversion of gradient retention times to CHI values for any compound in the same gradient system. Results are then suitable for interlaboratory comparison and database construction. The CH I scale (between 0 and 100) can be used as an independent measure of lipophilicity or also easily converted to a log P scale. 

The differential equations (Equation 5.2a or b) can be solved by integration after introducing the actual dependence of k on the time, t (or on the volume of the eluate, V, which has passed through the column) from the start of the gradient until the elution of the band maximum. Freiling [26] and Drake [27] were the first to introduce this approach, which has been used later to derive equations allowing calculations of gradient retention data in various LC modes [2,4-7,28-30]. 

In gradient elution of weak acids or bases, gradients of organic solvent (acetonitrile, methanol, or tetrahydrofuran) in buffered aqueous-organic mobile phases are most frequently used. The solvent affects the retention in similar way as in RPC of nonionic compounds, except for some influence on the dissociation constants, but Equations 5.8 and 5.9 usually are accurate enough for calculations of gradient retention volumes and bandwidths, respectively. 

Both the bandwidths and the differences in the elution volumes (times) of the compounds i and j with relative retention, a=k/ki are approximately constant during a reversed-phase gradient run, except for very early-eluting compounds [4-10]. With some simplification. Equation 5.8 can be adapted to describe approximately the differences in gradient retention volumes, AVg, of all but very early-eluting analytes ... 

Significant number of modern HPLC separation methods are based on gradient elution or continuous variation of the eluent composition during the analytical experiment. Theoretical description of the gradient separation is very complex and, to large extent, nonexistent. It is possible to use basic HPLC theory for phenomenological explanation of gradient retention dependencies. 

The correlation of isiK-ralic vs. gradient retention data of 76 structurally unrelated drug-ltkc (with permission from Ref. 4.t ). 

Krass et al. [45] have also reported a novel method for the determination of lipophilicity using a simple HPLC protocol based on gradient elution chromatography. They compared the gradient retention times with the traditional isocratic logkw param-... 

THE NORMALISED REGRESSION COEFFICIENTS OF THE SOLVATION EQUATIONS SET UP FOR THE GRADIENT RETENTION DATA (CHI) OBTAINED ON SEVERAL STATIONARY PHASES... 

The bandwidths Wg in gradient elution can be determined by introducing the appropriate instantaneous retention factor k at the elution of the peak maximum calculated using an appropriate gradient retention equation (e.g., Eq. 4 or Eq. 6) ... 

If the retention time is much higher than the time of the initial gradient (retention factor 10) the substances seem to be too lipophilic for RP separation on the initial adsorbent. The polarity of the adsorbent should be increased by the use of amino or diol phases. If the adsorbent is already polar the phase system must be changed to NP-chromatography (Section 4.3.2). 

Similarly, in gradient elution HP-RPC, resolution optimization can take advantage of the relationship between the gradient retention time of an analyte (expressed as the median retention factor k) and the median volume fraction of the organic solvent modifier, Ip, in regular HP-RPC systems based on the concepts of the linear solvent strength theory,1 31 such that... 

Calculation of the new gradient retention times tg Based on the knowledge of the S and the In k0 values, new gradient retention times can then be calculated. 

Displacement chromatography appears to be the method of choice if the retention factor (or the gradient retention factor) is larger than 5. Displacement... 

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