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High Performance Liquid Chromatography - Lough W.J.

Lough W.J. High Performance Liquid Chromatography - Blackie academic, 1977. - 282 p.
Download (direct link): highperoranceliquidchromatographi1977.pdf
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RECYCLE
Figure 12.10 Use of recycling in preparative LC with partial resolution.
12.2.7 Determination of physico-chemical parameters
12.2.7.1 Partition coefficients. Knowledge of partition coefficients, usually for the distribution of a compound between octanol and physiological buffer, is of value in pharmaceutical science because it provides information about the lipophilicity of a drug and from this, some idea of how it might act in the body. When partition coefficients are measured directly, (a) it is necessary to use milligram quantities of very pure compound; (b) the experiments are very time-consuming; (c) the experiments must be conducted with great care, and (d) the experimental procedure must be modified to accommodate compounds with very high or very low partition coefficients. It is much more convenient to estimate the partition coefficient (or log P value) of a compound by using LC. In the past some researchers used liquid-liquid partition LC with octanol coated on a solid support. Such lengths are not necessary as a perfectly adequate correlation .exists between logP and log A:' values for reversed phase LC on octadecylsilyl-silicas that are well end-capped. Within a set of analogues the correlation may be as good as r = 0.99. A set of compounds with a known log/* are chromatographed to create a calibration plot which is then used to estimate log P values from log k' values of sample compounds. Using such methodology, log P values can be determined very quickly with sub-milligram quantities of impure samples irrespective of the studied range of log P. Using fast LC (as mentioned earlier, high flow rates with short columns packed with 3 |am particles) the time taken to determine log P values of a large set of compounds can be reduced even further, as shown in Table 12.2. By carrying out reversed-phase LC over a wide range of pH values it is also possible to use reversed-phase LC to estimate pATa values. However pA^a values may be determined satisfactorily by other means.
12.2.7.2 Intermolecular interactions. LC can be used in two ways to determine intermolecular interactions; (a) by using one compound in the
Table 12.2 Example sample set of 30 compounds with k' values spread through the range 13-40.
Total chromatographic run time (h)
manual injection autosampler injection*
conventional LC (250 x 5 mm, 2.0 ml min'1) 2.31 25.6
fast LC (50 x 5 mm, 4.0 ml min'1) 0.45 4.9
*The next sample must not be injected until k' = 40.
Table 12.3 Chiral resolution of 2-aryl propionic acid non-steroidal anti-inflammatory drugs on the HSA-CSP
Solute Without octanoic acid With octanoic acid a
ak2' a [mM Acid] “k2'
Naproxen 63.50 1.32 2 15.06 1.15
Flurbiprofen * * 4 21.50 1.27
Ibuprofen 61.3 3.51 4 10.12 1.33
Benoxaprofen 59.18 1.16 4 19.90 1.12
Pirprofen 36.32 1.47 0.5 15.41 1.17
Suprofen 34.62 2.51 4 4.90 1.19
Ketoprofen 10.10 1.24 0 - -
Indoprofen 11.4 1.27 0 -
Fenoprofen 26.23 1.60 2 10.76 1.31
“k2' = the capacity factor of the more retained enantiomer
*In the absence of octanoic acid, the peaks for flurbiprofen were extremely ill-defined, eluting with capacity factors greater then 80. Reproduced from Nocter et al, (1991)
mobile phase at a range of concentrations and carrying out LC of the other compound, or (b) immobilising one molecule and carrying out LC of the other. The latter method is more difficult to set up and is more prone to giving misleading results because of the linking groups used in the immobilisation interfering with the interactions. However, for instance, the phase shown earlier (Figure 12.1) retains compounds through ë—˙ interactions and may be used to estimate the charge transfer properties of analytes by LC. Such information is occasionally used along with logP and pKa information in quantitative structure activity relationships to rationalise the pharmacological activity of drugs.
12.2.7.3 In vivo modelling. While the techniques described in section
12.2.7.2 are not frequently used for small molecules, they are increasingly being used, along with some other more sophisticated LC techniques, to study interactions between small molecules and large molecules, in particular to study drug-protein binding. Wainer and Nocter have been prominent in this field and have used an immobilised human serum albumin (HSA) phase to study the interaction of drugs such as benzodiazepines, warfarin and ibuprofen with HSA. Some of these workers’ data is shown in Table 12.3. The k! values of the non-steroidal anti-inflammatory drugs, when the mobile phase modifier is not used, are good indicators of the strength of their binding to HSA.
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