Lipophilicity reversed-phase HPLC

This highly lipophilic group is cleaved with isoamyl nitrite in Pyr/AcOH. The use of a lipophilic 5 -phosphate protective group aids in reverse-phase HPLC purification of oligonucleotides. 

Yamagami, C. Recent advances in reversed-phase-HPLC techniques to determine lipophilicity. In Pharmacokinetic Optimization in Drug Research. Biological, Physicochemical, and Computational Strategies, Testa, B., Van de Waterbeemd, H., Folkers, G., Guy, R. H. (eds.), VHCA, Zurich, 2001, pp. 383 00. 

Compounds that were included in the pharmacologic profile of [ H]MDA binding were subjected to reverse-phase HPLC analysis to assess their relative lipophilicity. Briefly, each compound (10 pg) was injected onto a Waters Nova-Pak C18 column and eluted with a linear gradient from 95 percent buffer A 5 percent buffer B to 20 percent buffer A 80 percent buffer B (buffer A=95 percent water, 5 percent acetonitrile, 0.1 percent ammonium acetate buffer B=20 percent water, 80 percent acetonitrile,... 

In reversed-phase HPLC separation of polyphenolics on the basis of polarity, the elution order of polyphenolics may be predicted. The more-polar polyphenolics are generally eluted first under reversed-phase conditions. Glycosylation in flavonoids increases their polarity and therefore their mobility in the re-versed-phase system. The elution order of benzoic acids, hydroxycinnamic acids, and agly-cones of flavonoids can normally be determined on the basis of the number of polar hydroxyl groups and lipophilic methoxyl groups. For additional information about elution order for various classes of polyphenolics, see Background Information. 

Normal phase (NP) separations are comparatively rarely used in environmental analysis. Again, the reasons lie in the range of analytes amenable to this mode of separation, and in the limited compatibility of typical normal phase HPLC (NP-HPLC) mobile phases with mass spectrometric detection (this also applies to IC). Not only for this reason has interest recently grown in hydrophilic-lipophilic interaction chromatography (HILIC), which represents a viable alternative to the separation of very polar compounds with mobile phases that have a much better compatibility with MS detection, for example, acetonitrile/water with a low water content, typically below 10%, 32 Nonetheless, NP chromato-graphy retains its important role in sample preparation, particularly for the cleanup of complex environmental samples. In the off-line approach, fractions are collected and the relevant one is injected into the reversed phase HPLC (RP-HPLC) system, often after solvent exchange. 

The lipophilicities of ( )-3-benzylidenethiochroman 4-one 251 and its 1-oxide, 1,1-dioxide, and the 2-phenyl derivative have been determined by reverse phase HPLC and a good linear correlation with calculated values is observed. The stronger polarizability of the sulfinyl and sulfonyl compounds results in decreased retention time, but the thioflavanone has the greatest lipophilicity <2005JCH(819)283>. 

As regards the lipids, two types of adsorbents are available, one of which is a form of silica gel and is utilized in normal-phase HPLC, and the other of which can be a silica gel bonded to a hydrophobic chain and is employed in reverse-phase HPLC. In normal-phase HPLC the phospholipids appear to be separated based on the molecular classes present (PE, PC, Sph, etc.), whereas in reverse-phase HPLC the separation is closely related to the lipophilic character of the acyl (fatty acyl, hydrocarbon chain) of the particular phospholipids. High-quality adsorbents suitable for HPLC are easily available from commercial companies. 

This is needed because of the mode of separation, with reversed-phase HPLC being based on a partitioning process. As the lipophilicity of the side-chain at the C5 position increases, so the barbiturate compound will partition preferentially into the stationary phase, but not re-enter the mobile phase. Increasing the proportion of acetonitrile in the solvent system will result in better mass transfer characteristics, and hence improved chromatography, for these more lipophilic barbiturates. 

Molnar and Horvath [140] proposed reversed-phase HPLC for characterisation of lipophilicity, by the use of log A- instead of log P. Later on, Biagi et al. 23] found a good correlation between log P. log A and Py/ values, determined by TLC. 

A comprehensive semi-empirical description of reversed-phase HPLC systems, for predicting the relative retention and selectivity within a series of analytes, has been developed by Jandera and co-workers [72,73]. The approach consists of determining the interaction indices and the structural lipophilic and polar indices. A suitable set of standard reference analytes is necessaiy to calibrate the retention (or selectivity) scale. 

The evaluation of alkamides, as an indicator for standardization, in Echinacea preparations is commonly completed using HPLC. The method of Bauer (1999b) illustrates the most common method to evaluate alkamides using reverse-phase HPLC. In this method, the lipophilic components are separated by gradient elution using water (eluent A) and acetonitrile (eluent B) linearly from 40% to 80% eluent B at 1 ml/min. The separation was completed on a C18 reverse-phase column and detection was at 254 nm. Thin-layer chromatography using silica 60 plates with... 

The overall strategy should take into consideration the nature of the matrix and the properties of the metabolites present. Relevant properties, to be investigated by appropriate analytical procedures Include hydrolytical stability, volatility, lipophilicity, solvent partition properties, electrical charge and susceptibility towards defined enzymes. A reliable analytical system providing efficient separation of individual metabolites present, needs to be established as early in the isolation procedure as possible. This analytical system is used to monitor the result of each single isolation step, and - of equal Importance - to recognize any artifact formation which may occur during the execution of that step. Two-dlrectlonal TLC and reversed phase HPLC are fast and, therefore, commonly used methods for this purpose. 

Measurement of labelling yield and subsequent radiochemical purity requires a suitable analytical technique, and the method of choice for radio-labelled peptides is reversed phase HPLC with on-line UV and radiometric detection. It is important to use as stringent a separation method as possible with isocratic or slow mobile phase composition gradients over the peptide peak. Ideally, more than one mobile phase system should be used (e.g. a phosphate buffer-methanol system in addition to the standard water-acetonitrile system), since these may show the presence of new impurities. It is important to recognize that HPLC analyses only measure those components that elute from the column. Insoluble, highly lipophilic or positively charged species may bind to the solid phase. It is very important to verify the absence of these species by a complimentary technique such as thin layer chromatography (TLC) and to ensure that the two techniques produce similar results. 

C]-Benzo[a]pyrene was administered to male germfree rats (Yang et al. 1994). Urine was collected 24 hours before and every 24 hours for 7 days after administration. Urinary metabolites, consisting of 9% ofthe administered radioactivity, were fractionated by lipophilic ion exchange chromatography, and characterized by reversed-phase HPLC, ultraviolet spectrometry, and gas... 

HPLC techniques have also been used in the determination of log P values. Lambert et al. (1990), for example, have described the development of a preformulation lipophilicity screen utilizing a C18 derivatized HPLC column. They appeared to prefer this column to the traditional reverse-phase HPLC columns, which may yield a poor correlation between log P and the capacity factor (k ). A potential problem with the use of HPLC retention data is that it is not a direct method and thus requires calibration. Futhermore, there may be problems with performing experiments above pH 8. 

Deoxy-4 -fluoro-DOXO was the object of studies, together with several other derivatives, for determination of the influence of lipophilicity on the cytotoxicity of anthracyclines in LoVo and LoVo/Dx human cell lines. The lipophilic character of selected anthracyclines was measured by means of reverse-phase HPLC, under appropriate experimental conditions. The results obtained in these in vitro models indicate that lipophilicity plays a role in anthracycline activity, influencing drug availability at the site of action. The introduction of a fluorine atom in the sugar moiety structure increases the lipophilicity and cytotoxicity of the drug [84]. 

---