Method development in HPLC

S. V. Galushko, Software for method development in HPLC, GIT Spezial Chro-matogr. 16 (1996), 88-93. 

Analytical method development in HPLC usually involves changing the composition of the mobile phase until the desired degree of separation of the targeted organic compounds has been achieved. One starts with a mobile phase which has a high solvent strength and moves downward in solvent strength to where a satisfactory resolution can be achieved. Recall the key relationship for resolution (3) ... 

Method development in HPLC is a challenging and interesting field of study. A number of factors need to be addressed in any method development exercise. We will deal with them on an individual basis in this chapter. 

It has been pointed out with the aid of an example how the factors that influence chromatographic separation can be systematically examined. The inclusion of the stationary phase as a factor offers an insight into the complexity of the interaction offactors in chromatography. With HEUREKA, a tool has been created with which method development in HPLC can be accomplished multifactorially. 

Thin-layer chromatography (TLC) is used both for characterization of alcohol sulfates and alcohol ether sulfates and for their analysis in mixtures. This technique, combined with the use of scanning densitometers, is a quantitative analytical method. TLC is preferred to HPLC in this case as anionic surfactants do not contain strong chromophores and the refractive index detector is of low sensitivity and not suitable for gradient elution. A recent development in HPLC detector technology, the evaporative light-scattering detector, will probably overcome these sensitivity problems. 

The identification and quantification of potentially cytotoxic carbonyl compounds (e.g. aldehydes such as pentanal, hexanal, traw-2-octenal and 4-hydroxy-/mAW-2-nonenal, and ketones such as propan- and hexan-2-ones) also serves as a useful marker of the oxidative deterioration of PUFAs in isolated biological samples and chemical model systems. One method developed utilizes HPLC coupled with spectrophotometric detection and involves precolumn derivatization of peroxidized PUFA-derived aldehydes and alternative carbonyl compounds with 2,4-DNPH followed by separation of the resulting chromophoric 2,4-dinitrophenylhydrazones on a reversed-phase column and spectrophotometric detection at a wavelength of378 nm. This method has a relatively high level of sensitivity, and has been successfully applied to the analysis of such products in rat hepatocytes and rat liver microsomal suspensions stimulated with carbon tetrachloride or ADP-iron complexes (Poli etui., 1985). 

Jimidar, M., and De Smet, M. (2007). HPLC method development in late phase pharmaceutical development. In HPLC Method Development For Pharmaceuticals (S. Ahuja, and H. Rasmussen, Eds), Vol. 8 of Separation Science and Technology, pp. 373—405, Academic Press, Elsevier, London, Chapter 13. 

GC has been used for process analysis for many decades, along with many spectroscopic tools and univariate sensors. In recent years, developments in HPLC have made it now also available for on-line monitoring It has the advantage over spectroscopic methods in being able to detect trace levels of compounds, such as... 

The unique structural character of these diterpenes and their exclusive distribution in the family Euphorblacae may allow their use as specific chemotaxonomlc markers within the family. Application of analytical HPLC methods developed in the Isolation of the jatrophane diterpenes to extracts of other E. esula accessions has revealed distinctively different unidentified jatrophane diterpenes (30) among the accessions. The rarity and exclusivity of these compounds within Euphorblacae warrants continued chemical examination and differentiation of E. esula accessions in relationship to successful Insect biological control. 

N. G. Mellish, Computer-assisted HPLC method development in a pharmaceutical laboratory, LC-GC, 9 845 (1990). 

Successful separations can be carried out only by planning and careful experimentation, the details of which are discussed extensively in Chapter 5. In one sense there are too many ways to achieve a separation in LC. But while this makes the first choice of where to start difficult, the good news is that there are many ways to achieve success. By looking at Figure 4-1 it is obvious that the use of the reverse-phase mode in LC has broad applicability and is, in fact, the most used mode of LC. Reverse phase is used for 80-85% of the separation problems encountered by users of HPLC. For this reason, the majority of Chapter 5, on developing methods, is devoted to reverse-phase examples. Additionally, Chapter 11 is a useful experiment to experience the method development aspect of this mode. Chapter 9 is a useful experiment to experience method development in the normal phase mode. 

Choice of the proper detection scheme is dependent on the properties of the analyte. Different types of detectors are available such as ultraviolet (UV), fluorescence, electrochemical, hght scattering, refractive index (RI), flame ionization detection (FID), evaporative light scattering detection (ELSD), corona aerosol detection (CAD), mass spectrometric (MS), NMR, and others. However, the majority of reversed-phase and normal-phase HPLC method development in the pharmaceutical industry is carried out with UV detection. In this section the practical use of UV detection will be discussed. 

Sample handling is a very important part of the method development for HPLC determination of phenolic acids in natural plants. Because of the great variability of phenolic acids (different polarity, acidity, number of hydroxyl groups, and aromatic rings), the various concentration levels of individual analytes, and the very complex natural matrix with many interfering components, the choice of the technique for their isolation and quantification differs from one described HPLC assay to the next. In some cases, only a one-step extraction and simple clean-up procedure are sufficient before the HPLC analysis, but the most often described HPLC assays include two or more steps of sample preparation, especially in the case of fruits and vegetable samples. It is obvious that each step contributes, on one hand, to the higher sensitivity and selectivity, but, on the other hand, it could increase the number of errors and decrease the recovery of the method. 

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