Applications of HPLC

Examples of the application of HPLC to the analysis of (a) acetaminophen, salicylic acid, and caffeine (b) chlorinated pesticides (c) tricyclic antidepressants and (d) peptides. (Chromatograms courtesy of Alltech Associates, Inc. Deerfield, IL). 

For selective estimation of phenols pollution of environment such chromatographic methods as gas chromatography with flame-ionization detector (ISO method 8165) and high performance liquid chromatography with UV-detector (EPA method 625) is recommended. For determination of phenol, cresols, chlorophenols in environmental samples application of HPLC with amperometric detector is perspective. Phenols and chlorophenols can be easy oxidized and determined with high sensitivity on carbon-glass electrode. 

In 1996, Mondello et al. (48) published a review article on the applications of HPLC-HRGC developed for food and water analysis over the period from 1986 to 1995. These authors cited 98 references, grouped by following a chronological order and by the subject of the application, as follows ... 

HPLC separations are one of the most important fields in the preparative resolution of enantiomers. The instrumentation improvements and the increasing choice of commercially available chiral stationary phases (CSPs) are some of the main reasons for the present significance of chromatographic resolutions at large-scale by HPLC. Proof of this interest can be seen in several reviews, and many chapters have in the past few years dealt with preparative applications of HPLC in the resolution of chiral compounds [19-23]. However, liquid chromatography has the attribute of being a batch technique and therefore is not totally convenient for production-scale, where continuous techniques are preferred by far. 

With notable exceptions, the application of HPLC to clinical chemistry has not as yet been extensive. This is somewhat surprising in view of the potential the method has for this area. This potential arises, in part, from the fact that HPLC is well suited to the types of substances that must be analyzed in the biomedical field. Ionic, relatively polar species can be directly chromatographed, without the need to make volatile derivatives as in gas chromatography. Typically, columns are operated at room temperature so that thermally labile substances can be separated. Finally, certain modes of HPLC allow fractionation of high molecular weight species, such as biopolymers. 

Aman, R. et al.. Application of HPLC coupled with DAD, APcI-MS and NMR to the analysis of lutein and zeaxanthin stereoisomers in thermally processed vegetables. Food Chem., 92 753, 2005. 

HPLC has proved to be fast and sensitive for the analyses of phenolic plant constit-nents, and is especially useful for the analysis of anthocyanins. The first application of HPLC to anthocyanin analyses was in 1975 by Manley and Shubiak and it has now become the method of choice for the separation of mixtures of anthocyanins and anthocyanidins. HPLC is now used for anthocyanin qualitative, quantitative, and preparative work, offering improved resolution compared to chromatographic procedures previously employed. It also allows for simultaneous rapid monitoring of the eluting anthocyanins. ... 

Several determination methods such as GC, HPLC, gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/mass spectrometry (LC/MS) are used for the analysis of neonicotinoid residues. The applications of GC/MS and LC/MS are of increasing importance. The application of HPLC to the determination of neonicotinoids residues is limited, especially when metabolites (such as acetamiprid and nitenpyram) can be easily determined by GC after derivatization. 

Bruns, A., Waldhoff, H., and Winkle, W., Application of HPLC with evaporative light-scattering detection in fat and carbohydrate chemistry, Chro-matographia, 27, 340, 1989. 

Four types of atomic spectrometry have been interfaced for chromatographic detection, namely AAS, FES, AFS and APES. Ebdon et al. [178] have discussed coupling of HPLC with AAS. HPLC-FAAS is relatively insensitive. Application of HPLC-GFAAS or... 

Application of HPLC-MS to the analysis of a black tea liquor was studied in a paper by Bailey 39 a great deal of useful information could be obtained without sample pretreatment. A tea liquor was applied to a wide-pore HPLC column connected to a mass spectrometer by a VG Plasmaspray interface. Pseudo-molecular ions were obtained from the flavanols, flavanol gallates, chlorogenic acids, 4-coumarylquinic acids, and caffeine, but the flavanol glycosides were extensively fragmented by the interface. Fragments were obtained from unresolved polymer that supported its previous designation as a flavanol polymer. 

Shibamoto, T., Harada, K., Mihara, S., Nishimura, O., Yamaguchi, K., Aitoku, A., Fukada, T., Applications of HPLC for evaluation of coffee flavor quality, in Charalambous, G., Inglett, G., Eds., Qual. Foods Beverages Chem Technol, Proc. Symp. Int. Flavor Conf. 2nd, Academic, New York, 1981, 311. (CA96 33486y)... 

Cannel, R.J.P., Rashid, T., Ismail, I.N. et al. (1997) Novel metabolites of warfarin produced by Beauveria bassiana and Streptomyces rimosus a novel application of HPLC-NMR. Xenobiotica The Fate of Foreign Compounds in Biological Systems, 27, 147-157. 

HPLC methods have been widely used for the analysis of OTC in different samples. As described above in the Section 2.3, the HPLC method is described in most of compendia [1,2,4,7] for determination of OTC in bulk drug substances and in some pharmaceutical preparations. The application of HPLC methods for the analysis of antibiotics including oxytetracycline has been recently reviewed by Diaz-Cruz et al. [37] and Lunn [38], A summary of HPLC method for the analysis of OTC is presented in Table 3. 

In the contemporary investigation of artworks and especially in the identification of natural organic dyestuffs the applicability of HPLC MS cannot be questioned. This technique allows recognition of almost all common colourants in one run , which decreases the probability of losing specific information (Table 13.3). In comparison with GC-MS, HPLC MS has wider application, as it is not limited by the presence of polar and nonvolatile compounds, and therefore it usually does not require the derivatization step. The number of published papers, which has doubled in the last 3 years in comparison with the period 2000 2004, proves that HPLC-MS performs a pivotal role in the analysis of the colourants discussed. 

The successful application of HPLC was made possible largely by (a) the development of pump systems that can provide constant flow rates at high pressure and (b) the identification of suitable pressure-resistant chromatographic media. Traditional soft gel media utilized in low-pressure applications are totally unsuited to high-pressure systems due to their compressibility. 

In order to study simultaneously the behaviour of parent priority surfactants and their degradation products, it is essential to have accurate and sensitive analytical methods that enable the determination of the low concentrations generally occurring in the aquatic environment. As a result of an exhaustive review of the analytical methods used for the quantification within the framework of the three-year research project Priority surfactants and their toxic metabolites in wastewater effluents An integrated study (PRISTINE), it is concluded that the most appropriate procedure for this purpose is high-performance (HP) LC in reversed phase (RP), associated with preliminary techniques of concentration and purification by solid phase extraction (SPE). However, the complex mixtures of metabolites and a lack of reference standards currently limit the applicability of HPLC with UV- or fluorescence (FL-) detection methods. 

This section covers only applications of HPLC with detection systems other than MS, giving special attention to specific issues related to the separation and detection of different groups of ionic and non-ionic surfactants. 

Results obtained for the application of HPLC—APCI—MS to the quantification of M2D—C3—O—(EO ) —CH3 recoveries from Chenopo-dium album plant foliage are shown in Table 2.8.5, as compared with HPLC-LSD analysis [29], The improvements in the sensitivity and reproducibility were obtained with the use of HPLC—APCI—MS as the analytical method and the HPLC—APCI—MS method also enabled detection of the n = 3 M2D-C3-0-(E0) -CH3 molecule. 

The various theoretical and practical aspects of the use of HPLC methods have been recently discussed in exquisite books, such as the application of HPLC-MS in drug analysis [59], the theory of chromatography [60], the fundamentals of chromatography [61, 62], the practice and theory of ion-chromatography [63], problem solving in HPLC [64], the... 

Cells are typically concentrated by filtration and extracted into an organic solvent (usually acetone) after which, pigments are detected by fluorescence or absorption spectroscopy, sometimes after chromatographic separation (Bidigare and Trees, 2000). The application of HPLC to phytoplankton pigment analysis has lowered the uncertainty in the measurement of Chi a and accessory carotenoids, since compounds are physically separated and individually quantified. 

The primary object of this book is to provide the HPLC practitioner with a handy guide to the use of HPLC for analyzing pharmaceutical compounds of interest. This means familiarizing the practitioner with the theory, instrumentation, regulations, and numerous applications of HPLC. This handbook provides practical guidelines using case studies on sample preparation, column or instrument selection, and summaries of best practices in method development and validation, as well as tricks... 

The application of HPLC in routine environments, like pharmaceutical, food, or environmental analysis and particularly quality assurance, makes not only great demands on the robnstness of HPLC hardware, comprising pumps, column thermostats, and detection units, bnt in addition to the column reproducibility. Column reproducibility can be investigated at different levels of complexity Run-to-run reproducibility compares consecutive chromatographic runs, whereas long-term stability describes the column variance over several hundreds of injections. Column-to-column (batch-to-batch) reproducibility finally explores the match of independently fabricated chromatographic columns. Column characteristics that are routinely consulted for the determination of the robustness are retention, selectivity, column efficiency, and peak symmetry. 

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