Analysis by Liquid Chromatography

Eor the separation, small amoimts of the lipid sample are spotted onto the TEC plate by means of a syringe, aroimd 2 cm from the bottom of the plate, which is then placed into a tank of an appropriate solvent, which serves as the mobile phase. Due to capillary forces, with time, the solvent moves up the plate taking the sample components with it at different rates, depending on their affinity to the adsorbent various combinations of aqueous stationary phases and organic mobile phases can be used to obtain differential migration of distinct lipid classes. The quality of the separation is further affected by the degree of hydration of the adsorbent, in a similar way as in column liquid chromatography. This in turn 

TLC can be successfully applied to the separation of some simple lipid classes, such as mono-, di-, and triacylglycerols, and their derivatives. Typical mobile phases for the separation of simple lipids contain hexane, diethyl ether, and formic or acetic acid in various proportions (e.g., 80 20 2, v/v) the latter ensures successful migration of the free fatty acids. 

Complex lipids (e.g., phospholipids and glycosphingolipids) do not migrate under these conditions, and they can then be detected or quantified only as if they were a single lipid class. Yet, TLC can be used as a rapid screening tool, prior to the application of more selective and sensitive methods. 

If a complex lipid sample cannot to be separated within a single TLC run, it may be resolved by rechromatography in a second direction. In the so-called two-dimensional TLC techniques, the sample is first applied in the bottom comer at the left-hand side of the plate and developed in the first selected solvent. After the first development is completed, the plate 

FIGURE 9.9 Schematic HP TLC separations of complex lipid mixtures from 

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B.G.M. Vandeginste, F. Leyten, M. Gerritsen, J.W. Noor, G. Kateman and J. Frank, Evaluation of curve resolution and iterative target transformation factor analysis in quantitative analysis by liquid chromatography. J. Chemom., 1 (1987) 57-71. 

Wuhrer, M., Koeleman, C.A., Hokke, C.H., and Deelder, A.M. 2005. Protein glycosylation analysis by liquid chromatography-mass spectrometry. Journal of Chromatography B 825, 124-133. 

King, R.C. et al. 2002. Description and validation of a staggered parallel high performance liquid chromatography system for good laboratory practice level quantitative analysis by liquid chromatography/tandem mass spectrometry. Rapid Commun. Mass Spectrom. 16 43. 

L6pez-Roldan P., de Alda M.J.L., and Barcelo D., 2004. Simultaneous determination of selected endocrine disrupters (pesticides, phenols and phthalates) in water by in-field solid-phase extraction (SPE) using the prototype PROFEXS followed by online SPE (PROSPEKT) and analysis by liquid chromatography-atomspheric pressure chemical ionization-mass spectrometry. Anal Bioanal Chem 378 599. 

J.E. Lawrence, in Organic Trace Analysis by Liquid Chromatography (J.E. Lawrence, Ed.), Academic Press, New York (1981). 

Mullen W, Boitier A, Stewart AJ, Crozier A. 2004. Flavonoid metabolites in human plasma and urine after the consumption of red onions Analysis by liquid chromatography with photodiode array and full scan tandem mass spectrometric detection. J Chromatogr A 1058 163-168. 

Kruve, A., A. Kunnapas, K. Herodes, et al. 2008. Matrix effects in pesticide multi-residue analysis by liquid chromatography-mass spectrometry. J. Chromatogr. A 1187 58-66. 

Ravelo-Perez, L.M., J. Hemandez-Borges, and M.A. Rodrfguez-Delgado. 2006. Pesticides analysis by liquid chromatography and capillary electrophoresis. J. Sep. Sci. 29 2557-2577. 

Nielen MWF, Brinkman UAT, Frei RW. 1985. Industrial waste-water analysis by liquid chromatography with precolumn technology and diode-array detection. Anal Chem 57 806-810. 

Microdialysis has also been used as a sampling method for measuring the concentration of drugs in human subcutaneous tissues for pharmacokinetic studies [50]. The microdialysates are simpler than other biological fluids and do not contain proteins, permitting their direct injection for analysis by liquid chromatography. 

Pajkovic, N. van Breemen, R. 2005. Analysis of carotenoids using atmospheric pressure chemical ionization mass spectrometry. In Modem Methods for Lipid Analysis by Liquid Chromatography (Byrdwell, C., Ed.). AOCS Publishing, Champaign, IL, pp. 413 30. 

J. F. Lawrence, Organic Trace Analysis by Liquid Chromatography, London, Academic Press, 1981. N. A. Parris, Instrumental Liquid Chromatography, 2nd Edn, Amsterdam, Elsevier, 1984. 

Hess, E, Nguyen, L., Aasen, I, Keogh, M., Kilcoyne, J, McCarron, P., and Aune, T. 2005. Tissue distribution, elFects of cooking and parameters affecting the extraction of azaspiracids from mussels, Mytilus edulis, prior to analysis by liquid chromatography coupled to mass spectrometry. Toxicon 46, 62—71. 

Lawrence, G.D. Yatim, N. Extraction of physostigmine from biologic fluids and analysis by liquid chromatography with electrochemical detection. J. Pharmacol. Meth. 1990, 24, 137-143. 

Tissue analysis by liquid chromatography-ion trap mass spectrometry 620... 

Cataldo F. Polyynes production in a solvent-submerged electric arc between graphite electrodes. 2. Analysis by liquid chromatography. Fullerenes, Nanotubes and Carbon Nanostructures 2004, 12, 619. 

King, R.C. Gundersdorf, R. Fernandez-Metzler, C.L. Collection of Selected Reaction Monitoring and Full Scan Data on a Time Scale Suitable for Target Compound Quantitative Analysis by Liquid Chromatography-Tandem Mass Spectrometry, Rapid Commun. Mass Spectrom. 17,2413-2422 (2003). 

Temperature has only a small effect on retention and resolution in liquid chromatography, since the enthalpy of solution from the mobile phase to the stationary phase is quite small unlike it is the case for gas chromatography. Thus analysis by liquid chromatography is most often carried out at room or relatively low temperature. 

Quantitative analysis by liquid chromatography should preferably be performed by isocratic elution, with samples dissolved in the mobile phase. This is not always possible, and in this case a somewhat lower, but for most applications quite acceptable, degree of reproducibility must be accepted. 

Saint-Jahn, Y, P. Moree-Testa, and A. Testa The volatile fraction of cigarette smoke condensate Analysis by liquid chromatography and gas chromatography-mass spectrometry Analysis 11 (1985) 12-18. 

Gago-Martmez, A. et al.. Effect of pH on the oxidation of paralytic shellfish poisoning toxins for analysis by liquid chromatography, J. Chromatogr. A., 905, 351, 2001. 

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