Liquid chromatography preparative HPLC

Equipment and expertise. Synthesis requires dedicated laboratory space and equipment. Protein synthesis is best done with the aid of a peptide synthesizer which is capable of optimal step-wise yields. Purification using reverse-phase high-performance liquid chromatography (RP-HPLC) is an integral part of the procedure (16), so at least one preparative and one analytical HPLC systems is needed. Access to electrospray mass spectrometry is essential. 

As a rule, a separation method should be used for both purification and concentration of the sample. The classic method for peptides and proteins is a reverse-phase liquid chromatography preparation of the sample, followed by a concentration step (often lyophiliza-tion) of the fraction of interest. During those steps performed on very small quantities of sample, loss on the sample can occur if care is not taken to avoid it. Lyophilization, for instance, can lead to the loss of the sample absorbed on the walls of the vial. The use of separation methods on-line with the mass spectrometer often are preferred. Micro- or nano-HPLC [32,33] and capillary electrophoresis [34], both coupled mainly to electrospray ionization/mass spectrometry (ESI-MS), are used more and more. 

CE, with its high resolving power, rapid method development, easy sample preparation, and low operational cost, is reported to be an excellent technique for resolving caseins (including different genetic variants), peptides derived from them, and whey proteins (16-19). Peptide profiles obtained by CE supplement the information obtained by reversed-phase high performance liquid chromatography (RP-HPLC) (17, 20). The application of CE to the assessment of proteolysis in milk and different cheese types has acquired an enormous importance in recent years. Reviews on the application of CE to this field can be found in papers by Otte et al. (21) and Redo et al. (22). 

FIGURE 2.10 Preparative reversed-phase high-performance liquid chromatography (RP-HPLC) separation of fatty acid methyl esters (FAME) (5-10 mg) from cheese on a Nucleosil C18 column (250 mm X 10 mm i.d.) with acetonitrile as mobile phase at a flow rate of 4 mL/min. Detection of FAME was by refractometry and the fraction containing conjugated linoleic acid (CLA) (also containing 14 0, 16 1, and 18 2) was monitored by ultraviolet (UV) detection at 234 nm. (Reproduced from Lavillonniere, F., Martin, J.C., Bougnoux, P., and Sebedio, J.-L., J. Am. Oil Chem. Soc., 75, 343-352, 1998.)... 

As a method of research, has been used high-performance liquid chromatography in reversed - phase regime (RP HPLC). The advantage of the present method is the following the additional information about AIST and FAS composition (homologous distribution) simple preparation of samples (dilution of a CS sample of in a mobile phase). 

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. 

Despite the difficulties caused by the rapidly expanding literature, the use of chiral stationary phases (CSPs) as the method of choice for analysis or preparation of enantiomers is today well established and has become almost routine. It results from the development of chiral chromatographic methods that more than 1000 chiral stationary phases exemplified by several thousands of enantiomer separations have been described for high-performance liquid chromatography (HPLC). 

High performance liquid chromatography (HPLC) has been by far the most important method for separating chlorophylls. Open column chromatography and thin layer chromatography are still used for clean-up procedures to isolate and separate carotenoids and other lipids from chlorophylls and for preparative applications, but both are losing importance for analytical purposes due to their low resolution and have been replaced by more effective techniques like solid phase, supercritical fluid extraction and counter current chromatography. The whole analysis should be as brief as possible, since each additional step is a potential source of epimers and allomers. 

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