Micro-purification method

As reported in the structural determination of BL, CS, DL, and typhasterol, MS is an essential technique for BRs isolated in pure form. However, in most cases, isolation of BRs in pure form is time-consuming and tedious work because of their very low concentration in plant materials. BRs are highly polar and involatile compounds. Therefore, conversion of BRs into volatile derivatives in gas phase makes it easy to characterize BRs in a partially purified bioactive fraction by GC/MS or GC/selected ion monitoring (SIM), which are analytical techniques most frequently used in natural products chemistry. The desired derivatives of BRs are BMBs or MB-TMSs. Another convenient and useful technique is HPLC. HPLC has now been routinely and effectively employed in the purification of natural BRs. Microanalysis of BRs by HPLC has recently been developed, which involves transformation of BRs into derivatives with a fluorophore or an electrophore by use of pre-labeling reagents. Immunoassay techniques to analyze plant hormones have recently advanced and are readily accessible by plant physiologists. RIA for BRs has also been developed. In this section, micro-analytical methods of BRs using GC/MS (SIM), HPLC, and RIA are described. 

Electrophoresis makes use of differences in the electrophoretic mobility of electrically charged particles (biomolecules, micro-organisms etc.) as a means to separate them. For this purpose, a homogeneous, rectified electrical field is used. Thanks to the excellent resolution and mild operating conditions, this is currently the best analytical method for protein separation, purification and characterization. It is also used as a preparative separation method which allows a few grams per hour to be purified. 

The ability of this ionization method for the determination of very high molecular weights is illustrated in Figure 1.26 [68], The spectrum displayed is obtained from assemblies of vanillyl alcohol oxidase containing respectively 16 and 24 proteins. The spectrum was obtained with a hybrid quadrupole TOF instrument, Q-TOF Micromass, equiped with a micro-ESI source. To obtain such a spectrum one needs not only a mass spectrometer with sufficient mass range and resolution, but also high skill in protein purification. 

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. 

When a product has been isolated from a reaction the next step is to purify it. The degree of purity required will depend on the use for which the sample is intended, a synthetic intermediate might only require rough purification, whereas a product for elemental analysis would require rigorous purification. This section describes the most important purification techniques, crystallization, distillation, sublimation, and chromatography. It is assumed that the reader is familiar with the basic principles of these methods, so the emphasis is on more demanding applications such as the purification of air-sensitive materials, and purifications on a micro-scale. 

Polyelectrolyte complexes are very promising materials for preparing semi-permeable membranes of definite permeability and selectivity The methods of preparation and the properties of membranes made of polyelectrolyte complexes based on strong polyelectrolytes, e.g. poly(sodium sterene sulfonate) and poly(vinylbenzyl-trimethyl ammonium chloride) were described These membranes may be applied for reverse osmosis in the desalting of sea-water, for dialysis and ultrafiltration in purifications and concentration of water solutions containing coUoids or micro-and macroparticles ... 

The classical methods of chemical and fermentative synthesis and purification are augmented by the powerful genetically modified cellular systems of production, by transgenic animals and plants, as well as by sophisticated techniques of syntliesis, analysis, purification, such as chual catalysts, micro-reactors, and process intensification. 

---