RP purification

Funke, C. et al., Harbor seal (Phoca vitulina) C-reactive protein (C-RP) purification, characterization of specific monoclonal antibodies and development of an immuno-assay to measure serum C-RP concentrations, Vet. Immunol. Immunopathol., 59, 151, 1997. 

Xanthopterin monohydrate (2-amino-4,6-dihydroxypteridine, 2-amino-pteridin-4,6(lff,5ff)-dione) [5979-01-1 (H2O), 119-48-8 (anhydr)] M 197.2, m <300", pK, 1.6 (basic), pKj 6.59 (acidic), PK3 9.31 (acidic)(anhydrous species), and pKj 1.6 (basic), pK2 8.65 (acidic), PK3 9.99 (acidic)(7,8-hydrated species). Purification as for isoxanthopterin. Crystd by acidifying an ammoniacal soln, and collecting by centrifugation followed by washing with EtOH, ether and drying at 100° in vacuo. Paper chromatography Rp 0.15 ( -PrOH, 1% aq NH3, 2 1), 0.36 ( -BuOH,AcOH, H2O, 4 1 1) and 0.47 (3% aq NH3). [Inoue and Perrin J Chem Soc 260 7962 Inoue Tetrahedron 20 243 I964 see also Blakley Biochemistry of Folic Acid and Related Pteridines North Holland Publ Co, Amsterdam 1969.]... 

A sequential analysis protocol includes three steps (1) extraction in water or other appropriate solvent for the colorant, (2) purification or concentration of the colorant, and (3) separation coupled with detection of the target molecule. Different methods of extracting synthetic colorants from foods have been developed using organic solvents followed by SPE protocols using as adsorption support RP-C18, amino materials, or Amberlite XAD-2. Eor qualitative evaluations, the easiest option for separating colorant molecules from unwanted ingredients found in an extract is SPE on polyamide or wool. 

Fukumori F, RP Hausinger (1993b) Purification and characterization of 2,4-dichlorophenoxyacetate/a-keto-glutarate dioxygenase. J Biol Chem 268 24311-24317. 

A clean-up process-scale RP-HPLC step has been introduced into production of human insulin prb. The C8 or C18 RP-HPLC column used displays an internal volume of 80 1 or more, and up to 1200 g of insulin may be loaded during a single purification run (Figure 11.4). Separation is achieved using an acidic (often acetic-acid-based) mobile phase (i.e. set at a pH value sufficiently below the insulin pi value of 5.3 in order to keep it fully in solution). The insulin is usually loaded in the water-rich acidic mobile phase, followed by gradient elution using acetonitrile (insulin typically elutes at 15-30 per cent acetonitrile). 

Figure 11.3 A likely purification scheme for human insulin prb. A final RP-HPLC polishing step yields a highly pure product. Refer to text for details...

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. 

The results of theoretical calculation using both general rate and transport-dispersive models were in good agreement with the overloaded band profiles determined experimentally, therefore, the method has been found to be suitable for the prediction of band profiles [88], Natural pigments were generally used as a complicated mixture of various compounds with chromophore substructure. Their separation by preparative RP-HPLC is not necessary, and the application of preparative RP-HPLC for the purification of one or more pigment fractions is not expected in the near future. 

The same technique has been employed for the purification of Food Colour Red No. 106 (Acid red). The chemical structure of the dye is shown in Fig. 3.118. The separation process was controlled by analytical RP-HPLC carried out in an ODS column (150 X 4.6 mm i.d. particle size 5 /.an). The mobile phase consisted of ACN-0.01 M TFA (27 73, v/v). The flow rate was 1 ml/min and analytes were detected at 254 nm. Equilibrated n-butanol and water were employed for CCC separation. OP was acidified with 40 mM of sulphuric acid, and 30 mM of aqueous ammonia was added to the LP. The coil was rotated at 800 rpm and LP was pumped at a flow rate of 1 ml/min. Fractions of 1 ml volume were taken from the effluent. The CCC profile of Food Colour Red No. 106 in CCC is shown... 

Several analytical techniques including capillary electrophoresis, thin layer chromatography (TLC), GC, lEC, and HPLC, have been proposed for the determination of biogenic amines in various foods. Among these, RP-HPLC is considered the most suitable one. HPLC methods used for amine determination usually involve two steps amine extraction from the matrix and analytical determination. Depending on the complexity of food matrix and the selectivity of the final analytical determination, a further purification step may be necessary prior to the analytical determination. To ensure adequate sensitivity, a derivatization step is generally required before injection [282]. 

For the final purification, a sequence of normal phase chromatography, size exclusion chromatography, reversed-phase (RP)-HPLC and other techniques are used. There are no general rules as to how to proceed but, due to the high capacity and low irreversible absorption of Sephadex, size exclusion should be used in the very beginning, whilst HPLC is better employed for the final purification steps. 

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