HPLC high performance liquid termination

The stable nonvolatile intermediate phenylthiocar-bamoyl derivatives are formed in basic media and can be analyzed directly by reverse-phase high-performance liquid chromatography (RP-HPLC). Their cyclization into hydantoins requires acid catalysis. This mode of derivatization is a very important supplement to the Edman s method of N-terminated sequencing of polypeptides. Before GC analysis, any hydantoins can be converted into N-trifluoroacetyl or enol-O-trimethylsilyl derivatives, which increases the selectivity of their determination in complex matrices. 

Activity of the purified en2ymes was assessed on methyl ferulate (methyl 4-hydroxy 3-methoxy cinnamate, MF). Assays were run for 30 min at 50 °C in 50 mM eitrate buffer at pH 4.8 with initial MF concentrations of 50 to 750 pM and an enzyme concentration of 50 nM. The reactions were terminated after 30 min by boiling for 10 min and analyzed for MF and ferulic acid content via C18 high performance liquid chromatography (HPLC) over a 0 to 100% acetonitrile gradient with 0.1% formic acid in all solutions. Preliminary isothermal titration calorimetry (ITC) on both ferulic acid esterase proteins was conducted in SEC buffer on a Microcal VP-ITC system. All reactions were carried out at 37 °C using MF as a substrate. 

Cmde dextransucrase was reacted with 100 mM sucrose for 1 h at 30 °C and then boiled for 5 min to terminate the enzyme reaction. One unit of dextransucrase activity was defined as that amount of enzyme releasing 1 p.M fructose per min from 100 mM sucrose. The fructose was determined by high-performance liquid chromatography (HPLC) using an Aminex FIPX 87K column (300 7.8 mm) and an HPLC analyzer coupled to a refiactive index detector. The column was maintained at 85 °C and 0.01 M K2SO4 was used as a mobile phase at a flow rate 0.6 ml/min. 

Fig. 10.3 Reversed-phase high-performance liquid chromatography (HPLC) of Me3C(CH=CH) jCMe3 prepared by the polymerization of acetylene (5 equivalents) in toluene, initiated by W(=CHCMe3)(=NAr)(OCMe3)2 (1 equivalent) in the presence of quinuclidine (5 equivalents) and terminated by Me3CCHO. The peak marked 3t3 is for the oligomer with jc = 3 and 3 trans double bonds likewise for the other marked peaks. The unmarked peaks refer to oligomers containing at least one cis double bond. The peak marked with an asterisk is the internal standard (guaiazulene) (Schlund 1989).

Terminal alkynes substituted with chiral substituents have been polymerized by using a rhodium catalyst, [RhCl(NBD)]2 (NBD = norbomadiene) [6]. As shown in Scheme 3, polymerization of a chiral (carbamoyloxy)phenylacetylene 4 forms a cis-substituted polyacetylene 5. Due to the bulkiness of the substituents, these polymers show a helical conformation with no extended conjugation in the polymer chain. These materials are potentially useful as enantioselective permeable membranes to separate racemic amino acids and alcohols in water or in methanol. They can be also used as chiral stationary phase for enantioselective high-performance liquid chromatography (HPLC) analysis. 

Furthermore, (AB)3-type star block copolymer was also prepared using ATRP, CGCP, and a click reaction (Scheme 33) [74]. ATRP of styrene was carried out in the presence of 2,4,6-tris(bromomethyl)mesitylene as a trifunctional initiator, and then the terminal bromines of the polymer were transformed to azide groups with NaNs. The azide-terminated polystyrene was then used for click reaction with alkyne-terminated aromatic polyether, obtained by CGCP with an initiator bearing an acetylene unit. Excess alkyne-terminated aromatic polyether was removed from the crude product by means of preparative high-performance liquid chromatography (HPLC) to yield the (AB)3-type star block copolymer. 

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