Reversed-phase HPLC gradient chromatography

Dibenzyl-14-crown-4 (lithium ionophore VI 6,6-dibenzyl-l,4,8,ll-tetra-oxa-cyclo-tetradecane) [106868-21-7] M 384.5, m 102-103°. Dissolve in CHCI3, wash with saturated aqueous NaCl, dry with MgSOa, evaporate and purify by chromatography on silica gel and gradient elution with C6Hg-MeOH followed by preparative reverse phase HPLC on an octadecyl silanised silica (ODS) column and eluting with MeOH. It can be crystd from MeOH (v Br 120 cm , C-O-C). [7 Chem Soc Perkin Trans 1 1945 1986.]... 

High-performance liquid chromatography (HPLC) techniques are widely used for separation of phenolic compounds. Both reverse- and normal-phase HPLC methods have been used to separate and quantify PAs but have enjoyed only limited success. In reverse-phase HPLC, PAs smaller than trimers are well separated, while higher oligomers and polymers are co-eluted as a broad unresolved peak [8,13,37]. For our reverse-phase analyses, HPLC separation was achieved using a reverse phase. Cl8, 5 (Jtm 4.6 X 250 mm column (J. T. Baker, http //www.mallbaker.com/). Samples were eluted with a water/acetonitrile gradient, 95 5 to 30 70 in 65 min, at a flow rate of 0.8 mL/min. The water was adjusted with acetic acid to a final concentration of 0.1%. All mass spectra were acquired using a Bruker Esquire LC-MS equipped with an electrospray ionization source in the positive mode. 

In reversed-phase HPLC, the E vitamers are eluted in the order <5-T-3, y-T-3, y3-T-3 (unresolved), a-T-3, <5-T, y-T, (3-T (unresolved), and a-T. This elution profile contrasts with that obtained using normal-phase chromatography, in which a-T is eluted first and 5-T-3 is eluted last. The positional (3 and y isomers of tocopherols and tocotrienols cannot be separated using reversed-phase columns of standard dimensions, even if gradient elution is used. a-Tocopheryl acetate elutes immediately in front of a-tocopherol, with baseline separation. 

Figure 1. Reversed-phase HPLC chromatograms of human relaxin side fraction before and after reduction with dithiothreitol. The chromatography was performed on a Vydac C4 column using TFA-containing mobile phases, and eluted with an acetonitrile linear gradient from 18 to 50%.

Reversed phase HPLC was performed using fused silica columns and solvent delivery systems developed and built in our laboratory (5,6). All chromatographies were carried out on Vydac S itm C18 RP support with or without an SDS removal precolumn. SDS removal resin was obtained from Poly-LC Solvent A was 0.1 % trifluoroacetic acid (TFA) in water and solvent B was 0.07 % TFA, 90 % acetonitrile in water. Water was obtained from a Milli-Q system. Samples were eluted with a gradient from 2% to 92 % solvent B in 45 minutes unless otherwise noted As a standard, cytochrome C digested with Lys-C (CCKCD) was used The separation of the peptides was carried out with or without SDS. 

The evaluation of alkamides, as an indicator for standardization, in Echinacea preparations is commonly completed using HPLC. The method of Bauer (1999b) illustrates the most common method to evaluate alkamides using reverse-phase HPLC. In this method, the lipophilic components are separated by gradient elution using water (eluent A) and acetonitrile (eluent B) linearly from 40% to 80% eluent B at 1 ml/min. The separation was completed on a C18 reverse-phase column and detection was at 254 nm. Thin-layer chromatography using silica 60 plates with... 

Measurement of labelling yield and subsequent radiochemical purity requires a suitable analytical technique, and the method of choice for radio-labelled peptides is reversed phase HPLC with on-line UV and radiometric detection. It is important to use as stringent a separation method as possible with isocratic or slow mobile phase composition gradients over the peptide peak. Ideally, more than one mobile phase system should be used (e.g. a phosphate buffer-methanol system in addition to the standard water-acetonitrile system), since these may show the presence of new impurities. It is important to recognize that HPLC analyses only measure those components that elute from the column. Insoluble, highly lipophilic or positively charged species may bind to the solid phase. It is very important to verify the absence of these species by a complimentary technique such as thin layer chromatography (TLC) and to ensure that the two techniques produce similar results. 

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