Vydac columns

Although some normal phase methods have been used, the majority of carotenoid separations reported in the literature were carried out by reversed phase HPLC. Among the Cjg columns employed for determination of complete carotenoid compositions in foods, the polymeric Vydac brand is preferably used for separation of cis isomers. Several examples of different C,g columns and mobile phases are cited in the literature, but not aU carotenoids are baseline separated in most systems. Table 6.2.1 shows some examples employing different brands of Cjg columns." Acetonitrile did not improve selectivity toward separation of carotene isomers in a Vydac 201TP column and resolution was strongly dependent on the Vydac column lot. ... 

Independently of the reversed phase column, the addition of TEA to the mobile phase increases carotenoid recovery from the column." Increased recoveries of 18% lutein, 33% zeaxanthin, 33% P-cryptoxanthin, 53% lycopene, 30% a-carotene, and 42% P-carotene in a Vydac column were observed after the addition of 0.1% TEA to the mobile phase. Recovery on a C30 column was also enhanced by the addition of 0.1% TEA to the mobile phase, with the peak area of lutein increasing by 26%, that of zeaxanthin by 42%, that of P-cryptoxanthin by 55%, that of lycopene by 21%, and those of a-carotene and P-carotene by 47 and 64%, respectively. ... 

FIGURE 16.8 HPLC chromatogram of cytochrome c and myoglobin digest, using a 250 cm x 4.6 mm ODS C18 Vydac column and a linear mobile-phase gradient, 5-50% B, in 50 min. Buffer A was 0.1% TFA in water and buffer B was 0.1 TFA in acetonitrile. UV detection was carried out at 214 nm, at room temperature (reprinted with permission from Electrophoresis). 

Instrumentation. HPLC isolations were performed on a Beckman 421A system using a Vydac column (C-18, 4.6 x 250 mm). Liquid secondary ion mass spectra (LSIMS) were recorded in the positive ion mode on a Kratos (Manchester, UK) MS-50S mass spectrometer equipped with a 23 kG magnet and post-acceleration detector. The LSIMS ion source has been described elsewhere (28). A Cs+ ion beam of energy 10 keV was used as the primary beam (21). Spectra were recorded (300 sec per decade) with a Gould ES-1000 electrostatic recorder. Tandem MS experiments were performed on a Kratos Concept IIHH (Manchester, UK) four sector instrument of EBEB... 

Figure 14.4 Comparison between plots of the HETP (cm) and of the SLT (cm) versus the mobile phase flow velocity. Same experimental conditions for both figures 5 cm long Vydac column. Mobile phase 50 50 methanol-water, monitored at 270 nm for both series of measurements. Sample 2-phenylethanol (fcg = 0.88). Height of the concentration step in frontal analysis 20 mg/mL. Sample size for linear elution peaks 40 fig (0.2 fiL of a 20 mg/mL solution). Top Figure Plot of the SLT versus the mobile phase flow velocity. Experimental data (symbols) and prediction of Eq. 14.33c (solid line). Bottom Figure Plot of the HETP versus the mobile phase velocity under linear conditions. Experimental data (symbols) and best fit to the Van Deemter equation (solid line). Reproduced with permission from /. Zhu and G. Guiochon, J. Chromatogr., 636 (1993) 189 (Fig. 2).

The Dionex column caused each of the metal ions to be completely retained. The authors reasoned that this behavior was due to the presence of unreacted surface-sulfonated PS-DVB macroparticles which were capable of adsorbing the metal cations. A mixed retention mechanism was theorized for the other three columns. In addition to anion-exchange of negatively charged metal-phthalate complexes, the authors suggest that adsorption of neutral metal-phthalate complexes might contribute to the retention of each metal ion. This theory was based on the fact that since the stationary phase of the Hamilton, Waters, and Vydac columns were comprised of different materials, these materials would have different hydrophobicity which would lead to differences in adsorption of the neutral metal-phthalate complexes on each column. This was corroborated experimentally, as the metal ions were retained more strongly on the more hydrophobic column (PS-DVB from Hamilton). The dominant mechanism of retention was similar for each column because there were no differences in metal ion elution orders between the three columns. 

Fig. 3. Cation-exchange chromatography of protein standards. Column poly(aspartic acid) Vydac (10 pm), 20 x 0.46 cm. Sample 25 pi containing 12.5 pg of ovalbumin and 25 pg each of the other proteins in the weak buffer. Flow rate 1 ml/min. Weak buffer 0.05 mol/1 potassium phosphate, pH 6.0. Strong buffer same +0.6 mol/1 sodium chloride Elution 80-min linear gradient, 0-100% strong buffer. Peaks a = ovalbumin, b = bacitracin, c = myoglobin, d = chymotrypsinogen A, e = cytochrom C (reduced), / = ribonuclease A, g = cytochrome C (oxidised), h = lysozyme. The cytochrome C peaks were identified by oxidation with potassium ferricyanide and reduction with sodium dithionite [47]...

Multiple electrodes have been used to obtain selectivity in electrochemical detection. An early example involved the separation of catecholamines from human plasma using a Vydac (The Separation Group Hesperia, CA) SCX cation exchange column eluted with phosphate-EDTA.61 A sensor array using metal oxide-modified surfaces was used with flow injection to analyze multicomponent mixtures of amino acids and sugars.62 An example of the selectivity provided by a multi-electrode system is shown in Figure 2.63... 

LC-MS-MS was also the method of choice for the analysis of UV filters in solid matrices. Both LC and UPLC have been applied in three out of the four methods available for the determination of UV filters in sludge. Separation was performed on C8 and C18 LC-chromatographic columns (Zorbax, Eclipse, Vydac, and Purosphere) using binary gradient elution of mobile phases consisting of water/ methanol or water/acetonitrile. MS-MS detection was performed in SRM with ESI and atmospheric pressure photoionization (APPI) in both positive and negative modes. For each compound, two characteristics transitions were monitored. In addition to MS and MS-MS, diode array detection (DAD) was occasionally applied to the determination of OT. Spectra were recorded between 240 and 360 nm and discrete channels at 310 nm. 

BA 3,4-dihydrodiol metabolites were isolated by a reversed-phase HPLC using a Vydac C18 column (Chiu et al., unpublished results). DMBA dihydrodiol metabolites were isolated as described (42). The enantiomeric composition was determined either by CD spectral data or by CSP-HPLC (7.19.20). 

Procedure The HPLC is carried out using (a) a Vydac C18 column, for proteins and peptides, maintained at 40 °C, (b) as the mobile phase at a flow rate of 1 ml per minute, a mixture of 48 volumes of mobile phase A and 52 volumes of mobile phase B prepared and maintained at a temperature of not less than 20 °C, and (c) a detection wavelength of 214 nm. 

Kele, M. and Guiochon, G. Repeatability and Reproducibility of Retention Data and Band Profiles on Reversed-phase Liquid Chromatography Columns. V. Results Obtained with Vydac 218TP C18 Columns,/. Chromatogr. A, 913 89—112, 2001. 

NP chromatography is unable to separate vitamin D2 from vitamin Dj. So it is usually used as semipreparative chromatography [527-531]. Instead, RP chromatography is able to resolve vitamin D2 and Dj, thus it is widely applied as aualy tical chromatography. Mattila et al. [532] describe a two dimensioual LC procedure. The sample is saponified and an NP semipreparative column is used before the quantification in a tandem column set (Zorbax ODS x Vydac 201 TP54 CIS). 

Figure 1 HPLC Profiles of Crude (a) and Purified (b) Fully Protected Segment 1. Column Vydac Diphenyl (250 x 4.6 mm) Eluant 80-90% Solution B in Solution A Linear Gradient for 25 min. HPLC Profiles of H-Asn103-Val124-OH Cys(Acm)110 (c) and (d) Obtained from Deprotection of Crude (a) and Purified (b) Fully Protected Segment 1, Respectively. Column Vydac C8(250 x 4.6 mm) Eluant 20 10% Solution B in Solution A Linear Gradient 25 min (Solution A 0.1% aq TFA and Solution B 90% MeCN/10% Solution A) Detection 215 nm...

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