Columns, reversed-phase HPLC

Mattila et al. (205) described a two-dimensional HPLC procedure for determining vitamin D3 and 25-hydroxyvitamin D3 in meat and milk products. Samples were saponified in the presence of vitamin D2 and 25-hydroxyvitamin D2 as internal standards, and the extracted un-saponifiable matter was subjected to normal-phase semipreparative HPLC to obtain a fraction containing 25-hydroxyvitamin D2 + 25-hydroxyvitamin D3 and a fraction containing vitamin D2 + vitamin D3. The collected fractions were evaporated and purified by reversed-phase HPLC. Fractions were again collected, after which vitamin D3 was quantified by tandem-column reversed-phase HPLC and 25-hydroxyvitamin D3 by tandem-column normal-phase HPLC. Analytical chromatograms of a purified extract of chicken are shown in Fig. 12. 

Reversed phase HPLC is only rarely used for the chromatographic resolution of saccharides however, in instances where only occasional separations are required the wide availability of reversed phase columns in many laboratories saves the purchase of a specialised carbohydrate column. Reversed phase HPLC has been used primarily for the separation of oligosaccharides and separation is dependent on the degree of polymerisation (Cheetham et al., 1981). Organic phase modifiers such as -alkylamines can be used to provide increased capacity and selectivity for saccharides (Lochmuller and Hill, 1983). 

Bondapak Cjg Column Reversed Phase HPLC. A method of quantitative determination of opium alkaloids on Bondapak Cig column was standardized by Akhila and Uniyal (1983) at our Institute. It operates under the conditions noted below ... 

CN Column Reversed Phase HPLC. Another improved method of straw analysis was recently developed by Verma et al. (1990) at our Institute. This method gives better recovery of alkaloids when the sample is extracted with 2.5% aqueous acetic acid in combination with lime, instead of aqueous acetic acid alone and when the mobile phase of 1% sodium acetate in water (pH adjusted to 7.4 with glacial acetic acid)-acetonitrile-tetrahydrofuran (80 16 4) is used rather than the mobile phase of 1% sodium acetate in water (pH adjusted to 6.78 with glacial acetic acid)-acetonitrile-l,4-dioxane (75 20 5), used by Srivastava and Maheshwari (1985). 

Vitamin K HPLC has provided the first assay of the phylloquinones and menaquinones that constitute vitamin K in plasma. Phylloquinone circulates bound to lipoproteins from which it can be extracted with hexane after ethanol protein precipitation. Removal of co-eluted lipids can be achieved with normal-phase cartridge columns. Reversed-phase HPLC is almost universally used for vitamin K measurement. Either UV (270 nm) or electrochemical detection is suitable. Electrochemical detection uses the reductive mode ( —1.3 V) to convert the quinone moiety to hydroquinone the main disadvantage being the need to remove oxygen from the mobile phase. 

If the hGH is exported to the culture medium the product can easily be collected by removal of the cells from the culture medium by centrifiigation. Purification of hGH from the culture medium is faciUtated by low amounts of contaminating proteins present. In fact, it has been shown that hGH can be purified on a laboratory scale by a single purification step on a reversed-phase hplc column (43). Mammalian cells growing in tissue culture have also been used as hosts to produce hGH, which is exported into the culture media (44). 

Reversed-phase hplc has been used to separate PPG into its components using evaporative light scattering and uv detection of their 3,5-dinitroben2oyl derivatives. Acetonitrile—water or methanol—water mixtures effected the separation (175). Polymer glycols in PUR elastomers have been identified (176) by pyrolysis-gc. The pyrolysis was carried out at 600°C and produced a small amount of ethane, CO2, propane, and mostiy propylene, CO, and CH4. The species responsible for a musty odor present in some PUR foam was separated and identified by gc (Supelco SP-2100 capillary column)... 

An on-line concentration, isolation, and Hquid chromatographic separation method for the analysis of trace organics in natural waters has been described (63). Concentration and isolation are accompHshed with two precolumns connected in series the first acts as a filter for removal of interferences the second actually concentrates target solutes. The technique is appHcable even if no selective sorbent is available for the specific analyte of interest. Detection limits of less than 0.1 ppb were achieved for polar herbicides (qv) in the chlorotriazine and phenylurea classes. A novel method for deterrnination of tetracyclines in animal tissues and fluids was developed with sample extraction and cleanup based on tendency of tetracyclines to chelate with divalent metal ions (64). The metal chelate affinity precolumn was connected on-line to reversed-phase hplc column, and detection limits for several different tetracyclines in a variety of matrices were in the 10—50 ppb range. 

PROBLEMS OF SIMILAR COLUMN IN QUANTITATIVE ANALYSIS BY REVERSED-PHASE HPLC... 

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.]... 

In 1993, Jorgenson s group improved upon then earlier reverse phase HPLC-CZE system. Instead of the six-port valve, they used an eight-port electrically actuated valve that utilized two 10-p.L loops. While the effluent from the HPLC column filled one loop, the contents of the other loop were injected onto the CZE capillary. The entii e effluent from the HPLC column was collected and sampled by CZE, making this too a comprehensive technique, this time with enhanced resolving power. Having the two-loop valve made it possible to overlap the CZE runs. The total CZE run time was 15 s, with peaks occurring between 7.5 and 14.8 s. In order to save separation space, an injection was made into the CZE capillary every 7.5s,... 

A six-port valve was first used to interface the SEC microcolumn to the CZE capillary in a valve-loop design. UV-VIS detection was employed in this experiment. The overall run time was 2 h, with the CZE runs requiring 9 min. As in the reverse phase HPLC-CZE technique, runs were overlapped in the second dimension to reduce the apparent run time. The main disadvantage of this yu-SEC-CZE method was the valve that was used for interfacing. The six-port valve contributed a substantial extracolumn volume, and required a fixed volume of 900 nL of effluent from the chromatographic column for each CZE run. The large fixed volume imposed restrictions on the operating conditions of both of the separation methods. Specifically, to fill the 900 nL volume, the SEC flow rate had to be far above the optimum level and therefore the SEC efficiency was decreased (22). 

Stopher and Gage used size-exclusion chromatography (SEL) coupled to reversed phase HPLC for the direct injection of plasma in the analysis of an antifungal agent, voriconazole (12). Their system consisted of three columns, i.e. first a size-exclusion... 

Purification of the activation products (PMs). The methylamine activation product dissolved in methanol is purified by chromatography, first on a column of silica gel using a mixed solvent of chloroform/ethanol, followed by reversed-phase HPLC on a column of divinylbenzene resin (such as Jordi Reversed-Phase and Hamilton PRP-1) using various solvent systems suitable for the target substance (for example, acetonitrile/water containing 0.15% acetic acid). 

Some authors have suggested the use of fluorene polymers for this kind of chromatography. Fluorinated polymers have attracted attention due to their unique adsorption properties. Polytetrafluoroethylene (PTFE) is antiadhesive, thus adsorption of hydrophobic as well as hydrophilic molecules is low. Such adsorbents possess extremely low adsorption activity and nonspecific sorption towards many compounds [109 111]. Fluorene polymers as sorbents were first suggested by Hjerten [112] in 1978 and were tested by desalting and concentration of tRN A [113]. Recently Williams et al. [114] presented a new fluorocarbon sorbent (Poly F Column, Du Pont, USA) for reversed-phase HPLC of peptides and proteins. The sorbent has 20 pm in diameter particles (pore size 30 nm, specific surface area 5 m2/g) and withstands pressure of eluent up to 135 bar. There is no limitation of pH range, however, low specific area and capacity (1.1 mg tRNA/g) and relatively low limits of working pressure do not allow the use of this sorbent for preparative chromatography. 

The purified protein was subjected to reversed-phase HPLC analysis by using a 150 X 1 mm Cig column with gradient elution from 0.1% aqueous trifluo-roacetic acid (TEA) to 0.1% TEA in acetonitrile, over a period of 55 min, at a flow... 

The stationary phase matrices used in classic column chromatography are spongy materials whose compress-ibihty hmits flow of the mobile phase. High-pressure liquid chromatography (HPLC) employs incompressible silica or alumina microbeads as the stationary phase and pressures of up to a few thousand psi. Incompressible matrices permit both high flow rates and enhanced resolution. HPLC can resolve complex mixtures of Upids or peptides whose properties differ only slightly. Reversed-phase HPLC exploits a hydrophobic stationary phase of... 

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. ... 

Compounds that were included in the pharmacologic profile of [ H]MDA binding were subjected to reverse-phase HPLC analysis to assess their relative lipophilicity. Briefly, each compound (10 pg) was injected onto a Waters Nova-Pak C18 column and eluted with a linear gradient from 95 percent buffer A 5 percent buffer B to 20 percent buffer A 80 percent buffer B (buffer A=95 percent water, 5 percent acetonitrile, 0.1 percent ammonium acetate buffer B=20 percent water, 80 percent acetonitrile,... 

Reversed-phase HPLC followed by post-column derivatization and subsequent fluorescence detection is the most common technique for quantitative determination of oxime carbamate insecticides in biological and environmental samples. However, for fast, sensitive, and specific analysis of biological and environmental samples, detection by MS and MS/MS is preferred over fluorescence detection. Thus, descriptions and recommendations for establishing and optimizing HPLC fluorescence, HPLC/ MS, and HPLC/MS/MS analyses are discussed first. This is followed by specific rationales for methods and descriptions of the recommended residue methods that are applicable to most oxime carbamates in plant, animal tissue, soil, and water matrices. 

HPLC with thermospray MS was reported by Hurst et al.40 where residues from an archeological site were analyzed for caffeine and theobromine using reversed phase HPLC coupled to a thermospray MS interface. Samples were extracted in water and separated on a reversed phase column. The presence of theobromine in this sample was confirmed by monitoring the MH+ ion at 181 for theobromine. 

Kobayashi, H., Kajiwara, W., Inui, Y., Hara, T., Hosoya, K., Ikegami, T., Tanaka, N. (2004). Chromatographic properties of monolithic silica capillary columns for polar and nonpolar compounds in reversed-phase HPLC. Chromatographia 60, S19-S25. 

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