Reversed phase HPLC

At the pH values commonly used in reversed phase HPLC the nucleotides are negatively charged and generally this mode is used for 

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The resulting oligonucleotide is often of surprising purity as judged by analytic HPLC or electrophoresis, and up to 30 mg of a deoxyeicosanucleotide (20-base DNA) can be routinely obtained. Nevertheless small amounts of short sequences, resulting from capping and from base-catalysed hydrolysis, must always be removed by quick gel filtration, repeated ethanol precipitation from water (desalting), reverse-phase HPLC, gel electrophoresis, and other standard methods. 

A reverse-phase HPLC separation is carried out using a mobile-phase mixture of 60% v/v water and 40% v/v methanol. What is the mobile phase s polarity index ... 

Solvent triangle for optimizing reverse-phase HPLC separations. Binary and ternary mixtures contain equal volumes of each of the aqueous mobile phases making up the vertices of the triangle. 

Bohman and colleagues described a reverse-phase HPLC method for the quantitative analysis of vitamin A in food using the method of standard additions. In a typical example, a 10.067-g sample of cereal is placed in a 250-mL Erlenmeyer flask along with 1 g of sodium ascorbate,... 

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)... 

Recovery and Purification. The dalbaheptides are present in both the fermentation broth and the mycelial mass, from which they can be extracted with acetone or methanol, or by raising the pH of the harvested material, eg, to a pH of 10.5—11 for A47934 (16) (44) and A41030 (41) and actaplanin (Table 2) (28). A detailed review on the isolation of dalbaheptides has been written (14). Recovery from aqueous solution is made by ion pair (avoparcin) or butanol (teicoplanin) extraction. The described isolation schemes use ion-exchange matrices such as Dowex and Amberlite IR, acidic alumina, cross-linked polymeric adsorbents such as Diaion HP and Amberlite XAD, cation-exchange dextran gel (Sephadex), and polyamides in various sequences. Reverse-phase hplc, ion-exchange, or affinity resins may be used for further purification (14,89). 

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. 

Numerous high pressure Hquid chromatographic techniques have been reported for specific sample forms vegetable oHs (55,56), animal feeds (57,58), seta (59,60), plasma (61,62), foods (63,64), and tissues (63). Some of the methods requite a saponification step to remove fats, to release tocopherols from ceHs, and/or to free tocopherols from their esters. AH requite an extraction step to remove the tocopherols from the sample matrix. The methods include both normal and reverse-phase hplc with either uv absorbance or fluorescence detection. AppHcation of supercritical fluid (qv) chromatography has been reported for analysis of tocopherols in marine oHs (65). 

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

The understanding of retention and selectivity behaviour in reversed-phase HPLC in order to control and predict chromatographic properties ai e interesting for both academic scientists and manufacturers. A number of retention and selectivity models are the subject of ongoing debate. The theoretical understanding of retention and selectivity, however, still lags behind the practical application of RP HPLC. In fact, many users of RP HPLC techniques very often select stationary phases and other experimental conditions by experience and intuition rather than by objective criteria. 

The teehniques of membrane extraetion permit an effieient and modern applieation of elassieal liquid-liquid extraetion (LLE) ehemistry to instmmental and automated operation. Various shorteomings of LLE are overeome by membrane extraetion teehniques as they use none or very little organie solvents, high enriehment faetors ean be obtained and there ai e no problems with emulsions. A three phase SLM system (aq/org/aq), where analytes are extraeted from the aqueous sample into an organie liquid, immobilized in a porous hydrophobie membrane support, and further to a seeond aqueous phase, is suitable for the extraetion of polar eompounds (aeidie or basie, ehai ged, metals, ete.) and it is eompatible with reversed phase HPLC. A two-phase system (aq/org) where analytes ai e extraeted into an organie solvent sepai ated from the aqueous sample by a hydrophobie porous membrane is more suitable for hydrophobie analytes and is eompatible with gas ehromatography. 

We proposed using MLC for assay of azithromycin in tablets and capsules. As alternative conventional reversed-phase HPLC method MLC was used for analysis of Biseptol (sulfamethoxazole and trimethoprim) tablets and injection. The MLC was proposed to assay of triprolydine hydrochloride and pseudoephedrine hydrochloride in tablets as alternative normal-phase HPLC method described in USP phamiacopoeia. 

Obtained P values for the description of benzodiazepine s retention in reversed-phase HPLC are used. The equation... 

A selective, sensitive and stability indicating reversed phase-HPLC method was developed for the determination of clarithromycin antibiotic in human plasma. 

A.M. Krstulovic and P.R. Brown, Reversed Phase HPLC. Theory, Practical and Biomedical Applications, J. Wiley Sons, New York, 1982. ISBN 0471053694. 

G. Szepesi, How to Use Reverse Phase HPLC, VCH, New York, 1992. ISBN 0895737663. 

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.]... 

Interleukin-2 (recombinant human) [94218-72-1] Mr-15,000, amorphous. Purified by reverse phase HPLC. [Weir and Sparks Biochem J 245 85 1 987 Robb et al. Proc Natl Acad Sci USA 81 6486 1984.]... 

Phenylurea herbicides (urons). Dinocap, Dinoseb, Benomyl, Carbendazim and Metamitron in Waters [e.g. determination of phenylurea herbicides by reverse phase HPLC, phenylurea herbicides by dichloromethane extraction, determination by GC/NPD, phenylurea herbicides by thermospray LC-MS, Dinocap by HPLC, Dinoseb water by HPLC, Carbendazim and Benomyl (as Carbendazim) by HPLC], 1994... 

This group, used for 5 -phosphate protection, has hydrophobicity similar to that of the dimethoxytrityl group and thus was expected to assist in reverse-phase HPLC purification of product from failure sequences in oligonucleotide synthesis. The group is cleaved with Bu4N F in DMSO at 70°. ... 

This highly lipophilic group is cleaved with isoamyl nitrite in Pyr/AcOH. The use of a lipophilic 5 -phosphate protective group aids in reverse-phase HPLC purification of oligonucleotides. 

Quantitation of the oral bronehodilator 2,5-diethyl-7-(tetrahydro-l,4-thiazin-4-yl)-l,2,4-triazolo[l,5-c]pyrimidine (R-836) (195) in plasma and urine of humans and experimental animals utilized reversed-phase HPLC and UV deteetion (88MI1). 

T. Hyotylainen, K. Grob, M. Biedermann and M-L. Riekkola, Reversed phase HPLC coupled on-line to GC by the vaporizer/precolumn solvent split/gas dischar ge analysis of phthalates in water , 7. High Resolut. Chromatogr. 20 410-416 (1997). 

Other groups have also used EC and CE to perform non-comprehensive multidimensional separations (15, 16). A three-dimensional separation was performed by Stromqvist in 1994, where size exclusion chromatography (SEC), reverse-phase HPLC, and CZE were used in an off-line manner to separate peptides (17). The most useful information gained from all of these non-comprehensive studies was knowledge of the orthogonality and compatibility of EC and CE. 

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,... 

The only other group to have performed comprehensive multidimensional reverse-phase HPLC-CZE separations is at Hewlett-Packard. In 1996, a two-dimensional LC-CE instrument was described at the Erederick Conference on Capillary Electrophoresis by Vonda K. Smith (21). The possibility for a commercial multidimensional instrument may have been explored at that time. 

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). 

Moore and Jorgenson eombined the rapid two-dimensional separation aehieved by LC-CZE with SEC to make the first eomprehensive three-dimensional separation involving an eleetrodriven eomponent in 1995. Size exelusion ehromatography separated the analytes over a period of several hours while the reverse phase HPLC-CZE eombination separated eomponents in only 7 min. A sehematie diagram of the three-dimensional SEC-reverse phase HPLC-CZE instrument is shown in Eigure 9.9 (18). A dilution tee was plaeed between the SEC eolumn and the reverse phase HPLC injeetion loop in order to dilute the eluent from the SEC eolumn, sinee it eon-tained more methanol than was optimal for the reverse phase HPLC eolumn. 

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