Reversed phase analytical column

Gundersen and Blomhoff (1999) used online dilution with online SPE to measure vitamin A (retinol) and other active retinoids in animal plasma. The intention of online dilution in this application was on optimizing SPE extraction conditions rather than on peak focusing during analytical separation. An SPE cartridge packed with Bondapak C18 materials (37 to 53 jt/M, 300 A, Waters, Milford, Massachusetts) and a reversed-phase analytical column (250 x 2.1 mm inner diameter, Superlex pkb-100, Supelco, Bellefonte, Pennsylvania) were controlled by a six-port switching valve (Rheodyne, Cotati,... 

Two analytical methods for priority pollutants specified by the USEPA (38) use HPLC separation and fluorescence or electrochemical detection. Method 605, 40 CFR Part 136, determines benzidine and 3,3-dichlorobenzidine by amperometric detection at +0.80 V, versus a silver/silver chloride reference electrode, at a glassy carbon electrode. Separation is achieved with a 1 1 (v/v) mixture of acetonitrile and a pH 4.7 acetate buffer (1 M) under isocratic conditions on an ethyl-bonded reversed-phase column. Lower limits of detection are reported to be 0.05 /xg/L for benzidine and 0.1 /xg/L for 3,3-dichlorobenzidine. Method 610, 40 CFR Part 136, determines 16 PAHs by either GC or HPLC. The HPLC method is required when all 16 PAHs need to be individually determined. The GC method, which uses a packed column, cannot adequately individually resolve all 16 PAHs. The method specifies gradient elution of the PAHs from a reversed-phase analytical column and fluorescence detection with an excitation wavelength of 280 nm and an emission wavelength of 389 nm for all but three PAHs naphthalene, acenaphthylene, and acenaphthene. As a result of weak fluorescence, these three PAHs are detected with greater sensitivity by UV-absorption detection at 254 nm. Thus, the method requires that fluores-... 

Colistin (COL) is a multicomponent antibiotic (polymyxins E) that is produced by strains of inverse Bacillus polymyxa. It consists of a mixture of several closely related decapeptides with a general structure composed of a cyclic heptapeptide moiety and a side chain acetylated at the N-terminus by a fatty acid. Up to 13 different components have been identified. The two main components of colistin are polymyxins El and E2 they include the same amino acids but a different fatty acid (216). A selective and sensitive HPLC method was developed for the determination of COL residues in milk and four bovine tissues (muscle, liver, kidney, and fat). The sample pretreatment consists of protein precipitation with trichloracetic acid (TCA), solid-phase purification on Cl 8 SPE cartridges, and precolumn derivatization of colistin with o-phthalaldehyde and 2-mercaptoethanol in borate buffer (pH 10.5). The last step was performed automatically, and the resulting reaction mixture was injected into a switching HPLC system including a precolumn and the reversed-phase analytical column. Fluorescence detection was used. The structural study of El and E2 derivatives was carried out by HPLC coupled with an electrospray MS. Recoveries from the preseparation procedure were higher than 60%. 

C18 reversed-phase analytical column Methanol-ammonium acetate buffer (50 50, v/v), pH 3.2 UV detection at 232 nm Determination in human plasma [15]... 

Foda et al. have developed a reversed-phase chromatography method using C18 reversed-phase analytical column. 

In another method, creams and ointments of a developmental corticosteroid are simply dissolved in tetrahydrofuran-isopropanol (30 60), clarified by centrifugation, and injected directly onto the HPLC column [331]. The automated switching valves direct the analytes plus an internal standard through the guard column to the reversed-phase analytical column,... 

Kerr el. al. [307] employed high performance liquid chromatography for the determination of uranium in groundwaters. The sample was passed through a small reversed phase enrichment cartridge, to separate the uranium from the bulk of the dissolved constituents. The uranium was then back flushed from the cartridge onto a reversed phase analytical column. The separated species were monitored spectrophotometrically after reaction with arsenazo(III). The detection limit was in the 1-2gg L 1 range with a precision of approximately 4%. 

The solution of [15N]r-metHuLeptin (0.3 mg/ml) in PBS (0.1 M sodium phosphate, 0.1 M sodium chloride, pH 7.2) was incubated with endoproteinase Asp-N at an enzyme-to-substrate ratio of 1 75 (w/w) at 25 °C for 5 h. The digestion was terminated by adding 5 )tL of 5% TFA to the reaction. Peptides were separated by a Vydac C4 reverse-phase analytical column (4.6x250 mm) using a Hewlett Packard HPLC (Model 1090), which is on-line connected to a PE-Sciex API-100 electrospray mass spectrometer. The column was initially equilibrated with 95% mobile phase A (0.1%TFA) and 5% mobile phase B (90% acetonitrile in 0.1% TFA). A linear gradient from 10 to 50% mobUe phase B was run over a period of 85 minutes at a flow rate of 0.5 raL/min. The splitting of the flow (9 1) was achieved post UV cell, allowing 50 iL/min of the eluent to be analyzed by the electrospray mass spectrometer. 

High-pressure liquid chromatography (HPLC) formats that use reverse-phase analytical columns dominate the bioanalytical field. Isocratic elution formats are used for traditional LC-MS/MS bioanalysis, since these separations do not require additional time for column reequilibration. However, gradient elution is a more effective approach for cassette analysis in highthrough-put screening methods for discovery applications because of its versatility for the separation of compounds that have a wide variety of lipophilicity. 

The LAS homologues were then separated by HPLC on an octadecyl reversed phase analytical column (u-Bondapak, Waters Associates or Spherisoib ODS2, Phase Sep) using gradient or isocratic elutioaDetection was made by either UV or fluorescence.Details of the HPLC conditions are summarised in Table 1... 

HPLC analytical column to a pH, ionic strength, and polarity that are appropriate for an lAC application buffer. This factor is especially important when using immunodetection with RPLC because an appreciable amount of organic modifier may be present in the mobile phase leaving the reversed-phase analytical column, which may damage the lAC column. One way to avoid this problem is to combine the analytical column eluent with a dilution buffer prior to sample application onto the lAC column. Immunodetection by the on-off mode also requires that the eluting analyte be present in a conformation that is recognized by the antibodies in the lAC column. 

For LC separation, various reverse-phase analytical columns can be used (Table 1). Most commonly. Cl8 columns are used. The LC column used for the analytical method described below is an XTerra C MS, 3.5 pm (150x2.1 mm I.D.) reversed-phase column (Waters, Milford, MA). 

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. 

Well water Extracted/concentrated using reverse phase SPE columns. Eluted analytes withmethanol. GC (detection not specified) No data 58-67 Hogmire et al. 1990... 

In reversed-phase HPLC, column temperature is a strong determinant of retention time and also affects column selectivity. A column oven is therefore required for most automated pharmaceutical assays to improve retention time precision, typically at temperatures of 30-50°C. Temperatures >60°C are atypical due to concerns about thermal degradation of the analytes and column lifetimes. Exceptions are found in high-throughput screening where higher temperatures are used to increase flow and efficiency. Ambient or snb-ambient operation is sometimes found in chiral separations to enhance selectivity. Column ovens... 

The injector, columns and valves reside in a low temperature chamber to minimize the loss of deuterium by back exchange (Fig. 12.2). The quenched protein solution is pumped in series through a column containing an immobilized protease and a trap column to capture the peptide fragments. The gradient pump is activated following digestion and the peptides captured on the trap column are eluted and separated over an analytical reverse-phase HPLC column directly into the mass spectrometer. 

Abidi and Mounts separated the molecular species of the neutral phospholipids PC and PE using A/M/W (70/22/8) containing tetra-alkyl ammonium phosphates (TAAPs). The competitive interaction of TAAPs and analyte solutes with the reversed-phase HPLC column resulted in... 

The determination of OCP residues in milk has always presented problems, because the most common approach has required the total extraction of fat, together with lipophilic compounds, including organochlorine pesticide residues. Only one procedure for the extraction and separation of OCPs directly into an HPLC system has been described (11). The direct procedure injects the samples into an internal-surface reversed-phase C18 column connected online with the analytical column. 

Analytes are often isolated from various sample preparations using reversed-phase extraction columns. However, extraction efficiency decreases as the organic solvent... 

Moore and Jorgenson combined the rapid two-dimensional separation achieved by LC-CZE with SEC to make the first comprehensive three-dimensional separation involving an electrodriven component in 1995. Size exclusion chromatography separated the analytes over a period of several hours while the reverse phase HPLC-CZE combination separated components in only 7 min. A schematic diagram of the three-dimensional SEC-reverse phase HPLC-CZE instrument is shown in Figure 9.9 (18). A dilution tee was placed between the SEC column and the reverse phase HPLC injection loop in order to dilute the eluent from the SEC column, since it contained more methanol than was optimal for the reverse phase HPLC column. 

Kang et al. [61] developed an advanced and sensitive HPLC method for the determination of omeprazole in human plasma. After omeprazole was extracted from plasma with diethylether, the organic phase was transferred to another tube and trapped back with 0.1N sodium hydroxide solution. The alkaline aqueous layer was injected into a reversed-phase C8 column. Lansoprazole was used as the internal standard. The mobile phase consisted of 30% of acetonitrile and 70% of 0.2 M potassium dihydrogen phosphate, pH 7. Recoveries of the analytes and internal... 

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