HPLC column connecting

Application of HPLC-MS to the analysis of a black tea liquor was studied in a paper by Bailey 39 a great deal of useful information could be obtained without sample pretreatment. A tea liquor was applied to a wide-pore HPLC column connected to a mass spectrometer by a VG Plasmaspray interface. Pseudo-molecular ions were obtained from the flavanols, flavanol gallates, chlorogenic acids, 4-coumarylquinic acids, and caffeine, but the flavanol glycosides were extensively fragmented by the interface. Fragments were obtained from unresolved polymer that supported its previous designation as a flavanol polymer. 

Although most Ag-HPLC analyses have traditionally been done using CLA methyl esters, improved separation and quantitation of CLA isomers has been noted when phenacyl or p-methoxyphenacyl ester rather than methyl ester derivatives were analyzed (58). Nikolova-Damyanova et al. (59) demonstrated improved separation of the p-methoxyphenacyl derivatives of the cis/trans 8,10- through 11,13-18 2 isomers in a commercial sample of CLA when compared with CLAME. Only a single Ag-HPLC column was required. Resolution of the cis/ trans isomers was similar to that obtained for CLAME using Ag-HPLC systems with two or three Ag-HPLC columns connected in series (60). A stepwise solvent gradient of 100% hexane/dichloromethane/ACN (40 60 0.2 vol/vol/vol 30 min) to 100% dichloromethane/ACN (100 1) over 10 min with UV detection at 270 nm (for phenacyl esters) yielded a semiquantitative estimation of fatty acid composition comparable to, but more detailed than that obtainable by GC analysis as FAME. This method was also applied to detamine the CLA isomer composition of a sample of beef/pig fat, but prefractionation by RP-HPLC was required before Ag-HPLC analysis. The prefractionation step was required to concentrate the CLA isomers and to remove octadecenoate and other fatty acids that might interfere with Ag-HPLC analysis. 

Ag-HPLC was applied recently to the analysis of a CLA-enriched TAG formulation. Utilizing two, three or four Ag-HPLC columns connected in series and isocratic solvent systems of 0.6-1.0% ACN in hexane, Adlof et al. (65) analyzed a commercially available CLA-enriched TAG formulation. A pattern of four minor and one major peak was obtained (Fig. 3.5), with peak 1 = mono-CLA/2 miscellaneous FA peak 2 = di-CLA/monosaturated FA peak 3 = di-CLA/9c-18 l peak 4 = Tri-CLA and peak 5 = di-CLA/mono cis/cis CLA isomer. (The term CLA is used in this instance for both the 9,11- and the 10,12-isomers.) A minimum of three columns in series was required to achieve at least 50% separation of peaks 3 and 4. The last-eluting part of peak 4 (peak 5) was also isolated and found to be composed predominately of TAG with the structure di-9c,lU-18 2/mono-c/5/c/5 -CLA (where cis/cis refers to 9c,llc- and 10c,12c-18 2). 

Ag-HPLC Semipreparative Applications. Ag-HPLC (4.6 x 250 mm colunms) may also be used for the semipreparative isolation of CLAME. Two Ag-HPLC columns connected in series were used to isolate miUigram quantities of CLAME isomers (Adlof, R.O., unpubUshed data Fig. 3.7) from a mixture of 78.8% 9/,ll/-18 2/21.2% 9c,ll/-18 2 (both 17,17,18,18-d4). Resolution of the two CLAME, which decreased with increasing weights of samples injected, was maintained at >95% of baseline by deaeasing the percentage of ACN in the isooadc hexane/ACN... 

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. 

The major advance in the way in which column eluate is deposited on the belt was the introduction of spray deposition devices to replace the original method which was simply to drop liquid onto the belt via a capillary tube connected directly to the outlet of the HPLC column. These devices, based on the gas-assisted nebulizer [5], have high deposition efficiencies, transfer of sample can approach 100% with mobile phases containing up to 90% water, and give constant sample deposition with little band broadening. 

The following operating guides are recommended for connecting HPLC columns ... 

Pharmacia MonoQ column 3. Eguilibrate a column connected to HPLC machine... 

ODS2 columns connected in series (Shandon HPLC, 120x4.6mm, 3pm),... 

The inlet line to the gas-liquid separator should be removed and connected to a plastic T-piece, which is also connected to the outlet from the HPLC column. The T-piece should then be connected to the gas-liquid separator with 7 cm of Teflon tubing. 

Using the coupling procedure described, 3-4 mg of affinity-purified anti-RIP antibody can be coupled to the HPLC column matrix The procedure requires a minimum of 6 mL of anti-RIP antibody solution to allow cycling of the solution via the liquid reservoir, prepump solvent filter, pump, precolumn filter, the column itself, and all the connecting tubing. 

Since FAB (or LSIMS) requires that the analyte be dissolved in a liquid matrix, this ionization technique was easily adapted for infusion of solution-phase samples into the FAB ionization source, in an approach known as continuous-flow FAB. Continuous-flow FAB was connected to microbore HPLC columns for LC/MS applications (Ito et al., 1985). Since this method is limited to microbore HPLC applications at flow rates of <10 pl/min... 

Mannio and Cosio (78) described a sensitive, specific, and rapid chromatographic procedure to determine BA and SA in different food products. Benzoic acid and SA were extracted from foods by a microdialysis probe connected online to an HPLC column that allows separation of BA and SA. Detection was done at 228 and 260 nm for BA and SA, respectively. The procedure was linear from 1 to 80 ppm, with a detection limit of 1 ppm for SA and 2 ppm for BA. The assay was successfully applied to soft drinks, fruit juices, and dairy products (cheese, yogurt, and cream). 

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. 

For the on-line SFE-NMR experiments, the set-up shown in Figure 7.2.17 can be used. A main pump serves an HP supercritical fluid chromatograph (G1205A), with analytical HPLC columns being used as extraction cells. The continuous-flow NMR cell is connected between the column outlet and the back-pressure regulator. 

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