HPLC, packings sample filters

Both GC and HPLC columns are expensive, so it is important not to clog them during analysis. GC columns will be clogged by nonvolatile compounds. For these materials, it is important to use HPLC. Samples must also be free of suspended particles that will clog the finely packed HPLC columns. Filtering samples, especially soil extracts, before injection is essential. 

If the mobile phase contains liquids that are not certified as HPLC grade solvents, they must be filtered ahead of time as well as degassed. The reason is that the packed bed of finely divided stationary phase particles through which the mobile phase percolates is itself an excellent filter. Particles in the mobile phase as small as 0.5 jim in diameter can be filtered out. The result of this is a decreased effectiveness of the column with time and possibly a blocked flow path. Unfiltered samples also may contain particles and cause this problem. 

Samples from the 40-mL VOA vials were split for chromate and PCE analyses and typically analyzed within 48 hours of collection. Chromate concentration was determined via an HPLC method using a Gilson Model 116 UV detector set at 365 nm and a 2- by 150-mm Waters Nova-Pak C18 60A HPLC column packed with 4-pm particles. The mobile phase consisted of 5-mM tert-butylammonium hydrogen sulfate buffered to pH 4.4 with NaOH with 10% acetonitrile (v/v) as a modifier. The eluent flow rate was 0.8 mL min-i Samples were filtered through a0.45-pm filter as they were injected by an Alcott 708 autosampler with a 0.1-mL sample loop. The typical run time was 4 min with a calibration range of 0.05 to 20 mg L 1 (0.001 - 0.38mmol L 1) Cr as chromate. 

HPLC was performed using Waters 600S solvent delivery system (Waters, Milford, MA, U.S.A.). 2487 UV dual channel detector of Waters was used and injector (20 fit sample loop) from Rheodyne. The data acquisition system was Millenium (Waters). Water filtered 1 Milipore ultra-pure water system (Milipore, Bedford, MA, USA). The wavelength was fixed at 254 nm and the experiment was performed at room temperature. The size of the analytical colunm packed by C g was lS0X4.6mm (Spm) (Alltech, USA). The mobile phase of 0.75% TFA in water and acetonitrile were used in this experiment. The flow rates of the mobile phase were fixed at I ml/min. The constant volume of 0(d, was injected. This experiment was implemented at room temperature. The gradient mode was employed to isolate peptides. The complete gradient condition was listed in Table I. 

A combination of SFE and SPE as a sample preparation technique for the ultratrace analysis of mebeverine alcohol in plasma has been proposed (123). Plasma containing the analyte was applied to conditioned octadecylsi-lane cartridges. After washing and drying, the packing was placed in an SFE system. Carbon dioxide with 5% methanol was used as the SCF at 40°C and 355 bar. The extract analysis was carried out by GC-MS. A similar method was used for the extraction of flavone, also from blood plasma, but the determination was performed by HPLC in this case (124). In other applications, plasma spiked with tritiated budesonide to 93 nM was deposited onto a filter paper placed in the extraction vessel, where the extraction was performed with pure CO2 for 30 min then, the budesonide in the extract was determined with a liquid scintillation counter. The recoveries were over 80% (125). " C-Fla-vone and C-ketorolac were extracted from plasma at various extraction pressures and times to test recovery (126). The SCF was CO2 at 60°C and 300 bar. The recoveries were determined by liquid scintillation counting and ranged from 85% to 98% for C-flavone (RSD = 3.8-5.8%, three replicates). 

The end fitting of an HPLC column has three functions (1) it should seal the fluid path at the high pressures used in HPLC, (2) it should distribute the mobile-phase flow and the sample evenly over the entire cross-section of the packed bed, and (3) it should retain the packing inside the column. Consequently, most column end fittings comprise three components the seal, the distributor, and the filter. 

Sample Distribndon All HPLC columns are terminated by filter assemblies, which often have incorporated some means to ensure that the sample is distributed uniformly over the cross section of the column. The proper functioning of these devices can be impaired by blockage. If this is the case, the sample band at the column top is distorted, which can result in a reduced column performance. The phenomenon is indistinguishable from a collapsed bed all peaks exhibit a similarly distorted profile independent of the retention (Fig. 17.6). Open the column and inspect the filter and the packed bed. If the packed bed appears intact, it is worthwhile to try to clean the filter assembly or replace it with a new one. But the appearance of an undisturbed bed does not guarantee that the column has not collapsed. Often, a bed collapse has occurred somewhere in the middle of the packed bed and is not visible at the ends. 

Another problem with even chemically pure solvents is that of particulates, which can clog np the column inlet or other components. Sometimes solvents are filtered before use but most commercial HPLC systems have a line filter installed between the pump and the sample injector. These filters are generally of porous stainless steel, with pore size of 2(xm for columns packed with larger particles, but 0.5 (xm pore-size filters are recommended for use with columns packed with particles of much less than 10 /xm. 

The ternary solubility phase diagram of (S) - and (R) - propranolol hydrochloride in a mixed solvent of methanol and acetone was measured by isothermal method [25]. For isothermal method, enough amount of powder, namely lOfttO.lmg, was dissolved in the solvent of methanol in a test tube. Saturated solution samples were carefully withdrawn and filtered, and the concentration of which were analyzed by the HPLC system with employment of above-mentioned self-packed column. 

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