Mobile phase oven, column

The sample is carried through the column (3) by the carrier gas/mobile phase. The column is enclosed in a thermostatically controlled oven (4). Depending on how firmly each component of the sample sticks to the stationary phase - i.e. its affinity for the stationary phase - the mobile phase carries it through the column more or less quickly. Components that stick least tightly to the stationary phase move fastest, while those that are held more firmly move through more slowly. The time taken for a component of a mixture to pass through the column is known as its retention time. The retention time of a substance depends on how its vapour is distributed between the mobile and stationary phases. 

Procedure (See Chromatography, Appendix BA.) Use a suitable high-performance liquid chromatograph equipped with differential refractometer, autosampler injection unit, mobile-phase degasser, column heating block or oven, and a computing integrator. The column is Lichrosorb RP-18 250-mm x 4.5 mm (id) (GL Science, Inc., or equivalent) and YMC-Pack ODA-A A-303 250-mm x 4.5 mm (id) (YMC Company, Ltd., or equivalent) connected in a series, or equivalent, maintained at 50°. Use 80 20 acetone acetonitrile as the eluent, at a flow rate of 2 mL/min. 

A JASCO HPLC system equipped with a JASCO Differential R1 detector and a Rheodyne 77251 injector with a 100 pi loop was used. The column used was a Waters Spherisorb silica column. A mixture of acetonitrile and water (90/10) was used as the mobile phase. The column was conditioned at 30°C using an external oven and the flow rate was 1.5 ml/min. 

For LC, temperature is not as important as in GC because volatility is not important. The columns are usually metal, and they are operated at or near ambient temperatures, so the temperature-controlled oven used for GC is unnecessary. An LC mobile phase is a solvent such as water, methanol, or acetonitrile, and, if only a single solvent is used for analysis, the chromatography is said to be isocratic. Alternatively, mixtures of solvents can be employed. In fact, chromatography may start with one single solvent or mixture of solvents and gradually change to a different mix of solvents as analysis proceeds (gradient elution). 

Figure 1.2 Chromatogram of coal-tar oil obtained by using the following conditions column, Waters Spherisorb PAH 5 mm in 250 p.m id X 30 cm fused silica column oven temperature, 100°C UV detector wavelength to 254 nm mobile phase, 100 to 300 bar CO2 and 0.10 to 1.00 p.L min methanol over 30 minutes.

The basis of chromatography is in the differential migration of chemicals injected into a column. The carrier fluid takes the solutes through the bed used for elution (mobile phase). The bed is the stationary phase. Based on mobility, the retention-time detectors identify the fast and slow-moving molecules. Based on internal or external standards with defined concentration, all unknown molecules are calculated in a developed method by software. GC columns are installed in an oven which operates at a specified temperature. A diagram of an oven with GC column is shown in Figure 7.16. 

The basic SFC system comprises a mobile phase delivery system, an injector (as in HPLC), oven, restrictor, detector and a control/data system. In SFC the mobile phase is supplied to the LC pump where the pressure of the fluid is raised above the critical pressure. Pressure control is the primary variable in SFC. In SFC temperature is also important, but more as a supplementary parameter to pressure programming. Samples are introduced into the fluid stream via an LC injection valve and separated on a column placed in a GC oven thermostatted above the critical temperature of the mobile phase. A postcolumn restrictor ensures that the fluid is maintained above its critical pressure throughout the separation process. Detectors positioned either before or after the postcolumn restrictor monitor analytes eluting from the column. The key feature differentiating SFC from conventional techniques is the use of the significantly elevated pressure at the column outlet. This allows not only to use mobile phases that are either impossible or impractical under conventional LC and GC conditions but also to use more ordinary... 

Salm et al.44 developed a high-throughput analytical method to measure cyclosporine in whole blood. They used a simple SPE procedure, followed by HPLC-MS/MS. An Agilent 1100 liquid chromatograph was coupled with an Agilent Zorbax Bonus C18 reversed-phase column (50 x 2.1 mm, 5 jt/rn particle size). The column temperature was maintained at 70°C in a column oven. The mobile phase consisted of 80% methanol and 20% 40mM ammonium acetate buffer (pH 5.1) delivered isocratically at a flow of 0.4 mL/min. D12 cyclosporine was the IS. 

A Waters Acquity UPLC system with a cooling autosampler and column oven was used. The stationary phase was a Waters Acquity BEH C18 column (50 x 2.1 mm, 1.7 /.un particle size). The column was maintained at 40°C. The mobile phase consisted of water and acetonitrile, each containing 0.3% formic acid and was delivered at 0.35 mL/min in a gradient mode at 60% water from 0 to 1.5 min, linearly decreased to 10% water in 0.5 min, and then returned to 60% water. Sample vials were maintained at 4°C. 

Lercanidipine — Kalovidouris et al.57 applied UPLC-MS/MS to the determination of lercani-dipine in human plasma after oral administration of lercanidipine. A Waters Acquity UPLC system with cooling autosampler and column oven was coupled with a Waters BEH C18 column (50 x 2.1 mm, 1.7 jum). The mobile phase was composed of 70% acetonitrile in water containing 0.2% v/v formic acid, delivered at a flow of 0.30 mL/min. The column temperature was maintained at 40°C and sample vials at 5°C. 

The various separation parameters should be adjusted to provide optimum resolution. These include mobile phase flow rate, stationary phase particle size, gradient elution, and column temperature (using an optional column oven). 

Qualitative and quantitative analyses with HPLC are very similar to those with GC (Sections 12.7 and 12.8). In the absence of diode array, mass spectrometric, and FTIR detectors that give additional identification information, qualitative analysis depends solely on retention time data, tR and C (Remember that tR is the time from when the solvent front is evident to the peak) Under a given set of HPLC conditions, namely, the mobile and stationary phase compositions, mobile phase flow rate, column length, temperature (when the optional column oven is used), and instrument dead volume, the retention time is a particular value for each component. It changes only when one of the above parameters changes. Refer to Section 12.7 for further discussion of qualitative analysis. 

Turn on the column oven if required. Rinse the column with strong solvent (e.g., 100% MeOH) first then equilibrate column for 5-10 min with the new mobile phase until the baseline and pressure are stable. 

An air-bath oven is an excellent choice for GPC in that a substantial number of columns may be accomodated by a single unit. Costs are low and temperature stability and reproducibility quite good. Some type of heat-exchange device should be placed in the oven to raise the temperature of the mobile phase to the desired point before it reaches the column this practice helps eliminate temperature gradients along the column axis. 

Several theoretical and empirical studies have been made of the impact of column ovens on separations [84]. The two most important factors that must be considered are the preheating of the mobile phase before it enters the column and the extra-column volume between the injector and the column. Work by Djordevic et al. [76] provides some of the most dramatic evidence for the impact of pre-heating the mobile phase. Their measurements of the axial temperature gradient in 4.6 mm columns at typical flow rates are shown in Table 9.2. 

The injection temperature can be a signiflcant issne for thermally unstable samples or where samples are stored for hours in an antosampler prior to injection. For this reason, most manufacturers sell autosamplers with optional thermostated sample compartments. This can be done either by placing the sample tray in an air bath oven or by a condnctive temperature control of the sample rack. The need to keep samples cool prior to injection when conpled with elevated temperature separation increases the complexity of the flow system reqnired. For such application, a separate mobile phase pre-heater with a low volnme placed between the injector and the column is a good choice. Alternatively, the injector valve wonld need to be monnted ontside the antosampler or in the column oven to insure preheating of the mobile phase before the colnmn. 

A gaseous mobile phase flows under pressure through a heated tube either coated with a liquid stationary phase or packed with liquid stationary phase coated onto a solid support. The analyte is loaded onto the head of the column via a heated injection port where it evaporates. It then condenses at the head of the column, which is at a lower temperature. The oven temperature is then either held constant or programmed to rise gradually. Once on the column separation of a mixture occurs according to the relative lengths of time spent by its components in the stationary phase. Monitoring of the column effluent can be carried out with a variety of detectors. 

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