Mobile phases, flow calibration

An HPLC column is a straight, stainless steel calibrated tube (sometimes coated on the inside with an inert material such as glass or PEEK 5 ) between typically 3 and 25 cm in length. The internal diameter of the column can vary from 0.5 to 5 mm (Fig. 3.6). The stationary phase is held in the column by one porous disc at each extremity. Dead volumes within the column are kept as small as possible. The flow of the mobile phase into the column must not exceed a few ml/min. Some manufacturers offer microcolumns that have an internal diameter in the order of 0.3 mm and are 5 cm in length. Typical mobile phase flow rates for these narrow columns can be as low as a few pl/min. Because complete elution of compounds with these microcolumns requires only a few drops of mobile phase, the stationary phase and pumps have to be adapted accordingly. These narrow columns not only substantially reduce the amount of mobile phase which has to be used but also improve resolution by diminishing the diffusion inside the column. They are well suited for HPLC-MS (cf. Chapter 16). 

Amantea and Narang [58] used a reversed-phase HPLC method for the quantitation of omeprazole and its metabolites. Plasma was mixed with the internal standard (the 5-methyl analog of omeprazole), dichlor-omethane, hexane, and 0.1 M carbonate buffer (pH 9.8). After centrifugation, the organic phase was evaporated to dryness and the residue was dissolved in the mobile phase [methanol-acetonitrile-0.025 M phosphate buffer of pH 7.4 (10 2 13)] and subjected to HPLC at 25 °C on a column (15 cm x 4.6 mm) of Beckman Ultrasphere C8 (5 ym) with a guard column (7 cm x 2.2 mm) of Pell C8 (30-40 /im). The mobile phase flow-rate was 1.1 ml/min with detection at 302 nm. The calibration graphs are linear for <200 ng/ml, and the limits of detection were 5, 10, and 7.5 ng/ml for omeprazole, its sulfone, and its sulfide, respectively. The corresponding recoveries were 96.42% and 96% and the coefficients of variation (n = 5 or 6) were 3.0-13.9%. 

Wad and Kramer [35] described an HPLC method for simultaneous determination of vigabatrin and gabapentin in serum and urine after precolumn derivatization with o-phthaldialdehyde and fluorimetric detection at 455 nm with excitation at 230 nm. A column (12.5 cm x 3.0 mm) of Superspher 60 RP-Selected B (5 /im) with acetonitrile in 20 mM 0.5 M KH7PO4 as a mobile phase (flow rate 0.7 ml/min). The day-to-day CV of vigabatrin in a pooled serum was 3.1%. The lower limit of detection was 0.5 /(mol/1 and the calibration graph was rectilinear from 1300 /tmol/1. 

Nimodipine was determined in human plasma using HPLC and methyltestosterone as an internal standard [26]. A Spherisorb ODS, 10 pm column (25 cm x 4.6 mm i.d.) with methanol/I I20/ -biitylamine (65000-35000-1) was used as the mobile phase (flow rate of 1.2 mL/min), with detection at 238 nm. The calibration graph was linear over the range of 5-100 ng/mL, the detection limit was 2 ng/mL, and the average recovery was 92%. 

The enantiospecific determination of nimodipine in human plasma could be established by liquid chromatography-tandem mass-spectroscopy [27]. The separation was effected using a 8 pm Chiral OJ MOD column (25 cm x 2 mm i.d.), operated at 35°C with ethanol-n-heptane (1 4) containing 2 mM ammonium acetate as the mobile phase (flow rate of 300 pL/min) and MS detection. Calibration graphs were linear over the range of 0.5-75 ng/mL, with a detection limit of 0.25 ng/mL for each enantiomer. 

The metabolism of nimodipine in rat liver microsomes was studied by HPLC [29]. The centrifugate of the reaction mixture was analyzed using a column (10 cm x 4 mm i.d.) of Spherisorb S3 ODS II at 40° C, with a mobile phase (flow rate of 0.5 mL/min) of 5 mM ammonium acetate buffer (pH 6.6)/methanol (2 3) and detection at 218 and 238 nm. Calibration graphs were linear over the range of 0.2-50 pM for nimodipine, and 0.2-20 pM for the three main metabolites with detection limits of 30-80 nM. 

Zhang et al. described a HPLC method for the simultaneous determination of six different dihydropyridine calcium antagonists (including nimodipine) in plasma [30]. After extraction with ethyl ether/ -heptane (1 1) and centrifugation, the organic phase was heated at 50°C and the residue dissolved in aqueous 60% acetonitrile (the mobile phase). An Ultrasphere 5 pm ODS column (25 cm x 4.6 mm i.d.) was used as the means of separation. The mobile phase flow rate was 1 mL/min, and detection was effected at 238 nm. The calibration graphs were linear over the range of 50 ng/mL-6.4 pg/mL, with a detection limit of 5 ng/mL. 

Detector calibration An intermediate step in concentration measurements during which the relationship between the detector signal and the composition of the mobile phase flowing through it is determined. If the relationship is linear, the result of the calibration is a response factor. Otherwise, it is a calibration curve. 

Figure 4.12 Analysis by the external standard method. The precision of this basic method is improved when several solutions of varying concentrations are used in order to create a calibration curve. For trace analyses by liquid chromatography it is sometimes advisable to replace the areas of the peaks by their heights as they are less sensitive to variations in the mobile phase flow rate.

Make a series of 0.5 pi injections of solvent to be analysed and optimise resolution for the components in the sample by adjusting column temperature and mobile phase flow-rate. Under the optimum conditions determined make at least three 0.5 pi injections of the standard solutions and of all sample solutions on both columns. Check the reproducibility of the area data. Repeat injections if greater than 5% deviation from mean of analyte peak. Check the linearity of detector response for both systems and by the method of external standardisation determine the concentration of water in the sample. Compare the order of elution of the analytes on the two columns and account for any differences (Figures 9.13 and 9.14). The experiment can be extended by using the technique of standard addition to determine the water content of methanol. A series of standard water solutions are prepared (e.g. 0.5, 1.0, 1.5 and 2.0% (v/v) water together with an accurate volume of methanol) these standards and the sample prepared in an analogous fashion are analysed as above. The water content in the solvent can be obtained by extrapolation of the calibration line the intercept with the x-axis giving the concentration of water in the sample. 

Review the description of the two different types of detector (mass detector or concentration detector) at the end of Section 11.4. Note well that the area that will be calculated for a peak measured by a mass detector is independent of mobile phase velocity, but when a concentration detector is used it is not. When calibrating against standards using a concentration detector, it is important that the analyte peaks in both the unknown and standard runs elute at the same time and under the same mobile phase flow conditions. Conversely, if quantitation is by peak height comparison using a mass detector, the same requirements apply. 

Evaporative fight scattering detection can be used as a imi-versal detector for LC. Its operation includes the nebufization of the eluent in the nebulizer, solvent evaporation in the drift tube, and scattered fight detection at the fight scattering chamber. Experimental conditions which can be adjusted in most ELSD systems to optimize the detector sensitivity are the nebulizer gas flow rate, mobile phase flow rate, and drift tube temperature. The detector response is non-linear, but can be used in quantitative work if a calibration curve is obtained. 

Experimental variables such as temperature, flow rate, sample concentration and mobile phase composition can cause changes in the elution volume of a polymer [439,457,460-464]. Chromatographic measurements made with modem equipment are limited more by the errors in the absolute methods used to characterize the molecular weight of the calibration standards than any errors Inherent in the measurements themselves, since the determination of molecular weights by SEC is not an absolute method and is dependent on calibration [462]. The Influence of temperature on retention in SEC is not very great, since no strong sorptive interactions are involved in the retention mechanism. Temperature differences between the column and solvent delivery... 

Figure 4.25 Protein calibration curves for Spherogel TSK-SN 2000 and TSK-SM 3000 colunn. Mobile phase phosphate buffer 0.2 M, pH 6.8, flow rate 1.0 sl/sin.

Figure 4 Calibration of external and internal standard method. Chromatographic conditions — column 30 cm x 3.9 mm p-Bondapak C18 (10-pm particle size) mobile phase water acetonitrile (50 50) flow rate 1.5 ml/min column temperature ambient detector wavelength 254 nm. (A) External standard method, (B) internal standard method.

Blank, calibrator, control, and patient whole-blood samples (50 /iL) were transferred into 1.5 mL conical test tubes, mixed with 100 /xL of the IS, vortexed for 10 sec, and centrifuged at 13,000 g for 5 min. Twenty-five microliters of supernatant were injected onto a Cohesive Technologies Cyclone polymeric turbulent flow column (50 x 1 mm, 50 /flushed with a mixture of methanol and water (10 90 v/v) at a flow of 5 mL/min. Column switching from the TFC to HPLC systems was via a Cohesive Technologies system. The analytical column was a Phenomenex Phenyl-Hexyl-RP (50 x 2.1 mm, 5 /.mi). The mobile phase consisted of methanol and ammonium acetate buffer (97 3 v/v). The buffer was 10mM ammonium acetate containing 0.1% v/v acetic acid. The flow rate was 0.6 mL/min. 

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