Flow rate of mobile phase

0 ml/min (1.H mm/s). Sample concentration was 0.2%. The minimum HETF for alanln was 33 (Jm which was obtained at a flow rate of 0.3 ml/mln. The value of HETP for alanine was almost unchanged or the change was small within this flow rate range, but the the values of HETP for ohymotrypsinogen (M = 2U500) and aldolase (M = 154-000) varied greatly with flow rate. For example, HETP for aldolase at 0.1 ml/mln was 0.15 mm and it increased to 0.445 mm at 

0 ml/min. A change in retention volume with increased flow rate was not observed within these experimental conditions. 

The change of HETP for small molecules is not large compared with the 

Sample 1 = aldolase 2 = ohymotrypsinogen 3 = alanine. For experimental conditions, see text. 

On the behavior of soft gels such as Bio-Gel P (polyacrylamide) as packing materials, retention volume is flow rate dependent and increases with the increase in flow rate [ref. 25). In such soft gels, the particle distribution of gels Influences the flow rate dependence of HETP. The values of HETP increased linearly with increasing flow rate on unfractionated Sepharose, 

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It is also possible for solute species to diffuse laterally (in a radial direction) across the column, and thus to move from one flow path into another. This effect reduces the amount of dispersion produced by the multiple path effect as it tends to equalise the speed of the solute species in the column. The longer the time a solute species spends in the column, the more lateral diffusion will occur, so that flow dispersion is reduced by using low flow rates of mobile phase. 

Longitudinal diffusion will become more serious the longer the solute species spend in the column, so this effect, unlike flow dispersion is reduced by using a rapid flow rate of mobile phase. 

You can see that these dispersion mechanisms are affected in different ways by the flow rate of mobile phase. To reduce dispersion due to longitudinal diffusion we need a high flow rate, whereas a low flow rate is needed to reduce dispersion due to the other two. This suggests that there will be an optimum flow rate where the combination of the three effects produces minimum dispersion, and this can be observed in practice if N or H (which measure dispersion) are plotted against the velocity or flow rate of the mobile phase in the column. The shape of the graph is shown in Fig. 2.3f. 

The instruments for polymer HPLC except for the columns (Section 16.8.1) and for some detectors are in principle the same as for the HPLC of small molecules. Due to sensitivity of particular detectors to the pressure variations (Section 16.9.1) the pumping systems should be equipped with the efficient dampeners to suppress the rest pulsation of pressure and flow rate of mobile phase. In most methods of polymer HPLC, and especially in SEC, the retention volume of sample (fraction) is the parameter of the same importance as the sample concentration. The conventional volumeters— siphons, drop counters, heat pulse counters—do not exhibit necessary robustness and precision [270]. Therefore the timescale is utilized and the eluent flow rate has to be very constant even when rather viscous samples are introduced into column. The problems with the constant eluent flow rate may be caused by the poor resettability of some pumping systems. Therefore, it is advisable to carefully check the actual flow rate after each restarting of instrument and in the course of the long-time experiments. A continuous operation— 24h a day and 7 days a week—is advisable for the high-precision SEC measurements. THE or other eluent is continuously distilled and recycled. 

A low flow rate of mobile phase minimises the dispersion of compounds, which occurs inevitably throughout the instrument. Detection limits are also improved. The laminar flow within the column follows Poiseuille s law, the velocity of the mobile phase being at its maximum in the centre of the tube and zero at the wall. 

Mobile phase. Hexane is the major component of mobile phase in the normal-phase HPLC method. The percentage of hexane is up to 99% for silica normal-phase columns. Ethyl acetate, acetic acid, methanol, and isopropanol are used as modifier components (Shin and Godber, 1994). The flow rate of mobile phase is usually controlled at 1 to 1.5 ml/min to completely separate all eight tocopherols and... 

Equipment with different characteristics (e.g., delay volume of an HPLC system) Variations in material and instrument conditions (e.g., in HPLC, mobile phases composition, pH, flow rate of mobile phase)... 

Fig. 8.3. Isocratic (a) and gradient (b) separation of PTH amino acids. Column, 250 x 0.075 mm i.d. packed with 3.5 p.m/80 A Zorbax ODS eluents, (A) 2 mmol/1 ammonium acetate, pH 7.0, (B) 2 mmol/1 ammonium acetate, pH 7.0, 90% acetonitrile isocratic elution with 30% B in (a) gadient elution with 30-80% B in 5 min, followed by 80% for 5 min in (b) flow rate of mobile phase through inlet reservoir, 100 pl/min applied voltage, 15 kV Detection, ESI-MS, m/z 100-2000, 0.5 s/spectrum integration time sheath liquid, 1 mmol/1 ammonium acetate, pH 7.0, 90% methanol, 3 pl/min injection, electrokinetic, 2 kV, 2 s sample, PTH-asparagine, PTH-glutamine, PTH-threonine, PTH-glycine, PTH-tyrosine, PTH-alanine (in order of elution). (Reproduced from ref. [82] with permission of Elsevier Sciences B. V.).

Figure 14.3 Chromatograms of excipients in film-former class under different mobile phase pH. In both plots, the curves from the bottom are blank, HPMC, acacia, sucrose NF, HPC, povidone and Eudragit EPO, respectively. The sample solvent, mobile phase and column used are (a) 20% ACN-80% pH 2, 25 mM phosphate buffer, a gradient from 30% ACN-70% pH 2, 25 mM phosphate buffer to 80% ACN-20% pH 2, 25 mM phosphate buffer and a Zorbax SB-C8, 4.6 x 150 mm, 3.5 p.m column at 35°C, respectively and (b) 20% ACN-80% pH 7, 25 mM phosphate buffer, a gradient from 30% ACN-70% pH 7, 25 mM phosphate buffer to 80% ACN-20% pH 7, 25 mM phosphate buffer and a Zorbax XDB-Cg, 4.6 x 150 mm, 3.5 p,m column at 35°C, respectively. In both cases, samples were injected at 1800 p,L, the flow rate of mobile phase was 1 mL/min and the detection was at 210 nm. An on-bench examination of the mixture of either 80% ACN-20% pH 2, 25 mM phosphate buffer or 80% ACN-20% pH 7, 25 mM phosphate buffer revealed no precipitation, so they were suitable as the mobile phase.

The elution point of an unretained substance occurs at the dead time (t ) and the volume of mobile phase that has passed through the column between the injection point and the dead point is the dead volume (VJ. The dead volume is given by (Q tj, where (Q) is the flow rate of mobile phase through the column. The volume of mobile phase that passes through the column between the injection point and the peak maximum is called the retention volume (V,), which is given by (Q f.) where (k) is the time that has elapsed between the injection point and the peak maximum. The difference between the retention volume and the dead time - VJ is called the corrected retention volume which is also equal to the product of the corrected retention time (t ) and the flow rate (Q). 

Nemutlu et al. [22] determined lomoxicam in pharmaceutical preparations by a liquid chromatographic method. The separation was achieved on a reversed phase (Nucleosil 100-5 Cis 25 cm x 4.6 mm, 5 gm) column kept at room temperature. The flow rate of mobile phase was 1 ml / min. The mobile phase consisted of 0.1 M phosphate buffer (pH 6)-acetonitrile (60 40) and UV detection at 293 nm. The retention times for the drug and the internal standard, metronidazole, were 5.65 and 3.95 min, respectively. Quantitative analysis of the drug in tablets and injections were performed. The method is fast, simple, inexpensive and applicable over a wide range of concentrations with high precision and accuracy. 

A stainless column packed with Lichrospher lOORP-18 was used to analyze Acanthoside-D from the solvent extraction. The composition of mobile phase in analytical HPLC was experimentally determined and it was water/acetonitrile/methanoi=80/14/6 vol.%. From the chromatogram, retention time of Acanthoside-D was found to be 12 min. Figure 1 shows the analysis of Acanthoside-D from the extraction of the trunk of Acanthopanax senticosus. The flow rate of mobile phase and injection volume were 1 nt(/min and 20pl, respectively. 

Reproducibility, as defined by ICH, represents the precision obtained between laboratories with the objective of verifying if the method will provide the same results in different laboratories. The reproducibility of an analytical method is determined by analyzing aliquots from homogeneous lots in different laboratories with different analysts, and by using operational and environmental conditions that may differ from, but are still within the specified, parameters of the method (interlaboratory tests). Various parameters affect reproducibility. These include differences in room environment (temperature and humidity), operators with different experience, equipment with different characteristics (e.g., delay volume of an HPLC system), variations in material and instrument conditions (e.g., in HPLC), mobile phases composition, pH, flow rate of mobile phase, columns from different suppliers or different batches, solvents, reagents, and other material with different quality. 

Analytical Detector, and a Rheodyne injector (SO-pL sample loop). The data acquisition system was a Chromate (Ver. 3.0 Interface Engineering, South Korea) installed in a PC. The flow rate of mobile phase was fixed at 4, 2, and 1 mL/min with CIM QA, QlOO, and HiTrap Q, respectively. The wavelength was fixed at 260 and 280 nm and the injection volume was fixed at 20 pL. The experiment was performed at room temperature. 

Equilibrate the columns with 500 mL of HPLC mobile phase flow-rate of mobile-phase is 1 mL/min. 

A more rigorous technique involves the use of internal standards. An internal standard is a compound that is similar in chemical structure and physical properties to the sample being analysed. The internal standard should be added to the sample in question before extraction or assay commences and is then present in the sample matrix throughout the subsequent assay In the assay of complex samples, some sample pre-treatment is usually required and the recovery of the sample from the extraction process may not be 100%. If an internal standard is used, losses in sample will be mirrored by similar losses in the standard and the ratio of sample to standard should remain constant. Internal standards are particularly used in chromatographic analysis (especially gas chromatography and high-performance liquid chromatography), where fluctuations in instrumental parameters (e.g. flow rate of mobile phase) affect accuracy... 

In these equations, CfJ and Cfj are the concentrations of component i at the outlet and the inlet of column /, respectively. Qj is the actual volumetric flow rate of mobile phase through column j. It is related to the liquid phase velocity, Uj, by Qj = eAuj, where A is the column cross-section area, which is assumed, without loss of generality, to be the same for all the columns including the feed and drawoff columns. 

Sedimentation Field Flow Fractionator. The chromatography-related principle of this particle size and size distribution analyzer is based upon the interaction of the particle suspension under centrifugal field motion in a thin channel. The elution time of the particles is a function of particle size, particle density, flow rate of mobile phase, density of mobile phase, and the centrifugal force applied. After the size separation has occurred, the particles are detected in the mobile phase using a turbidity detection system. The dynamic range of the instrument is dependent on particle density and operating conditions and is typically within 0.03 /rm— 1 /rm range. 

Ac peak area for component C peak height for component A he peak height for component B he peak height for component C /fo.s peak half height ho.i 10% peak height Wb width at the base of a peak Wb width of a peak at half height Fc flow-rate of mobile phase a, b the forward, rear part of a peak at 10% h... 

The volume of mobile phase required to carry a band of component molecules through the system to the detector is termed the retention volume, Kr, and is measured from the start of the chromatography to the peak maxima (Figure 2.2). However, as stated earlier it is difiScult to accurately measure volume flow rates of the mobile phase in column chromatography systems such as GC and HPLC. Therefore, a constant flow rate of mobile phase is maintained and the time taken by a component band to pass through the column is recorded as the retention time, /r. If the flow rate of the mobile phase through the column is Fq then retention voliune is calculated from... 

Dg Diffusion rate in the stationary phase Fc Flow-rate of mobile phase through the column Fg Flow-rate through split vent of split-splitless GC injector... 

Originally the electrospray interface, like the thermospray interface, was limited to use with very low flow rates of mobile phase from capillary or microbore HPLC columns or capillary electrophoretic separations. The acceleration of droplet evaporation by... 

For the tests of flow deviation, flow reproducibility, and pressure deviation the above-mentioned column is installed. Connect a calibrated flow and pressure meter and set the flow rate of mobile phase 1 to 1.5mlmin . Let the whole HPLC system stabilize for 10-15 min. Then read and note the indicated data of the meter and the pump in intervals of 1 min for 6 min. Special attention needs to be given to possible flow and pressure deviations. If the deviations of flow... 

There are many parameters which control the enantiomeric resolution by HPLC. The most important of them include parameters of the stationary phase, such as particle size of CSP, pore size of column, and kind of chiral selector, composition, and pH of the mobile phase, flow rate of mobile phase, and temperature. Systematic variation of column temperature should be considered as one way to improve chiral separations in HPLC. From the practical point of view, it is easier to vary column temperatures than mobile phase composition. In addition, variable temperature runs can provide useful information concerning the thermodynamic parameters for the CSP-analyte interactions. The effect of temperature on the resolution... 

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