Flow-rate of the mobile-phase

Similarly, with molecules, their speed of movement through the chromatographic column depends on the time spent in the mobile phase compared with that in the stationary one and on the flow rate of the mobile phase. 

The concentration of biodiesel (fetty acid methyl esters) and glycerides were analyzed by liquid chromatography (Shimadzu-lOA HPLC). An ODS-2 column (250x4.6mm) was used for the separation. The flow rate of the mobile phase (acetone acetonitrile=l l) was set to 1 ml/min. Peaks were identified by comparison with reference standards. Standards of methyl esters, monoglycerides, digjycerides and triglycerides were bought from Fluka. 

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. 

Aboul-Enein and Ali [78] compared the chiral resolution of miconazole and two other azole compounds by high performance liquid chromatography using normal-phase amylose chiral stationary phases. The resolution of the enantiomers of ( )-econazole, ( )-miconazole, and (i)-sulconazole was achieved on different normal-phase chiral amylose columns, Chiralpak AD, AS, and AR. The mobile phase used was hexane-isopropanol-diethylamine (400 99 1). The flow rates of the mobile phase used were 0.50 and 1 mL/min. The separation factor (a) values for the resolved enantiomers of econazole, miconazole, and sulconazole in the chiral phases were in the range 1.63-1.04 the resolution factors Rs values varied from 5.68 to 0.32. 

For example, hexane (see Table 15.1) can be injected as a sample to determine t0 if chloroform is being used as the mobile phase in liquid-solid chromatography. As long as the flow rate of the mobile phase through the column remains unchanged, t0 is the same for any mobile phase. If flow rate changes by some factor x, t0 will change by the factor 1/x. 

When k is already within the optimum range of values and the resolution is still marginal, the best solution is to increase N. This is generally achieved by increasing column length or by decreasing the flow rate of the mobile phase. 

Based on the previous analysis of the different transport phenomena, which determine the overall mass transport rate, the structure of the solid phase matrix is of extreme importance. In the case of any chromatographic process, the different diffusion restrictions increase the time required for separation, since any increase of the flow rate of the mobile phase leads to an increase of the peak broadening [12]. Thus, the improvement of the existing chromatographic separation media (column packing of porous particles) and hence the speed of the separation should enable the following tasks ... 

Figure 3.11 illustrates the effect of varying the flow rate of the mobile phase on the efficiency of the separation process and provides a standard method of determining the optimum flow rate for a specific column and mobile phase system. 

The beneficial effect of the change of the flow rate of the mobile phase has also been exploited for the improvement of CCC purification of the components of the dye Quinoline yellow (Colour Index No. 47005). The chemical structures of the components of Quinoline yellow are shown in Fig. 3.121. The two-phase system used for the purification consisted of tm-butyl methyl ether-l-butanol-ACN-0.1 M TFA (1 3 1 5 v/v). The column... 

H. Oka, K.-I. Harada, M. Suzuki, K. Fujii, M. Iwaya, Y. Ito, T. Goto, H. Matsumoto andY. Ito, Purification of quinoline yellow components using high-speed counter-current chromatography by stepwise increasing the flow-rate of the mobile phase. J. Chromatogr.A, 989 (2003) 249-255. 

Molecules tend to diffuse randomly, in no particular direction, within any fluid, independently of the flow rate of the mobile phase. Their diffusion rate is determined by the type of molecule, the nature of the mobile phase, and the temperature, and is expressed quantitatively by their diffusion constants. 

If a column with different diameter, but the same length is used, the column hold-up volume changes in proportion to the second power of the column diameters. To keep the separation time constant, the flow rate of the mobile phase should be adjusted in the proportion of changing V , see Equation 5.28. Another possibility of compensation is by adjusting the gradient time to keep the ratio VJitQ-FJ constant [8,9]. 

RI detectors measure this deflection, and are sensitive to all analytes that have a different R1 than the mobile phase. There are two major limitations First, Rl detectors are very sensitive to changes in the temperature, pressure, and flow rate of the mobile phase, and so these measurement conditions must be kept stable in order to obtain low background levels. Second, Rl detectors are incompatible with chromatographic separations using gradient elution. Furthermore, because Rl detectors are nonselective, they must be used in conjunction with other detection methods if specificity is required. Nevertheless, they have found wide application in isocratic chromatographic analysis for analytes that do not have absorptive, fluorescent, or ionic properties, such as polymers and carbohydrates. 

Zogg, G.C., N dredy, Sz., and Sticher, O., Operating conditions in preparative medium pressure liquid chromatography (MPLC). II. Influence of solvent strength and flow rate of the mobile phase, capacity and dimensions of the column, J. Liq. Chromatogr., 12, 2049, 1989. 

The solution of 0.0001 K SO should be pumped through the column at 0.5 ml mim for 30 min to allow the column to equilibrate. After this time, raise the flow rate of the mobile phase to 1.2 ml min k... 

A 1.0 cm-i.d. and 50 cm-long chromatography column is packed with gel beads that are 100 pm in diameter. The interparticle void fraction e is 0.27, and the flow rate of the mobile phase is 20 cm h. A retention volume of 20 cm and a peak width W of 1.8 cm were obtained for a protein sample. 

Equation (1.27), which has been expanded to different types of liquid chromatography (Knox equation), shows that there is an optimum flow rate for each separation and that this does indeed correspond to the minimum on the curve represented by equation (1.27). The loss in efficiency that occurs when the velocity is increased represents what occurs when trying to rush the chromatographic separation by increasing the flow rate of the mobile phase. However, intuition can hardly predict the loss in efficiency that occurs when the flow is too slow. To explain this phenomenon, the origins of the terms A, B and C have to be considered. Each of these parameters has a domain of influence that can be seen in Fig. 1.9. Essentially, this curve does not depend on the nature of the solute. 

The present performance of high field NMR instruments allows the recording of a spectrum in quantities of micrograms. Under these conditions, it is possible to install a flow cell of only a few microlitres into the magnet of the instrument which allows the spectra of the analytes to be recorded. The experiment is conducted with a very small flow rate of the mobile phase (D20 or CD3CN) or in the stop-flow mode. In this mode, the mobile phase is momentarily stopped in order to record the spectrum. This technique, which requires very expensive materials, is of limited use. It is used mainly to isolate and identify very unstable compounds that cannot be isolated through classical means. 

A mixture of proteins is separated on a column with a stationary phase of carboxymethylated cellulose. The internal diameter of the column is 0.75 cm and its length is 20 cm. The dead volume is 3 ml. The flow rate of the mobile phase is 1 ml/min. The pH of the mobile phase is adjusted to 4.8. Three peaks appear upon the chromatogram corresponding to the elution volumes V, V2 and V2 at 12 ml, 18 ml and 34 ml respectively. 

In summary, the efficiency TV of a TLC plate is variable. The height equivalent to a theoretical plate has a minimum value, as in HPLC. However, it is not possible, unlike in HPLC, to vary the flow rate of the mobile phase in order to increase separation efficiency. 

A solution in THF of a set of polystyrene standards of known molecular mass was injected onto a column whose stationary phase is effective for the range 400-3000 daltons. The flow rate of the mobile phase (THF) is 1 ml/min. The chromatogram below was obtained. 

Flowrate. Fc- The volumetric flow rate of the mobile phase (carrier gas), in cm3/min, measured at the column temperature and outlet pressure. 

The peak height is thus proportional to N and is highest when the optimum flow rate is achieved. Any comparison between quantitative data should indicate what the unretained solute was, what the flow rate of the mobile phase was, and what the plate count was. 

Postcolumn derivatization with iodine The procedure has been developed by Tuinstra and Haasnoot (44) a freshly prepared aqueous saturated solution of iodine (2 g in 400 ml of bidistilled water) is pumped by an auxiliary HPLC pump simultaneously with the mobile phase (commonly water/methanol/acetonitrile). The iodine solution must be kept away from UV light. The reaction is carried out in Teflon tubing (0.5 mm X 3000 mm) thermostated at 60°C. A silicone oil bath is preferred to a water bath for a more stable temperature. The flow rates of the mobile phase and the derivatizing agent are generally 0.8 ml/min. and 0.7 ml/min, respectively. A scheme of the derivatization system is shown in Fig. 2. 

Chiral resolution on polysaccharide-based CSPs is sensitive, and therefore, the optimization of HPLC conditions on these phases is very important. The most important factors that control enantiomeric resolution are the composition, pH, and flow rate of the mobile phase and parameters, including temperature and solute structure. The optimization of these parameters on polysaccharide-based CSPs is discussed next. 

Temperature and pressure are rarely optimized in HPLC, but these parameters are very important in SFC, hence can alter retention, selectivity, and resolution. Toribio et al. [149] presented the chiral separation of ketoconazole and its precursors on Chiralpak AD and Chiralcel OD CSPs. The authors also reported that alcohol modifiers provided better separation than acetonitrile. Further, Wilson [143] studied the effects of composition, pressure, temperature, and flow rate of the mobile phase on the chiral resolution of ibuprofen on a Chiralpak AD CSP. It was observed that temperature affords the greatest change in resolution, followed by pressure and composition. An increase in methanol concentration, pressure, and temperature has resulted in poor chiral resolution. At first chiral resolution increased with an increase of flow rate (up to 1.5 mL/min) but then started to decrease. Contrary to this, Biermann et al. [135] described the... 

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