Mobile phase column selectivity

In general, the majority of separations are achieved by exploiting dispersive interactions in the stationary phase and modifying and controlling the absolute and relative retention of the solutes by adjusting the composition of the mobile phase. It is far easier to adjust the mobile phase by selecting different mixtures of water and the solvents methanol, acetonitrile and/or tetrahydrofuran than change from column to column. 

Omura et al.21 used a reverse phase high performance liquTcT cEromatographic column, JASCO PACK SV-02-500, for macrolide antibiotics with methanol, M/15 acetate buffer pH 4.9, and acetonitrile (35 60 5) as solvent. A variable wavelength UV detector using the absorption of the individual compounds gave the required sensitivity. Alterations of buffer pH and the composition ratio of the mobile phase gave selectivity for separation of individual macrolide antibiotics. 

In the development and optimization of a comprehensive LCxLC method, many parameters have to be taken in acconnt in order to accomplish snccessfnl separations. First of all, selectivity of the columns used in the two dimensions must be different to get maximum gain in peak capacity of the 2D system. For the experimental setup, column dimensions and stationary phases, particle sizes, mobile-phase compositions, flow rates, and second-dimension injection volumes should be carefully selected. The main challenges are related to the efficient coupling of columns and the preservation of mobile phase/column compatibility. 

Mobile phases for SEC fall into two broad categories aqueous buffers for GFC and organic solvents for GPC. In SEC, the mobile phase is selected not to control selectivity but for its ability to dissolve the sample. In addition, the mobile phase should have a low viscosity and be compatible with the detector and column packing. For example, polar solvents such as methanol... 

Martin et al. published a paper on the theoretical limits of HPLC which is well worth reading.They used relatively simple mathematics to calculate pressure-optimized columns for which the length L, particle size and flow rate u of the mobile phase were selected such that a minimum pressure Ap is required to solve a separation problem. It has been shown that these optimized colunms are operated at their van Deemter curve minima. Some astonishing facts have emerged from the study, provided that the chromatography is performed on well packed columns (reduced plate height h = 2-3 see Section 8.5). 

During initial method development, a set of initial conditions (detector, column, mobile phase) is selected to obtain the first scouting chromatograms of the sample. In most cases, these are based on reversed-phase separations on a Cl 8 column with UV detection. A decision on developing either an iso-cratic or a gradient method should be made at this point. 

The above sequence will stop very quickly unless a mobile phase is added to force the process to continue. The molecules of the mobile phase are selected so as to be more strongly adsorbed to the active sites than either W or S. When the mobile phase molecules reach an active site, they have two distinct advantages (1) they are more strongly adsorbed to the site than either W or S, and (2) they are much more concentrated. The net result is that they displace the S molecules, and the S molecules in turn displace the W molecules. This displacement sequence continues along the length of the column as shown in Figure 14-IB. In order to complete the separation, M is added until W and S are eluted from the column. 

The first step in the selection of the size-exclusion separation system is the choice of the mobile phase. We need to select a mobile phase in which the analytes, usually polymers, are soluble. This, in turn, determines the selection of the stationary phase, spedfically, whether we select a padring designed for organic or aqueous size-exclusion chromatography (the term aqueous may include polar solvents). If the goal of the separation is a molecular-weight determination, the requirements for the mobile phase-column combination are quite stringent ... 

If our analytes are retained on the column for too long, we need to decrease the polarity of the mobile phase by selecting a solvent with a lower polarity index. For example, if we started our separation using methanol and our analytes were retained for too long, we could adjust the elution power... 

The retention characteristics of 29 aza-arenes (e.g., pyridine, acridine, quinoline, benz[a]acridine, and numerous substituted analogs) were studied on a diol column using a 97.5/2.5 iso-octane/ethanol mobile phase [611]. Dimethylbenz[a]acridine was least retained k < 3) and indole had the greatest retention (k > 9). The study also worked with 29 phenols (e.g., numerous alkyl-substituted phenols, nitrophenols, and halogenated phenols). They were studied in detail on the diol column using a 50/50 iso-octane/dichloromethane mobile phase. Appropriate selection of solvent composition provided baseline resolution of isomeric groups (e.g., dimethylphenols using hexane/ethyl acetate). 

GPC columns can be selected that have a wide spectrum of pore sizes (and thus separate a wide range of molecular weights-from thousands to millions). Alternatively, columns with more narrow pore size distributions can be used to get a better separation of polymers that have similar molecular weights—say from 1000 to 20,000. These columns are designed to be used with organic solvents (required for most polymers) or water that flow through as the mobile phase. The selection of which columns to use depends on the polymer structure and the expected molecular weight distribution. 

While it is possible to use a number of volatile solvents as the mobile phase for SCF chromatography, the most commonly used mobile phase is carbon dioxide. However, carbon dioxide is not a good solvent for polar compounds so it is common to add a small amount of some additional polar organic liquid such as an alcohol or even water as a modifier. However, the modifier needs to be miscible with carbon dioxide. Much of the other technology associated with either GC or HPLC in terms of sample inlets and types of pumps are adapted to specific applications but the key attribute of the SCF-type chromatography is the maintenance of T, P) conditions near the critical point of the mobile phase. A selection of columns is available just as for GC or HPLC. 

After the column and mobile phase are selected, the next step in method transfer is to adjust the flow rate and gradient profile to keep the same retention factor for the analytes of interest. The conversion between HPLC and UHPLC for isocratic methods has been reviewed before (24,25). Here, we will focus on gradient elution, which is more commonly used. The gradient retention factor (k ) is determined by gradient time (to), flow rate (F), gradient step or the difference in organic composition... 

Production processes are invariably developed initially at the bench scale where stationary and mobile phases are selected and sample volumes determined. In the majority of cases the application is then simply scaled up by increasing the diameter of the column while maintaining the bed height constant, and increasing all volumes, including sample, elution, and regeneration solutions in direct proportion to the increase in column cross sectional area. Linear flow rate is maintained the same so that volumetric flow also increases in proportion to the increase in colunm cross sectional area, and process times remain constant. 

From equation 12.1 it is clear that resolution may be improved either by increasing Afr or by decreasing wa or w-q (Figure 12.9). We can increase Afr by enhancing the interaction of the solutes with the column or by increasing the column s selectivity for one of the solutes. Peak width is a kinetic effect associated with the solute s movement within and between the mobile phase and stationary phase. The effect is governed by several factors that are collectively called column efficiency. Each of these factors is considered in more detail in the following sections. 

Use of column selectivity to improve chromatographic resolution showing (a) the variation in retention time with mobile phase pH, and (b) the resulting change in alpha with mobile phase pH. 

To minimize the mobile phase s contribution to conductivity, an ion-suppressor column is placed between the analytical column and the detector. This column selectively removes mobile-phase electrolyte ions without removing solute ions, for example, in cation ion-exchange chromatography using a dilute solution of HCl as... 

The analysis of cigarette smoke for 16 different polyaromatic hydrocarbons is described in this experiment. Separations are carried out using a polymeric bonded silica column with a mobile phase of 50% v/v water, 40% v/v acetonitrile, and 10% v/v tetrahydrofuran. A notable feature of this experiment is the evaluation of two means of detection. The ability to improve sensitivity by selecting the optimum excitation and emission wavelengths when using a fluorescence detector is demonstrated. A comparison of fluorescence detection with absorbance detection shows that better detection limits are obtained when using fluorescence. 

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