Mobile phase resolution

Analytical Properties Ionically bonded to silica, this phase provides good resolution of enantiomeric quasiequatorial frans-dehydriols of unsubstituted and methyl- and bromo-substituted benz[a]anthracene derivatives covalently bonded to silica, this phase provides good resolution of enantiomeric pairs of quasidiaxial frans-dihydrodiols of unsubstituted and methyl- and bromo-substituted benz[a]anthracene derivatives by addition of a third solvent (chloroform) to the classical binary mixture (hexane-alcohol) of the mobile phase, resolution of enantiomers of tertiary phosphine oxides is possible Reference 31-33, 36, 37... 

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. 

Fig. 2. Tic densitometer scans showing the resolution of isoproterenol on a hpflc siUca-gel plate obtained using a mobile phase consisting of 6.8 mM (1 R)-(—)-ammonium-10-camphorsu1fonic acid in 75 25 (v/v) methylene chioride methano1. (a) 254 nm, (b) 275 m, (c) 300 nm.

The dependence of chiral recognition on the formation of the diastereomeric complex imposes constraints on the proximity of the metal binding sites, usually either an hydroxy or an amine a to a carboxyHc acid, in the analyte. Principal advantages of this technique include the abiHty to assign configuration in the absence of standards, enantioresolve non aromatic analytes, use aqueous mobile phases, acquire a stationary phase with the opposite enantioselectivity, and predict the likelihood of successful chiral resolution for a given analyte based on a weU-understood chiral recognition mechanism. 

Achiral Columns Together with Chiral Mobile Phases. Ligand-exchange chromatography for chiral separation has been introduced (59), and has been appHed to the resolution of several a-amino acids. Prior derivatization is sometimes necessary. Preparative resolutions are possible, but the method is sensitive to small variations in the mobile phase and sometimes gives poor reproducibiUty. 

Retention and stereoselectivity on the BSA columns can be changed by the use of additives to the aqueous mobile phase (30). Hydrophobic compounds generally are highly retained on the BSA, and a mobile-phase modifier such as 1-propanol can be added to obtain reasonable retention times. The retention and optical resolution of charged solutes such as carboxyUc acids or amines can be controlled by pH and ionic strength of the mobile phase. 

Appllca.tlons. MCA is used for the resolution of many classes of chiral dmgs. Polar compounds such as amines, amides, imides, esters, and ketones can be resolved (34). A phenyl or a cycloalkyl group near the chiral center seems to improve chiral selectivity. Nonpolar racemates have also been resolved, but charged or dissociating compounds are not retained on MCA. Mobile phases used with MCA columns include ethanol and methanol. 

The stationary phase is selected to provide the maximum selectivity. Where possible, the retention factor is adjusted (by varying the mobile phase composition, temperature, or pressure) to an optimum value that generally falls between 2 and 10. Resolution is adversely affected when k 2, while product dilution and separation time... 

Errors in the molecular weight data from HPSEC are usually due to improperly prepared samples, column dispersity, or flow rate variations. The sample to be analyzed should be completely dissolved in the mobile phase and filtered prior to injection onto the column. A plugged column inlet frit will invalidate results. In addition, do not load the column with excess sample. Column overloading affects the accuracy of data by broadening peaks, reducing resolution, and increasing elution volume. For best results, the concentration of the injected sample should be as low as possible while still providing adequate... 

The ionic species of the mobile phase will also affect the separation. This is shown in Table 4.3 by the difference in resolution values for magnesium chloride buffer compared to sodium sulfate buffer. In addition, calibration curves for proteins in potassium phosphate buffers are shallower than those generated in sodium phosphate buffers. The slope of the curve in Sorenson buffer (containing both Na and ) is midway between the slopes generated with either cation alone (1). Table 4.4 illustrates the impact of different buffer conditions on mass recovery for six sample proteins. In this case, the mass recovery of proteins (1,4) is higher with sodium or potassium phosphate buffers (pH 6.9) than with Tris-HCl buffers (pH 7.8). 

Figure 4.21 demonstrates the effect of temperature on the resolution of PEOs on a TSK-GEL G6000PWxi. and G3000PWxl in series. Increased temperature will decrease mobile phase viscosity and improve diffusion, which will improve resolution. 

The hydrophilic surface characteristics and the chemical nature of the polymer backbone in Toyopearl HW resins are the same as for packings in TSK-GEL PW HPLC columns. Consequently, Toyopearl HW packings are ideal scaleup resins for analytical separation methods developed with TSK-GEL HPLC columns. Eigure 4.44 shows a protein mixture first analyzed on TSK-GEL G3000 SWxl and TSK-GEL G3000 PWxl columns, then purified with the same mobile-phase conditions in a preparative Toyopearl HW-55 column. The elution profile and resolution remained similar from the analytical separation on the TSK-GEL G3000 PWxl column to the process-scale Toyopearl column. Scaleup from TSK-GEL PW columns can be direct and more predictable with Toyopearl HW resins. 

Figure 13.21 shows the resolution of a dozen polymer additives at very high resolution using chloroform as the mobile phase. Tinuvin 622 will elute in pure chloroform whereas Chimassorb 944 and many other hindered amine light stabilizers (HALS) will not. With the addition of 1% triethyl amine to the chloroform, however, virtually all HALS will elute. 

Figure 13.22 shows the resolution of the surfactants Tween 80 and SPAN. The high resolution obtained will even allow the individual unreacted ethylene oxide oligomers to be monitored. Figure 13.23 details the resolution of many species in both new and aged cooking oil. Perhaps the most unique high resolution low molecular weight SEC separation we have been able to obtain is shown in Fig. 13.24. Using 1,2,4-trichlorobenzene as the mobile phase at 145°C with a six column 500-A set in series, we were able to resolve Cg, C, Cy, Cg, C9, Cio, and so on hydrocarbons, a separation by size of only a methylene group. Individual ethylene groups were at least partially resolved out to Cjg. This type of separation should be ideal for complex wax analysis.

Comparison of the separation efficiency between two columns in the same mobile phase or one column in two mobile phases is based on the extent of resolution of the peaks of the PEO standards in the respective chromatograms of the PEO A, B, and C group. Due to the limitation of space, only the TSK PEO A chromatograms for the four columns in water and water/methanol are... 

Other specifications of the porous materials that affect the performance of HOPC include pore volume. A larger pore volume, or equivalently closer packing, of the porous materials increases the ratio of the volume of the stationary phase to the volume of the mobile phase. The difference causes a shift in the segregation boundary in the partitioning and a change in the resolution. 

Thus, a 2-D separation can be seen as 1-D displacement operating in two dimensions. The 2-D TLC separation is of no interest if selection of the two mobile phases is not appropriate. With this in mind, displacement in either direction can be either selective or non-selective. A combination of two selective displacements in 2-D TLC will lead to the application of different separating mechanisms in each direction. As an extreme, if the solvent combinations are the same (5ti = 5t2 5vi = va) or very similar (5ti 5vi 5ya), the compounds to be separated will be poorly resolved or even unresolved, and as a result a diagonal pattern will be obtained. In such circumstances, a slight increase in resolution might occur, because of an increase by a factor of V2 in the distance of migration of the zone (4). 

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