![]() Taking advantage of a secondary equilibrium reaction is a useful strategy for improving a separation. There are two commonly used approaches for increasing α: add a reagent to the mobile phase that reacts with the solutes in a secondary equilibrium reaction, or try a different mobile phase. To effect a better separation between two solutes we must improve the selectivity factor, α. As we learned in Section 12C.1, however, a change in k is not an effective method for improving resolution when its initial value is greater than 10. Because the silica substrate may undergo hydrolysis in basic solutions, the pH of the mobile phase must be less than 7.5.Ĭhoosing a Mobile Phase–Adjusting SelectivityĬhanging the mobile phase’s polarity index changes a solute’s retention factor. Most reversed-phase separations are carried out using a buffered aqueous solution as a polar mobile phase, or with other polar solvents, such as methanol and acetonitrile. The most common nonpolar stationary phases use an organochlorosilane where the R group is an n-octyl (C 8) or n-octyldecyl (C 18) hydrocarbon chain. In reversed-phase chromatography, which is the more common form of HPLC, the stationary phase is nonpolar and the mobile phase is polar. The combination of a polar stationary phase and a nonpolar mobile phase is called normal-phase chromatography. Because the stationary phase is polar, the mobile phase is a nonpolar or moderately polar solvent. Examples of polar stationary phases include those where R contains a cyano (–C 2H 4CN), a diol (–C 3H 6OCH 2CHOHCH 2OH), or an amino (–C 3H 6NH 2) functional group. If R is a polar functional group, then the stationary phase is polar. The properties of a stationary phase depend on the organosilane’s alkyl group. To prevent unwanted interactions between the solutes and any remaining –SiOH groups, Si(CH 3) 3Cl is added, converting the unreacted sites to –SiOSi(CH 3) 3 such columns are designated as end-capped. Bonded stationary phases are created by reacting the silica particles with an organochlorosilane of the general form Si(CH 3) 2RCl, where R is an alkyl, or substituted alkyl group. To prevent the loss of stationary phase, which shortens the column’s lifetime, it is covalently bound to the silica particles. ![]() Because the stationary phase may be partially soluble in the mobile phase, it may elute, or bleed from the column over time. In liquid–liquid chromatography the stationary phase is a liquid film coated on a packing material, typically 3–10 μm porous silica particles. Stationary Phases for Gas–Liquid Chromatography Although reducing particle size by 2× increases efficiency by a factor of 1.4, it also produces a 4-fold increase in back pressure. 11įigure 12.40 The packing of smaller particles creates smaller interstitial spaces than the packing of larger particles. Monolithic rods made of a silica-gel polymer typically have macropores with diameters of approximately 2 μm and mesopores-pores within the macropores-with diameters of approximately 13 nm. ![]() A monolithic column-which usually is similar in size to a conventional packed column, although smaller, capillary columns also are available-is prepared by forming the monolithic rod in a mold and covering it with PTFE tubing or a polymer resin. Monolithic columns, in which the solid support is a single, porous rod, offer column efficiencies equivalent to a packed capillary column while allowing for faster flow rates. Because the tubing and fittings that carry the mobile phase have pressure limits, a higher back pressure requires a lower flow rate and a longer analysis time. One limitation to a packed capillary column is the back pressure that develops when trying to move the mobile phase through the small interstitial spaces between the particulate micron-sized packing material (Figure 12.40). Capillary columns packed with 3–5 μm particles have been prepared with column efficiencies of up to 250 000 theoretical plates. These columns are made from fused silica capillaries with internal diameters from 44–200 μm and lengths of 50–250 mm. This particular column has an internal diameter of 4.6 mm and a length of 150 mm, and is packed with 5 μm particles coated with stationary phase.Ĭapillary columns use less solvent and, because the sample is diluted to a lesser extent, produce larger signals at the detector. You can use equation 12.16 to estimate a column’s peak capacity.įigure 12.39 Typical packed column for HPLC.
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