8.9: Other Forms of Chromatography
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\(\newcommand{\avec}{\mathbf a}\) \(\newcommand{\bvec}{\mathbf b}\) \(\newcommand{\cvec}{\mathbf c}\) \(\newcommand{\dvec}{\mathbf d}\) \(\newcommand{\dtil}{\widetilde{\mathbf d}}\) \(\newcommand{\evec}{\mathbf e}\) \(\newcommand{\fvec}{\mathbf f}\) \(\newcommand{\nvec}{\mathbf n}\) \(\newcommand{\pvec}{\mathbf p}\) \(\newcommand{\qvec}{\mathbf q}\) \(\newcommand{\svec}{\mathbf s}\) \(\newcommand{\tvec}{\mathbf t}\) \(\newcommand{\uvec}{\mathbf u}\) \(\newcommand{\vvec}{\mathbf v}\) \(\newcommand{\wvec}{\mathbf w}\) \(\newcommand{\xvec}{\mathbf x}\) \(\newcommand{\yvec}{\mathbf y}\) \(\newcommand{\zvec}{\mathbf z}\) \(\newcommand{\rvec}{\mathbf r}\) \(\newcommand{\mvec}{\mathbf m}\) \(\newcommand{\zerovec}{\mathbf 0}\) \(\newcommand{\onevec}{\mathbf 1}\) \(\newcommand{\real}{\mathbb R}\) \(\newcommand{\twovec}[2]{\left[\begin{array}{r}#1 \\ #2 \end{array}\right]}\) \(\newcommand{\ctwovec}[2]{\left[\begin{array}{c}#1 \\ #2 \end{array}\right]}\) \(\newcommand{\threevec}[3]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \end{array}\right]}\) \(\newcommand{\cthreevec}[3]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \end{array}\right]}\) \(\newcommand{\fourvec}[4]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \\ #4 \end{array}\right]}\) \(\newcommand{\cfourvec}[4]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \\ #4 \end{array}\right]}\) \(\newcommand{\fivevec}[5]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \\ #4 \\ #5 \\ \end{array}\right]}\) \(\newcommand{\cfivevec}[5]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \\ #4 \\ #5 \\ \end{array}\right]}\) \(\newcommand{\mattwo}[4]{\left[\begin{array}{rr}#1 \amp #2 \\ #3 \amp #4 \\ \end{array}\right]}\) \(\newcommand{\laspan}[1]{\text{Span}\{#1\}}\) \(\newcommand{\bcal}{\cal B}\) \(\newcommand{\ccal}{\cal C}\) \(\newcommand{\scal}{\cal S}\) \(\newcommand{\wcal}{\cal W}\) \(\newcommand{\ecal}{\cal E}\) \(\newcommand{\coords}[2]{\left\{#1\right\}_{#2}}\) \(\newcommand{\gray}[1]{\color{gray}{#1}}\) \(\newcommand{\lgray}[1]{\color{lightgray}{#1}}\) \(\newcommand{\rank}{\operatorname{rank}}\) \(\newcommand{\row}{\text{Row}}\) \(\newcommand{\col}{\text{Col}}\) \(\renewcommand{\row}{\text{Row}}\) \(\newcommand{\nul}{\text{Nul}}\) \(\newcommand{\var}{\text{Var}}\) \(\newcommand{\corr}{\text{corr}}\) \(\newcommand{\len}[1]{\left|#1\right|}\) \(\newcommand{\bbar}{\overline{\bvec}}\) \(\newcommand{\bhat}{\widehat{\bvec}}\) \(\newcommand{\bperp}{\bvec^\perp}\) \(\newcommand{\xhat}{\widehat{\xvec}}\) \(\newcommand{\vhat}{\widehat{\vvec}}\) \(\newcommand{\uhat}{\widehat{\uvec}}\) \(\newcommand{\what}{\widehat{\wvec}}\) \(\newcommand{\Sighat}{\widehat{\Sigma}}\) \(\newcommand{\lt}{<}\) \(\newcommand{\gt}{>}\) \(\newcommand{\amp}{&}\) \(\definecolor{fillinmathshade}{gray}{0.9}\)At the beginning of Section 12.5, we noted that there are several different types of solute/stationary phase interactions in liquid chromatography, but limited our discussion to liquid–liquid chromatography. In this section we turn our attention to liquid chromatography techniques in which partitioning occurs by liquid–solid adsorption, ion-exchange, and size exclusion.
Liquid-Solid Chromatography
In liquid–solid adsorption chromatography (LSC) the column packing also serves as the stationary phase. In Tswett’s original work the stationary phase was finely divided CaCO3, but modern columns employ porous 3–10 μm particles of silica or alumina. Because the stationary phase is polar, the mobile phase usually is a nonpolar or a moderately polar solvent. Typical mobile phases include hexane, isooctane, and methylene chloride. The usual order of elution—from shorter to longer retention times—is
olefins < aromatic hydrocarbons < ethers < esters, aldehydes, ketones < alcohols, amines < amide < carboxylic acids
Nonpolar stationary phases, such as charcoal-based absorbents, also are used. For most samples, liquid–solid chromatography does not offer any special advantages over liquid–liquid chromatography. One exception is the analysis of isomers, where LSC excels.
Ion-Exchange Chromatography
In ion-exchange chromatography (IEC) the stationary phase is a cross-linked polymer resin, usually divinylbenzene cross-linked polystyrene, with covalently attached ionic functional groups (see Figure 8.9.1 and Table 8.9.1 ). The counterions to these fixed charges are mobile and are displaced by ions that compete more favorably for the exchange sites. Ion-exchange resins are divided into four categories: strong acid cation exchangers; weak acid cation exchangers; strong base anion exchangers; and weak base anion exchangers.
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Size-Exclusion Chromatography
We have considered two classes of micron-sized stationary phases in this section: silica particles and cross-linked polymer resin beads. Both materials are porous, with pore sizes ranging from approximately 5–400 nm for silica particles, and from 5 nm to 100 μm for divinylbenzene cross-linked polystyrene resins. In size-exclusion chromatography—which also is known by the terms molecular-exclusion or gel permeation chromatography—the separation of solutes depends upon their ability to enter into the pores of the stationary phase. Smaller solutes spend proportionally more time within the pores and take longer to elute from the column.
Size-exclusion chromatography provides a rapid means for separating larger molecules, including polymers and biomolecules. It is also capable of providing an estimation of a solute’s molecular weight (MW). Calibration curves are prepared using a series of standards of known molecular weight and measuring each standard’s retention volume. As shown in Figure 8.9.3 , a plot of log(MW) versus Vr is roughly linear between the column parameters called the exclusion limit and the inclusion limit. Because a solute’s retention volume is influenced by both its size and its shape, a reasonably accurate estimation of molecular weight is possible only if the standards are chosen carefully to minimize the effect of shape.
Size-exclusion chromatography is carried out using conventional HPLC instrumentation, replacing the HPLC column with an appropriate size-exclusion column. A UV/Vis detector is the most common means for obtaining the chromatogram.
Supercritical Fluid Chromatography
Although there are many analytical applications of gas chromatography and liquid chromatography, they can not separate and analyze all types of samples. Capillary column GC separates complex mixtures with excellent resolution and short analysis times. Its application is limited, however, to volatile analytes or to analytes made volatile by a suitable derivatization reaction. Liquid chromatography separates a wider range of solutes than GC, but the most common detectors—UV, fluorescence, and electrochemical— have poorer detection limits and smaller linear ranges than GC detectors, and are not as universal in their selectivity.
For some samples, supercritical fluid chromatography (SFC) provides a useful alternative to gas chromatography and liquid chromatography. The mobile phase in supercritical fluid chromatography is a gas held at a temperature and pressure that exceeds its critical point (Figure 8.9.4 ). Under these conditions the mobile phase is neither a gas nor a liquid. Instead, the mobile phase is a supercritical fluid.
Analysis time and resolution, although not as good as in GC, usually are better than in conventional HPLC. The density of a supercritical fluid, on the other hand, is much closer to that of a liquid, which explains why supercritical fluids are good solvents. In terms of its separation power, a mobile phase in SFC behaves more like the liquid mobile phase in HPLC than the gaseous mobile phase in GC.
The most common mobile phase for supercritical fluid chromatography is CO2. Its low critical temperature of 31.1oC and its low critical pressure of 72.9 atm are relatively easy to achieve and maintain. Although supercritical CO2 is a good solvent for nonpolar organics, it is less useful for polar solutes. The addition of an organic modifier, such as methanol, improves the mobile phase’s elution strength. The instrumentation for supercritical fluid chromatography essentially is the same as that for a standard HPLC. The only important additions are a heated oven for the column and a pressure restrictor downstream from the column to maintain the critical pressure. Gradient elutions are accomplished by changing the applied pressure over time. The resulting change in the mobile phase’s density affects its solvent strength. Detection is accomplished using standard GC detectors or HPLC detectors. Supercritical fluid chromatography has many applications in the analysis of polymers, fossil fuels, waxes, drugs, and food products.