Chromatography Notes Separations Chromatography separation of components on the basis of differential affinities for a stationary phase relative to a mobile phase Figure 23-6 Mechanisms of chromatographic interactions Adsorption solid stationary phase Partition liquid stationary phase Ion-exchange covalently attached anions or cations on a resin Molecular (Size) exclusion -gel Affinity anti-bodies attached to a solid support Column separation techniques GC HPLC CE Sample type volatiles nonvolatiles ionic compounds sample introduction vaporize/he injection loop electrophoretic (apply voltage) mobile phase He liquid liquid stationary phase liquid coating varies none reverse phase not chomatog. on surface of column covalently bound or solid phase particles to particles
Separation vapor pressure reverse phase charge/size Polarity polarity/mw varies Mechanism adsorption, partition all none detector FID/MS UV/MS UV/MS theoretical plates high moderate very high advantages resolution versatility high resolution compat. w/ MS sample load simplicity disadvantages limited to complexity poor conc. Volatiles sensitivity DAY 2 Chromatographic parameters Retention time - tr Adjusted retention time tr` = tr-tm Capacity factor k` = (tr-tm)/tm (fairly indep. of flow rate) = time solute spends in stat. phase/ time solute spends in mobile phase fraction of time spent in mobile phase = tm/tr = 1/(k`+1) Relative retention - α = t`r 1 /t`r 2 = k`1/k`2 Resolution (how well are two peaks separated) R = tr/w av = V r /w av Band spreading
σ = standard deviation of the band H = σ 2 /x - the height of a theoretical plate (stages of separation) x = the distance traveled down the column N (number of theoretical plates) = L/H = 16tr 2 /w 2 = tr 2 /σ 2 R = (N 1/2 /4) ((α-1)/α) (k`2/(1+k`av )) GC vs HPLC N can be increased by using a longer column α depends on the stationary phase and/or mobile phase increase k`2 shallow gradient Why band spread? van Deemter Equation H A + B/v + Cv v = linear flow rate A = multiple paths B = longitudinal diffusion C = equilibrium term Capillary GC no A term C term small (diffusion fast for gas, helps establish equilibrium quickly B large, diffusion coefficients of gases are large H = 40 plates/cm for a typical 0.25-0.32 mm ID column N = 120,000 plates (for a 30 m column) HPLC A term decreases with particle size and uniformity B term is small (diffusion for liquids is small) C term, equilibrium is difficult to maintain at high flow rates H = 700 plates/cm for typical 15 cm column with 5 µm ID particles N = 10,000 plates CE No A term No C term (no stationary phase, no equilibrium) B term is small (diffusion for liquids is small), short analysis times H = 50,000-500,000 plates/cm depending on ID of column N = 2,500 25,000 plates (for a 20 cm column) DAY 3
GC Injector Carrier gas H 2, He, or N 2 Column - open tubular fused silica with a coated liquid support ID 0.1 0.53 mm Injection Split - 1/100 of the sample go onto the column Splitless - most of sample goes on to column (better quantitation, worse separation) On-column all of sample goes onto column The more narrow the initial plug the better the separation Fast GC Richard Smith University of Michigan Oven Low molecular weight / highly volatile cmpds elute from column first Isothermal vs Temp. program (fig 24-7) The more narrow the initial plug the better the separation Fast GC Richard Smith University of Michigan Detectors FID burn sample in flame to produce CHO + ions only detects hydrocarbons molar sensitivity increases with the number of carbons quantitative, but provides no qualitative data on component mass spectrometry ionizes gaseous sample molecules with a 70 ev electron beam, imparts excess energy, induces fragmentation. Fragmentation pattern is reproducible and distinctive for different compound. The fragment ions are then separated on the basis of their mass-to-charge ratio, and are detected sequentially. A mass spectrum is then constructed. Libraries of mass spectra are automatically searched to provide identification.