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Ch 21 – Principles of Chromatography and Mass Spectrometry Ch 22 – Gas and Liquid Chromatography. What is Chromatography? – Sec 21-1. Chromatography = a process where compounds in a mixture are separated by passing it through a column that retains some compounds longer than others.
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Ch 21 – Principles of Chromatography and Mass Spectrometry Ch 22 – Gas and Liquid Chromatography
What is Chromatography? – Sec 21-1 Chromatography = a process where compounds in a mixture are separated by passing it through a column that retains some compounds longer than others
Mobile phase = Stationary phase = Elution =
Adsorption chromatography • stationary phase = solid (analyte adsorbs onto) • mobile phase = gas or liquid • Partition chromatography • stationary phase = thin liquid film coating inside surface of column or coats the solid support (SiO2) • mobile phase = gas or liquid • Ion-exchange chromatography • stationary phase = charged resin with covalently bound ionic groups such as -SO3- or -N(CH3)3+; electrostatically attracts ionic analytes • mobile phase = liquid • Molecular exclusion chromatography • e.g. gel filtration, gel permeation, molecular sieve • smaller molec ules trapped in pores while the larger ones elute faster • Affinity chromatography • most selective, covalently bonded antibody binds a specific protein
Theoretical Plates N = 5.55 tr2/W21/2 H = L/N
Why Do Bands Spread? – Sec 21-3 1. Longitudinal Diffusion (B/u)
Practical Control of Separation • Find optimum flow rate (uopt) • Decrease the stationary phase thickness • Temperature programming increases Ds • Choose carrier gas with higher Dm • Decrease solid support particle size • Narrow-bore columns
Internal Standards – Sec 5-4 Area concentration X = unknown S = standard (Area X) / (Area S) unknown(Area X) / (Area S) standard [X] / [S] unknown [X] / [S] standard =
ASK YOURSELF (5-D, p. 103) - Using an Internal Standard A mixture containing 52.4 nM iodoacetone (X) and 38.9 nm p-dichlorobenzene (S) gave the relative detector response (area of X)/(area of S) = 0.644. A solution containing an unknown quantity of X plus 742 nM S gave a relative detector response (area of X)/(area of S) = 1.093. Find the concentration of X in the unknown.
Gas Chromatography – Sec 22-1 • Sample volatilized and injected into a column along with an inert carrier gas or MOBILE PHASE • Mixture separated by differential retention by the STATIONARY PHASE (some molecules hjeld up longer than others) • Components separate according to boiling point (lowest first) • Match analyte polarity to stationary phase polarity (“Like dissolves Like”)
Detectors A. Thermal Conductivity
Applications of Gas Chromatography • Routine separation (e.g. after a synthesis) • Identification of an unknown by comparing retention time to a known standard • Quantization using an INTERNAL STANDARD
Example Separations A. Environmental – EPA Methods