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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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Mobile phase =
Stationary phase =
Elution =
Different Types of Chromatography
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1. Adsorption chromatography
• stationary phase = solid (analyte adsorbs onto)
• mobile phase = gas or liquid
2. Partition chromatography
• stationary phase = thin liquid film coating inside surface of column or coats the solid support (SiO2)
• mobile phase = gas or liquid
3. 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
4. Molecular exclusion chromatography
• e.g. gel filtration, gel permeation, molecular sieve
• smaller molec ules trapped in pores while the larger ones elute faster
6. Affinity chromatography
• most selective, covalently bonded antibody binds a specific protein
How We Describe a Chromatogram – Sec 21-2
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N = 5.55 tr2/W2
1/2
H = L/N
Theoretical Plates Resolution = tr/Wav
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1. Longitudinal Diffusion (B/u)
Why Do Bands Spread? – Sec 21-32. Equilibration Time (C·u)
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3. Eddy Diffusion (A)
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The Van Deemter Equation 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
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Internal Standards – Sec 5-4
Area concentration
(Area X) / (Area S) unknown(Area X) / (Area S) standard
= [X] / [S] unknown [X] / [S] standard
X = unknown
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.
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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”)
Basic Instrumentation
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The Injection Port Capillary Columns
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Capillary Columns
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Stationary Phases Temperature Programming
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Detectors
A. Thermal Conductivity
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B. Flame Ionization Detector (FID)C. Electron Capture Detector (ECD)
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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
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B. Biological