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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