Transcript Powerpoint

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 =
Different Types of Chromatography
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
Theoretical Plates
N = 5.55 tr2/W21/2
H = L/N
Resolution = tr/Wav
Why Do Bands Spread? – Sec 21-3
1. Longitudinal Diffusion (B/u)
2. Equilibration Time (C·u)
3. Eddy Diffusion (A)
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
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”)
Basic Instrumentation
The Injection Port
Capillary Columns
Capillary Columns
Stationary Phases
Temperature Programming
Detectors
A. Thermal Conductivity
B. Flame Ionization Detector (FID)
C. Electron Capture Detector (ECD)
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
B. Biological