Chromatography

ENVH 432
Reading: Chapters 23-25 in Harris,
Quantitative Chemical Analysis
 Chromatography
• The separation of analytes for individual
detection in a complex matrix

GC

LC

Sample  Column  Peaks with area

(or Extract) proportional to
concentration
 Chromatography
• Chromatographic Principles
• Peak Separation and Quantification
• Gas Chromatography
• Liquid Chromatography
• Detectors
 Principles of Chromatography
• Stationary phase is fixed in place in a column or on a
planar surface
• Mobile phase (or Eluent) is moving over or through the
stationary phase, carrying the analyte along with it
• Chromatography: components of a mixture are
separated based on rates at which they are carried
through the stationary phase by a gaseous or liquid
mobile phase
• Elution: process of an analyte being washed through a
stationary phase by the movement of a mobile phase
• Chromatogram: the plot of analyte signal (function of
concentration) versus elution time
 Thin Layer Chromatography (TLC)
• Sample mixture applied to bottom of plate
• TLC plate: Thin layer of silica (or other solid stationary
phase) on metal plate backing
• Liquid mobile phase carries sample through solid
phase by capillary action
• Separate fractions can be scraped off and further
analyzed
• TLC used to optimize conditions for liquid column
chromatography or to purify
synthesized standards

TLC Separation of Chlorophyll

Analyte Quantification Peak Height

• Chromatogram
• Retention Time
• Resolution Threshold
– Peak Shape Noise

– Peak Width
• Signal-to-Noise ratio Peak Area:
• Threshold Integral of signal

Peak Width
 Quantitation methods in
chromatography
• External standard calibration
– Uses calibrant external to sample
– Requires precise control of injection volumes
• Internal standard calibration
– Uses a standard in each sample and calibrant
at a constant mass/sample
– Does not require precise control of injection
volumes
 IS
Analyte Quantification Analyte

External Standard

Area of Analyte
Calibration

Concentration

Internal Standard

Relative Area
Calibration
Relative Area = Area of Analyte
Area of Internal Standard

Concentration
 Chromatographic Parameters
• Stationary Phase (solid or solid-liquid)
– Adsorption
– Partition GC

– Molecular exclusion HPLC

– Ion-Exchange
– Affinity (protein lock to antibody key)
• Mobile Phase (gas or liquid)
– Flow rate
– Composition
• Column Dimensions
– Length, width (ID), particle size or film thickness
 Peak Resolution
Change Column phase or
Signal

dimensions (e.g., Length)

Change Flow
• Mobile phase (gas or liquid)
1 Change Gradient
• Temperature or pressure
• Mobile Phase Composition
Time (minutes)

Gas Chromatography

Oven Temperature (C)

1

2
2

Time (minutes)
 GC vs HPLC
GC
• Highly efficient separations
• Analytes must be (semi)
volatile
• Analytes must be thermally
stable
• Non-polar, small molecules
• Limited control over mobile
and stationary phases
HPLC
• (Slightly) less efficient
separations
• Small and large molecules,
polar and non-polar
• Useful for thermally
unstable analytes
• Many choices available for
mobile and stationary
phases
Gas Chromatograph (GC)
Chromatogram
 Gas Chromatography
• Mobile Phase (gas)
– N2, He, H2
• Stationary Phase
– Packed Columns
• Silica (short & wide)
– Open Tubular Columns
• Liquid-bonded siloxanes
R O Si R
• Long & narrow CH3
R O Si R
CH3
GC: Partition
Chromatography
• Liquid film with siloxane
backbone

• Choose attached functional

groups that match the
polarity of your analytes

• For separation of nonpolar

trihalomethanes, EPA
Method 551.1 recommends
GC columns with
1% Diphenylpolysiloxane or
6% cyanopropylphenyl-
polysiloxane phases
 GC Detectors
• Quantitative
– Thermal Conductivity Detector (TCD)
– Flame Ionization (FID)
– Electron Capture (ECD)
– Flame Photometric (FPD)
• Qualitative and Quantitative
– Infrared Spectrometer (FTIR)
– Mass Spectrometer (MS or MS/MS)
 GC Detector
Detector Detection Limit Linear range
TCD 400 pg/mL (propane) > 105
FID 2 pg/s > 107
ECD 5 fg/s 104
FPD 1 pg/s P, 10 pg/s S >104, >103
FTIR 200 pg to 40 ng 104
MS 25 fg to 100 pg 105

Reference: Table 24-5 in Harris, Quantitative Chemical Analysis

 GC Detector Pros/Cons
• TCD and FID
– Generic detectors that give relatively uniform
response to all carbon-containing compounds
– Not very sensitive
• ECD
– Very sensitive for electron-capturing species, e.g.
halogens and aromatics
– More sensitive than full scan MS
– Not structure specific
– Wide variation in response from analytes
• FPD: specific to N, S, P analytes
 GC Detector Pros/Cons
• FTIR
– Gives info on functional groups
– Not as sensitive as MS
• MS or MS/MS
– Gives structural info
– Responds to all compounds
– Can be very sensitive (SIM, SRM)
– expensive
High Performance Liquid
Chromatography (HPLC)
High Performance Liquid Chromatograph
(HPLC)

Fluorescence
detector

Diode array (UV)

detector

Column heater + Solvents

micro switching
valve
Solvent
degasser
Autosampler

High pressure
Sampler pump unit
cooling unit
 HPLC Parameters
• Stationary Phase (solid or solid-liquid)
– Adsorption
– Partition
– Molecular exclusion
– Ion-Exchange
– Affinity (protein lock to antibody key)
• Mobile Phase (liquid)
– Flow rate
– Composition
• Column Dimensions
– Length, width (ID), particle size
Choice of Separation Mechanism
 Adsorption Chromatography
• Liquid-solid chromatography
• Normal Phase
• Separation of neutral species
on the basis of polarity
• Stationary phase examples
– Bare Silica
– Calcium carbonate
• Adsorption process similar to trapping air samples on
charcoal or sample clean-up with granular activated
carbon (GAC)
 Partition Chromatography
• Liquid-liquid chromatography
• Siloxane bonded-phase
coverings to silanol backbone
– Reverse Phase
• C18 most common (ODS)
• Separation of neutral species on the basis of
hydrophobicity
– Ion-Pair Chromatography for separation of ionic species
– Chiral Separation of optically active isomers
(enantiomers)
 Ion-Exchange Chromatography (IC)

• Separation of ionic
analytes on the basis
of charge, on ion-
exchange stationary
phases
Ion-Exchange Chromatography
• Anion exchangers:
RX+A- + M+B- ⇔ RX+B- + M+ + A-
– contain bound positive groups
• Strongly basic (SAX): Aryl–N(CH3)3+Cl-
• Weakly basic (WAX): R–NH(R)2+Cl-
– exclude neutrals and positive ions (elute first)
• Cation exchangers:
RX-C+ + M+B- ⇔ RX-M+ + C+ + B-
– contain bound negative groups
• Strongly acidic (SCX): Aryl–SO3-H+
• Weakly acidic (WCX): R–CO2-H+
– exclude neutrals and negative ions
 Ion-Exchange Chromatography (IC)

• Suitable for both inorganic analytes and organic

analytes (retains charged and elutes neutrals)
• Eluent suppressor technique heightens
sensitivity for inorganic analytes, decreases
background signal
• Examples:
– Post-Column Reaction with UV detection of bromate
as a disinfection byproduct of ozone water treatment
– Chelating microcolumn used for metal (Al) speciation
in soils and sediments
– Analysis of organic acids in foods and beverages
Size-Exclusion Chromatography (SEC)

• Separation of molecules on
the basis of differences in
molecular size, not by
partitioning behavior
• Ideal for separation of heavy
natural product molecules from
lighter analytes and salts
• Gel-Permeation SEC for nonpolar analytes
– aqueous solvents and hydrophilic packings
• Gel-Filtration SEC for polar or ionic analytes
– hydrophilic solvents and aqueous packings
Size-Exclusion Chromatography (SEC)
 Affinity Chromatography
• Lock and Key mechanism
– Enzymes and substrates
– Antibodies and antigens
– Receptors and hormones

• Isolates a single compound from a complex mixture

– Interfering compounds elute first
• Specific binding of compound to stationary phase
– A molecular imprinted polymer is polymerized in the presence
of a template molecule
 HPLC Mobile Phase
• Composition of solvent
– water, phosphate buffer, methanol, acetonitrile
• Isocratic elution
– single solvent or constant solvent mixture
• Solvent gradient
– Increase eluent strength to elute the strongly retained
analytes
– Reversed phase column: start with 100% aqueous
phase (A) and gradually increase the % of organic
solvent phase (B) running through the column as
mobile phase
– Normal phase…..starts with non-polar solvent,
gradually increase % of polar solvent (e.g. methanol)
 HPLC Detectors
• Quantitative
– Ultraviolet (UV) – Refractive Index
– Conductivity – Light-Scattering
– Fluorescence Post-column
– Electrochemical Derivatization

• Qualitative and Quantitative

– Photodiode array (DAD): UV spectrum of
each peak
– Mass spectrometer (MS) and MS/MS
 HPLC detectors
Detector Limit of detection (ng) specificity

Evaporative light 0.1-1 none

scattering
Ultraviolet 0.1-1 low

Refractive Index 100-1,000 none

Electrochemical 0.01-1 low

Fluorescence 0.001-0.01 high

Mass spectrometry 0.0001-1 Very high

Adapted from Harris, D.C Quantitative Chemical Analysis

Chromatographic Image Resources
• Skoog, D.A.; West, M.W.; Holler, F.J. Fundamentals of
Analytical Chemistry, 7th Ed, Saunders College
Publishing, 1997.
• www.chem.ubc.ca/courseware/121/tutorials/exp3A/colu
mnchrom/
• www.agilent.com
• www.waters.com
• www.dionex.com
• www.wikipedia.org
– Chromatography, Gas chromatography, Liquid chromatography,
Size-exclusion chromatography (Isaac Yonemoto)