Air, Water and Land Pollution: Chromatographic Methods For Environmental Analysis

Air, Water and Land Pollution
Chapter 10:
Chromatographic Methods for
Environmental Analysis
Copyright © 2010 by DBS

Contents
• Introduction to Chromatography
• Instruments of Chromatographic Methods
• Common Detectors for Chromatography
• Applications of Chromatographic Methods in Environmental
Analysis
• Practical Tips to Chromatographic Methods
Chromatographic Methods…
Introduction to Chromatography
Types of Chromatography and Separation Columns
• Chromatography – separates chemical mixtures by distributing them between a

stationary phase and a mobile phase
• All chromatographic analyses are based on the establishment of equilibrium between
the two phases
e.g. Gas Chromatography (GC), Liquid Chromatography (LC), and Supercritical Fluid
Chromatography (SFC) have gas, liquid and supercritical fluid mobile phases
Types of Chromatography and Separation Columns
• GC – gas mobile phase / liquid stationary phase (GLC) or gas mobile phase / solid
stationary phase (GSC)
• LC – separation is achieved on the basis of molecular polarity partitioning between

liquid mobile phase / liquid film adsorbed on a solid support material (LLC) or liquid
mobile phase / solid stationary phase (LSC)
• Also separation via molecular size (size exclusion chromatography) or molecular

charge (IC)
• Separation in modern analyses is performed in a column

• Column chromatography has stationary phase in a narrow tube through which mobile
phase flows under pressure
LC Columns
• Packed Column: stainless steel tube 3-25 cm long with packing materials inside the
tube
GC Columns (2 types)
• Packed Column: glass or stainless steel coil (1-5 m long, 5 mm i.d.) filled with
stationary phase or packing coated with stationary phase
• Capillary Column: thin fused-silica tube (10-100 m long, 250 µm i.d.) with stationary
phase inside – tubular structure – no packing material inside, coated inside in one of
3 ways:
GC Columns (2 types)
• Capillary Column:
1. “wall-coated open-tubular” (WCOT) has a thin wall coated

liquid film stationary phase
2. “support-coated open-tubular” (SCOT) has microparticles

attached to the wall of the capillary, coated with liquid
stationary phase
3. “porous layer open-tubular” (PLOT) has solid-phase

microparticles attached to the capillary walls
• Open tubular structure has low resistance to gas flow, column can be made much
longer (up to 100 m) than packed columns
• Long length permit very efficient separations of samples of complex mixtures
• Used in ~ 80 % of environmental analyses

Common Stationary Phases: The Key to Separation
• Thousands of commercially available stationary phases

• Choice of stationary phase has greatest influence on the separation obtained
GC Column Stationary Phases

• Gas-Solid chromatography (GSC) stationary phase is alumina (Al 2O3) or porous
polymers
• Based on adsorption of gaseous chemicals onto the stationary phase and limited to
low molecular weight gas species (H2S, CS2, CO, CO2 etc.)
• Gas-Liquid Chromatography (GLC) – various liquid stationary phases


• Gas-Liquid Chromatography (GLC) – various liquid stationary phases with general
structure of polydimethyl siloxane and polyethylene glycol
• e.g. Polydimethyl siloxane: -R groups are all hydrophobic (R = CH3), least polar stationary
phase, other stationary phases contain different functional groups altering their polarity

• Must chose nonpolar column for a nonpolar mixture and a polar column for a polar
mixture
HPLC Column Stationary Phases

• Adsorption-based LSC and partition based LLC
• LSC stationary phases are limited to either silica or alumina
• Most liquid chromatography is performed via LLC – partition between liquid mobile
phase and liquid stationary phase (adsorbed or chemical bonded)
• Bonded phase packing can be either ‘normal’ or ‘reverse’

Other Parameters Important to Compound Separation
• Column related parameters: length, i.d., stationary film thickness, particle size of
packing, etc.)
• Operational parameters: flow velocity of mobile phase, temperature, selection of
mobile phase
• e.g. size of sample (capacity) increases on using a longer or wider column with a
thicker film of stationary phase
– Increases analytical time
– Improves resolution
• Temperature control for GC and mobile phase selection for HPLC
Temperature for GC
• Column temperature is a compromise between speed, sensitivity and resolution
• High temperatures – analytes spend most of their time in the gas mobile phase
(eluted quickly)
– Resolution is poor
– Sensitivity is increased (peaks are closer together)
• Easy parameter to control so not considered as important as choice of column
Mobile Phase for HPLC

• Polarity is important since it is the basis for separation in HPLC
Terms and Theories of Chromatogram
• Chromatogram – output of a chromatographic analysis

• Plot of detector signal vs. time
tm = mobile-phase holdup time – time required for a molecule of the mobile phase to pass
through the column – may be due to air or methanol in GC, or solvent in HPLC
tr = retention time – time taken after sample injection for analyte to reach detector
A = first eluted compound eluted at t = trA
B = second eluted compound eluted at t = trB
• 3 important features of a chromatogram:

– Unretained compound peak (air or solvent)
– As time increases peaks broaden (due to diffusion in the column)
– Symmetrical nature of eluting peaks (normal distribution)
• Ideal chromatogram has narrow, symmetrical, well-spaced peaks
• Number of concepts related to separation principles

Distribution Constant (Kc)
Kc = Cs / Cm
• Where Cs = conc. Solute in stationary phase, Cw = conc. Solute in mobile phase

• Larger the Kc, the more solute is sorbed or partitioned in stationary phase
• Temperature dependent, compounds must have different Kc values in order to be
resolved
Retention Factor (k)
k = Ms / Mm
• Where Ms = amount of solute in stationary phase, Mm = amount of solute in mobile

phase
• Larger the value of k the larger the amount in the stationary phase, longer solute will
be in the column
• Depends on both mobile and stationary phases and temperature
• Can be calculated from a chromatogram, k = ( tr – tm) / tm
• Ideal k values 2-10, analytical time too long if k > 20
Separation Factor (α)

• Ratio of distribution constants of two solutes
• Can be calculated as the ratio of retention factors (k) of two solutes
α = kB = trB – tm
kA trA – tm
• Larger value of α the easier the separation, compounds can only be separated if α >
1.0
Resolution (R)
• R is the measure of a column’s ability to separate two peaks
R = peak separation / average peak width = trB – tRA / [(wA + wb)/2]
• Where wA and wB are the baseline width of two peaks

• When R = 1.0 two peaks of equal widths have a 2.3 % overlap which is the minimum
for quantitative separation
• Typically R > 1.5 is needed (depends on height ratio of the peaks)
Use of Chromatograms for Qualitative and Quantitative Analysis
• Qualitative analysis: compare retention time of analyte to that of a pure standard

(libraries)
• Quantitative analysis: measurement of peak area (proportional to analyte

concentration)
• Standard calibration curve relates area of peak to concentration

GC of a mixture containing benzene, methyl
toluene and chlorotoluene
Calculate:
Retention
factor (k),
Separation
factor (α), and
Resolution (R)
Instruments of Chromatographic Methods
Gas Chromatography
• Components
– Carrier gas
– Flow control
– Sample injection port
– Separation column
– Oven
– Detector
– Data-acquisition system
Gas Chromatography
Gas Chromatography
• Carrier gas: carry analytes through the GC

– He and N2 most common
– Chemically inert and of high purity
– Impurities such as O2 and H2O attack the liquid stationary phase in the columns
– Pressure us controlled by gas regulator
• Sample port
– Manual or autosampler
– Sample size is typically very small in GC
– Microsyringe used to introduce gaseous or liquid samples
– Inlet transfers the sample to the column
Gas Chromatography
• Column and oven:

– Oven contains heaters for the injection port and the detector
– 3 components (oven, injection port and detector) have independent temperature
controls
– Temperature control is critical for all 3
– Injection port: 50 ºC higher than Bpt. of sample, high enough to vaporize the
sample rapidly but low enough not to thermally decompose the analytes
– Column temperature should be ± 0.5 ºC in order to optimize chromatographic
separation
– Detector temperature: must be hot enough to prevent condensation of sample
and/or liquid phase
• Detector: see later

High Performance Liquid Chromatography (HPLC)
• Components:
– Solvent reservoirs
– Solvent pump
– Sample injection system
– Column (guard and analytical)
– Detector
– Data acquisition system
• Solvent reservoir:
– Solvent (neat liquid, mixture, buffered solutions)
– HPLC grade (purity), free from particles and dissolved gases
– Inlet filter inside each solvent container to remove particles
– Degassing unit or degassing unit removes dissolved gases
• Solvent pump:
– High pressure, pulse free, accurate flow rate
– Mobile phase solvent is passed through the column for separation
– Typical pressure 1000-3000 psi
• Sample injection system

– Manual (syringe) or autosampler
– Located between pump and column
• Column:
– Analytical column and guard column
– Guard column contains same packing material as analytical column
• Detector:
– See later: Ultraviolet, differential refractometers, fluorescence, conductivity
• Solvent mobile phase is the most often changed component in HPLC
• Given a set of compounds and a given column, what solvent mixture should be
chosen to maximize separation efficiency?
– Isocratic – solvent or mixed solvent at constant composition over time

– Gradient – solvent or mixed solvent composition changes over time
• Gradient elution is used to improve separation similar to changing temperature in GC

to improve separation
• Solvent properties?
– Viscosity
– UV cutoff
– Refractive index
– Boiling point
– Polarity
• UV cutoff:
– If UV detector is used, solvents should be transparent at required wavelength
• Volatility (Bpt.):
– Important for evaporative light-scattering detectors (refractometers)
– Solvents with high v.p. produce bubbles in the detector
• Refractive index (Solvent’s property in changing the speed of light):

– Important for evaporative light-scattering detectors
– Should be as large as possible for both solvent and sample
• Polarity:
– More polar solvents cause increased retention in reverse phase HPLC and reduced
retention in normal phase HPLC
Ion Chromatography
• Similar components to HPLC

– Eluent reservoir (in place of solvent reservoir)
– Pump
– Sample injection system
– Column (analytical and suppressor)
– Detector
– Data-acquisition system
Dionex ICS-1000
• Chromeleon software
• Continuous electrolytic
suppression
Ion Chromatography
• Separation in IC is based on ion exchange of ionic species rather than partitioning for
HPLC
• Mobile phase, column, stationary phase, and detector are all different from those
used in HPLC
• IC uses ion exchange, eluent suppression, and conductivity detection
• IC uses acid/base or salt buffer solutions as the eluent (mobile phase)
• Type and strength of eluent affects the retention times of ionic analytes
• Eluent carries the mixture through the column

Ion Chromatography
IC uses 2 columns:
• Analytical – separates anions or cations
– If anions (A-) are to be analyzed column contains
cationic exchange resin (+ve charge –N(CH3)3+)
Called anion exchange column
– If cations (M+) are to be analyzed column contains

anionic exchange resin (-ve charge –SO3H)
Called cation exchange column
• Suppressor – reduces the background conductivity of

the eluent to low levels
– For A- analysis suppressor is anionic exchange
resin used to retain cations and convert anions to HA
– For M+ analysis suppressor is cationic exchange
resin used to retain anions and convert cations to MOH
Ion-Chromatographic Separation
Principles
1. Mobile phase – eluent carries the mixture through the column
2. Stationary phase – compounds adsorb to ion-exchange resin in the column.
Stickier compounds require more elution time
3. Force pushes the mixture through the column – pump
4. Separation depends on retention time (ion charge and size)
e.g. Anion analysis
• Column is covered in +ve binding sites - N(CH 3)3+

• As sample passes over the resin, anions in the sample replace bicarbonate ions
bound to the resin
[Resin]+-HCO3- + Cl- → [Resin]+-Cl- + HCO3-
• Eventually sample anions are displaced again by new eluent (bicarbonate) and
washed off the column
Ion Chromatography
• Na2CO3 and NaHCO3 is most common eluent, suppessor reactions
2R-SO3H + Na2CO3 ⇌ 2R-SO3Na + H2CO3 (retains cation, anion converted to HA)
R-SO3H + NaHCO3 ⇌ R-SO3Na + H2CO3
• Suppressor removes all buffer ions via ion exchange - Na + replaced by H+, eluent
converted to carbonic acid, then CO2
• Cations are retained in suppressor column and separated anions in their acid form
(HA) are measured using conductivity detector
Kegley, 1998
IC Chart
Conductivity
Conductivity ~ concentration
Compare peak area of unknown with that of a standard
Common Detectors for Chromatography
• Detectors:
– Recovers chemical information from column effluent:
e.g. presence, concentration, mass, structure

Detectors for Gas Chromatography
• More than 60 different types for GC
• Environmental applications use:
– Thermal Conductivity Detector (TCD)

– Flame Ionization Detector (FID)
– Electron Capture Detector (ECD)
Thermal Conductivity Detector (TCD)

• Electrically heated filaments in a temperature-controlled cell
• Two gas streams (column flow = carrier gas + sample, reference flow = carrier gas) pass over two separate
temperature sensitive resistors (filaments)
• Under normal conditions there is a stable heat flow from the filament to the detector body
• Temperature of electrically heated filaments depends on the thermal conductivity of the surrounding gas
• When an analyte elutes and the thermal conductivity of the column effluent is reduced, the filament heats up and
changes resistance
• This resistance change is often sensed by a Wheatstone bridge circuit which produces a measurable
current/voltage change.
• Current change is compared to current in a reference cell with carrier gas only
Thermal Conductivity Detector (TCD)

• TCD is relatively universal detector with a low sensitivity
• Limited use
• Measurement of major constituents of air (H2O, CO, CO2, H2)
Flame Ionization Detector (FID)

• Uses H2/air flame to burn organic compounds
• Sample effluent from the column is mixed with H 2/air

and ignited electrically at a small metal jet
• Number of ions produced ~ number of reduced carbon

atoms in flame ~ number of molecules
• Electrode collects ions formed at the flame,

produces electrical signal
• GC-FID requires 3 gases: He (carrier), H2 and air

Flame Ionization Detector (FID)

• FID can be used for all organics
• Exceptions include non-hydrogen containing organics (e.g. hexachlorobenzene), and
compounds with carbonyl, alcohol, halogen, and amine functionality that yield fewer
ions or none at all in a flame
• No response to H2O, CO2, SO2, NOx, and other non-combustible gases
Electron Capture Detector (ECD)

• ECD uses a β-emitting radiochemical
• Ionizes the carrier gas (N2) to produce electrons
Ni63 → β-
β- + N2 → 2e- + N2+
• Electrons generate an electrical current between a pair of

electrodes
• When organic molecules containing electronegative
functional groups (F, Cl, Br etc.) are present the e-
are captured and reduce the measured current
M + e - → M-
Electron Capture Detector (ECD)

• ECD is very sensitive but not a universal detector
• Cannot detect amines, alcohols or hydrocarbons
• 10-100 x more sensitive than FID, 1,000,000 x than TCD
• Invention of ECD in 1961 revolutionized our understanding of global fate and

transport of pesticides
Comparison of Major GC Detectors
• Photo Ionization Detectors (PID): similar to FID, uses UV light for ionization instead of a
flame
• Nitrogen-Phosphorous Detector (NPD): similar to FID, uses ceramic bead containing

alkaline metal that emits positive ions when heated in a gas stream containing certain
analytes (N + P compounds)
• Flame Photometric Detector (FPD): GC effluent is mixed with H2/O2, chemiluminescence

emitted from combustion of S- or P- compounds is measured spectrophotometrically
• Hall or Electrolytic Conductivity (ELCD): S-, N-, and halogen compounds converted to
ions under a Ni catalyst, conductivity of dissolved ion is measured
ECD sensitive to
TCD and MS detect all halogen compounds
FID sensitivity is low for NPD only detects N-, P-

non-H containing containing compounds
organics
PID uses UV, saturated
halogens not detected
Detectors for High Performance Liquid Chromatography
• Limited compared to GC detectors:

– UV detectors
– Fluorescence detectors
– Refractive index detectors
UV Detectors
• Relies on strong absorbance of analyte in UV region

• Same UV detector as used in UV-VIS spectrometers
• Most powerful UV detectors are photodiode array (PDA) detectors
• Simultaneous detection of spectral data in a range of wavelengths
Fluorescence Detectors
• Design similar to UV-VIS units

• Requires two wavelengths of emitted radiation
• UV lamp emits a range of wavelengths
• Filters or monochromator acquires the needed exciting
beam λexcitation
• Emitted light (fluorescence) is measured at a 90º angle to the
exciting light beam at a wavelength λemission
Fluorescence Detectors
• Not universal, limited to certain chemicals
• Typically 1 order of magnitude more sensitive than UV
• Most intense fluorescence is found in aromatic compounds
e.g. PAHs
Refractive Index Detectors
• Refractive index of solutions is changed by the presence of solutes

• RI detector responds to all solutes (universal)
Detectors for Ion Chromatography

• Conductivity detector is most commonly used
• Conductivity – ability of a solution containing a salt to conduct electricity across two
electrodes
• 3 related terms are used for “conductivity”:

– Conductance (G)
– Specific conductance or conductivity (k)
– Equivalent conductance (Λ)
• Conductance (G) (ohm-1 or mho) measured between two electrodes of area A (cm2) and
spacing L (cm) is the reciprocal of resistance R (ohms)
G = 1/R
G = 1/R
• G has international units of Siemens (S), 1 S = 1 Ω-1
• Conductivity (k) (Ω-1 cm-1) is also called “specific conductance”
k = GK
• Where K (cm-1) is called the cell constant (=L/A)

• Equivalent conductance (Λ), conductivity of an ion with a valence z in dilute solution

at 25 ºC relates conductance to concentration:
Λ = 1000 k
Cz
• Where C = molar concentration of ion (mol/L) and is equivalent conductance (S cm2

mol-1)
• Combining previous 2 equations:
k = GK
Λ = 1000 k
Cz
G = ΛCz / 1000K
• Conductivity detector: for a given ion (constant Λ and z), the conductance G is
proportional to C

Applications of Chromatographic Methods in Environmental Analysis
• Key instrument for organic analysis:
– Petroleum and petrochemicals

– Clinical and pharmaceuticals
– Forensic and crime labs
– Environmental monitoring
Gases, Volatile, and Semivolatile Organics with GC
• Analysis with GC methods
• Direct analysis:
– Gases
– Volatile compounds
– Semivolatile compounds
• Sample may require extraction and clean-up steps depending on matrix (chp. 7)
• Volatility can be compared based on Henry’s Law constants (H)

– Highly volatile: H > 10-3 atm.m3/mol
– Volatile: 10-3 < H < 10-5 atm.m3/mol
– Semivolatile: 10-5 < H < 3 x 10-7 atm.m3/mol
– Nonvolatile: H < 3 x 10-7 atm.m3/mol
Volatile
Volatile
volatile
volatile
Highly
Semi-
Non-
X(aq) ⇌ X(g) KH= P (atm)
M (mol/L)
Many pollutants have low VP but high KH,
Preference for gaseous phase over water

• 3 main categories of GC-based US EPA methods:

– Air,
– Water and wastewater
– Waste
1. Determination of toxic organic compounds in air (TO series): sampled by absorbent trap made of
Tenax, molecular sieve, activated charcoal, XAD-2 resin, or graphited carbon, desorption by thermal or
solvent. VOCs analyzed via GC-FID, GC-ECD, or GC-MS
2. Water/wastewater:
500 series (1988) published to support Safe Drinking Water Act of 1974. Low concentrations in drinking
water. 12 methods total, 6 for VOCs and 6 for specific synthetic organics and pesticides. 5 use purge-
and-trap, 6 use L-L extraction, 1 uses L-S extraction.
600 Series published for Clean Water Act and National Pollutant Discharge Elimination System
(NPDES). 624 – GC/MS for purgable organic compounds (includes compounds from 601, 602, 603,
612)
625 – Semivolatile organics (includes compounds from 604, 606, 607, 609, 610, 611, 612)
608 – Pesticides and PCBs
613 – Dioxin
3. Waste:
8000 series published in SW-846 (test Methods for Evaluating Solid Waste), all use a variety
of detectors, constantly updated year to year.
Semivolatile and Nonvolatile Organics with HPLC
• 85% of known organics are not sufficiently volatile or stable enough to be analyzed
via GC
• 85% of known organics are not sufficiently volatile or stable enough to be analyzed
via GC
• HPLC should have greater potential for analysis
• Number of HPLC methods developed by US EPA is significantly fewer than GC
– GC has many more detectors
– HPLC more expensive
– More trouble shooting is required
• 3 main categories of LC-based US EPA methods:
1. Determination of toxic organic compounds in air (TO series): aldehyde, ketone, phogene, phenol, cresols, formaldehyde and PAHs
2. Water/wastewater:
500 series (1988) published to support Safe Drinking Water Act of 1974. Low concentrations in drinking water.
531: Analysis of N-methylcarbomoyloximes and N-methyl carbamates
600 Series published for Clean Water Act and National Pollutant Discharge Elimination System (NPDES).
605: Benzidines and 610: PAHS
3. Waste:
8000 series published in SW-846 (test Methods for Evaluating Solid Waste), all use a variety of detectors, constantly updated year to year.
Carbonyl compounds, acrylamide, acrylonitrile, acrolein, N-methylcarbamates, nitroaromatics, nitramines, tetrazene, nitroglycerine
• Some SVOCs can also be analyzed via GC

Ionic Species with IC
• Separates anions and detects at ppb levels

– F-, Cl-, Br-, I-, NO2-, NO3-, SO42-, HPO42-, PO43-, SCN-, IO3-, ClO4-
US EPA method 300 for Cl-, F-, NO3-, NO2-, PO43-, SO42- in water
US EPA method 9056 and 7199 for anions in waste
– IC is method of choice for anions

– Sensitive and powerful alternative methods for cations (atomic spectroscopy)
– Recent advances include the separation of various species of Hg, Se and As

(differences in toxicity and bioavailability drive the requirements)
Practical Tips to Chromatographic Methods
What Can and Cannot be Done with GC and HPLC
• All gases and volatile compounds can be analyzed via GC not by HPLC
• Nonvolatiles and thermally unstable compounds cannot be analyzed via GC unless
their structures are changed through derivatization
• For some semivolatile compounds (e.g. PAHs, nitroaromatics and explosives) both
GC and HPLC can be used
• HPLC is preferred when direct analysis of aqueous sample is needed to avoid time
consuming extractions
• In other cases, GC is instrument of choice due to variety of detection methods
• Volatility is related to Bpt. And molecular size

• Smaller nonionic organics tend to be more volatile
– Organics containing up to 25 carbons can liekly be analyzed via GC
– HPLC can handle larger molecular weight (MW > 1000) (biomolecules – amino
acids, proteins, HCs, carbohydrates, drugs, steroids etc.)
– Most chemicals of environmental concern have MW < 500
• Advantages of GC:
– Low cost, fast analysis, ease of operation
– More sensitive and higher resolution
– Variety of columns and detectors
– Can use mass spectrometry (see chp. 12)
• Advantages of HPLC:
– Direct analysis of aqueous samples (can’t be done on GC)
– Mobile phases of various polarities provide versatility
• HPLC is usually more expensive, less sensitive and slower, operationally more
problematic hardware
Development for GC and HPLC Methods
• p280-284
Overview on Maintenance and Troubleshooting
• General Guidelines on Maintenance

• HPLC Maintenance
• GC maintenance
• General Guidelines on Troubleshooting
• Baseline Problems
• Peak Problems
• Retention Time Problems
References
• McNair, H.M., and Miller, J.M. (1998) Basic Chromatography. John Wiley & Sons, Inc.,
New York.
• O’Dell et al (1984)
Questions
1. Calculate: Retention factor (k), Separation factor (α), and Resolution (R) for slide 29.
Comment on how these may be adjusted to give optimum values.
11. Explain why resolution and speed of analysis is always a compromise during a
chromatographic analysis
26. Describe the principles of measuring anions in water by anionic ion-chromatography with
Na2CO3-NaHCO3 as the eluting solvents.
31. Describethe following: (a) What chemical functionalities will contribute fluorescence and
can be measured by fluorescence detetcors? (b) How halogen substitution affects a
compound’s ability to fluoresce?
35. For the following compound and the sample matrix, which chromatographic instrument
(GC, HPLC, IC) and detetcor would you recommend? Suggest an EPA standard method for
each chemical as well.
(a) trichloroethene (volatile) in air, (b) PAHs in solid wastes, (c) BTEX in soil, (d) acid rain
composition (sulfate, nitrate, chloride), (e) species of chromium (Cr6+) in water.

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Air, Water and Land Pollution

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Types of Chromatography and Separation Columns

• Chromatography – separates chemical mixtures by distributing them between a

Types of Chromatography and Separation Columns

• LC – separation is achieved on the basis of molecular polarity partitioning between

• Also separation via molecular size (size exclusion chromatography) or molecular

• Separation in modern analyses is performed in a column

1. “wall-coated open-tubular” (WCOT) has a thin wall coated

2. “support-coated open-tubular” (SCOT) has microparticles

3. “porous layer open-tubular” (PLOT) has solid-phase

• Long length permit very efficient separations of samples of complex mixtures

• Used in ~ 80 % of environmental analyses

Common Stationary Phases: The Key to Separation

• Thousands of commercially available stationary phases

GC Column Stationary Phases

• Gas-Liquid Chromatography (GLC) – various liquid stationary phases

Common Stationary Phases: The Key to Separation

GC Column Stationary Phases

Common Stationary Phases: The Key to Separation

GC Column Stationary Phases

Common Stationary Phases: The Key to Separation

HPLC Column Stationary Phases

• Bonded phase packing can be either ‘normal’ or ‘reverse’

Other Parameters Important to Compound Separation

Other Parameters Important to Compound Separation

Other Parameters Important to Compound Separation

• Temperature control for GC and mobile phase selection for HPLC

Mobile Phase for HPLC

Terms and Theories of Chromatogram

• Chromatogram – output of a chromatographic analysis

Terms and Theories of Chromatogram

Terms and Theories of Chromatogram

• 3 important features of a chromatogram:

• Ideal chromatogram has narrow, symmetrical, well-spaced peaks

• Number of concepts related to separation principles

Terms and Theories of Chromatogram

Distribution Constant (Kc)

• Where Cs = conc. Solute in stationary phase, Cw = conc. Solute in mobile phase

Terms and Theories of Chromatogram

Retention Factor (k)

• Where Ms = amount of solute in stationary phase, Mm = amount of solute in mobile

Terms and Theories of Chromatogram

Separation Factor (α)

Terms and Theories of Chromatogram

R = peak separation / average peak width = trB – tRA / [(wA + wb)/2]

• Where wA and wB are the baseline width of two peaks

Use of Chromatograms for Qualitative and Quantitative Analysis

• Qualitative analysis: compare retention time of analyte to that of a pure standard

• Quantitative analysis: measurement of peak area (proportional to analyte

• Standard calibration curve relates area of peak to concentration

• Carrier gas: carry analytes through the GC

• Column and oven:

• Detector: see later

High Performance Liquid Chromatography (HPLC)

High Performance Liquid Chromatography (HPLC)

High Performance Liquid Chromatography (HPLC)

• Sample injection system

High Performance Liquid Chromatography (HPLC)

• Solvent mobile phase is the most often changed component in HPLC

– Isocratic – solvent or mixed solvent at constant composition over time

• Gradient elution is used to improve separation similar to changing temperature in GC

High Performance Liquid Chromatography (HPLC)