Gas Chromatography and Supercritical Fluid Chromatography docx

1
Gas and Supercritical Fluid
Chromatography
Lecture Date: April 7
th
, 2008
Gas and Supercritical Fluid Chromatography
 Outline
– Brief review of theory
– Gas Chromatography
– Supercritical Fluid Extraction
– Supercritical Fluid Chromatography
 Reading (Skoog et al.)
– Chapter 27, Gas Chromatography
– Chapter 29, Supercritical Fluid Chromatography
 Reading (Cazes et al.)
– Chapter 23, Gas Chromatography
– Chapter 24, Supercritical Fluid Chromatography
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GC and SFC: Very Basic Definitions
 Gas chromatography – chromatography using a gas as
the mobile phase and a solid/liquid as a stationary phase
– In GC, the analytes migrate in the gas phase, so their
boiling point plays a role
– GC is generally applicable to compounds with masses
up to about 500 Da and with ~60 torr vapor pressure
at room temp (polar functional groups are trouble)
 Supercritical fluid chromatography – chromatography
using a supercritical fluid as the mobile phase and a
solid/liquid as a stationary phase
– In SFC, the analytes are solvated in the supercritical

fluid
– SFC is applicable to a much wider range of molecules
Review of Chromatography
 Column/separation performance:
Plates:
HLN /

Selectivity:
AB
KK /

 Important concepts/equations to remember:
Retention volume:
tFV

m
tLu /
 Linear velocity of mobile phase:
3
Review of Chromatography
 Terminology and
equations from
Skoog:
GC Theory
 Mobile-phase flow rates are
much higher in GC (pressure
drop is much less for a gas)
 The effect of mobile-phase flow
rate on the plate height (H) is
dramatic

– Lower plate heights yield
better chromatography
– However, much longer
columns can be used with
GC
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GC Instrumentation
 Basic layout of a GC:
Injector
Column
Oven
Detector
Carrier Gas
 See pg 703 of Skoog et al. for a similar diagram
GC Instrumentation
 A typical modern GC – the Agilent 6890N:
Diagram from Agilent promotional literature.
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GC Instrumentation
 Typical carrier gases (all are chemically inert): helium,
nitrogen and hydrogen. The choice of gas affects the
detector.
 Injectors: most desirable to introduce a small “plug”,
volatilize the sample evenly
– Most samples introduced in solution: microflash injections
“instantly” volatilize the solvent and analytes and sweep them into
the column
 Splitters: effectively dilute the sample, by splitting off a
portion of it (up to 1:500)
 Ovens: Programmable, temperature ranges from 77K

(LN
2
) up to 250 C.
 Detectors: wide variety, to be discussed shortly…
Headspace GC
 A very useful method for analyzing
volatiles present in non-volatile solids
and liquids
 Sample is equilibrated in a sealed
container at elevated temperature
 The “headspace” in the container is
sampled and introduced into a GC
Needle
Liquid/solid
Headspace
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Columns for GC
 Two major types of columns
used in GC
– Packed
– Open
 Open columns
work better at
higher mobile
phase velocities
Columns for GC
 Open tubular columns: most
common, also known as capillary
columns (inner diameters of
<0.25 mm)

– up to 150 m long
– 1000-3000 plates/m
– pressure limits particle size in packed
columns
– No “A” term (Eddy or multipath) in van
Deemter equation
– N up to 600000
 Packed columns: contain packing, like HPLC columns
– typical particle sizes 100-600 um
– 3 m long
– 1000-3000 plates/m
– difficult to overload
– N up to 12000
A Phenomenex Zebron capillary GC column
www.phenomenex.com
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Types of Columns for GC
 GLC: Gas-liquid chromatography (partition) – most
common
 GSC: Gas-solid chromatography (adsorption)
 FSWC: fused-silica wall-coated open tubular columns,
very popular in modern applications (a form of WCOT
column)
 WCOT (GLC): wall-coated open tubular – stationary
phase coated on the wall of the tube/capillary
 SCOT (GLC): support-coated open tubular – stationary
phase coated on a support (such as diatomaceous earth)
– More capacity that WCOT
 PLOT (GSC): porous-layer open tubular
 Packed columns

Mobile Phases for GC
 Common mobile phases:
– Hydrogen (fast elution)
– Helium
– Argon
– Nitrogen
– CO
2
 The longitudinal diffusion (B)
term in the van Deemter
equation is important in GC
– Gases diffuse much faster than
liquids (10
4
-10
5
times faster)
 A trade-off between velocity
and H is generally observed
– This is equivalent to a trade-off
between analysis time and
separation efficiency
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Columns and Stationary Phases for GC
 Modern column design emphasizes inert, thermally stable
support materials
– Capillary columns are made of glass or fused silica
 The stationary phase is designed to provide a k and  that
are useful. Polarities cover a wide range (next slide).
– Stationary phases are usually a uniform liquid coating on the wall

(open tubular) or particles (packed)
– When the polarity of the stationary phase matches that of the
analytes, the low-boilers come off first…
– Bonded/cross-linked phases – designed for more robust life, less
“bleeding” – often these phases are the result of good polymer
chemistry
 Adsorption onto silicates (via free silanol groups) on the
silica column itself: avoided by deactivation reactions,
usually leaving an OCH
3
group instead.
Stationary Phases for GC
 Target: uniform liquid coating of thermally-stable, chemically
inert, non-volatile material on the inside of the column or on
its particles.
 Polysiloxanes
– Polydimethylsiloxane
 (R = CH
3
)
– phenyl polydimethylsiloxane
 (R = C
6
H
5
, CH
3
)
– trifluoropropyl polydimethylsiloxane
 (R = C

3
H
6
CF
3
, CH
3
)
– cyanopropyl polydimethylsiloxane
 (R = C
3
H
6
CN, CH
3
)
– polyethylene glycol
 Chiral
– amino acids, cyclodextrins
Backbone structure of
polydimethylsiloxane
(PDMS)
HO
O
OH
n
R Si
R
R
O Si

R
R
O Si
R
R
R
n
structure of polyethylene
glycol (PEG)
9
Common Stationary Phases for GC
 High-temperature columns work to 400C, include Agilent’s
DB-1ht (100% polydimethylsiloxane), DB-5ht (5% phenyl).
Stationary
phase
polarity
Stationary Phase
Common Trade
Name
Maximum
Temperature
(C)
Common Applications
polydimethylsiloxane OV-1, SE-30 350 General-purpose nonpolar
phase; hydrocarbons,
steroids, PCBs
5% phenyl
polydimethylsiloxane
OV-3, SE-52 350 Fatty acid methyl esters,
alkaloids, drugs,

halogenated compounds
50% phenyl
polydimethylsiloxane
OV-17 250 Drugs, steroids, pesticides,
glycols
50% trifluoropropyl
polydimethylsiloxane
OV-210 200 Chlorinated aromatics,
nitroaromatics, alkyl-
substituted benzenes
polyethylene glycol Carbowax 20M 250 Free acids, alcohols,
ethers, essential oils,
glycols
50% cyanopropyl
polydimethylsiloxane
OV-275 240 Polyunsaturated fatty acids,
rosin acids, free acids,
alcohols
Temperature Effects in GC
 Temperature programming can be used to speed/slow
elution, help handle compounds with a wide boiling point
range
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Comparison of GC Detectors
 See pg. 793 of Skoog et al. 6
th
Ed.
Detector Sensitivity
Selective or
Universal

Common Applications
Flame ionization (FID) 1 pg
“carbon”/sec
Universal Hydrocarbons
Thermal conductivity (TCD) 500 pg/mL Universal Virtually all compounds
Electron capture (ECD) 5 fg/sec Selective Halogens
Mass spectrometry (MSD) 0.25 to 100 pg Universal Ionizable species
Thermionic (NPD) 0.1 pg/s (P)
1 pg/s (N)
Selective Nitrogen and phosphorus
compounds (e.g. pesticides)
Electrolytic conductivity
(Hall)
0.5 pg/s (Cl)
2 pg/s (S)
4 pg/s (N)
Selective Nitrogen, sulfur and halogen-
containing compounds
Photoionization 2 pg/s Universal Compounds ionized by UV
Fourier transform IR (FTIR) 0.2 to 40 ng Universal Organics
GC Detectors: FID
 The flame ionization detector
(FID), the most common and
useful GC detector
 Process: The column effluent
is mixed with hydrogen and air
and is ignited. Organic
compounds are pyrolyzed to
make ions and electrons,
which conduct electricity

through the flame (current is
detected)
 Advantages: sensitive (10
-13
g), linear all the way up to 10
-4
g), non-selective
 Disadvantages: Destructive,
certain compounds (non-
combustible gases) don’t give
signals in the FID.
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GC Detectors: Thermal Conductivity
 Thermal conductivity
detector (TCD): a non-
selective detector like the
FID
 Also known as the
katherometer
(catherometer) or “hot
wire”
– Works by detecting the
changes in thermal
conductivity (also the
specific heat) of a gas
containing an analyte
– About 1000x < sensitive
than FID
– Non-destructive
GC Detectors: Electron Capture Detector

 Electron capture: selectively detects halogen-containing compounds
(e.g. pesticides)
– Works by ionizing a sample using a radioactive material (
63
Ni). This material
ionizes the carrier gas – but this ionization current is quenched by a
halogenated compound
– Detects compounds via electron affinity – e.g. I (most sensitive) > Br > Cl > F
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GC Detectors: Other
 Atomic emission detector: plasma systems (like ICP, but
often using microwaves) – elemental analysis
 Sulfur chemiluminescence detector (SCD): reaction
between sulfur and ozone, follows an FID-like process
 Thermionic detector: like an FID, optimized and
electrically charged to form a low-temp (600-800 C)
plasma on a special bead. Leads to large ion currents for
phosphorous and nitrogen – a selective detector that is
500x as sensitive as FID
 Flame photometric detector: specialized form of UV
emission from flame products
 Photoionization detector: UV irradiation used to ionize
analytes, detected by an ion current.
 And, of course, the mass spectrometer (MS)…
Examples of GC Detection: Petroleum Analysis
 An example of atomic
spectroscopy, using
microwave-induced
plasma (MIP), to
selectively detect lead

(Pb) containing
compounds in gasoline
 See pg 710 of Skoog for
an example of oxygen
(O) and carbon (C)
detection for separating
hydrocarbons…
13
Examples of ECD Detection: Pesticide Analysis
Data from Agilent, />Interpretation of GC Data
 Common use: develop a method to separate compounds
of interest by spiking, and use retention times to determine
whether a compound is present or not in unknowns
– Watch out for compounds with the same retention time!
– GC can function as a negative test – e.g. “rule out the presence of
ethyl acetate in my sample”….
 Relative retention time:
 Quantitative – Kovats’ retention index (I) – based on
normal alkanes
– the retention index of these compounds is independent of
temperature and packing
– I = 100z (z is the number of carbons in a compound)
– Relative retention index:
stdRAR
ttr )/()(




 

zRzR
zRBR
tt
tt
zI
)log()log(
)log()log(100
100
1








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Purge and Trap GC for Volatile Organic Compounds
 Invented 30 years ago by T. A. Bellar at the US EPA
 Principle:
– Inert gas is bubbled through an aqueous sample
– Gas carries analytes to headspace above sample, through to a
sorbent trap
– After a collection period, the sorbent trap is heated to desorb the
analytes
– The desorbed analytes are injected into a GC
 Results:
– ppb detection of VOC’s like benzene, decane, halomethanes,
etc… in water samples

 Commercialized by Teledyne Tekmar (e.g. the Velocity
XPT) and used worldwide
 Legally-mandated for water analysis in many areas
See C&E News December 12
th
, 2005, page 28, for more info on the 30
th
anniversary of Purge and Trap GC
Chemical Derivatization for GC Analysis
 GC is only applicable to lower molecular weight
compounds with significant (> ~60 torr) volatility
– Polar functional groups reduce volatility
– For other compounds, another separations approach can be used
(LC, etc…) or derivatization can be explored
 Derivatization: chemical reaction(s) that modify an analyte
so that it is easier to separate or detect
 Advantages:
– Can lower LOD (increase sensitivity)
– Can stabilize heat-sensitive compounds
– Can avoid tailing in GC caused by on-column reactions (carbonyl,
amino, imino)
– Can improve the separation of closely-related molecules
 Disadvantage:
– Requires running a reaction, with all its complexities
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Chemical Derivatization for GC Analysis
 A typical derivitization reactions – silylation of an alcohol:
 Common derivatives that reduce polarity:
Groups Derivative
Alcohol (–OH) Alkyl ester, alkyl ether, silyl ether

Carboxylic acid (–COOH) Alkyl ester, silyl ester
Amino (-NH
2
) Acyl derivative, silyl derivative
Imino (=NH) Silyl derivative
Aldehyde (COH) Dimethyl acetal
Thiol (SH) Thioether, silylthioether
OH
+
Si
CH
3
CH
3
Cl
+
HCl
CH
3
Si
CH
3
CH
3
CH
3
O
 Other derivatives contain halogens for ECD detection
S. Ahuja, “Derivatization for Gas and Liquid Chromatography”, in Ultratrace Analysis of Pharmaceuticals and Other Compounds of Interest, Wiley, 1986.
Applications of Derivatization and GC in Doping

 Example: derivatization of androgens (like testosterone)
for GC-MS analysis. Detection limits can be as low as 0.2
ng/mL
 In one procedure, derivitization with TMS is used in
conjunction with a series of pretreatment and extraction
steps, followed by GC-MS:
O
OH
H
H
H
testosterone
K. Shimada , K. Mitamura, T. Higashi, J. Chrom. A., 935, 2001, 141–172.
O
O
H
H
H
Si
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Hyphenation of GC and MS
 The first useful “hyphenated” method?
 Continuous monitoring of the column effluent by a mass
spectrometer or MSD
 Very easy to interface – capillary GC columns have low
enough flow rates, and modern MS systems have high
enough pumping rates, that GC effluent can be fed directly
into the ionization chamber of the MS (for EI or CI, etc…)
– Larger columns require a “jet separator”
 Most common systems use quadrupole or ion trap mass

analyzers (MSD)
Supercritical Fluids
 Phase diagrams show
regions where a
substance exists in a
certain physical state
 Beyond the “critical
point”, a gas cannot
be converted into the
liquid state, no matter
how much pressure is
applied!
17
Supercritical Fluids
 Supercritical properties of CO
2
 The fluid – intermediate between
a liquid and a gas
 Obtained in a not-so-sudden
manner (there is no real
transition)
Supercritical Fluids
 Photos of CO
2
as it goes from a gas/liquid to a supercritical fluid
Images from />1
2
3
4
Meniscus

Increasing
temp
18
Extractions with Supercritical Fluids
 Why use supercritical fluid extraction (SFE)?
 Supercritical fluids can solvate just as well as organic
solvents, but they have these advantages:
– Higher diffusivities
– Lower viscosities
– Lower surface tensions
– Inexpensive
– Pure
– Easy to dispose of….
 Basic utility – many of the same features apply to SFC, so
we introduce them here with SFE.
Extractions with Supercritical Fluids
 Pure CO
2
is able to extract a wide range of non-polar and
moderately polar analytes.
 Modifiers (such as methanol) at v/v% of 1-10% can be
used to help solubilize polar compounds.
 Other supercritical fluids can be used (note that NH
3
is
reactive and corrosive, while N
2
O and pentane are
flammable)
See S. B Hawthorne, Anal. Chem., 62, 633A (1990).

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Some Uses of SFE
 Environmental analysis:
– total petroleum hydrocarbons
– polyaromatic hydrocarbons
– organochloropesticides in soils
 Food industry:
– Extraction of fats
– Extraction of caffeine
 Density-stepping SFE – used as a form of “mini-
chromatography”
See M. McHugh and V. Krukonis, Supercritical Fluid Extraction: Principles and Practice, Butterworth, Stoneham, MA, 1987.
Supercritical Fluid Chromatography (SFC)
 SFC is the next logical step from SFE
 A supercritical fluid is used as the mobile phase –
hardware is otherwise similar to GC.
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Control of Pressure in SFC
 Pressure affects the retention
(capacity) factor k
 Why? The density of the SF
mobile phase increases with
more pressure
 More dense mobile phase
means more solvating power
(more molecules)
 More solvating power means
faster elution times
 Changing the pressure in SFC
is somewhat analogous to

changing the solvent gradient in
LC
Detectors for SFC
 Detectors are generally similar to those used in GC and
LC
 Major advantage of SFC over HPLC: SFC can use the
“universal” FID as a detector
 SFC can also use UV, IR, and fluorescence detectors
 SFC is compatible with MS hyphenation
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Applications of SFC
 Why use SFC over other techniques? Consider speed
and capability as well as expense
Study Problems and Further Reading
 For more information about SFC, see:
– M. McHugh and V. Krukonis, Supercritical Fluid Extraction:
Principles and Practice, Butterworth, Stoneham, MA, 1987.
 Study problems:
– 27-1, 27-12
– 29-3, 29-4
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Further Reading
M. McHugh and V. Krukonis, Supercritical Fluid Extraction: Principles and
Practice, Butterworth, Stoneham, MA, 1987.