gas and supercritical fluid chromatography
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Gas and Supercritical Fluid Chromatography. Lecture Date: April 17 th , 2013. Gas and Supercritical Fluid Chromatography. Outline Brief review of theory Gas Chromatography Supercritical Fluid Extraction Supercritical Fluid Chromatography Reading (Skoog et al.) - PowerPoint PPT PresentationTRANSCRIPT
Gas and Supercritical Fluid Chromatography
Lecture Date: April 17th, 2013
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
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
mtLu /
Linear velocity of mobile phase:
Summary of Chromatographic Relationships From Skoog:
GC Theory
Mobile-phase flow rates are higher in GC (pressure drop is much less for a gas) than in LC
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
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.
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 (LN2) 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 (usually) equilibrated in a sealed container at elevated temperature
The “headspace” in the container is sampled and introduced into a GC
Needle
Liquid/solid
Headspace
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 columnwww.phenomenex.com
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– CO2
The longitudinal diffusion (B) term in the van Deemter equation is important in GC
– Gases diffuse much faster than liquids (104-105 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
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 OCH3 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 = CH3)
– phenyl polydimethylsiloxane (R = C6H5, CH3)
– trifluoropropyl polydimethylsiloxane (R = C3H6CF3, CH3)
– cyanopropyl polydimethylsiloxane (R = C3H6CN, CH3)
– polyethylene glycol
Chiral– amino acids, cyclodextrins
Backbone structure of polydimethylsiloxane
(PDMS)
HOO
OH
n
R Si
R
R
O Si
R
R
O Si
R
R
R
n
structure of polyethylene glycol (PEG)
Common Stationary Phases for GC
High-temperature columns work to 400C, include Agilent’s DB-1ht (100% polydimethylsiloxane), DB-5ht (5% phenyl).
Stationaryphase
polarityStationary 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 (normally a ramp) can be used
to speed/slow elution. It helps handle compounds with a wide boiling point range. Newer “low thermal mass” systems allow for fast ramps.
Comparison of GC Detectors
See pg. 793 of Skoog et al. 6th 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.
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 (63Ni). 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
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…
Examples of ECD Detection: Pesticide Analysis
Data from Agilent, http://www.chem.agilent.com/cag/graphics/445a.jpg
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
ttttzI)log()log()log()log(100100
1
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 areasSee C&E News December 12th, 2005, page 28, for more info on the 30th 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
Chemical Derivatization for GC Analysis A typical derivitization reaction – 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 (-NH2) Acyl derivative, silyl derivative
Imino (=NH) Silyl derivative
Aldehyde (COH) Dimethyl acetal
Thiol (SH) Thioether, silylthioether
OH + Si
CH3
CH3
Cl + HClCH3 Si
CH3
CH3
CH3O
Other derivatives contain halogens for ECD detectionS. 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
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!
Supercritical Fluids
Supercritical properties of CO2
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 CO2 as it goes from a gas/liquid to a supercritical fluid
Images from http://www.chem.leeds.ac.uk/People/CMR/criticalpics.html
1
2
3
4
Meniscus
Increasing temp
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 CO2 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 NH3 is reactive and corrosive, while N2O and pentane are flammable)
See S. B Hawthorne, Anal. Chem., 62, 633A (1990).
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.
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
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
Further Reading
M. McHugh and V. Krukonis, Supercritical Fluid Extraction: Principles and Practice, Butterworth, Stoneham, MA, 1987.