SKA6014 ADVANCED ANALYTICAL CHEMISTRY TOPIC 15 Gas Chromatography and Supercritical Fluid Chromatography

Azlan Kamari, PhD

Department of Chemistry Faculty of Science and Mathematics Universiti Pendidikan Sultan Idris

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

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

GC Instrumentation
Basic layout of a GC:
Injector Detector

Carrier Gas

Column Oven

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 equilibrated in a sealed container at elevated temperature The headspace in the container is sampled and introduced into a GC
Needle

Headspace

Liquid/solid

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

A Phenomenex Zebron capillary GC column www.phenomenex.com

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

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
R R Si R O R Si R n O R Si R R

Backbone structure of polydimethylsiloxane (PDMS)

HO O n

OH

structure of polyethylene glycol (PEG)

Common Stationary Phases for GC

Stationary phase polarity
Stationary Phase polydimethylsiloxane Common Trade Name OV-1, SE-30 Maximum Temperature (C) 350 Common Applications General-purpose nonpolar phase; hydrocarbons, steroids, PCBs Fatty acid methyl esters, alkaloids, drugs, halogenated compounds Drugs, steroids, pesticides, glycols Chlorinated aromatics, nitroaromatics, alkylsubstituted benzenes Free acids, alcohols, ethers, essential oils, glycols Polyunsaturated fatty acids, rosin acids, free acids, alcohols

5% phenyl polydimethylsiloxane 50% phenyl polydimethylsiloxane 50% trifluoropropyl polydimethylsiloxane polyethylene glycol

OV-3, SE-52

350

OV-17

250

OV-210

200

Carbowax 20M

250

50% cyanopropyl polydimethylsiloxane

OV-275

240

High-temperature columns work to 400C, include Agilents DB-1ht (100% polydimethylsiloxane), DB-5ht (5% phenyl).

Temperature Effects in GC
Temperature programming can be used to speed/slow elution, help handle compounds with a wide boiling point range

Comparison of GC Detectors
Detector
Flame ionization (FID) Thermal conductivity (TCD) Electron capture (ECD) Mass spectrometry (MSD) Thermionic (NPD) Electrolytic conductivity (Hall)

Sensitivity
1 pg carbon/sec 500 pg/mL 5 fg/sec 0.25 to 100 pg 0.1 pg/s (P) 1 pg/s (N) 0.5 pg/s (Cl) 2 pg/s (S) 4 pg/s (N)

Selective or Universal
Universal Universal Selective Universal Selective Selective

Common Applications
Hydrocarbons Virtually all compounds Halogens Ionizable species Nitrogen and phosphorus compounds (e.g. pesticides) Nitrogen, sulfur and halogencontaining compounds

Photoionization
Fourier transform IR (FTIR)

2 pg/s
0.2 to 40 ng

Universal
Universal

Compounds ionized by UV
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 (noncombustible gases) dont give signals in the FID.

GC Detectors: Thermal Conductivity

Thermal conductivity detector (TCD): a nonselective 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:

r (t R ) A /( t R ) std

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:

100log( t R ) B log( t R ) z I 100 z log( t R ) z 1 log( t R ) z

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 VOCs 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

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 reactions silylation of an alcohol:
CH3 OH CH3 CH3 O Si CH3 CH3

Cl

Si CH3

HCl

Common derivatives that reduce polarity:

Groups Alcohol (OH) Carboxylic acid (COOH) Amino (-NH2) Imino (=NH) Aldehyde (COH) Thiol (SH) Derivative Alkyl ester, alkyl ether, silyl ether Alkyl ester, silyl ester Acyl derivative, silyl derivative Silyl derivative Dimethyl acetal Thioether, silylthioether

Other derivatives contain halogens for ECD detection

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:
OH

Si O

H
H O H

testosterone
O

K. Shimada , K. Mitamura, T. Higashi, J. Chrom. A., 935, 2001, 141172.

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-sosudden manner (there is no real transition)

Supercritical Fluids
Photos of CO2 as it goes from a gas/liquid to a supercritical fluid

1
Meniscus

Increasing temp

Images from http://www.chem.leeds.ac.uk/People/CMR/criticalpics.html

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)

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 minichromatography

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