1

Gas and Supercritical Fluid Chromatography

Lecture Date: April 7th, 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

2

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:

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

4

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.

5

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

Liquid/solid

Headspace

6

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 capillarycolumns (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

7

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

8

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)

9

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 can be used to speed/slow

elution, help handle compounds with a wide boiling point range

10

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.

11

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

12

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, 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

14

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

15

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 (-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

16

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 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

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 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).

19

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.

20

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

21

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

22

Further Reading

M. McHugh and V. Krukonis, Supercritical Fluid Extraction: Principles and Practice, Butterworth, Stoneham, MA, 1987.