Gas Chromatography and Supercritical Fluid Chromatography docx

Gas and Supercritical Fluid Chromatography Lecture Date: April 7 th , 2008 Gas and Supercritical Fluid Chromatography  Outline – Brief review of theory – Gas Chromatography – Supercriti

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

GC and SFC: Very Basic Definitions

 Gas chromatography – chromatography using a gasas

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 fluidas the mobile phase and a

solid/liquid as a stationary phase

– In SFC, the analytes are solvated in the supercritical

u  /

 Linear velocity of mobile phase:

 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

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

 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

 Open tubular columns: most

columns (inner diameters of

 Packed columns: contain packing, like HPLC columns

– typical particle sizes 100-600 um

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

 The longitudinal diffusion (B)

term in the van Deemter

equation is important in GC

– Gases diffuse much faster than

 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,

Stationary Phases for GC

 Target: uniform liquid coating of thermally-stable, chemically

inert, non-volatile material on the inside of the column or on

HO

O

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 400C, include Agilent’s

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

alkyl-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

Comparison of GC Detectors

 See pg 793 of Skoog et al 6thEd

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

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

certain compounds

(non-combustible gases) don’t give

signals in the FID.

GC Detectors: Thermal Conductivity

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

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

(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:

std R A

R

t t

t t

z I

)log(

)log(

)log(

)log(

100100

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

– 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,

Chemical Derivatization for GC Analysis

 A typical derivitization reactions – silylation of an alcohol:

 Common derivatives that reduce polarity:

 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 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

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 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 CO2as it goes from a gas/liquid to a supercritical fluid

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:

– 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 CO2is 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 NH3is

reactive and corrosive, while N2O and pentane are

flammable)

See S B Hawthorne, Anal Chem., 62, 633A (1990).

Some Uses of SFE

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

 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.