1. Gas and Supercritical Fluid Chromatography Lecture Date: April 7 th, 2008

2. 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

3. 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

4. Review of Chromatography  Column/separation performance: Plates: Selectivity:  Important concepts/equations to remember: Retention volume:  Linear velocity of mobile phase:

5. Review of Chromatography  Terminology and equations from Skoog:

6. 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

7. GC Instrumentation  Basic layout of a GC: Injector Column Oven Detector Carrier Gas  See pg 703 of Skoog et al. for a similar diagram

8. GC Instrumentation  A typical modern GC – the Agilent 6890N: Diagram from Agilent promotional literature.

9. 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…

10. 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

11. Columns for GC  Two major types of columns used in GC –Packed –Open  Open columns work better at higher mobile phase velocities

12. 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

13. 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

14. 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

15. 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.

16. 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) structure of polyethylene glycol (PEG)

17. 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 polydimethylsiloxaneOV-1, SE-30350General-purpose nonpolar phase; hydrocarbons, steroids, PCBs 5% phenyl polydimethylsiloxane OV-3, SE-52350Fatty acid methyl esters, alkaloids, drugs, halogenated compounds 50% phenyl polydimethylsiloxane OV-17250Drugs, steroids, pesticides, glycols 50% trifluoropropyl polydimethylsiloxane OV-210200Chlorinated aromatics, nitroaromatics, alkyl- substituted benzenes polyethylene glycolCarbowax 20M250Free acids, alcohols, ethers, essential oils, glycols 50% cyanopropyl polydimethylsiloxane OV-275240Polyunsaturated fatty acids, rosin acids, free acids, alcohols

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

19. Comparison of GC Detectors  See pg. 793 of Skoog et al. 6 th Ed. DetectorSensitivity Selective or Universal Common Applications Flame ionization (FID)1 pg “carbon”/sec UniversalHydrocarbons Thermal conductivity (TCD)500 pg/mLUniversalVirtually all compounds Electron capture (ECD)5 fg/secSelectiveHalogens Mass spectrometry (MSD)0.25 to 100 pgUniversalIonizable species Thermionic (NPD)0.1 pg/s (P) 1 pg/s (N) SelectiveNitrogen and phosphorus compounds (e.g. pesticides) Electrolytic conductivity (Hall) 0.5 pg/s (Cl) 2 pg/s (S) 4 pg/s (N) SelectiveNitrogen, sulfur and halogen- containing compounds Photoionization2 pg/sUniversalCompounds ionized by UV Fourier transform IR (FTIR)0.2 to 40 ngUniversalOrganics

20. 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.

21. 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

22. 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

23. 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)…

24. 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…

25. Examples of ECD Detection: Pesticide Analysis Data from Agilent, http://www.chem.agilent.com/cag/graphics/445a.jpg

26. 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:

27. 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

28. 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

29. Chemical Derivatization for GC Analysis  A typical derivitization reactions – silylation of an alcohol:  Common derivatives that reduce polarity: GroupsDerivative 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  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.

30. 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: K. Shimada, K. Mitamura, T. Higashi, J. Chrom. A., 935, 2001, 141–172.

31. 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)

32. 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!

33. 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)

34. Supercritical Fluids  Photos of CO 2 as it goes from a gas/liquid to a supercritical fluid Images from http://www.chem.leeds.ac.uk/People/CMR/criticalpics.htmlhttp://www.chem.leeds.ac.uk/People/CMR/criticalpics.html 1 2 3 4 Meniscus Increasing temp

35. 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.

36. 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).

37. 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.

38. 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.

39. 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

40. 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

41. Applications of SFC  Why use SFC over other techniques? Consider speed and capability as well as expense

42. 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

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