1 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 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  m tLu /  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 (LN 2 ) 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 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 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 – 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 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 OCH 3 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 = 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) HO O 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 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 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. 6 th 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. [...]... 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 1 3 Meniscus 2 Increasing temp 4 Images from http://www.chem.leeds.ac.uk/People/CMR/criticalpics.html 17 Extractions with Supercritical Fluids  Why use supercritical fluid extraction (SFE)?  Supercritical fluids can solvate just as well as organic solvents,... Extraction of fats – Extraction of caffeine  Density-stepping SFE – used as a form of “minichromatography” 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 19 Control of... 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! 16 Supercritical Fluids  Supercritical properties of CO2  The fluid – intermediate between a liquid and a gas  Obtained in a not-so-sudden manner... 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.,... “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... 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 20 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. .. 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 21 Further Reading M McHugh and V Krukonis, Supercritical Fluid Extraction: Principles and Practice, Butterworth, Stoneham, MA, 1987 22 ... independent of temperature and packing – I = 100z (z is the number of carbons in a compound) – Relative retention index: 100 log(t  )  log(t  ) I  100 z   R B R z    log(t R ) z 1  log(t R ) z  13 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... 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… 12 Examples... GC-MS: OH Si O H H H H O H H testosterone O K Shimada , K Mitamura, T Higashi, J Chrom A., 935, 2001, 141–172 15 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 . 1 Gas and Supercritical Fluid Chromatography Lecture Date: April 7 th , 2008 Gas and Supercritical Fluid Chromatography  Outline – Brief review of theory – Gas Chromatography – Supercritical. 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. Chromatography – Supercritical Fluid Extraction – Supercritical Fluid Chromatography  Reading (Skoog et al.) – Chapter 27, Gas Chromatography – Chapter 29, Supercritical Fluid Chromatography  Reading