Gas Chromatography (GC)

1. Gas Chromatography (GC) By Yours Truly: Niyam Shah

2. What is GC? • Gas chromatography is the process where various elements of a compound are separated into their distinct parts for individual analysis Schematic of a GC

3. What is GC Used for? • Determine the number of component compounds in a mixture (separation of a mixture) • Determine whether or not a specific compound is present in a sample (qualitative analysis) • Determine the specific amount of a given compound in a sample (quantitative analysis) • Used to monitor industrial processes

4. Value to Forensic Science • Chromatographic techniques are particularly valuable when combined with other analytical techniques • Ex: Mass spectrometry in GC/Mass spectrometry (workhorse technique in forensic chemistry) • Mainly a confirmatory test (esp. for drugs) • Color reagent tests performed prior to GC analysis

5. Uses in Forensic Science • Forensic pathology • Crime scene testing • Arson investigations • GC can be a low-cost alternative of detecting flammable liquids, on-site. • Drug analysis • Forensic toxicology • Paint analysis • Food and fragrance analysis

6. What Can Be Analyzed? • Biological Specimens • Drugs • Synthetic, designer, OTC • Petrol • Lighter fluid • Alcohol • Pollutants

7. Blood-Alcohol Analysis (DUI Cases) • Human blood is a mixture of various substances and left alone, it is very difficult to analyze • Some components in blood are volatile organic compounds • With use of method called “head space”, various foreign components of the blood can be measured and identified • Lab analyst must prepare a standard mix • Allows lab to determine specific retention times of various volatiles of interest • Calibration curve also produced • Extracts blood and places it, with a very small amount of an internal standard, into a separate testing vial • Internal standards are alcohols that would not be expected to occur in human blood except in minute quantities

8. DUI Cases • The vial is shaken and heated to produce a gas • A syringe is used to introduce the sample into the GC • Uses flame ionization detector (FID), which is located at the end of the column. • As the chemicals exit from the end of the column, the FID incinerates them, and this combustion produces an electronic charge in the form of ions. • These ions are measured by the detector and subsequently converted by the instrument’s computer into a graph which usually contains two peaks. One of these peaks represents the internal standard and the other the ethyl alcohol.  The retention time of the peak for the ethyl alcohol must match the expected retention time in order to qualitatively confirm its identity • The area beneath this peak is compared with the peak for the ethyl alcohol.  This ratio is compared with the calibration curve and converted into the driver’s blood alcohol level.

9. Heroin Analysis • If heroin is being illicitly synthesized from raw opium, the first step is to extract morphine from the opium latex. • Extraction • Lime method • Involves use of calcium hydroxide (lime) to precipitate out a crude morphine base, which is then dissolved in warm hydrochloric acid. • Diacetylmorphine results from the reaction with morphine and acetic anhydride at elevated temperatures and the subsequent addition of sodium carbonate. The free base form of diacetylmorphine is produced. • Sample Preparation • Derivatisation • Used to modify the chemical structure of the analytes of interest in order to produce less thermally labile forms of the analyte(s) and/or to produce better peak shape • Samples will be loaded into the auto sampler of the GC-flame ionization detector (FID)

10. Molecular Structure of diacetylmorphine

11. Casey Anthony Murder Case • Air inside Anthony’s car was collected and placed into an airtight container • Trying to determine if odor is of human decomposition • Vital to case b/c people who came in contact with Anthony after the disappearance of Caylee Anthony claim her car smelled like death • Analysts were actually given a sealed can containing a scrap of upholstery from the car, and used a syringe to extract the air from inside • Ran it through GC and cross referenced the results against a database of more than 400 chemical traces of decomposition • According to the analyst, the air contained an “overwhelmingly strong” scent of decomposition • Problem? Yes… • The GC technique has never been used before in court • Up to jury to take in consideration

12. Chemical Principles (General) • 2 phases • Stationary phase (does not move) • Mobile phase (moves through stationary phase) • Properties of compounds are a function of their structure • Compounds that differ in structure will differ in the rates at which they travel (ball analogy)

13. GC Chemical Principles • Mobile phase = Carrier gas • Inert gas (nonreactive) • Stationary phase = High-boiling liquid in a column (termed gas-liquid chromatography (GLC))

14. Types of Columns Packed Capillary • Walls are coated with liquid stationary phase • More efficient than packed columns • Contains a finely divided, inert, solid support material (commonly based on diatomaceous earth) coated with liquid stationary phase.

15. What Happens Inside a GC? • Gas chromatography includes three basic operational steps. They are injection, separation, and detection • A standard sample must be processed before and after the collected specimen under identical conditions • Separation begins when mixture is injected from a syringe through a rubber septum • For optimum efficiency, the sample should not be too large • Should be introduced as a “plug” of vapor (rapid injection) • Slow injection = band broadening and loss of resolution • Leads to a column, which is heated by an oven • Sample vaporizes • Carrier gas enters column near injection port and sweeps sample through column • The various compounds in the mixture vary in their solubility in the stationary liquid phase. Thus, the compounds in the mixture will travel through the column according to their solubility in the liquid phase.

17. Separation • One of three things might happen to a particular molecule in the mixture injected into the column: • It may condense on the stationary phase. • It may dissolve in the liquid on the surface of the stationary phase. • It may remain in the gas phase. • None of these are necessarily permanent.

18. What Happens Inside a GC? (cont.) • As it travels through the column, the components go back and forth at different rates between the gas phase and dissolution in the high-boiling liquid, and thus separate into pure components • The time it takes for a given compound to pass through the column from the time it is injected to the time it is detected is called the retention time. • The greater the compound’s affinity for the column, the longer its retention time. • Just before each compound exits the instrument, it passes through a detector. • When the detector sees a compound, it sends an electronic message to the recorder, which responds by plotting a peak. • The strip chart of the entire sample, called a chromatogram provides documentation of the analysis.

19. Retention Time Factors • Volatility of compound: Low boiling (volatile) components will travel faster through the column than will high boiling components • Polarity of compounds: Polar compounds will move more slowly, especially if the column is polar. • Column temperature: Raising the column temperature speeds up all the compounds in a mixture. • Column packing polarity: Usually, all compounds will move slower on polar columns, but polar compounds will show a larger effect. • Flow rate of the gas through the column: Speeding up the carrier gas flow increases the speed with which all compounds move through the column. • Length of the column: The longer the column, the longer it will take all compounds to elute. Longer columns are employed to obtain better separation. • #1 factor to consider = boiling points of the different components (“fingerprint” of compounds)

20. Detectors • Flame photometric Detector (FPD) • Measures photons or light • Light produced when sample burns in hydrogen flame • Thermal Conductivity Detector (TCD) • Involves conduction of heat • Compares thermal conductivity of carrier gas to that of the carrier gas enriched in a sample • Electron Capture Detector (ECD) • Involves “capture” of electrons • Electrons come from a radioactive source • These are only 3, there are many more

21. Safety and Hazards • When handling biological specimens, ensure to wear gloves and protective gear • Could be potentially exposed to communicable diseases, HIV, and hepatitis if not protected • Many parts of the GC operate at temperatures high enough to cause serious burns • Be careful when working behind the instrument. During cool-down cycles, the GC emits hot exhaust which can cause burns. • Make sure work area is ventilated • Eliminate from your laboratory as many ignition sources as possible (open flames, devices that can spark, sources of static electricity, etc.).

22. Safety and Hazards (cont.) • If hydrogen gas is used as a carrier gas, there is a possibility for its accumulation and: • Combustion of leaking hydrogen • Combustion due to rapid expansion of hydrogen from a high-pressure cylinder • Accumulation of hydrogen in the GC oven and subsequent combustion • Do not excessively heat the column • May cause the stationary film to coalesce into uneven “hills and valleys” on the inner column wall.

23. Difficulties/Sources of Interference • DO NOT intentionally shut off the GC when it’s in the process of analysis, this can damage the column and machinery. • The sample will be stuck within the column and will require tedious cleaning • Power outages • Ensuring all plugs are in correct place (It may seem obvious, but it can get confusing) • If not, the GC will operate inefficiently and hazards will be created • Using hydrogen as a carrier gas • Can alter the sample • Leaks in column • Ensure to perform inspections • Not cleaning GC or syringe properly • Injecting large amounts of sample • Injecting too slowly

24. Limitations • Microcontaminants may gradually deposit at the beginning of the column and interact with solutes, which could lead to adsorption and possibly to catalytic decomposition or rearrangement. • Fundamental limitation = substances must be volatile, so that a finite fraction of it is distributed in the gaseous phase. • High temperatures of up to 300°C enhance volatility, but decomposition of the substance can be the result. • Temperature of column limited to 380 °C • Analytes should have boiling points below 500 °C • Results using one detector will probably differ from results obtained using another detector.  • Comparing analytical results to tabulated experimental data using a different detector does not provide a reliable identification of the specimen.

25. Limitations (cont.) • An injection port septum should last between 100 and 200 injections.   Higher injection port temperatures shorten the septum's lifespan.  • The temperature of the GC injection port must be high enough to vaporize a liquid specimen instantaneously.   • Temp too low = separation is poor and broad spectral peaks should result or no peak develops at all.  • Temp too high = the specimen may decompose or change its structure.   • Technical expertise required • Although retention times are characteristic for a given compound under a specific set of analytical conditions, you must have a reference for comparison and identification. • The area under a chromatographic peak can be used to establish the relative abundance of a component, but not its exact concentration.

26. Limitations (cont.) • Neither carrier gases nor mobile phases affect solute retention, but they do affect: 1.) Desired efficiency for the GC System (Van Deemter) - low molecular weight gases (He, H2) = larger diffusion coefficients - low molecular weight gases = faster, more efficient separations 2.) Stability of column and solutes - H2 or O2 can react with functional groups on solutes and stationary phase or with surfaces of the injector, connections and detector 3.) Response of the detector - thermal conductivity detector requires H2 or He - other detectors require specific carrier gas

27. Advantages Disadvantages • Limited to volatile samples • Some samples may require intensive preparation • Samples must be soluble and not react with the column • Requires spectroscopy (usually MS) to confirm the peak identity • Does not necessarily provide definitive proof in the ID of a substance • Fast analysis • Typically minutes (even seconds) • Automated • Small samples required (µl or µg) • High resolution • Reliable, relatively simple and cheap • Sensitive detectors • Highly accurate quantification

28. Works Cited • Barone, Patrick T. "Gas Chromatography in DUI Cases – Theory and Operation." Criminal Law Blog. James Publishing, 11 Nov. 2009. Web. 29 Mar. 2015. • Carlin, Michelle, and Dean, John Richard. Forensic Applications of Gas Chromatography. London, GBR: CRC Press, 2013. ProQuestebrary. Web. 29 March 2015. • Clark, Jim. "Gas-Liquid Chromatography." Gas-Liquid Chromatography. N.p., 2007. Web. 29 Mar. 2015. • Dillow, Clay. "In Caylee Anthony Case, New Forensic Technique Sniffs Crime Scene Air Samples for the Lingering Scent of Death." Popular Science. Popular Science, 8 June 2011. Web. 29 Mar. 2015. • Douglas, Frederic. "GC/MS." GC/MS. Scientific Testimony, 2015. Web. 29 Mar. 2015. • "Gas Chromatography." Gas Chromatography. Sheffield Hallam University, 2015. Web. 29 Mar. 2015. • "Gas Chromatography." Gas Chromatography. University of Colorado at Boulder, 10 Jan. 2015. Web. 29 Mar. 2015. • Gilman, Jessica, and Jose-Luis Jimenez. "Gas Chromatography." Journal of Chromatography 57 (1971): 338-41. Colorado EDU. University of Colorado at Boulder, Fall 2004. Web. 29 Mar. 2015. • Gross, Ray A., and Indravandan Shah. An Introduction to Instrumental Analysis (2004): 1-38. Prince George’s Community College, Spring 2014. Web. 29 Mar. 2015. • "How Is Gas Chromatography Used in Forensics?" Chromatography Today. International Labmate Limited, 26 May 2014. Web. 29 Mar. 2015. • Khan, JaVed I., Kennedy, Thomas J., and Christian, Donnell R.. Basic Principles of Forensic Chemistry. New York, NY, USA: Springer, 2012. ProQuestebrary. Web. 29 March 2015. • Patnaik, Pradyot. Dean's Analytical Chemistry Handbook. New York, NY, USA: McGraw-Hill Professional Publishing, 2004. ProQuestebrary. Web. 29 March 2015. • Spencer, James T. "Chapter 11." An Introduction to Forensic Science: The Science of Criminalistics. N.p.: Cengage Learning, 2012. III.11.1-II.11.44. Print. • Thet, Kyaw, and Nancy Woo. "Gas Chromatography." Chemwiki. UC Davis, 2015. Web. 29 Mar. 2015.