Introduction to Gas Chromatography

A Quick Historical Perspective Gas Chromatography A Quick Historical Perspective 1903 Adsorption chromatography 1952 Gas chromatography (GLC) 1968 High performance liquid chromatography (HPLC)

HPLC GC Gas Chromatography Mobile phase changes Constant temperature Compounds partition from the mobile phase based on solubility. Elution is generally time or volume dependent Mobile phase is constant Increasing temperature Compounds partition from the mobile phase based on volatility. Elution is generally temperature dependent

Gas Chromatography

Gas Chromatography Sample Injected Low Temperature High Temperature

Gas Chromatography Typical Volatile Profile of Strained Carrots (Common Terpenoids Marked in Blue)

Sample Preparation Gas Chromatography How do we obtain a volatile sample from foods?

Sample Preparation Gas Chromatography 1) Headspace Methods 2) Distillation Methods 3) Solvent Extraction 4) Solid-Phase Microextraction

Gas Chromatography Direct Headspace In a closed container, take a sample of the air above the sample and inject it. While direct injection of gasses can be used, many volatile analytes are often in too low of a concentration to be quantified using most common detectors.

Gas Chromatography Dynamic Headspace Typically the sample is placed in a closed container and a flow of inert gas such as nitrogen is used to purge the headspace onto an adsorbent or a cryogenic trap. The trap can be eluted with solvent, the solvent can be concentrated and injected.

Gas Chromatography Solvent Extraction Blend sample and mix with non-miscible solvent. (a solvent that doesn’t mix with water). Centrifuge to unmix solvent and food sample

Gas Chromatography Solvent Extraction Separate the solvent containing the analytes of interest and concentrate by evaporation for injection into GC.

Gas Chromatography Distillation Methods Produces a dilute aqueous sample which is not the best for GC. Sample can be re-extracted with solvent. Food Sample Sample

Gas Chromatography Solid Phase Microextraction Sampling devices (manual and autosampler) consist of a coated silica fiber inside a hollow needle. The coating on the fiber adsorbs and concentrates volatiles from the headspace inside the sample container. The needle protects the fiber which can be inserted through the septa of the GC injector for direct analysis. Probably the easiest method of sample prep.

Gas Chromatography Hardware and Columns

Gas Chromatography Mobile Phase: He, Ar, N2, H2 Injection Port: Rubber septum barrier (usually maintained at a higher temperature than the boiling point of the least volatile component in the sample mixture)…why? Column: (fused silica with a thin coating of stationary phase on the inner surface) Oven: Thermostat controlled forced air oven Detector: Many types Data System

Gas Chromatography Split/Splitless Injector Splitless Injection, (where the split vent is closed) attempts to transfer all of the sample to the column and is used for trace analysis. Split Mode, only a small portion (maybe 1-10% of the sample moves into the column, and the rest is sent to waste. This is used when the analytes are in high concentration and would overload the column. Sample is injected through the septum with a syringe.

Gas Chromatography The oven Inside here Column

Gas Chromatography Question: The injection port is at a HIGH temperature 250oC is common. What happens when the hot, volatized sample hits a “cold” (50oC) GC column?

Gas Chromatography The volatile compounds condense at the front end of the column. Raising the temperature of the column allows for the separation of the compounds as they boil away at different temperatures. If two compounds have the same volatility, the compound with the least affinity for the stationary phase will volatilize first.

Gas Chromatography Gas chromatography separates molecules mostly based on: Volatility (Oven) Polarity (Column)

Oven Temperature Program Gas Chromatography Oven Temperature Program Increase Temp. Linearly or Stepwise Temperature Time

Gas Chromatography COLUMNS GC column nomenclature can be confusing. Carbowax, DB-1, DB-5, PDMS ….. Columns are often referred to by their polarity, like most things with chromatography. The most non-polar stationary phase is polydimethyl siloxane (PDMS). Polarity of a column is increased by adding phenyl groups to PDMS (1% = DB-1; 5% = DB-5). For more polar analytes, polyethylene glycol (carbowax) is used as the stationary phase

Gas Chromatography COLUMNS About a dozen different types in common usage.

GC Applications with Food Gas Chromatography GC Applications with Food Analysis of foods is concerned with the assay of lipids, proteins, carbohydrates, preservatives, flavors, colorants, vitamins, steroids, drugs, and pesticide residues. Most of the components are non-volatile (thus the use of HPLC) but with modification, GC can be effectively used. Derivatization of lipids and fatty acid to their methyl esters Proteins are acid hydrolyed followed by esterification (N-propyl esters) Carbohydrates derivatized by silylation to produce a volatile compound