CHROMATOGRAPHY

HISTORY
First developed by the Russian botanist Mikhail Tswett in 1903. Produced a colorful separation of plant pigments through a column of calcium carbonate. Chromatography has since developed into an invaluable laboratory tool for the separation and identification of compounds. Although color usually no longer plays a role in the process, the same principles of chromatography still apply.

CHROMATOGRAPHY
The word Chromatography is derived from two Greek words Chroma means colour and graphein to write. Chromatography is the collective term for a family of laboratory techniques for the separation of mixtures into their components in order to analyze, identify, and purify the mixture or components

TERMS
Mobile phase: It is the phase which moves in a definite direction.
Stationary phase (bounded phase): It is a phase that is covalently bonded to the support particles or to the inside wall of the column tubing. Analyte (Sample): It is the substance to be separated during chromatography. Eluent: It is the solvent that will carry the analyte

Chromatograph: It is equipment that enables a sophisticated Separation. Chromatogram: It is the visual output of the chromatograph.
Retention time: It is the characteristic time it takes for a particular analyte to pass through the system (from the column inlet to the detector) under set conditions.

PRINCIPLE
Chromatography involves a sample (or sample extract) being dissolved in a mobile phase (which may be a gas, a liquid or a supercritical fluid). The mobile phase is then forced through an immobile, immiscible stationary phase (may be solid/porous particles or walls of a capillary tube) The phases are chosen such that components of the sample have differing solubilities in each phase. As a result of these differences in mobilities, sample components will become separated from each other as they travel through the stationary phase.

CHROMATOGRAM
The visual output of the chromatograph. Different patterns on the chromatogram correspond to different components of the separated mixture

X axis Retention time; Y axis Solute concentration leaving chromatographic column.

GAS CHROMATOGRAPHY
Separation of gaseous or volatile substances GC can consist of GSC (gas solid chromatography) GLC (gas liquid chromatography) Where, Gas = M.P and Solid / Liquid = S.P GSC principle is ADSORPTION GLC principle is PARTITION GSC not used because of limited no. of S.P

Sample to be separated is converted into vapour and mixed with gaseous mobile phase. Component more soluble in the S.P travels slower Component less soluble in the S.P travels faster Components are separated according to their Partition Coefficient (K) = Cs/Cg Where, Cs is the concentration of analyte in sample phase; Cg is the concentration of analyte in gas phase
Criteria for compounds to be analyzed by G.C 1.VOLATILITY 2.THERMOSTABILITY

WHY IS G-C USED IN FORENSICS?

Separation of chemical components - vital in any type of chemical analysis. To identify - the sample must be simplified into its constituent compounds.

The unknown can then be identification by its parts.

Separated compounds standards. are compared to known

It is important to have an idea of what compounds are being searched for in the first place.

HOW DOES THE G-C WORK?

First, a vaporized sample is injected onto the chromatographic column. Second, the sample moves through the column through the flow of inert gas.

Third, retention time is determined by each component reaching the detector at a characteristic time.
Fourth, the components are recorded as a sequence of peaks as they leave the column. Fifth, The peaks can then be read and analyzed by a forensic scientist to determine the exact components of the mixture.

PRACTICAL REQUIREMENTS
Carrier gas Flow regulators & Flow meters Injection devices Columns Temperature control devices Detectors Recorders & Integrators

 Properties of Carrier gas

CARRIER GAS

Inertness Suitable for the detector High purity Easily available Cheap Should not cause the risk of fire Should give best column performance

Examples : Helium, Hydrogen or Nitrogen

FLOW REGULATORS & FLOW METERS

Flow regulators - deliver the gas with uniform pressure/flow rate Flow meters tell the flow rate of the mobile phase Examples : Rota meter & Soap bubble meter (float attached to spring or soap bubble formed indicates the flow rate of the carrier gas)

INJECTION DEVICES
Gases can be introduced into the column by valve devices Liquids can be injected through loop or septum devices

COLUMNS
Important part of GC Made up of glass or stainless steel DEPENDING ON ITS NATURE, 2 TYPES : 1.Packed column:
Columns are available in a packed manner 1.5 10 m long 2-4 mm diameter Glass, stainless steel - solid packing

2.Open tubular/Capillary column /Golay column

Long capillary tubing 30-90m in long Uniform & narrow diameter - 0.025 - 0.075 cm Made up of stainless steel - in the form of a coil Disadvantage: more sample cannot loaded

TEMPERATURE CONTROL DEVICES

Preheaters: Convert sample into its vapour form. Are present along with injecting devices Thermostatically controlled oven: Temperature maintenance in a column is highly essential for efficient separation.

DETECTORS F.I.D
Destructive detector The effluent from the column is mixed with H & air, and ignited. Organic compounds burning in the flame produce ions and electrons, which can conduct electricity through the flame. A large electrical potential is applied at the burner tip The ions are collected on collector or electrode and recorded on recorder due to electric current produced.

RECORDERS & INTEGRATORS

RECORDERS : Record the baseline and all the peaks obtained INTEGRATORS : Record the individual peaks with Rt, height etc

PARAMETERS USED IN G-C

Retention time (Rt) It is the difference in time b/w the point of injection & appearance of peak maxima. Measured in minutes or seconds. Retention volume (Vr) It is the volume of carrier gas which is required to elute 50% of the component from the column. Retention volume = Retention time x Flow rate Separation factor (S) Ratio of partition co-efficient of the two components to be separated.

Resolution (R) The true separation of 2 consecutive peaks on a chromatogram is measured by resolution

Tentative Identification of Unknown Compounds

FORENSIC ANALYSIS WITH G-C

Determination of explosives If a forensic scientist can identify the type of explosive then he/she could possibly identify the source Ammonium nitrate Gasoline analysis 10,000 fires intentionally set each year Used to check for arson Fire debris evidence is collected in paint cans to keep the vapors inside

Urine Analysis
Cocaine Major cause of crime in the United States Amphetamines Uses similar procedure as cocaine Quinine in horse urine Prohibited in race horses Need to use mass spectrometer with gas chromatography

 Blood analysis
To check the concentration of alcohol Used if death is thought to be the result of intoxication Used for breath analysis (Must be confirmed by gas chromatography in the United Kingdom and Europe)

RESULTS
Qualitative
If the sample is suspected to be a certain compound, the sample can be spiked with said compound. In the read out, if there is no new peak for the spiked compound, the sample and the compound are the same.

Quantitative
In the read out, the area under the curve is the amount of the compound (integrate the peak)

 Thus, GC is capable of separating, detecting & partially characterizing organic compounds, particularly when present in small quantities.
1. Qualitative analysis Rt & RV are used for the identification & separation 2. Quantitative analysis It is necessary to measure the peak area or peak height of each component 3. Checking the purity of a compound Compare the chromatogram of the std. & that of the sample 4. Used for analysis of drugs & their metabolites, blood, explosives.

ADVANTAGES & DISADVANTAGES

ADVANTAGES :
Speed Good resolution Qualitative analysis Quantitative analysis Inexpensive Sensitive Simple Small sample is needed - ml

DISADVANTAGES :
Can be slow Only volatile samples can be used Thermally stable sample Destructive (Sample cannot be obtained back once separated) Requires caution while injecting samples Ghost peaks due to contaminants