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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
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
It is important to have an idea of what compounds are being searched for in the first place.
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
CARRIER GAS
Inertness Suitable for the detector High purity Easily available Cheap Should not cause the risk of fire Should give best column performance
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
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.
Resolution (R) The true separation of 2 consecutive peaks on a chromatogram is measured by resolution
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.
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