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“color writing” Is a technique used to separate and
identify the components of a mixture. Ettre & Zlatkis, 1967 introduced “The
Practice of Gas Chromatography. Gas chromatography is a technique used
for separation of volatile substances where the mobile phase is gas and stationary phase may be solid/liquid.
A sample containing the materials to be separated is injected into the gas chromatograph. A mobile phase (carrier gas) moves through a column that contains a wall coated or granular solid coated stationary phase. As the carrier gas flows through the column, the components of the sample come in contact with the stationary phase. The different components of the sample have different affinities for the stationary phase, which results in differential migration of solutes, thus leading to separation
Syringe
Injector
Detector
Carrier Gas Cylinder
Column
To Waste or Flow Meter
Flow Controller
Two-Stage Regulator
Hydrogen
Air
Capillary tube (column)
Platinum jet
Collector
Sintered disk
Teflon insulating ring
Flame
Gas outletCoaxial cable to Analog to Digital converterIons
Why do we need hydrogen?
Responds to compounds that produce ions when burned in an H2-air flame◦ all organic compounds
Little or no response to (use a Thermal Conductivity Detector for these gases)◦ CO, CO2, CS2, O2, H2O, NH3, inert gasses
Linear from the minimum detectable limit through concentrations times the minimum detectable limit
107
Thermal Conductivity Detector ◦Difference in thermal conductivity
between the carrier gas and sample gas causes a voltage output
◦Ideal carrier gas has a very low thermal conductivity (He)
Electron Capture Detector◦Specific for halogenated organics
Requires only very small samples with little preparation
Good at separating complex mixtures into components
Results are rapidly obtained (1 to 100 minutes)
Very high precision Only instrument with the sensitivity to detect
volatile organic mixtures of low concentrations Equipment is not very complex (sophisticated
oven)
Only volatile samples or the sample which can be made volatile are separated by this method.
During injection of the gaseous sample proper attention is required.
The sample of gas which is about to inject must be thermally stable so that it does not get degraded when heated.
gas Compound must exist as a ____ at a temperature that can be produced by the GC and withstood by the column (up to 450°C)
Alcohols in blood Aromatics (benzene, toluene, ethylbenzene,
xylene) Flavors and Fragrances Permanent gases (H2, N2, O2, Ar, CO2, CO, CH4) Hydrocarbons Pesticides, Herbicides, PCBs, and Dioxins Solvents
Originally referred to as High-Pressure Liquid Chromatography
Now more commonly called High Performance Liquid Chromatography HPLC is a form of liquid chromatography
used to separate compounds that are dissolved in solution.
Mobile phase – liquid Stationary phase- solid
1. Solvent Reservoir: To carry sample into the column2. Pumps: To produce an appropriate pressure to push
solvent into the sample.3.Sample Injection System: Syringe/injector4.Columns:straight, 15 to 150 cm in length; 2 to 3 mm id
and packing - silica gel, alumina.5.Detectors:UV/Vis,Refractive
index,Fluorescence,Evaporative light scattering (ELSD),MS,Diode Array Detector (DAD).
6.Data Processing: Receive the information from HPLC machine and present it as a graph using softwares.
The graph describes about qualitative data (Retention time) and quantitative data (area under curve)
7.Waste
HPLC COLUMN
The solvent is pumped from the solvent reservoir through the pump passing through the filters and the sample is injected through the sample port and gets mixed with the solvent and is passed into the HPLC column and the sample runs on the silica gel like paper chromatography and the eluted sample at the end of the column is detected by the detector and the signal is passed to a computer where the signal is converted to a chromatogram and displayed.
Column Parameters Column Material Stationary Phase Coating Material
Instrument Parameters Temperature Flow Signal Sample Sensitivity Detector
Sample Parameters
• Concentration• Matrix• Solvent Effect• Sample Effect
Speed in minutes High resolution High accuracy and reproducibility. Automated
Need a skill to run the instruments High cost Complexity Low sensitivity for few compounds.
1. Pharmaceuticals industry To control the drug stability Quantity of drug determination from
pharmaceutical dosage forms, ex. Paracetamol determination in panadol tablet
Quantity of drug determination from biological fluids, ex: blood glucose level
2. Analysis of natural contamination- Phenol & Mercury from sea water
3. Forensic test- Determination of steroid in blood, urine & sweat.- Detection of psychotropic drug in plasma
4. Clinical test- Monitoring of hepatic chirosis patient through aquaporin 2 in the urine.
5. Food and essence manufacture- sweetener analysis in the fruit juice- preservative analysis in sausage.
1930s,first developed by A.WILHELM TISELUIS-a Scottish biochemist won the Nobel prize in 1948.
Used to study enzymes and other proteins.
Relies on the affinity of various biochemical compounds with specific properties.
Antigen antibody Antibody antigen Substrate enzyme DNA HISTON Hormone receptor
Specificity is based on three aspects of affinity :
1. Matrix-for ligand attachment2. Spacer arm-used to bind ligand to matrix3. Ligand-molecule that binds reversibly to a
specific target molecule
The sample is injected into the equilibrated affinity chromatography column.
Only the substance with affinity for the ligand are retained on the column.
The substance with no affinity to the ligand will elute off.
The substance retained in the column can be eluted off by changing the pH of salt or organic solvent concentration of the eluent.
Used in Genetic Engineering for nucleic acid purification
Vaccine production-antibody purification from blood
Basic metabolism research-protein or enzyme purification from cell free extracts.
1. Extremely high specificity2. High degrees of purity can be obtained3. The process is reproducible
Expensive ligands Leakage of ligand Degradation of the solid support Limited lifetme Non specific adsorption Relatively low productivity