high performance-liquid-chromatography-hplc

“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