Basics of Chromatography
Chromatography is a combination of two words;
* Chromo Meaning color
* Graphy Representation of something on paper
Introductory Principles
Chromatography, literally "color writing", was first employed by Russian scientistMikhail Tswettin 1903/1906. He continued to work with chromatography in the first decade of the 20th century, primarily for the separation of plantpigments such aschlorophyll,carotenes and xanthophylls. Since these components have different colors (green, orange, and yellow,respectively) they gave the technique its name.
History of Chromatography
Chromatograph: Instrument employed for a chromatography
Eluent: Fluid entering a column
Eluate: Fluid exiting the column
Elution: The process of passing the mobile phase through the column
Flow rate: How much mobile phase passed / minute (ml/min)
Linear velocity: Distance passed by mobile phase per 1 min in the column (cm/min)
Analyte: Itis the substance to be separated during chromatography.
Mobile Phase: Gas or liquid that carries the mixture of components through the stationary phase.
Stationary Phase (Immobilized phase): The part of the apparatus that holds the components as they move through it, separating them.Retention time: Time taken for particular analytes to pass through the system (from the column inlet to the detector) under set conditions.
Retardation factor: Fraction of an analyte in the mobile phase of a chromatographic system.
Chromatography is used by scientists to:
Analyze to examine a mixture, its components, and their relations to one another
Identify to determine the identity of a mixture or components based on known components
Purify to separate components in order to isolate one of interest for further study
Quantify to determine the amount of the a mixture and/or the components present in the sample
Based on the physical means by which stationary (SP) and mobile phase (MP) comes into contact
3 phases solid , liquid & gas.
MP can be either gas or liquid
SP can be either solid or liquid
Column chromatography: SP held in a narrow tube through which MP is forced under pressure.Planar chromatography: SP supported on a flat paper. MP moves through SP due to gravity.Contd.
Based on MP used chromatography is classified as
Gas Chromatography separates vaporized samples with a carrier gas (mobile phase) and a column composed of a liquid or of solid beads (stationary phase) performed only on the columns.Liquid Chromatography: separates liquid samples with a liquid solvent (mobile phase) and a column composed of solid beads (stationary phase) performed either on surface or column.Gas Chromatography
Gas Chromatography: To separate stable and volatile organic and inorganic compounds.
It involves a sample being vaporized and injected onto the head of the chromatographic column. The sample is transported through the column by the flow of inert, gaseous mobile phase. The column itself contains a liquid stationary phase which is adsorbed onto the surface of an inert solid.
Types : Gas Solid Chromatography (GSC)Gas Liquid Chromatography (GLC) or Gas Chromatography
Introduction
Here, the mobile phase is a gas while the stationary phase is a solid.
Used for separation of low molecular gases, e.g., air components, H2S, CS2 ,CO2 , rare gases, CO and oxides of nitrogen
Gas-liquid chromatography:
The mobile phase is a gas while the stationary phase is a liquid retained on the surface as an inert solid by adsorption or chemical bonding
TYPES OF GC
Principle of Operation
Carrier gas has pressure regulator and flow monitor for constant flow of carrier gas- available in form of compressed gas.
Sample injection port to introduce sample vapours into gas stream - micro syringe
Columns with appropriate length of stationary phase
Thermal Compartment or thermostat to maintain column at appropriate temperature
Detectors to detect the sample components as they come out of the column.
Microprocessor or recorder to provide signal proportional to amount of each component present in analyte.
Instrumentation of GC
Carrier Gas
Contd.
Nitrogen less expensive but reduced sensitivityHelium used due to the excellent thermal conductivity, greater density & allows more flow rate.Hydrogen better thermal conductivity and lower density - hazardousLiquid Samples : Microsringe can be used. Sample is introduced into hot zone of the column. So that liquid gets transferred into gaseous phase.
Gas Samples : Tight syringe to deliver 0.1 10 ml of the sample. Rotary sample valve can be used.
Solid Samples : Dissolved into volatile liquids for introduction.
SAMPLE INJECTION SYSTEM
Packed column:
glass or metal is used for packingLength is 2 cm longer column is difficult to packForms can be U or helix or straight shaped columns.Inert gas support can be takenChromatographic Column
Capillary Column
Short response time
Non-destructive of sample
Sensitivity, stability & linear response
Insensitive to flow rate of the samples
The detector then produces electrical signals proportional to the concentration of the components of solute. The signals are amplified and recorded as peaks at intervals on the chromatograph.Detectors
Thermal conductivity Detector
Contd.
When only carrier gas flows heat loss to metal block is constant, filament T remains constant.
When an analyte species flows past the filament generally thermal conductivity changes, thus resistance changes which is sensed by Wheatstone bridge arrangement.
The imbalance between control and sample filament temperature is measured and a signal is recorded.
Molecules of compounds, which posses affinity for electrons, differ in their electron absorbing capacities. This difference is utilized in this detector for identification of the compounds.
Working- A foil made up of a radioactive metal like Ni63 (-emitter) is placed inside a Teflon coated cell which also contains a cathode and an anode.
Electron Capture Detector
In the absence of organic species, the produced electrons migrate towards positive electrode and produce a certain constant standing current.
When a sample/eluent is present it captures the electrons, elutes from column, there is a drop in this constant current.
The potential across two electrodes is adjusted to collect all the ions and a steady saturation current, is therefore, recorded.
Flame Ionization Detector
Flame Ionization Detector
HPLC
HPLC stands for High-performance liquid chromatography(sometimes referred to as High-pressure liquid chromatography).
HPLC is a physical separation technique in which a sample dissolved in a liquid is injected into a column packed with small particles and it is separated into its constituent components
HPLC is probably the most important and widely used analytical technique for quantitative analysis of organics and biomolecules
Most useful for pharmaceuticals, biomolecules, and labile organics
Contd.
HPLC is a separation technique that involves
Injection of a small volume of liquid sample into a tube packed with tiny particles (3 to 5 (m) in diameter called the stationary phase)Individual components of the sample are moved down the packed tube (column) with a liquid (mobile phase).It is forced through the column by high pressure delivered by a pump.These components are separated from one another by the column packing that involves various chemical and/or physical interactions between their molecules and the packing particles.HPLC System
Contd
These separated components are detected at the exit of this tube (column) by a flow-through device (detector) that measures their amount. The output from the detector is called a liquid chromatogramIn principle, LC and HPLC work the same way except the speed , efficiency, sensitivity and ease of operation of HPLC is vastly superiorHPLC Instrumentation Overview
*
Principle Pattern
An Example
Detector
Thermostatted
Column Compartment
Autosampler
Binary Pump
Vacuum Degasser
Solvent Cabinet
Solvent Reservoirs
Controller
HPTLC
HPTLC is a sophisticated form of TLC.
Fastest of all chromatographic techniques.
Any combinations of stationary and mobile phases can be used.
Analytical HPTLC is used for micro preparative analysis (ie., separation of milligram scale for analysis of fraction )
Gives more sharper and compact bands with minimum distance of migration.
Used for both qualitative and quantitative analysis.
HPLC Vs. HPTLC
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