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

It is a physical separation method in which the components of a mixture are separated by differences in their distribution between two phases, one of which is stationary (stationary phase) while the other (mobile phase) moves through it in a definite direction. The substances must interact with the stationary phase to be retained and separated by it.The stationary phase may be a solid, or a liquid supported on a solid or gel, the mobile phase may be either a gas or a liquid.

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.

Mixture of various components enters into system at various rate of speed.Mixture moves over the absorptive materials & provides separation.Repeated absorption takes place over stationary bed which determines the rate.Smaller the affinity of molecule to stationary phase shorter time spent in a column.

Separates complex mixtures with precision.Similar components can be separated easily . Ex : proteins that vary by a single amino acidPurify any soluble substances if proper conditions are employed.Separates very delicate products.Used in chemical or bio-processing field, manufacturing plants.

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

Gas-solid chromatography:

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

The principle of separation in GC is partitionThe mixture of component to be separated is converted to vapor and mixed with gaseous mobile phase.The component which is more soluble in stationary phase travel slower and eluted later. The component which is less soluble in stationary phase travels faster and eluted out first.Partition coefficient is the ratio of solubility of a substance distributed between two immiscible liquids at a constant temperature.No two components has same partition coefficient conditions. So the components are separated according to their partition coefficient.

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

The gas is conducted from cylinder through pressure and flow regulator to sample injection port at a temperature TMolecular sieve to remove water & other impurities.Flow rates monitored by rotameter.Hydrogen, helium, argon, nitrogen, carbon-dioxide and air are used.Hydrogen is advantageous but dangerousHelium second best but expensive

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 - hazardous

To determine the efficiency of the column.Method of sample injection depends on pressure in the column during introduction.

Liquid 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

Constructed using glass or metal tubing.Types : Packed column and Capillary column

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 taken

Chromatographic Column

Open tubular columns made of fused silica.Length 30 to 300 m and diameter of 1 mmThin walled column used for higher efficiency due to reduced diameter that reduces diffusion of molecules. advantage is better separation of components at lower temperature with short time period.

Capillary Column

Located at the exit of separation column to sense the presence of individual component as they leave the 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

TCD is based upon the fact that the heat lost from a filament depends upon the thermal conductivity of the stream of surrounding gas as well as its specific heat.

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.

This employs hydrogen flame that is maintained in a small cylindrical jet made up of platinum or quartz.Effluent from the column with helium or nitrogen as carrier gas are fed into the hydrogen flame, gets ignited and undergoes pyrolysis to produce ions.For detection of these ions, two electrodes are used that provide a potential difference. The ions produced are repelled by the positive electrode which hit the collector plate. The current produced in doing so is amplified and fed to an appropriate recorder.

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 superior

HPLC 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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