UNIT II CHROMATOGRAPHY

Different techniques – Gas chromatography – Detectors – Liquid chromatographs – Applications – High-pressure liquid chromatographs – Applications. CHROMATOGRAPHY

• In any chemical or bioprocessing industry the need to separate and purify product from a complex mixture is necessary and important step in the production line.

• CHROMATOGRAPHY is a very special separation process for many process. They are It can separate complex mixture with great precision Very similar components It can purify any soluble or volatile substance if the right adsorbent material carrier fluid and

operating conditions are employed It can be used to separate delicate product since the condition under which it is performed

are not typically severe. Chromatography is a physical method of separation of the components of a mixture by

distribution between two phases of which one is a stationary phase and another one is mobile phase.

The process of Chromatographic separation involves the transport of a sample of the mixture through a column.1. The mixture can be in liquid or gaseous state 2. Stationary phase may be in solid adsorbent or a liquid partitioning agent . 3. Mobile phase is usually gas or liquid. The mobile phase carries the sample (mixture of component) through the column.BASIC OPERATION OF CHROMATOGRAPHY

• The process of a Chromatographic separation takes place within a Chromatography column.• The column made of glass or metal is either packed bed or open tubular column .• The stationary phase is usually a viscous liquid coated on the surface of solid particle which

are packed into the column or solid particle can also be taken as a the stationary phase.• The mobile phase is typically a solvent moving through the column which carries the mixture

to be separated. • The basic operation involves 4 main functions

1.Injection Feed.2. Separation in the column3. Elution from the column4. Detection of components

Injection Feed.It consists of mobile phase and sample whose components to be separated. The mobile phase flow through the system by the action of pumping.Separation in the columnAs the sample flow through the column the components of a mixture (sample) distribute themselves between the two phases.

Different component will absorb to the stationary phase to varying degree. Those strong attraction to the support move more slowly than those with weak attraction.

Elution from the column After the sample is flushed or displaced from the stationary phase . The different component

will elute from the column at different times. The component with least affinity from the stationary phase (the most weakly adsorbed) will

elute first. The greatest affinity from the stationary phase (the most strongly adsorbed) will elute last.

Detection of components The different components are collected as they emerge from the column. The detector is placed at the end of the column and its signal is plotted as a function of time or

of volume of added mobile phase , a series of peaks is obtained and this is called chromatogram. Useful in qualitative and quantitative analysis. The position of peaks on the time axis to identify the component of sample(quality). The area under the peak provide a measure of the amount of each component(quantity).

BASIC DEFINITIONS Retention Time(tR)

It is the total time that a compounds spends in both the mobile phase and stationary phase or the time between sample injection and an analyte peak reaching a detector at the end of the column is termed the retention time.

Dead Time(tM)It is a non-retained compound spends in the mobile phase which is also the amount of time the non-retained compound spends in the column. It is expressed in minutes.

Adjusted Retention Time It is the time that a compound spends in the stationary phase . It is the difference between the dead time and the retention time for a compound.

Capacity Factor( or Partition Ratio)It is the ratio of the mass of compound in the stationary phase relative to the mass of compound in the mobile phase

Phase ratio It relates the column diameter and film thickness of the stationary phase . The phase ratio is unit less and constant for a particular column and represent the volume ratio β β = r/2.df

Distribution constant KD = Concentration of compound in stationary phase/ Concentration of compound in mobile phase

Selectivity It is the ratio of the capacity factors of two peaks. The selectivity is always equal or greater than one. If the selectivity equals one , the two compounds cannot be separated. The higher the selectivity the more will be separation between two compounds or peaks. α = KA/KB

LINEAR VELOCITY It is the speed at which the carrier gas or mobile gas travels through the column. It expressed in centimeter per second. u=L/tm

EFFICIENCY It is related to the number of compounds that can be separated by the column. GAS CHROMATOGRAPH

BASIC PARTS OF A GAS CHROMATOGRAPHThe basic parts of GC are

1. Carrier gas supply along with pressure regulator and flow meter2. Sample injection system3. Chromatographic column4. Thermal compartment or thermostat5. The detection system6. RecorderCarrier gas supply along with pressure regulator and flow meter

The carrier gas normally N2, Ar, or He , compressed in cylinder with suitable pressure regulator. The sample injection port is maintained at temp T1, which ensures rapid vaporization but not

thermal degradation of the solute. The gas and liquid sample are injected by syringe through a self-sealing silicon rubber

diaphragm in the injection port. Sample vapour mixes with carrier gas and is swept into the chromatographic column. Different component in the voporized sample are separated from each other. Column is maintained at another temp T2. At the end of the column the solute emerging individually enter the detector.

Carrier gas• The carrier gas must be chemically inert. Commonly used gases include nitrogen, helium, argon,

and carbon dioxide. • The choice of carrier gas is often dependant upon the type of detector which is used. • The carrier gas system also contains a molecular sieve to remove water and other impurities. • He and H2 are preferred for thermal conductivity detector• Argon used with argon ionization gauge detector.

PURITY OF GASThe presence of contaminants in the carrier gas may affect column performance and detector response particularly when ionization detector are used.GAS FLOW RATE

The gas flow rate to be used in a analysis depends upon column diameter. Generally in the range of 10-400ml/min The flow rate of the carrier gas also affects the detector signals because fluctuations would

produce variable heat removal from katherometer filament . SAMPLE INJECTION SYSTEM

• For optimum column efficiency, the sample should not be too large, and should be introduced onto the column as a "plug" of vapour - slow injection of large samples causes band broadening and loss of resolution.

• The most common injection method is where a micro syringe is used to inject sample through a rubber septum into a flash vapouriser port at the head of the column. The temperature of the sample port is usually about 50°C higher than the boiling point of the least volatile component of the sample.

• For packed columns, sample size ranges from tenths of a microliter up to 20 microliters. Capillary columns, on the other hand, need much less sample, typically around 10 -3 mL.

• For capillary GC, split/splitless injection is used.

• The injector can be used in one of two modes; split or splitless. The injector contains a heated chamber containing a glass liner into which the sample is injected through the septum.

• The carrier gas enters the chamber and can leave by three routes (when the injector is in split mode). The sample vapourises to form a mixture of carrier gas, vapourised solvent and vapourised solutes.

A proportion of this mixture passes onto the column, but most exits through the split outlet. The septum purge outlet prevents septum bleed components from entering the columnCHROMATOGRAPHIC COLUMN

• The column is the heart of a gas chromatograph wherein the fundamental process of separation takes place.

• There are two general types of column, packed and capillary (also known as open tubular). • Packed columns contain a finely divided, inert, solid support material (commonly based on

diatomaceous earth) coated with liquid stationary phase. Most packed columns are 1 and 50m in length and have an internal diameter of 4 - 8mm.

• Packed columns are made from Glass, stainless steel or copper.CAPILLARY COLUMN

• Open tube column, 0.25mm dia. Lengths from 30 to 300m• Very high efficiency have been achieved.• It contain no packing and stationary phase is coated directly on the inside of the tubing.• It cannot handle samples more than 0.1µl.

Capillary column are 2 types1. Wall coated open tubler (WCOT)

2. Support coated open tubler (SCOT)• Wall coated column consists of a capillary tube whose walls are coated with a liquid stationary

phase.• In support coated column the inner wall of the capillary is lined with a thin layer of support

material such as diatomaceous earth, onto which the stationary phase has been adsorbed. Fused Silica Open Tubular (FSOT) column

These have much thinner walls than the glass capillary columns, and are given strength by the polyimide coating. These columns are flexible and can be wound into coils. They have the advantages of physical strength, flexibility and low reactivity. THERMAL COMPARTMENT

• The column is not operated at room temp because it would then be suitable only for the analysis of gases or extremely volatile liquids.

• It is desirable to keep the column at precisely constant temp.This is essential because the quantitative response of the detector is often affected by column temp.

• The temp is controlled to an accuracy of 0.1deg C.• The temp of the oven can be controlled accurately by using a proportional temp controller with

a platinum resistance thermometer as sensing element. • The oven is thermally insulated, so that heat loss to the atm is minimized.• The optimum column temperature is dependant upon the boiling point of the sample.

In designing column oven the following consideration should be taken into account.1. The oven must have minimum thermal gradient so that temp is uniform over the whole column.

The oven must have a fast rate of heatingKATHAROMETER OR THERMAL CONDUCTIVITY DETECTOR

• The TCD consists of an electrically heated filament in a temperature-controlled cell. Under normal conditions there is a stable heat flow from the filament to the detector body.

• When an analyte elutes and the thermal conductivity of the column effluent is reduced, the filament heats up and changes resistance.

• This resistance change is often sensed by a Wheatstone bridge circuit which produces a measurable voltage change. The column effluent flows over one of the resistors while the reference flow is over a second resistor in the four-resistor circuit.

• Since all compounds, organic and inorganic, have a thermal conductivity different from helium, all compounds can be detected by this detector.

• The TCD is often called a universal detector because it responds to all compounds. Also, since the thermal conductivity of organic compounds are similar and very different from helium, a TCD will respond similarly to similar concentrations of analyte.

FLAME IONIZATION DETECTOR

• An FID consists of a hydrogen/air flame and a collector plate. The effluent from the GC column passes through the flame, which breaks down organic molecules and produces ions.

• The ions are collected on a biased electrode and produce an electrical signal. The FID is extremely sensitive with a large dynamic range, its only disadvantage is that it destroys the sample.

• It responds with high sensitivity to almost all organic compounds .FLAME PHOTOMETRIC DETECTOR

• The instrumental requirements for 1) a combustion chamber to house the flame, 2) gas lines for hydrogen (fuel) and air (oxidant), and 3) an exhaust chimney to remove combustion products, the final component necessary for this instrument is a thermal (bandpass) filter to isolate only the visible and UV radiation emitted by the flame.

• Without this the large amounts of infrared radiation emitted by the flame's combustion reaction would heat up the PMT and increase its background signal.

• The PMT is also physically insulated from the combustion chamber by using poorly (thermally) conducting metals to attach the PMT housing, filters, etc.

• The physical arrangement of these components is as follows: flame (combustion) chamber with exhaust, permenant thermal filter (two IR filters in some commercial designs), a removable phosphorus or sulfur selective filter, and finally the PMT.

PHOTO IONIZATION DETECTOR

• The selective determination of aromatic hydrocarbons or organo-heteroatom species is the job of the photoionization detector (PID).

• This device uses ultraviolet light as a means of ionizing an analyte exiting from a GC column. The ions produced by this process are collected by electrodes.

• The current generated is therefore a measure of the analyte concentration. • The lamp energies range from 8.3 to 11.7ev and wavelength range from 150nm to 106nm.

ELECTRON CAPTURE DETECTOR

• The ECD uses a radioactive Beta emitter (electrons) to ionize some of the carrier gas and produce a current between a biased pair of electrodes.

• When organic molecules that contain electronegative functional groups, such as halogens, phosphorous, and nitro groups pass by the detector, they capture some of the electrons and reduce the current measured between the electrodes.

• The ECD is as sensitive as the FID but has a limited dynamic range and finds its greatest application in analysis of halogenated compounds.

ATOMIC-EMISSION DETECTOR (AED)

• The components of the AED include • 1) an interface for the incoming capillary GC column to the microwave induced plasma chamber• 2) the microwave chamber itself • 3) a cooling system for that chamber, • 4) a diffraction grating and associated optics to focus then disperse the spectral atomic lines• 5) a position adjustable photodiode array interfaced to a computer. The microwave cavity

cooling is required because much of the energy focused into the cavity is converted to heat.• The strength of the AED lies in the detector's ability to simultaneously determine the atomic

emissions of many of the elements in analytes that elute from a GC capillary column (called eluants or solutes in some books).

• As eluants come off the capillary column they are fed into a microwave powered plasma (or discharge) cavity where the compounds are destroyed and their atoms are excited by the energy of the plasma.

CHEMILUMINESCENCE SPECTROSCOPY

• Uses quantitative measurements of the optical emission from excited chemical species to determine analyte concentration.

• Chemiluminescence is usually emission from energized molecules instead of simply excited atoms.

• If the excitation energy for analytes in chemiluminescence doesn't come from a source lamp or laser, where does it come from? The energy is produced by a chemical reaction of the analyte and a reagent.

• The light emission (represented as Planck's constant times nu-the light's frequency) is produced by the reaction of an analyte (dimethyl sulfide in the above example) and a strongly oxidizing reagent gas such as fluorine (in the example above) or ozone, for instance.

• The reaction occurs on a time scale such that the production of light is essentially instantaneous; therefore, most analytical systems simply mix analytes and the reagent in a small volume chamber directly in front of a PMT.

• High Pressure Liquid Chromatography

Partition ChromatographyAdsorption ChromatographyIon ChromatographySize-Exclusion Chromatography