Modern chromatographic technique.pptx

chromatography

1. MODERN CHROMATOGRAPHIC TECHNIQUE

2. INTRODUCTION Technique used to separate and identify the components of a mixture PRINCIPLE: Works by allowing the molecules present in the mixture to distribute themselves between a stationary and a mobile medium. Molecules that spend most of their time in the mobile phase are carried along faster. The factors effective on this separation process include molecular characteristics related to adsorption (liquid-solid), partition (liquid-solid ), and affinity or differences among their molecular weights.

3. Gas Chromatography Also called as Gas liquid chromatography (GLC) Based on a partition equilibrium of analyte between a solid stationary phase (often a liquid silicone based material) and a mobile gas (most often Helium). The stationary phase is adhered to the inside of a small-diameter glass tube (a capillary column) or a solid matrix inside a larger metal tube (a packed column). Well suited for use in the petrochemical, environmental monitoring, and industrial chemical fields. It is also used extensively in chemistry research.

4. GC involves vaporizing a sample and injecting it onto the head of the column. Under a controlled temperature gradient , the sample is transported through the column by the flow of an inert, gaseous mobile phase . Volatiles are then separated based on several properties, including boiling point, molecular size, and polarity. Working Principle:

5. Instrumentation: Mobile Phase: He, Ar, N2, H2 Flow regulators: deliver the gas with uniform pressure/flow rate. Injection Port: Rubber septum barrier ( usually maintained at a higher temperature than the boiling point of the least volatile component in the sample mixture ) Column: fused silica with a thin coating of stationary phase on the inner surface Oven: Thermostat controlled forced air oven Detector : Data System: recorders & integrators

6. Columns: Columns in GC are two types P acked column- Glass or metals. Densely packed with packing materials or solid support coated with thin layer of stationary liquid phase 2) Capillary column- Better resolution – efficient mass transfer between gas and SP Tubing – fused silica, glass, copper, stainless steel Packed column Capillary column (Open tubular column)

7. GC detectors: The eluted solute particles enter the detector Then produces electrical signals proportional to the concentration of the components of solute. This signals are amplified and recorded as peaks at intervals on the chromatograph. Different types of detectors are: Thermal conductivity detector Flame ionization Detector Electron Capture Detector

8. Gas chromatography-Mass spectroscopy (GC-MS) Gas chromatography-Mass spectroscopy (GC-MS) is one of the so-called hyphenated analytical technique. I t is actually two techniques that are combined to form a single method of analyzing mixtures of chemicals. Gas chromatography is a technique capable of separating , detecting and partially characterizing the organic compounds particularly when present in small quantity . Mass spectroscopy provides some definite structural information from in small quantity.

9. Principle of GC-MS: The sample solution is injected into the GC inlet where it is vaporized and swept onto a chromatographic column by the carrier gas ( usually helium ). The sample flows through the column and the compounds comprising the mixture of interest are separated by virtue of their relative interaction with the coating of the column (stationary phase) and the carrier gas (mobile phase). The latter part of the column passes through a heated transfer line and ends at the entrance to ion source where compounds eluting from the column are converted to ions.

10. Gas chromatography: Carrier gas- Served as mobile phase supplied in the steel tank under high pressure. Eg :-nitrogen , helium; hydrogen and argon can also be used Pneumatic Control- Gas supply is regulated to the correct pressure and then fed to the required part of instrument . Oven- Temperature programmable, typically range from 5°C - 400°C but can go as low as - 25°C with cryogenic cooling Sample Injection Port- Sample is made to vaporized rapidly before entering to column Column- Two kinds of column used :Packed column & Capillary column. Gas chromatograph GC-MS utilizes capillary column Detectors- Thermal Conductivity Detector (TCD) Flame Ionization Detector ( FID) Electron Capture Detector (ECD)

11. Interface: Pressure incompatibility problem between GC and MS was solved by inserting an interface. Mainly three types of interfaces are there commercially available : Jet/Orifice separator Direct capillary infusion interface Watson- Biemann effusion separator

12. Mass Spectrometer: Ion source- it converts the components of a sample into ions by bombardment with electrons, ions and molecules. The gas molecules exiting the GC are bombarded by high energy electron beam. Different types are: Electron Impact Ionization (EI )- Electrons produced by tungsten filament move to ion source chamber to produce magnetic flux in parallel to the electron beam. Chemical Ionization (CI )- Involves ionization of reagent gas like methane at high pressure & this collides with the analyte molecules producing ions. Fast Atom Bombardment (FAB)- Technique in which the analyte is bombareded by a high energy beam of inert gas such as Argon or Xenon.

13. Mass Selective Analyzer They deflects ions down a curved tubes in a magnetic fields based on their kinetic energy determined by the mass, charge and velocity. The magnetic field is scanned to measure different ions . Quadrupole Analyzer- consists of four parallel metal rods with different charges. For given DC and AC voltage, only ions of certain mass-to-charge ration pass through the analyzer. Ion Trap Mass Analyzer- The ion trap mass analyzer operates by similar principles where it consists of circular ring electrode and two end caps that form a chamber . The ions entering into the chamber are trapped by electromagnetic fields & they oscillates in concentric trajectories. Time-of-flight Analyzer - analyser uses an electric field to accelerate the ions through the same potential. Then it measures the time take to reach the detector. If the particles all have the same charge, the kinetic energies will be identical and their velocities will depend upon only on their masses.

14. Mass spectrometers employ computer control of same functions and also use a computerized display and output . It is used to identify and measure the concentration of one or more analytes in a complex mixture Data Handling :

15. High Performance Liquid Chromatography (HPLC) HPLC has the ability to separate, identify, and quantitate the compounds that are present in any sample that can be dissolved in a liquid Principle: The stationary phase is usually an inert solid or a liquid held on the inert solid Mobile phase travels through the column forcibly with the aid of the high pressure pump Solutes of the sample separated on column and eluted with mobile phase The technique is applicable to thermally fragile samples, e.g . amino acids, proteins, nucleic acids, hydrocarbons, antibiotics , steroids, drugs, inorganic and may organic substances .

16. NORMAL-PHASE HPLC: Adsorption of analytes on the polar, weakly acidic surface of silica gel Stationary Phase.: Silica (pH 2-8), Alumina (pH 2 - 12 ), Bonded Diol and NH2 Mobile Phase: Non-polar solvents (Hexane, CHCl3) Polar solutes elute later than non-polar lyophilic ones REVERSED-PHASE HPLC: Separation of non-polar analytes by partitioning onto a non-polar, bonded stationary phase . Stationary Phase: Hydrophobic surfaces of moieties bonded on silica Mobile phase: Methanol or Acetonitrile and Water.

17. Instrumentation: Solvent/ mobile phase reservoirs- Glass or stainless-steel containers capable of holding up to 1 liter mobile phase (pure organic solvents or aqueous solutions of salts and buffers) Degassing & filtration of mobile phase- Degassing is done to prevent formation of gas bubbles in the pump or detector ( Mobile phases are degassed by stirring of the mobile phase under vacuum , sonication or sparing with helium gas) The mobile phase are filtered to remove particulate matter that may clog the system Pump- The solvents or mobile phase must be passed through a column at high pressures at up to 6000 psi.. Pulse free output & all materials in the pump should be chemically resistant to solvents. Flow rates ranging from 0.1 to 10 mL/min

18. Sample injector system- Several injector devices are available either for manual or auto injection of the sample . Septum Injector- used for injecting the sample through a rubber septum Stop flow- the flow of mobile phase is stopped for a while & the sample is injected through a valve Rheodyne injector - This has a fixed volume of loop, for holding sample until its injected into the Column Rheodyne injector

19. Column- Precolumn - It contains a packing chemically identical to that in analytical column. Mainly used to remove the impurities from the solvent and thus prevents contamination of the analytical column, it can protect analytical column. It is also called as guard column or protective column. It is having large particle size. It is having short length of 2 to 10 cm, so does not affect separation

20. Analytical column- Actual separation is carried out here. Column is filled with small particles 5 – 10 micron. The solid support can be silica gel, alumina. The separation is result of different components adhering to or diffusion into the packing particles when the mobile phase is forced through column. HPLC Column

21. Detectors: Used upon the property of the compounds to be separated. Different detectors available are: Refractive index detectors- detects the change in turbidity U.V detector- based on electronic transitions within molecules Fluorescence detectors- based on emission of excited state molecules Photo diode array detector- allows for the recording of the entire spectrum of each solute as it passed trough the diode array detector.

22. High Performance Thin Layer Chromatography (HPTLC) HPTLC- sophisticated form of thin layer chromatography it involves the same theoretical principle of thin layer chromatography Key feature of HPTLC: Simultaneous processing of sample and Standard Several analysts work simultaneously. Lower analysis time and less cost per analysis. Low maintenance cost. Simple sample preparation No prior treatment for solvents like filtration and degassing. Low mobile phase consumption per sample Visual chromatogram

23. Principle: The mobile phase flows through the plate because of capillary action. the components move according to their affinities toward the adsorbent . The component with more affinity towards stationary phase travels slower. the component lesser affinity towards stationary phase travel faster

24. Steps Involving in HPTLC Selection Of Chromatography Plates Sample And Standard Preparation Pre Washing Of Pre Coated Plates Activation Of Pre-coated Plates Application Of Standard And Sample Selection Of Mobile Phase Chromatographic Development And Drying Detection and visualization

25. Selection Of Chromatography Plates: Precoated plates- different supporting materials – different solvents available 80% of analysis takes place by silica gel Aluminium oxide: Basic substances ,alkaloid and steroids . Sample And Standard Preparation: Sample and standard should dissolved in the same solvent to ensure comparable distribution at stationary zones. For normal phase chromatography solvent for dissolving the sample should be non polar . For reverse phase chromatography polar solvents are used.

26. Pre Washing Of Pre Coated Plates: To avoid any possible interference due to impurities it is recommended to wash the plates is called pre washing Solvents used for washing are: Chloroform in methanol(1:1) Methylene chloride – methanol(1:1) 1%ammonia or 1% acetic acid Activation Of Pre-coated Plates: Freshly open box of plates do not require activation. Plates exposed to high humidity or kept on hand for long time to be activated by placing in oven at 110-120ºc for 30 minutes prior to spotting . Aluminium sheets should be kept in between two glass plates and placing in oven at 110-115ºc for 15 minutes

27. Application Of Standard And Sample: Selection of sample application and devices used depends on:- Sample volume Number of samples to be applied Samples is applied by use of automatic devices and graduated capillaries. Volume recommended for HPTLC 0.5-5μl Capillary tubes- samples applied in form of spots Micro syringes- sample can apply either as spot or bands Capillary tubes Micro syringes

28. Selection Of Mobile Phase: Normal phase - Stationary phase is polar Mobile phase is non polar Non polar compounds eluted first because of lower affinity with stationary phase . P olar compound retained because of higher affinity with the stationary phase Reversed phase- Stationary phase is non polar Mobile phase is polar P olar compound eluted first because of lower affinity with stationary phase N on polar compounds retained because of higher affinity with the stationary phase

29. Chromatographic Development And Drying: After development remove the plate and mobile phase is removed from the plate –to avoid contamination of lab atmosphere. Dry in vacuum desiccator Detection and visualization: Detection under UV light is first choice –non destructive spots of fluorescent compounds can be seen at 254nm(near UV range)

30. The Liquid Chromatography-Mass Spectrometry (LC-MS) is combination of Liquid Chromatography and Mass Spectrometry which is used with separation power of HPLC with detection power of Mass Spectrometry Basic Principle of LC/MS: High Performance Liquid Chromatography (HPLC ): Liquid chromatography involves two main phases : Mobile phase: Liquid (solvents i.e. ethanol, Acetonitrile ). Stationary phase: Column packed with very small particles 2. Mass Spectrometry: Analytical technique that measures the mass to charge ratio of charged particles Liquid Chromatography-Mass Spectrometry (LC-MS )

31. Instrumentation: Instrumentation of HPLC Solvents (mobile phase) Degasser Pump Injector Column (Stationary phase) Detector

32. Instrumentation of MS: Ion source- which can convert gas phase sample molecules into ions . Different methods of ionization are: Electrospray Ionization:- the eluate stream leaving the chromatographic column is introduced into the ionization source through the capillary. At the outlet of the capillary, the sample dissolved in the solvent is exposed to a strong nebulizing gas (typically, nitrogen) and a very strong electric field , which results in the atomization of the sample into charged microdroplets . Electrospray Ionization

33. Atmospheric Pressure Chemical Ionization:- the eluate leaving the chromatographic column is heated and sprayed from the capillary and then captured, in gaseous form, by a stream of gas and carried to the electrode where ions are formed. Atmospheric Pressure Photoionization:- uses photons to excite and ionize molecules after nebulisation . Mass Analyzer- Its task is to separate ions in terms of their mass-to charge ratio and to direct the beam of focused ions to the detector . Different types of mass analyser are:

34. Quadrupole Analyzer:- consists of four metal electrodes in the form of symmetrically arranged rods. It works like a mass filter with the specific parameters of the electromagnetic field , allows the passage of ions with the selected mass-to-charge ratio values. T ime-of-flight mass analyzer:- The analyzer separates ions accelerated by an electric field according to their velocity which depends on their mass and charge. Quadrupole Analyzer Time-of-flight mass analyzer

35. Detectors- The detector is used to count the ions emergent from the mass analyzer, and may also amplify the signal generated from each ion. Following are three different kinds of detectors are used in Mass Spectrometry:- Electron Multipliers :- used to convert either – ve , + ve ions into electrons, that will be amplified and detected. Dynolyte Photomultiplier:- converts the charged ions into electrons. These electrons stick to a phosphor and emit photons, and that photons are made to strike the photomultiplier to achieve multiplied signals for recording.

36. CONCLUSION Initially chromatographic techniques were used to separate substances based on their color as was the case with herbal pigments With time its application area was extended considerably. Nowadays, chromatography is accepted as an extremely sensitive, and effective separation method These modern chromatographic techniques got attention in the field of pharmaceutical, chemical, food, agrochemical industries, environmental ,forensic applications, drug research and quality control