CHROMATOGRAPHY A laboratory technique in which the components of a sample are separated based on how they distribute between two chemical or physical phases, one of which is stationary and other of which is allowed to travel through the separation system.
CHROMATOGRAPHY Introduced first by the Russian botanist Mikhail Semenovich Tswett. Mixtures of solutes dissolved in a common solvent are separated from one another by a differential distribution of the solutes between two phases.
PRINCIPLE Fractionalism of mixtures of substances In the operation of the chromatogram, a mobile gaseous or liquid phase is use to wash the substances to be separated through a column of a porous material.
PRINCIPLE The rate of migration of the solute depends upon the rate of interaction of the solute with the two phases, one being the mobile phases and the other stationary phase as the compounds travel through the supporting medium.
DEFINITION OF TERMS: Capillary Action – the movement of liquid within the spaces of a material due to the forces of adhesion, cohesion, and surface tension. Retention time-the average time of the substance that is bound by the stationary phase and will travel slowly through the column and exit at some later time.
DEFINITION OF TERMS: Peak-the width of the region that contains each compound Viscosity- measure of the resistance of a fluid which is being deformed by either shear stress or tensile stress. VOCs – volatile organic compounds made up of large group of small organic compounds with boiling point below 200 oC.
DEFINITION OF TERMS: Stationary phase- fixed phase that is coated or bonded within the column Mobile phase-phase that is flowing through the column and causes sample components to move towards the column’s end.
DEFINITION OF TERMS: Partition Coefficient- ratio of concentrations of a compound in the two phases of a mixture of two immiscible solvents at equilibrium. Volatility- measure of the tendency of a substance to vaporize
DEFINITION OF TERMS: Eluate- the separated components Elution- Eluent- the “carrier” portion of the mobile phase.
COMPONENTS OF A CHROMATOGRAPH MOBILE PHASE – A phase that is flowing through the column and causes sample components to move toward the column’s end. STATIONARY PHASE- A fixed phase that is coated or bonded within the column; it always remain in the system. It is responsible for delaying the movement of compounds as they travel through the column. SUPPORT- onto which the SP is coated or attached.
COMPONENTS OF A CHROMATOGRAPH ORIGINAL SAMPLE AND MOBILE PHASE COLUMNSUPPORT ANDSTATIONARYPHASE Receiving vessel
TYPES OF CHROMATOGRAPHY CAN BE CLASSIFIED ACCORDING TO: ∞MOBILE PHASE ∞STATIONARY PHASE ∞SUPPORT
GAS CHROMATOGRAPHY A type of Chromatography in which the mobile phase is a GAS. First GC system was developed by Erika Cremer The presence of a gas mobile phase makes GC valuable for separating substances like VOCs that occur naturally as gases that can easily be placed into gaseous phase.
GAS CHROMATOGRAPHY • It can separate nanograms or picograms of volatile substances. • It is principally a method for the separation and quantitative determination of gases and volatile liquids and substances.
GAS CHROMATOGRAPHY HOW IS IT PERFORMED? A System called Gas Chromatograph is used to perform GC. COMPONENTS: GAS SOURCE INJECTION SYSTEM COLUMN DETECTOR
GAS CHROMATOGRAPHY∞ GAS SOURCE- supplies the mobile phase. It is typically a gas cylinder equipped with pressure regulators to deliver the mobile phase at a controlled state.∞ TWO STAGE REGULATOR- for the pressure
GAS CHROMATOGRAPHY∞ INJECTION SYSTEM- consists of a heated loop or port into which the sample is placed and converted into a gaseous form.∞ COLUMN- contains the stationary phase and support material for the separation of components in the sample. This column is held in an enclosed area known as the column oven
GAS CHROMATOGRAPHY∞ COLUMN OVEN- maintains the temperature at a well-defined value.∞ DETECTOR- monitors sample components as they leave the column.∞ DATA SYSTEM
GAS CHROMATOGRAPHY FACTORS THAT AFFECT GC: Requirements for the Analyte Volatility and Thermal Stability Chemical Derivatization
GAS CHROMATOGRAPHYCommon Mobile Phases in GC: Hydrogen Helium Nitrogen Argon
GAS CHROMATOGRAPHYELUTION METHODS IN GC: THE GENERAL ELUTION PROBLEM x Works well if the sample is relatively simple or has only a few known compounds. x Generally used for samples containing only relatively volatile analytes x Difficulty to find a single set of conditions that can separate all the components of a complex sample with adequate resolution and in a reasonable amount of time.
GAS CHROMATOGRAPHYELUTION METHODS IN GC: Temperature Programming *vary the temperature of the column over time. *temperature increases; retention decreases
GAS CHROMATOGRAPHY GC SUPPORT MATERIALS Packed Column filled with small support particles that acts as an adsorbent or that are coated with the desired stationary phase.
GAS CHROMATOGRAPHY GC SUPPORT MATERIALS Packed Column Diatomaceous earth is a common support placed on packed columns. This material is formed from fossilized diatoms and mainly consists of Silicon dioxide or silica.
GAS CHROMATOGRAPHY Open- Tubular Columns stationary phase coated on or attached to its interior surface.
GAS CHROMATOGRAPHY Open- Tubular Columns A coating of polyimide is present on the outside of this column to give it better strength and flexibility for handling and storage.
GAS CHROMATOGRAPHYThree types of open-tubular columns in GC Based on how the stationary phase is placedin the column.
GAS CHROMATOGRAPHY1.Wall-Coated Open-Tubular (WCOT) Column Thin film of a liquid stationary phase is placeddirectly on the wall of the column.
GAS CHROMATOGRAPHY2. Support- Coated Open-Tubular (SCOT) column Has an interior wall that is coated with a thinlayer of a particulate support, plus a thin film of aliquid stationary phase that is coated onto thissupport layer.
GAS CHROMATOGRAPHY3. Porous-layer Open-Tubular (PLOT) column It also contains a porous material that isdeposited on the column’s interior wall, but thesurface of this material is now used directly as thestationary phase without any additional coating.
GAS CHROMATOGRAPHY GC STATIONARY PHASES Gas- Solid Chromatography ( solid adsorbents)Gas-Liquid Chromatography (liquids coated on solids)Bonded phases
GAS CHROMATOGRAPHY◊ Gas-Solid Chromatography o Solid adsorbent is used as a stationary phase. o Uses the same material as both the support and stationary phase, with retention occurring through the adsorption of analytes to the support’s surface. o Example of support is a MOLECULAR SIEVE. o Other supports include: o ORGANIC POLYMERS - porous polystyrene o INORGANIC SUBSTANCES – Silica or Alumina
GAS CHROMATOGRAPHYo Increasing the surface area of the GSC support will increase the phase ratio and result in higher retention for analyteso Pore size is important because only compounds smaller than the pores are able to contact the surface are within the space.o Polarity of Support and its functional groups will also affect how analytes will bind to them.
GAS CHROMATOGRAPHY◊ Gas-Liquid Chromatography o A chemical coating or layer is placed onto the support and used as the stationary phase. o Most Common types of GC. o 100% dimethylpolysiloxane, 5%phenyl-95% methyl polysiloxane – Examples of liquids that are used as Stationary phase.
GAS CHROMATOGRAPHY◊ Gas-Liquid Chromatography o All of these liquids have High boiling points and low volatilities, which allows them to stay within the column at relatively high temperatures that are often used in GC for sample injection and elution. o Liquids are also wettable- easy to place onto a support in a thin, uniform layer.
GAS CHROMATOGRAPHY◊ Bonded Phases o Column Bleed- most nonvolatile liquid will slowly vaporize or break apart and leave the column over time. o Column bleed changes the retention characteristics of the column. o It can also cause some GC detectors to have a high background and noisy signal as the stationary phase leaves the column and enters the detector. o Use of bonded phase minimize column bleed.
GAS CHROMATOGRAPHY◊ Bonded Phases o Produced by reacting groups on a polysiloxane stationary phase with silanol groups that are located on the surface of a silica support.
GAS CHROMATOGRAPHYTypes of Gas Chromatography Detectors General Detectors x Thermal Conductivity Detector -used for both organic and inorganic compounds -measures the ability of the eluting carrier gas and analyte mixture to conduct heat away from hot-wire filament-”thermal conductivity”. -example: Wheatstone bridge
GAS CHROMATOGRAPHY Flame Ionization Detector - detects organic compounds by measuringtheir ability to produce ions when they are burnedin flame.
GAS CHROMATOGRAPHY Selective Detectors x Nitrogen-Phosphorous Detector - selective for the determination of nitrogen- or phosphorous containing compounds. - Similar to FID, but does not use a flame for ion production.
GAS CHROMATOGRAPHY Electron capture detector - detects compounds that have electronegativeatoms or groups in their structure, such as halogen atoms( I,Br,Cl and F) and Nitro Groups. -can also be used to detect polynuclear aromaticcompounds, anhydrides and conjugated carbonylcompounds.
GAS CHROMATOGRAPHY/MASS SPECTROMETRY A POWERFUL TOOL for both measuring and identifying analytes as they elute from a GC column. Converts a portion of the eluting analytes into gas-phase ions that can be separated and detected. “electron-impact ionization” or “chemical ionization” can be used to create ions.
SAMPLE INJECTION AND PRETREATMENTTGAS SAMPLES Natural candidates for this technique Should be possible to directly sample and inject this gas onto a system. It is often necessary to collect and concentrate these analytes before they are examined by GC. Another way of collecting is using a cold trap.
SAMPLE INJECTION AND PRETREATMENTTLIQUID SAMPLES Direct injection can be used for a liquid sample which uses a calibrated microsyringe to apply the desired volume of sample to the system.
SAMPLE INJECTION AND PRETREATMENTTSolid samples Extraction of compounds of interest from the solid material. Can be accomplished by using liquid-liquid extraction or super critical fluid extraction.
GAS CHROMATOGRAPHYADVANTAGES:o Ability to provide qualitative information and quantitative informationo FAST ANALYSIS, HIGH SPEEDo Efficient, providing high resolutiono Sensitiveo Requires small sampleso Inexpensiveo EASY, WELL KNOWN
GAS CHROMATOGRAPHYDISADVANTAGES: o LIMITED to volatile samples o Dirty samples require clean up o Some training/experience is necessary o Most use another instrument for confirmation of Identity (e.g Mass Spectrometry)
GAS CHROMATOGRAPHYApplications: Most effectively used for analyses of organic compounds, space related, complex mixtures of volatile substances at column temperature of less than -40 °C to greater than 550° C. Geochemical research projects such as determination of various environmental pollutants at extremely low concentrations.
LIQUID CHROMATOGRAPHY√ A Chromatographic technique in which the mobile phase is a liquid.√ Originally developed by Russian botanist Mikhail Tswett in 1903.√ Works directly with liquid samples, which makes it valuable in such areas as food testing, environmental testing and biotechnology.
LIQUID CHROMATOGRAPHY√ Components of the LC System: Support – enclosed in a Column Stationary phase- enclosed in a Column Liquid mobile phase-delivered to Column by means of Pump Injection device- on analytical applications it is being used, to apply samples to the column. Collection Device (optional)- placed after the column to capture analytes as they elute.
LIQUID CHROMATOGRAPHYFACTORS THAT AFFECT LIQUID CHROMATOGRAPHY:*requires both a difference in retention and good efficiencyfor it to separate two given chemicals*Sample*Analyte Requirements*Column Efficiency*Role of the Mobile phase
LIQUID CHROMATOGRAPHY1. ADSORPTION CHROMATOGRAPHY A chromatographic technique that separates solutesbased on their adsorption to the surface of a support. Also known as Liquid-Solid Chromatography Equivalent method in GC is Gas-Solid Chromatography Uses the same material as both stationary phase and support. Retention process can be explained on the ff below: A+ n M-Surface A-Surface + n M
LIQUID CHROMATOGRAPHY Elutropic strength- strength of a mobile phase in adsorption chromatography It is a measure of how strongly a particular solvent or liquid mixture will absorb to the surface of a given support. Examples: silica and Alumina supports A liquid with large elutropic strength will strongly adsorb to the given support, which will prevent the analyte from binding to the support.
LIQUID CHROMATOGRAPHY Stationary Phases and Mobile Phases Silica (SiO2) - most popular support since it is polar in nature, thus it will strongly retain polar compounds. Alumina (Al2O3) – general-purpose support, but can retain some polar solutes so strongly that they are irreversibly absorbed onto its surface. Carbon-based Compounds- used as nonpolar supports that retain nonpolar solutes and have a strong mobile phase that is nonpolar.
LIQUID CHROMATOGRAPHY Applications Help purify new compounds Separation of geometrical isomers and chemicals that belong to a given class of substances Remove undesired side-products during synthesizing AZT or other drugs
LIQUID CHROMATOGRAPHY2. PARTITION CHROMATOGRAPHYo It is a Liquid Chromatographic technique in which solutes are separated based on their partitioning between a liquid mobile phase and a stationary phase coated on a solid support.o Support used is usually Silicao Originally, it involves coating of support with a liquid stationary phase that was immiscible with the mobile phase
LIQUID CHROMATOGRAPHYTwo Main Categories of Partition Chromatography:• Normal-phase- uses polar stationary phase• Reversed phase-uses nonpolar stationary phase
LIQUID CHROMATOGRAPHYStationary Phases and Mobile Phases Liquid Stationary Phases coated onto solid supports Bonded Phases – widely used due to their better stability and efficiency compared to liquid stationary phases. Silica - often used as the support. To place bonded phases on this support, silanol groups on the surface of silica are first treated with an organosilane that contains the desired stationary phase as a side chain. Agents like triethylamine and trifluoroacetic acid can also be added to the mobile phase to prevent silanol groups from binding to analytes.
LIQUID CHROMATOGRAPHYApplications: Used in analytical laboratories Separation of analytes in organic solvents and chemicals that contain polar functional groups. Employed in pharmaceutical industry as a method for separating and analyzing drugs during their testing and development.
LIQUID CHROMATOGRAPHY3. ION- EXCHANGE CHROMATOGRAPHY Solutes are separated by their adsorptiononto a support containing fixed charges on itssurface. Routinely used in Industry for the removal or replacement of Ions in products. Used for the separation of charged compounds( inorg. Ions, org. ions, AA, Proteins and NucleicAcids)
LIQUID CHROMATOGRAPHYStationary Phases and Mobile Phases Cation-exchanger – has negatively charged group and is used to separate positive ions Anion-exchanger – has positively charged group and is used to separate negative ions
LIQUID CHROMATOGRAPHY Silica (SiO2) - most popular support since it is polar in nature, thus it will strongly retain polar compounds. Polystyrene – used for small inorganic and organic ions Carbohydrate-based Gels – useful in separation of biological compounds, which can have very strong, undesirable binding to organic polymeric resins like polysterene. Example: Agarose
LIQUID CHROMATOGRAPHY Applications Removal of certain types of ions from samples such as in water-purification Direct separation and analysis of samples
Factors to be considered in Ion-Exchange ChromatographyBufferspHSalt Gradient
LIQUID CHROMATOGRAPHY4. SIZE-EXCLUSION CHROMATOGRAPHY It is a LC technique that separates substancesbased on different abilities of analytes to accessthe mobile phase within the pores of a support.. o No true stationary phase is present in this system o Uses a support that has a certain range of pore sizes. o A separation based on size or molar mass.
LIQUID CHROMATOGRAPHYStationary Phases and Mobile Phases Ideal support consists of a porous materialthat does not interact directly with the injectedsolute. o Carbohydrate-based supports like dextran and and agarose in their underivatized form can be used in SEC for biological compounds and aqueous-based samples. o Polyacrylamide gel can also be used o For organic solvents, Polystyrene can also be used o Silica containing diol-bonded phase can be used on aqueous samples
LIQUID CHROMATOGRAPHYAPPLICATIONS: Used in biological samples Transfer of large analytes from one solution to another Removal of salts from sample Separation of biomolecules and polymers Estimation of molar mass
LIQUID CHROMATOGRAPHY5. AFFINITY CHROMATOGRAPHY A Technique based on biologically relatedinteractions. o Makes use of the selective, reversible interactions that characterize most biological systems. o Example: binding of enzyme with its substrate.
LIQUID CHROMATOGRAPHYStationary Phases and Mobile Phases Stationary phase is represented by theaffinity ligand. o High specificity ligands- bind to only one or a few very closely related molecules o Examples:antibodies for binding to foreign agents and single-stranded Nucleic Acids for separating and binding to complemetary strands o General ligands- bind to a family or class of related molecules
LIQUID CHROMATOGRAPHY Carbohydrate gels like agarose or cellulose are commonly used with affinity ligands serve as supports. Silica can also be used with affinity ligands
LIQUID CHROMATOGRAPHY APPLICATIONS: oLarge-Scale Purification method for enzymes and proteins oSample preparation oDirect analysis of complex biological samples. oStudy of biological interactions.
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY Uses a pressure for the pumping of aqueous or organic solution through a column. The mobile phase is forced under pressure through a long, narrow column, yielding an excellent separation in a relatively short time. Highly sensitive and specific.
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY Become the primary means of monitoring the use of drugs and of detecting drug abuse. Also used to separate the compounds contributing to the fragrance of the flowers.
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY ADVANTAGES:An automated process that takes only a few minutes to produce results.Uses gravity instead of high speed pump to force compounds through the densely packed tubing.Results are of high resolution and are easy to read.Can be reproduce easily via automated process.
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY DISADVANTAGES:Difficult to detect coelution, which may lead to inaccurate compound categorization.High cost for equipment needed to conduct HPLC.Operation is complex, requiring a trained technician to operate.Equipment has low sensitivity to some compounds because of the speed of the process.
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY Applications: Use in biomedical research, routine clinical determination and drug researching programs
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY Instruments Fraction Collector Auto Sampler Pumping systems Columns & Packing Detectors Control Data & Processing
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY Applications: Use in monitoring the use of therapeutic drugs and detecting drug abuse. also use to separate compounds contributing to the fragrance of flowers
OLD TECHNIQUES IN CHROMATOGRAPHYA separation that takes place on a flatsurface or a plane: PAPER CHROMATOGRAPHY THIN LAYER CHROMATOGRAPHY
PAPER CHROMATOGRAPHY Based on nature of solvent,solubility of soluteand rate of diffusion. Uses paper as thestationary phase anda solvent as themobile phase.
PAPER CHROMATOGRAPHY Solvent moves through the paper by a capillary action Separation depends on the solubility of solute and solvents, the polarity of solvent, and polarity of solutes in the sample.
PAPER CHROMATOGRAPHYVisualization of theseparated sampleoccurs by chemicalreaction, whichproduces a colorchange.
PAPER CHROMATOGRAPHY Considered to be the simplest and the most widely used of the chromatographic techniques because its APPLICABILITY TO THE FOLLOWING: ISOLATION IDENTIFICATION AND QUANTITATIVE DETERMINATION OF ORGANIC AND INORGANIC COMPOUNDS
THIN LAYER CHROMATOGRAPHY Used as a semi-quantitative screening test screening test Uses as thin layer of silica gel, alumina gel, polyacrylamide gel, or starch gel attached to glass plate as stationary phase and the mobile phase is liquid solvent.
THIN LAYER CHROMATOGRAPHY Fractions in the sample are generally quite soluble in the solvent and move with it up the stationary phase by capillary action. Separated fractions are also developed in TLC by applying a chemical reaction with the separated fractions to produce color changes
THIN LAYER CHROMATOGRAPHYADVANTAGES: DISADVANTAGES: Simple and economical Spots are often faint Easy to perform since it TLC is difficult to only involves spotting the stationary phase reproduce with the sample & Not typically placing one edge of the automated stationary phase plate in the mobile phase reservoir.
General Categories of Chromatographic Methods Gas Chromatography Type of Stationary Phase Gas-Solid Chromatography Solid, Underivatized Support Gas-Liquid Chromatography Liquid-coated support Liquid Chromatography Type of Stationary Phase Adsorption Chromatography Solid, Underivatized Support Partition Chromatography Liquid-coated support or derivatized supportIon-Exchange Chromatography Support Containing Fixed Charges Exclusion Chromatography Porous, Inert Support Affinity Chromatography Support with Immobilized Biological Ligand
Prepared by:Algarne, Den MarionArtates, Michael JosanHosillos, Ianniel EddynLipardo, Fredrik ThomasTapiculin, JamaicaBMLS3A