Gas Chromatography.

Gas Chromatography

Gas Chromatography an analytical separations technique useful for separating volatile organic compounds consists of : Flowing mobile phase (inert gas - Ar, Ne, N) Injection port ( rubber septum - syringe injects sample) kept at a higher temperature than the boiling point

Principles Separation due to differences in partitioning behavior
selective retardation

Key Information organic compounds separated due to differences in their participating behavior between the mobile gas phase and the stationary phase in the column in contrast to other types of chromatography, the mobile phase does not interact with molecules of the analyte; its only function is to transport the analyte through the column

Gas Chromatography Separation column containing stationary phase
since partitioning behavior independent of temperature - kept in thermostat - controlled oven Detector

Schematic of a gas Chromatograph

The Beginning concept of GC announced in 1941 by Martin and Synge (also did liquid partition chromatography) 10+ years later GC used experimentally 1955, first commercial apparatus for GC appeared on the market

Today estimate : 200, 000 gas chromatographs are currently used through out the world. 30+ instrument manufactures 130 different models cost 1,500 to 40,000 dollars improvements: computers- automatic control open tubular columns-separate a multitude of analytes in relatively short times

Uses of Gas Chromatography
Determination of volatile compounds (gases & liquids) Determination of partition coefficients and absorption isotherms Isolating pure components from complex mixtures

Instrumentation

Instrumentation flowing mobile phase injection port separation column
detector

GC detectors another powerpoint

Liquid Chromatography much slower diffusion in liquid as compared to gas

Liquid liquid extraction repeated extraction is basis for LC

Retardation of solutes in liquid onto a solid phase

Elution chromatography
Increasing polarity of pure solvents hexane ether acetone methanol water acetic acid Solvents mixed %hexane and % methanol miscible can be mixed continuously (solvent programming)

Types of Liquid Chromatography
Liquid-solid: adsorption on solid which is generally polar (silica gel, alumina, magnesium silicates) or reverse phase (cellulose, poly amides) Ion exchange: specific interactions with ionic species (change relative strengths of acid or base)

Types of Liquid Chromatography
Liquid-liquid: partition between 2 bulk phases (one immobilized) is highly selective Liquid exclusion: molecular sieve separates molecules on basis of ability to diffuse into immobile support

Retardation based on size of molecule as it diffuses into porous solid

High Performance Liquid Chromatography
Once called High Pressure Liquid Chromatography

Outline What is HPLC? Types of HPLC An Overview
Partition Chromatography Adsorption Chromatography Ion Chromatography Size-Exclusion Chromatography

What is HPLC? The most widely used analytical separations technique
Utilizes a liquid mobile phase to separate components of mixture uses high pressure to push solvent through the column Popularity: sensitivity ready adaptability to accurate quantitative determination suitability for separating nonvolatile species or thermally fragile ones 1. HPLC like classical chromatography uses liquid as the mobile phase to separate components of a mixture 2. HPLC is the most widely used of all of the analytical separation techniques, with annual sales of HPLC equipment approaching the billion dollar mark 3. The reasons for the popularity of this method are:

HPLC is…. Popularity: widespread applicability to substances that are of prime interest to industry, to many fields of science, and to the public Ideally suited for separation and identification of amino acids, proteins, nucleic acids, hydrocarbons, carbohydrates, pharmaceuticals, pesticides, pigments, antibiotics, steroids, and a variety of other inorganic substances HPLC can be widely applied to substances that are of prime interest to industry, to many fields of science, and to the public

History lesson Early LC carried out in glass columns
diameters: 1-5 cm lengths: cm Size of solid stationary phase diameters: m Flow rates still low! Separation times long! Eureka! Decrease particle size of packing causes increase in column efficiency! diameters 3-10 m This technology required sophisticated instruments new method called HPLC -Early LC carried out in glass columns -to assure reasonable flow rates, diameters… -BUT were still low (a few tenths of a mL/min) separation times long, taking several hours -attempts to speed up the classic procedure by application of vacuum or by pumping were not effective, because increases in flow rates acted to increase plate heights beyond the minimum -early on, scientists realized that major increases in column efficiency could be brought about by decreasing the particle size of packings -1960s particle sizes 3-10 m----required sophisticated instruments

Advantages to HPLC Higher resolution and speed of analysis
HPLC columns can be reused without repacking or regeneration Greater reproducibility due to close control of the parameters affecting the efficiency of separation Easy automation of instrument operation and data analysis Adaptability to large-scale, preparative procedures

Advantages to HPLC Advantages of HPLC are result of 2 major advances:
stationary supports with very small particle sizes and large surface areas appliance of high pressure to solvent flow The increased resolution achieved in HPLC compared to classical column chromatograph is the result of adsorbents of very small paraticle sizes (less than 20 m) and large surface areas BUT the smaller the particle size, the lower the flow rat In HPLC, increased flow rates are obtained by applying a pressure differential across the column. A combination of high pressure and adsorbents of small particle size leads to high resolving power and short analysis times characteristic of HPLC

Liquid chromatography
Instrumentation Mobile Phase Reservoir Pumping Systems Sample Injection Systems Liquid-Chromatographic Columns Detectors

Schematic of liquid chromatograph

LC column LC injector

Types of HPLC Liquid-solid (adsorption) chromatography
Liquid-liquid (partition) chromatography Ion-exchange chromatography Size exclusion chromatography

Partition Chromatography
Most widely used Bonded-phase Chromatography Silica Stationary Phase: OH OH OH OH O O O Si Si Si Si Siloxanes: O CH3 Si O Si R R= C8, C18 O CH3

Partition Chromatography II
Reverse Phase Chromatography Nonpolar Stationary Phase Polar Mobile Phase Normal Phase Chromatography Polar Stationary Phase Nonpolar Mobile Phase Column Selection Mobile-Phase Selection

Partition Chromatography III
Research Applications Parathion in Insecticides: O CH3CH2O P O NO2 CH3CH2O Cocaine in Fruit Flies: A Study of Neurotransmission by Prof. Jay Hirsh, UVa

Adsorption Chromatography
Classic Solvent Selection Non-polar Isomeric Mixtures Advantages/ Disadvantages Applications

What is Ion Chromatography?
Modern methods of separating and determining ions based on ion-exchange resins Mid 1970s Anion or cation mixtures readily resolved on HPLC column Applied to a variety of organic & biochemical systems including drugs, their metabolites, serums, food preservatives, vitamin mixtures, sugars, pharmaceutical preparations Ion chromatography was first developed in the mid-1970s, when it was shown that anion or cation mixture can be readily resolved on HPLC columns packed with anion-exchange or cation-exchange resins.

The Mobile Phases are... Aqueous solutions
containing methanol, water-miscible organic solvents also contain ionic species, in the form of a buffer solvent strength & selectivity are determined by kind and concentration of added ingredients ions in this phase compete with analyte ions for the active site in the packing

Properties of the Mobile Phase
Must dissolve the sample have a strong solvent strength leads to reasonable retention times interact with solutes in such a way as to lead to selectivity

Ion-Exchange Packings
Types of packings pellicular bead packing large (30-40 µm) nonporous, spherical, glass, polymer bead coated with synthetic ion-exchange resin sample capacity of these particles is less coating porous microparticles of silica with a thin film of the exchanger faster diffusion leads to enhanced efficiency

Ion-Exchange Equilibria
Exchange equilibria between ions in solution and ions on the surface of an insoluble, high molecular-weight solid Cation exchange resins sulfonic acid group, carboxylic acid group Anion exchange resins quaternary amine group, primary amine group CM Cellulose Cation Exchanger DEAE Cellulose Anion Exchanger

Eluent Suppressor Technique
Made possible the conductometric detection of eluted ions. Introduction of a eluent suppressor column immediately following the ion-exchange column. Suppressor column packed with a second ion-exchange resin Cation analysis Anion analysis Development of HPLC began in the late 1960s, but the application was delayed by the lack of a sensitive general method of detecting eluted ionic species. In 1975, the development of an eluent suppressor technique made possible the conductometric detection of eluted ions. Ions in solution can be detected by measuring the conductivity of the solution. In ion chromatography, the mobile phase contains ions that create a background conductivity, making it difficult to measure the conductivity due only to the analyte ions as they exit the column. This problem can be greatly reduced by selectively removing the mobile phase ions after the analytical column and before the detector. This is done by converting the mobile phase ions to a neutral form or removing them with an eluent suppressor, which consists of an ion-exchange column or membrane.

Size Exclusion Chromatography(SEC)
Gel permeation(GPC), gel filtration(GFC) chromatography Technique applicable to separation of high-molecular weight species Rapid determination of the molecular weight or molecular-weight distribution of larger polymers or natural products Solute and solvent molecules can diffuse into pores -- trapped and removed from the flow of the mobile phase Size exclusion chromatography allows molecules to be separated by their ability to permeate a sieve-like structured stationary phase that is commonly made up of a cross-linked polymeric material or gel. gel w/ exclusion limit of several thousand can cleanly separate proteins from a.a & low molecular weight peptides. No chemical or physical interaction between analytes and the stationary phase, and such interactions are avoided due to impaired column efficiencies determination of the molecular weight-- Here the elution volumes of the sample are compared with elution volumes for a series of standard compounds that possess the same chemical characteristics

SEC(continued) Specific pore sizes.average residence time in the pores depends on the effective size of the analyte molecules larger molecules smaller molecules intermediate size molecules

SEC Column Packing Small (~10 µm) silica or polymer particles containing a network of uniform pores Two types (diameters of 5 ~ 10 µm) Polymer beads silica-based particles

Advantages of Size Exclusion Chromatography
Short & well-defined separation times Narrow bands--> good sensitivity Freedom from sample loss, solutes do not interact with the stationary phase Absence of column deactivation brought about by interaction of solute with the packing

Disadvantages Only limited number of bands can be accommodated because the time scale of the chromatogram is short Inapplicability to samples of similar size, such as isomers. At least 10% difference in molecular weight is required for reasonable resolution

Instrumentation Instruments required: Mobile phase reservoir Pump
Injector Column Detector Data system

Schematic of liquid chromatograph

Mobile phase reservoir
Glass/stainless steel reservoir Removal of dissolved gases by degassers vacuum pumping system heating/stirring of solvents sparging vacuum filtration 1. The solvent chamber should have a capacity of at least 500 mL for analytical purposes 2. In order to avoid bubblesin the column and detector, the solvent must be degassed. Methods to remove unwanted gases: refluxing filtration through a vacuum filter ultrasonic vibration sparging: (purging with an inert gas) in which dissolved gases are swept out of soln. By fine bubbles of an inert gas with low solubility

Elution methods Isocratic elution Gradient elution
single solvent of constant composition Gradient elution 2 or more solvents of differing polarity used isocratic elution: single solvent of constant composition gradient elution: can enhance separation efficiency 2 solvent systems that differ significantly in polarity are employed the ratio of solvents is varied in a programmed way, sometimes continuously and sometimes in a series of steps. Modern HPLC equipment is often equipped with devices that introduce solvents from 2/more reservoirs into a mixing chamber at rates that vary continuously; the volume ratio of the solvents may then be altered linearly or exponentially with time

Pumping System I Provide a continuous constant flow of the solvent through the injector Requirements pressure outputs up to 6000 psi pulse-free output flow rates ranging from mL/min flow control and flow reproducibility of .5% or better corrosion-resistant components

Pumping System II Two types: Reciprocating pumps constant-pressure
constant-flow Reciprocating pumps motor-driven piston disadvantage: pulsed flow creates noise advantages: small internal volume ( L), high output pressures (up to 10,000 psi), ready adaptability to gradient elution, constant flow rates Although neither type of pump meets all these criteria, constant-volume pumps maintain a more accurate flow rate and a more precise analysis is obtained Reciprocating pumps -currently used in 90% of commercially available HPLC systems -usually consist of a small chamber in which the solvent is pumped by the back/forth motion of a motor-driven piston

Pumping System III Displacement pumps
syringe-like chambers activated by screw-driven mechanism powered by a stepper motor advantages: output is pulse free disadvantage: limited solvent capacity (~20 mL) and inconvenience when solvents need to be changed Flow control and programming system computer-controlled devices measure flow rate increase/decrease speed of pump motor Displacement pumps -usually consist of large, syringe-like chambers equipped with a plunger that is activated by a screw-driven mechanism powered by a stepper motor Flow control and programming systems -part of pumping system -many commercial instruments are equipped with computer-controlled devices for measuring the flow rate by determining the pressure drop across a restrictor located at the pump outlet. -Any difference in signal from a preset value is then used to increase/decrease the speed of the pump motor -Most instruments also have a means for varying the composition of the solvent either continuously or in a stepwise fashion

Sample Injection Systems
For injecting the solvent through the column Minimize possible flow disturbances Limiting factor in precision of liquid chromatographic measurement Volumes must be small L Sampling loops interchangeable loops (5-500 L at pressures up to psi)

LC column LC injector

Liquid Chromatographic Column
Smooth-bore stainless steel or heavy-walled glass tubing Hundreds of packed columns differing in size and packing are available from manufacturers ($200-$500) Add columns together to increase length

Liquid Chromatographic Columns II
Column thermostats maintaining column temperatures constant to a few tenths degree centigrade column heaters control column temperatures (from ambient to 150oC) columns fitted with water jackets fed from a constant temperature bath Guard Columns -introduced before analytical column to increase the life of the analytical column by removing particulate matter and contaminants from solvents -similar in composition to analytical column, except particle size large to minimize pressure drop Column Thermostats -often better chromatograms obtained by maintaining column temperatures constant to a few tenths degree centigrade. -temperature control...

Detector Mostly optical Equipped with a flow cell
Focus light beam at the center for maximum energy transmission Cell ensures that the separated bands do not widen

Some Properties of Detector
Adequate sensitivity Stability and reproducibility Wide linear dynamic range Short response time Minimum volume for reducing zone broadening Properties of Detector Adequate sensitivity. Usually, the higher the sensitivity, the better the detector. However, depending on the application, the sensitivity may not be needed to be extremely high. Stability and reproducibility. If you run HPLC on the same sample for several times, you want the results to be consistent; they should agree to each other. The data should be clear enough that it can be reproduced. Wide linear dynamic range. The concentration of sample used should be proportional to the results linearly. This simplifies quantitation. Short response time. You want good results in as little time as possible. Minimum volume for reducing zone broadening. The cell should has minimum volume so the separated bands would not mix together again.

More Properties of Detector
High reliability and ease of use Similarity in response toward all analytes Selective response toward one or more classes of analytes Non-destructive Properties of Detector (con’t) High reliability and ease of use. You don’t want the HPLC equipment to break in the middle of an analysis; you don’t want to spend all the time learning how to use the equipment because it is so complicated to use Similarity in response toward all analytes. This is a desirable property because you don’t want your detector to behave different toward different analytes. The lack of this property would make comparison between different sets of data a lot harder. Selective response toward one or more classes of analytes. Usually, the higher selectivity, the lower noise and drift level, which yields better results. Non-destructive. You may want to reuse the sample for more HPLC runs or other types of analysis.

Types of Detector Refractive index UV/Visible Fluorescence
Conductivity Evaporative light scattering Electrochemical

Refractive Index I Measure displacement of beam with respect to photosensitive surface of dectector

Refractive Index II Advantages universal respond to nearly all solutes
reliable unaffected by flow rate low sensitive to dirt and air bubbles in the flow cell RI Advantages RI detectors have the advantage of responding to nearly all solutes. In fact, RI index is the most universal detector for HPLC that we are going to discuss here. EI detectors are reliable, and they are unaffected by flow rate, because the measurement is purely obtained from the displacement of the beam. EI detectors are not sensitive to dirt and air bubbles which may be present in the sample.

Refractive Index III Disadvantages expensive
highly temperature sensitive moderate sensitivity cannot be used with gradient elution RI Disadvantages They are expensive They are highly temperature sensitive, because the change of temperature within a compound can change its refractive index value. Therefore, they must be maintained at a constant temperature to a few thousandths of a degree centigrade. They are moderate sensitive only, which is not as sensitive comparing to other types of detectors They cannot be used with gradient elution Gradient Elution Two (and sometimes more) solvent systems that differ significantly in polarity are used. After elution is begun, the ratio of the solvents is varied in a programmed way, either continuously or in steps. Volume ratio of the solvents can be altered linearly or exponentially with time. Advantage of GE: shorten time without sacrificing resolution

UV/Visible I Mercury lamp  = 254nm
 = 250, 313, 334 and 365nm with filters Photocell measures absorbance Modern UV detector has filter wheels for rapidly switching filters; used for repetitive and quantitative analysis UV/Visible Utilize the different absorbance of different compounds at a certain wavelength. A = log (Ao / A) Simplest UV absorption detectors are filter photometers with a mercury lamp as the source. 254nm, etc….. With the use of filters Deuterium or tungsten filament sources with interference filters can also be used to vary wavelengths. Restricted to solutes that absorb at one of these wavelengths. Several organic functional groups and some inorganic species can be analyzed using this method. Modern UV detectors has filter wheels for rapid switching…….from slide….. Filter changes are computer controlled.

UV/Visible II

UV/Visible III Advantages high sensitivity
small sample volume required linearity over wide concentration ranges can be used with gradient elution UV Advantages Absorbance provides high sensitivity For absorbance testing, only a small sample volume is required This analysis behaves lineraly over a wide concentration ranges, which is one of the desirable properties of an ideal detector It can be used with gradient elution to shorten run-time!

UV/Visible IV Disadvantage
does not work with compounds that do not absorb light at this wavelength region UV Disadvantage There are some compounds that do not absorb light at this wavelength. Therefore, if such compounds are encountered, this analytical method is useless. Infrared Absorbance Detector Low transparency of many useful solvents. i.e. the broad infrared absorption bands for water and alcohol largely prelude the use of IR detector in many applications

Fluorescence I For compounds having natural fluorescing capability
Fluorescence observed by photoelectric detector Mercury or Xenon source with grating monochromator to isolate fluorescent radiation

Fluorescence II Advantages Disadvantage extremely high sensitivity
high selectivity Disadvantage may not yield linear response over wide range of concentrations Fluorescence Advantages Extremely high sensitivity….better than UV/Visible in orders of magnitude Highly selective, only works with compounds that have fluorescence capabilities. Fluorescence Disadvantage Over a wide range of concentration, it may not yield linear response, which is one of the undesirable properties of an ideal detector.

Conductivity Measure conductivity of column effluent
Sample indicated by change in conductivity Best in ion-exchange chromatography Cell instability

Evaporative Light Scattering I
Nebulizer converts eluent into mist Evaporation of mobile phase leads to formation of fine analyte particles Particles passed through laser beam; scattered radiation detected at right angles by silicon photodiode Similar response for all nonvolatile solutes Good sensitivity Evaporative Light Scattering ELSD Column effluent is passed into a nebulizer where it is converted into a mist by a flow of nitrogen or air Fine droplets are carried through a controlled temperature drift tube, where evaporation of mobile phase occurs leading to formation of fine particles of analyte. The cloud of analytes particles is passed through a laser beam. Scattered radiation is detected at right angles to the flow by a silicon photodiode. Advantages Response same for all nonvolatile solutes. A lot more sensitive than RI detector

Evaporative Light Scattering II

Electrochemical I Based on reduction or oxidation of the eluting compound at a suitable electrode and measurement of resulting current

Electrochemical II Advantages Disadvantages high sensitivity
ease of use Disadvantages mobile phase must be made conductive mobile phase must be purified from oxygen, metal contamination, halides Electrochemical Advantages high sensitivity simplicity convenience widespread applicability Potential for fulfilling a long-time need of HPLC as a sensitive general or universal detector. Disadvantages Mobile phase must be made conductive with the addition of a suitable salt. Mobile phase must be purified from oxygen, metal contamination, and halides in order to reduce background current, so noise and drift in the baseline is minimized.

Data System For better accuracy and precision Routine analysis
pre-programmed computing integrator Data station/computer needed for higher control levels add automation options complex data becomes more feasible software safeguard prevents misuse of data system Data system To minimize the work of a human operator, data system is used; it has an advantage of increasing accuracy and precision that can be caused by human errors. For routine analysis, the data system is just a pre-programmed computing integrator. For high control levels, a data station or computer is needed. The use of computer allows the analyst to add automation options by programming it. It makes complex data more feasible with the use of a computer. With software safeguard features on a computer, the opportunity of misusing the system is minimized.

Electrophoresis…charged species migrate in electric field Separation based on charge or mobility

Capillary electrophoresis higher voltages can be used as the heat can be dissipated

Capillary electrophoresis

The End

Gas Chromatography.

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Gas Chromatography.

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