Adamson University

Chromatographic Methods of Analysis

Analytical Chemistry for ChE

INTRODUCTION TO CHROMATOGRAPHY Chromatography Derived from the Greek words chromos (color) and graphein (to write). First developed by the botanist M. Tswett in 1903 Used to separate components of a given mixture Separation is based upon the rates by which different components of a given mixture are carried through a stationary phase by a mobile phase. Used to separate, identify, and quantify components of a mixture.

Components of Chromatography Stationary phase none moving component (solid or liquid bonded to a solid support) Mobile phase moving component (liquid or gas)

Types of Chromatography Based on the configuration of the Stationary phase Planar chromatography o Stationary phase is supported on a flat plate or in pores of paper o Mobile phase moves by capillary action or gravity Column chromatography o Stationary phase packed inside a narrow tube o Mobile phase is forced through the tube under pressure or gravity.

Based on the Phase of the Mobile Phase Liquid Chromatography Gas Chromatography Supercritical Fluid Chromatography

Based on the mechanism of interaction of the solute with the stationary phase Adsorption chromatography A solid stationary phase and a liquid or gaseous mobile phase are used. Solute is adsorbed on the surface of the solid particles. The more strongly a solute is adsorbed, the slower it travels through the stationary phase. Partition chromatography A liquid stationary phase bonded to a solid support matrix. Solute equilibrates between the stationary liquid and the mobile phase, which is usually a flowing gas such as in gas chromatography.
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J. Garcia 2011

Adamson University

Chromatographic Methods of Analysis

Analytical Chemistry for ChE

Ion-exchange chromatography Anions (such as SO3-) and cations (such as N(CH3)3+) are covalently attached to the stationary phase. The mobile phase is a liquid. Molecular exclusion chromatography Also called size exclusion, gel filtration, or gel permeation chromatography, this technique separates molecules by size, with the larger solutes passing through most quickly. In the ideal case of molecular exclusion, there is no attractive interaction between the stationary phase and the solute. Rather, the liquid or gaseous mobile phase passes through a porous gel. The pores are small enough to exclude large solute molecules but not small ones. Large molecules stream past without entering the pores. Small molecules take longer time to pass the porous gel since they fit inside the pores, therefore taking a much longer route to elute. Affinity chromatography The most selective type of chromatography that employs specific interactions between one kind of solute molecules and a second molecule that is covalently immobilized to the stationary phase.

Separation Process Elution solutes are washed through a stationary phase by movement of a mobile phase Eluent Solvent used to carry components of mixture through the stationary phase Components of the mixture distribute (or partition) themselves between the 2 phases Addition of solvent carry solute molecules down the column in a continuous series of transfers between phases Rate of solute migration depends on the fraction of time the solute spends in the mobile phase Components that are retained strongly by the stationary phase will take more time to elute (and vice versa)

Elution process can be monitored by: o Visual Inspection (colored substances) o Instrumental methods (Use of detectors)

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Adamson University

Chromatographic Methods of Analysis

Analytical Chemistry for ChE

The Chromatogram A graph of the detection signal with time Used to interpret chromatographic data

Migration Rate of Solutes

The partition coefficient K relates the ration of the concentration of the solute in the stationary phase (CS) and in the mobile phase (CM). Different partition coefficients of solutes in the mixture leads to different rates of elution leads to separation.

Retention Time

Retention time (tR) The time it takes after the sample injection for the analyte peak to reach the detector Dead time (tM) The time it takes for unretained species to reach the detector. Average rate of motion of mobile phase ~ rate of migration of unretained species

Rate of solute Migration

Capacity factor (k) is an experimental parameter used to describe migration of solutes in a column. The lower the k, the faster the elution process is.
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Adamson University

Chromatographic Methods of Analysis

Analytical Chemistry for ChE

If k < 1 elution occurs very rapidly If k is large (~ 20-30), elution occurs very slowly Ideal k value is between 1 & 5 In gas chromatography, k can be varied by changing the temperature and column packing In liquid chromatography, k can be varied by changing the composition of the mobile phase and stationary phase.

Relative Migration Rates: the Selectivity Factor

( ( ) )

The selectivity factor of a column for two species A and B provides a measure of how well the column separates two solutes from a mixture. It can be used to compute for the resolving power of a column.

Parameters Evaluation for Chromatographic Data Band Broadening occurs when a band moves down a column when more time is allowed for spreading to occur Methods for Reducing Band Broadening Use smaller packing diameter Use smaller column diameter or narrower column Use lower temperature Use thinner layer of stationary phase Optimize flow rate

Objectives of Optimizing Column Performance Reduce band broadening Alter relative migration rates of components

Evaluating the Efficiency of Separation

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Adamson University

Chromatographic Methods of Analysis

Analytical Chemistry for ChE

RS provides a quantitative measure of columns ability to separate two analytes. RS > 1.5: complete resolution with only 0.3 % overlap. RS < 0.75: incomplete separation. RS = 1.0: around 4% overlap. RS can be improved by increasing the column length at the expense of analysis time.

GAS CHROMATOGRAPHY Gas chromatography an analytical separation technique useful for separating volatile organic compounds. An inert gas is used as the mobile phase while the stationary phase consists of a packed solid column or a liquid phase bonded on a solid support. 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 Separation due to differences in partitioning behavior selective retardation

Instrumentation

A. Carrier gas Should be inert (commonly used: He, Ar, N2) Should be compatible with the detector Little interaction between sample molecules and mobile phase; simply serves as carrier of sample Flow rate controlled by pressure regulators B. Column Packed column glass or stainless steel tubes (i.d. ~ 1.5 mm) with length of about 1 3 meters, coiled to fit inside the oven
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Adamson University

Chromatographic Methods of Analysis

Analytical Chemistry for ChE

Open tubular (or capillary) columns Made form fused silica (i.d. 30 500 m). Length 3 10 m. It has greater separation efficiency than packed columns. C. Column oven GC column is placed inside a thermostated oven for control of column temperature Optimum temperature is determined by boiling point of sample and the required resolution Temperature equal to or slightly above average boiling point of the sample will give a reasonable elution time. Types of temperature control i. Isothermal elution constant temperature ii. Temperature programmed elution column temperature is gradually increased as separation proceeds. Used when two or more components are unresolved at a fixed temperature, and/or have long elution times, or if the sample has components with a wide range of molecular weight To improve resolution for all components. D. Sample Injection System The sample is injected as a discreet plug of vapor Normally uses a microsyringe to inject the sample (0.1 1.0 L) through a selfsealing silicone rubber septum into an injection port at the head column Injection port temperature = at least 50C above boiling point of lowest boiling component Ensures that the sample is vaporized onto the column. E. Types of GC Detectors Flame ionization Detector (FID) i. Effluent from column mixed with H2 + air, then ignited to produced ionize organic compounds ii. Applications: most organic samples; DOES NOT respond to noncombustible gases such as H2O, CO2, SO2 and NOx iii. Features: sensitivity ~ 1x10-12 g/s; large linear response and low noise, possesses selectivity, sample destructive Electron capture detector (ECD) i. Ionizes organic compounds to produce positive ions and radicals that capture electrons from the detector. Decrease in current (amount of electrons) from the detector signals presence of organic analyte. ii. Application: selective towards molecules which contains electronegative functional groups (halogens, peroxides, quinines, nitro geoups) but are insensitive towards amines, alcohols, and hydrocarbons. Commonly used for the analysis of halogenated pesticides and pesticide residues.
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Adamson University

Chromatographic Methods of Analysis

Analytical Chemistry for ChE

iii. Highly sensitive and non destructive Thermal Conductivity Detector (TCD) or Katharometer i. The conductivity (or resistance) of a heated filament changes when an analyte passes through the detector. ii. General type of indicator. Responsive towards organic and inorganic species iii. Features: relatively low sensitivity (1x10-3 g/mL); non-destructive, not suitable for capillary columns Thermionic Ionization Detector (TID) / Flame Thermionic Detector (FTD) / Nitrogen-Phosphorus Detector (NPD) i. Converts analyte into plasma which produces a large number of analyte ions and therefore large ion currents in the presence of compounds containing nitrogen and phosphorus ii. Features: approximately 500 times more sensitive for species containing phosphorus and 50 times more sensitive for nitrogen containing species than the FID. iii. Sensitive towards P and N containing organic compounds; determination of pesticides and drugs that contain P and N. Flame photometric detector (FPD) i. Selective for compounds containing S and P ii. Application: analysis of air and water pollutants, pesticides, and coal hydrogenation products. Mass Spectrometer (MS) i. Provides information regarding the structure of complex organic compounds ii. Done by fragmenting organic compounds and measuring the mass/charge ratio of each fragments iii. Most sensitive; lowest LOD and LOQ

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY High Performance Liquid Chromatography (HPLC) Uses high pressure to force the mobile phase through closed columns containing packed fine particles of the stationary phase that gives high resolution. Separation of nonvolatile or thermally unstable compounds. Should have some degree of solubility with the mobile phase. Separation of mixtures is based on the degree by which the solute is distributed between the mobile phase and the stationary phase.
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J. Garcia 2011

Adamson University

Chromatographic Methods of Analysis

Analytical Chemistry for ChE

Types of HPLC analysis (See page 1-2: Based on the mechanism of interaction of the solute with the stationary phase)

Retardation of components in the stationary phase

Instrumentation

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Adamson University

Chromatographic Methods of Analysis

Analytical Chemistry for ChE

Solvent Reservoirs o Contains the mobile phase Degasser o Removes dissolved gases from the mobile phase can cause bubbles to form inside the column at high pressure operations Pumps o Uses high pressure to force the mobile phase into the column Isocratic Elution Uses only one mixture of MP Gradient Elution Changes the proportion of mixtures in the mobile phase over time Uses 2 3 pump heads Used to increase resolution Pre-column / Guard column o Protects the main column from being contaminated by unwanted components in the mobile phase Sample Injection Port o Allows user to introduce the sample into the chromatograph o Manual or Automatic samplers are available Column o Contains the stationary phase o Available in different packing material, packing particle diameter, column internal diameter, and column length. Detectors
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J. Garcia 2011

Adamson University

Chromatographic Methods of Analysis

Analytical Chemistry for ChE

o Spectrophotometric detectors (absorbance detectors) Relies on the absorption of light (Uv-visible range) of the components being separated Useful for most chromophore containing compounds o Fluorescence detectors Relies on the emission of light by the sample after exposure to a certain wavelength of light More sensitive by 2 or 3 digits compared to absorbance detectors o Refractive Index detector Measures change in refractive index of the eluent as the solute passes through the detector Cheapest detector Most useful when analyzing sugars and polymers Least sensitive; highest LOD o Conductimetric detectors Measures the change in the conductivity of the eluent as the solute passes through the detector Useful for charged analytes o Mass spectrometer Determines the structural identity of the analyte / solute Most sensitive; lowest LOD Important Notes on HPLC analysis The correct choice of mobile phase stationary phase pairs will determine the resolution of components in a mixture Types of HPLC method based on MP-SP pairs: o Reverse HPLC o Normal HPLC o Ion-exchange HPLC o Size-exclusion HPLC Mobile phase should be degassed and filtered with micrometer pore size filter membrane to remove any impurities that will clog up the column The column is the lifeline of the HPLC system. Handle it with care Choose the right method for your analyte o Polar analytes reverse HPLC o Non-polar analytes normal HPLC o Charged analytes / ions ion exchange HPLC o Polymers, proteins, macromolecules Size exclusion HPLC

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