High Performance High Performance Liquid ChromatographyLiquid Chromatography

Chem. 331Chem. 331

IntroductionIntroduction HPLC is a form of liquid chromatography used to HPLC is a form of liquid chromatography used to

separate compounds that are dissolved in solution. separate compounds that are dissolved in solution. HPLC instruments consist of a reservoir of mobile phase, HPLC instruments consist of a reservoir of mobile phase, a pump, an injector, a separation column, and a a pump, an injector, a separation column, and a detector.detector.

Compounds are separated by injecting a sample mixture Compounds are separated by injecting a sample mixture onto the column. The different component in the mixture onto the column. The different component in the mixture pass through the column at differentiates due to pass through the column at differentiates due to differences in their partition behavior between the mobile differences in their partition behavior between the mobile phase and the stationary phase. The mobile phase must phase and the stationary phase. The mobile phase must be degassed to eliminate the formation of air bubbles. be degassed to eliminate the formation of air bubbles.

HPLC systemHPLC system

FOUR TYPES OF LIQUID FOUR TYPES OF LIQUID CHROMATOGRAPHYCHROMATOGRAPHY

Partition chromatographyPartition chromatography

Adsorption, or liquid-solid Adsorption, or liquid-solid

chromatographychromatography

Ion exchange chromatographyIon exchange chromatography

Size exclusion, or gel, chromatographySize exclusion, or gel, chromatography

COMPOSITION OF A LIQUID COMPOSITION OF A LIQUID CHROMATOGRAPH SYSTEMCHROMATOGRAPH SYSTEM

SolventSolvent Solvent Delivery System (Pump)Solvent Delivery System (Pump) InjectorInjector SampleSample ColumnColumn Detectors (Diode Array)Detectors (Diode Array) Waste CollectorWaste Collector Recorder (Data Collection)Recorder (Data Collection)

Picture of HPLC instrumentPicture of HPLC instrument

Uses of HPLC Uses of HPLC This technique is used for chemistry and biochemistry This technique is used for chemistry and biochemistry

research analyzing complex mixtures, purifying chemical research analyzing complex mixtures, purifying chemical compounds, developing processes for synthesizing chemical compounds, developing processes for synthesizing chemical compounds, isolating natural products, or predicting physical compounds, isolating natural products, or predicting physical properties. It is also used in quality control to ensure the purity properties. It is also used in quality control to ensure the purity of raw materials, to control and improve process yields, to of raw materials, to control and improve process yields, to quantify assays of final products, or to evaluate product quantify assays of final products, or to evaluate product stability and monitor degradation. stability and monitor degradation.

In addition, it is used for analyzing air and water pollutants, for In addition, it is used for analyzing air and water pollutants, for monitoring materials that may jeopardize occupational safety monitoring materials that may jeopardize occupational safety or health, and for monitoring pesticide levels in the or health, and for monitoring pesticide levels in the environment. Federal and state regulatory agencies use HPLC environment. Federal and state regulatory agencies use HPLC to survey food and drug products, for identifying confiscated to survey food and drug products, for identifying confiscated narcotics or to check for adherence to label claims.narcotics or to check for adherence to label claims.

HPLC Chromatograph injectorsHPLC Chromatograph injectors The function of the injector is to place the sample into The function of the injector is to place the sample into

the high-pressure flow in as narrow volume as possible the high-pressure flow in as narrow volume as possible so that the sample enters the column as a so that the sample enters the column as a homogeneous, low-volume plug. To minimize spreading homogeneous, low-volume plug. To minimize spreading of the injected volume during transport to the column, the of the injected volume during transport to the column, the shortest possible length of tubing should be used from shortest possible length of tubing should be used from the injector to the column. the injector to the column.

When an injection is started, an air actuator rotates the When an injection is started, an air actuator rotates the valve: solvent goes directly to the column; and the valve: solvent goes directly to the column; and the injector needle is connected to the syringe. The air injector needle is connected to the syringe. The air pressure lifts the needle and the vial is moved into pressure lifts the needle and the vial is moved into position beneath the needle. Then, the needle is lowered position beneath the needle. Then, the needle is lowered to the vial. to the vial.

HPLC columnsHPLC columns The column is one of the The column is one of the

most important components most important components of the HPLC chromatograph of the HPLC chromatograph because the separation of because the separation of the sample components is the sample components is achieved when those achieved when those components pass through components pass through the column. The High the column. The High performance liquid performance liquid chromatography apparatus chromatography apparatus is made out of stainless is made out of stainless steel tubes with a diameter steel tubes with a diameter of 3 to 5mm and a length of 3 to 5mm and a length ranging from 10 to 30cm. ranging from 10 to 30cm.

Normally, columns are filled Normally, columns are filled with silica gel because its with silica gel because its particle shape, surface particle shape, surface properties, and pore structure properties, and pore structure help to get a good separation. help to get a good separation. Silica is wetted by nearly every Silica is wetted by nearly every potential mobile phase, is inert potential mobile phase, is inert to most compounds and has a to most compounds and has a high surface activity which can high surface activity which can be modified easily with water be modified easily with water and other agents. Silica can be and other agents. Silica can be used to separate a wide variety used to separate a wide variety of chemical compounds, and of chemical compounds, and its chromatographic behavior is its chromatographic behavior is generally predictable and generally predictable and reproducible. reproducible.

Picture of an HPLC columnPicture of an HPLC column

WHAT AFFECTS SYSTEMWHAT AFFECTS SYSTEM Column ParametersColumn Parameters

Column MaterialColumn Material DeactivationDeactivation Stationary PhaseStationary Phase Coating MaterialCoating Material

Instrument ParametersInstrument Parameters

TemperatureTemperature FlowFlow SignalSignal Sample SensitivitySample Sensitivity DetectorDetector

WHAT AFFECTS SYSTEMWHAT AFFECTS SYSTEM

Sample ParametersSample Parameters

ConcentrationConcentration MatrixMatrix Solvent EffectSolvent Effect Sample EffectSample Effect

Several column typesSeveral column types(can be classified as ) (can be classified as )

Normal phaseNormal phase

Reverse phaseReverse phase

Size exclusionSize exclusion

Ion exchangeIon exchange

Normal phaseNormal phase

In this column type, the retention is In this column type, the retention is governed by the interaction of the polar governed by the interaction of the polar parts of the stationary phase and solute. parts of the stationary phase and solute. For retention to occur in normal phase, the For retention to occur in normal phase, the packing must be more polar than the packing must be more polar than the mobile phase with respect to the sample mobile phase with respect to the sample

Reverse phaseReverse phase In this column the packing material is relatively In this column the packing material is relatively

nonpolar and the solvent is polar with respect to nonpolar and the solvent is polar with respect to the sample. Retention is the result of the the sample. Retention is the result of the interaction of the nonpolar components of the interaction of the nonpolar components of the solutes and the nonpolar stationary phase. solutes and the nonpolar stationary phase. Typical stationary phases are nonpolar Typical stationary phases are nonpolar hydrocarbons, waxy liquids, or bonded hydrocarbons, waxy liquids, or bonded hydrocarbons (such as C18, C8, etc.) and the hydrocarbons (such as C18, C8, etc.) and the solvents are polar aqueous-organic mixtures solvents are polar aqueous-organic mixtures such as methanol-water or acetonitrile-water.such as methanol-water or acetonitrile-water.

Size exclusionSize exclusion In size exclusion the HPLC column is In size exclusion the HPLC column is

consisted of substances which have consisted of substances which have controlled pore sizes and is able to be controlled pore sizes and is able to be filtered in an ordinarily phase according to filtered in an ordinarily phase according to its molecular size. Small molecules its molecular size. Small molecules penetrate into the pores within the packing penetrate into the pores within the packing while larger molecules only partially while larger molecules only partially penetrate the pores. The large molecules penetrate the pores. The large molecules elute before the smaller molecules.elute before the smaller molecules.

Ion exchangeIon exchange In this column type the sample In this column type the sample

components are separated based upon components are separated based upon attractive ionic forces between molecules attractive ionic forces between molecules carrying charged groups of opposite carrying charged groups of opposite charge to those charges on the stationary charge to those charges on the stationary phase. Separations are made between a phase. Separations are made between a polar mobile liquid, usually water polar mobile liquid, usually water containing salts or small amounts of containing salts or small amounts of alcohols, and a stationary phase alcohols, and a stationary phase containing either acidic or basic fixed sites.containing either acidic or basic fixed sites.

Selectivity FactorSelectivity Factor

K’ values tell us where bands elute relative K’ values tell us where bands elute relative to the void volume. These values are to the void volume. These values are unaffected by such variables as flow rate unaffected by such variables as flow rate and column dimensions. The value tell us and column dimensions. The value tell us where two peaks elute relative to each where two peaks elute relative to each other. This is referred to as the selectivity other. This is referred to as the selectivity factor or separation factor (now and then factor or separation factor (now and then as the chemistry factor). as the chemistry factor).

Types of Liquid Column Types of Liquid Column ChromatographyChromatography

(LCC)(LCC) LLC (Liquid Liquid)LLC (Liquid Liquid)

LSC LSC (Liquid Solid - (Liquid Solid - adsorption) adsorption)

SEC (SEC (Size Exclusion)Size Exclusion)

GLC GSCGLC GSC

SFC (Supercritical SFC (Supercritical Fluid)Fluid)

Types of DetectorsTypes of Detectors Absorbance (UV Absorbance (UV

with Filters, UV with with Filters, UV with Monochromators)Monochromators)

IR AbsorbanceIR Absorbance

FluorescenceFluorescence

Refractive-Index Refractive-Index

Evaporative Light Evaporative Light Scattering Detector Scattering Detector (ELSD)(ELSD)

ElectrochemicalElectrochemical

Mass-Mass-SpectrometricSpectrometric

Photo-Diode ArrayPhoto-Diode Array

EVALUATION PARAMETERSEVALUATION PARAMETERS EFFICIENCYEFFICIENCY RESOLUTIONRESOLUTION INERTNESSINERTNESS RETENTION INDEXRETENTION INDEX COLUMN BLEEDCOLUMN BLEED CAPACITY FACTORCAPACITY FACTOR

ReferencesReferences http://192.215.107.101/ebn/942/tech/techfocus/1071main.htmlhttp://192.215.107.101/ebn/942/tech/techfocus/1071main.html http://www.chem.usu.edu/~sbialk/Classes/565/opamps/http://www.chem.usu.edu/~sbialk/Classes/565/opamps/

opamps.htmlopamps.html Skoog, Holler, and Neiman. Skoog, Holler, and Neiman. Principles of Instrumental AnalysisPrinciples of Instrumental Analysis. .

5th ed. Orlando: Harcourt Brace & Co., 1998. 5th ed. Orlando: Harcourt Brace & Co., 1998. http://weather.nmsu.edu http://weather.nmsu.edu http://elchem.kaist.ac.kr/vt/chem-ed/sep/lc/hplc.htmhttp://elchem.kaist.ac.kr/vt/chem-ed/sep/lc/hplc.htm http://www.chemistry.nmsu.edu/Instrumentation/Lqd_Chroma.htmlhttp://www.chemistry.nmsu.edu/Instrumentation/Lqd_Chroma.html http://weather.nmsu.edu/Teaching_Material/SOIL698/http://weather.nmsu.edu/Teaching_Material/SOIL698/

Student_Material/HPLCHP1090/HPLCINJ.HTMStudent_Material/HPLCHP1090/HPLCINJ.HTM http://test-equipment.globalspec.com/LearnMore/http://test-equipment.globalspec.com/LearnMore/

Labware_Scientific_Instruments/Analytical_Instruments/Labware_Scientific_Instruments/Analytical_Instruments/Chromatographs/HPLC_ColumnsChromatographs/HPLC_Columns

http://www.chemistry.adelaide.edu.au/external/soc-rel/content/lc-http://www.chemistry.adelaide.edu.au/external/soc-rel/content/lc-col.htmcol.htm