about liquid chroatography

1. High Performance LiquidHigh Performance Liquid
ChromatographyChromatography
Chem. 331Chem. 331

2. IntroductionIntroduction
 HPLC is a form of liquid chromatography used toHPLC 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 aa pump, an injector, a separation column, and a
detector.detector.
 Compounds are separated by injecting a sample mixtureCompounds are separated by injecting a sample mixture
onto the column. The different component in the mixtureonto the column. The different component in the mixture
pass through the column at differentiates due topass through the column at differentiates due to
differences in their partition behavior between the mobiledifferences in their partition behavior between the mobile
phase and the stationary phase. The mobile phase mustphase 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.

3. HPLC systemHPLC system
HPLC systemHPLC system

4. FOUR TYPES OF LIQUIDFOUR TYPES OF LIQUID
CHROMATOGRAPHYCHROMATOGRAPHY
 Partition chromatographyPartition chromatography
 Adsorption, or liquid-solidAdsorption, or liquid-solid
 chromatographychromatography
 Ion exchange chromatographyIon exchange chromatography
 Size exclusion, or gel, chromatographySize exclusion, or gel, chromatography

5. COMPOSITION OF A LIQUIDCOMPOSITION 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)

6. Picture of HPLC instrumentPicture of HPLC instrument

7. Uses of HPLCUses of HPLC
 This technique is used for chemistry and biochemistryThis technique is used for chemistry and biochemistry
research analyzing complex mixtures, purifying chemicalresearch analyzing complex mixtures, purifying chemical
compounds, developing processes for synthesizing chemicalcompounds, developing processes for synthesizing chemical
compounds, isolating natural products, or predicting physicalcompounds, isolating natural products, or predicting physical
properties. It is also used in quality control to ensure the purityproperties. It is also used in quality control to ensure the purity
of raw materials, to control and improve process yields, toof raw materials, to control and improve process yields, to
quantify assays of final products, or to evaluate productquantify 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, forIn addition, it is used for analyzing air and water pollutants, for
monitoring materials that may jeopardize occupational safetymonitoring materials that may jeopardize occupational safety
or health, and for monitoring pesticide levels in theor health, and for monitoring pesticide levels in the
environment. Federal and state regulatory agencies use HPLCenvironment. Federal and state regulatory agencies use HPLC
to survey food and drug products, for identifying confiscatedto 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.

8. HPLC Chromatograph injectorsHPLC Chromatograph injectors
 The function of the injector is to place the sample intoThe function of the injector is to place the sample into
the high-pressure flow in as narrow volume as possiblethe high-pressure flow in as narrow volume as possible
so that the sample enters the column as aso that the sample enters the column as a
homogeneous, low-volume plug. To minimize spreadinghomogeneous, low-volume plug. To minimize spreading
of the injected volume during transport to the column, theof the injected volume during transport to the column, the
shortest possible length of tubing should be used fromshortest 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 theWhen an injection is started, an air actuator rotates the
valve: solvent goes directly to the column; and thevalve: solvent goes directly to the column; and the
injector needle is connected to the syringe. The airinjector needle is connected to the syringe. The air
pressure lifts the needle and the vial is moved intopressure lifts the needle and the vial is moved into
position beneath the needle. Then, the needle is loweredposition beneath the needle. Then, the needle is lowered
to the vial.to the vial.

9. HPLC columnsHPLC columns
 The column is one of theThe column is one of the
most important componentsmost important components
of the HPLC chromatographof the HPLC chromatograph
because the separation ofbecause the separation of
the sample components isthe sample components is
achieved when thoseachieved when those
components pass throughcomponents pass through
the column. The Highthe column. The High
performance liquidperformance liquid
chromatography apparatuschromatography apparatus
is made out of stainlessis made out of stainless
steel tubes with a diametersteel tubes with a diameter
of 3 to 5mm and a lengthof 3 to 5mm and a length
ranging from 10 to 30cm.ranging from 10 to 30cm.
 Normally, columns are filledNormally, columns are filled
with silica gel because itswith silica gel because its
particle shape, surfaceparticle shape, surface
properties, and pore structureproperties, and pore structure
help to get a goodhelp to get a good
separation. Silica is wetted byseparation. Silica is wetted by
nearly every potential mobilenearly every potential mobile
phase, is inert to mostphase, is inert to most
compounds and has a highcompounds and has a high
surface activity which can besurface activity which can be
modified easily with watermodified easily with water
and other agents. Silica canand other agents. Silica can
be used to separate a widebe used to separate a wide
variety of chemicalvariety of chemical
compounds, and itscompounds, and its
chromatographic behavior ischromatographic behavior is
generally predictable andgenerally predictable and
reproducible.reproducible.

10. Picture of an HPLC columnPicture of an HPLC column

11. 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

12. WHAT AFFECTS SYSTEMWHAT AFFECTS SYSTEM
Sample ParametersSample Parameters
 ConcentrationConcentration
 MatrixMatrix
 Solvent EffectSolvent Effect
 Sample EffectSample Effect

13. 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

14. Normal phaseNormal phase
 In this column type, the retention isIn this column type, the retention is
governed by the interaction of the polargoverned 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, theFor retention to occur in normal phase, the
packing must be more polar than thepacking must be more polar than the
mobile phase with respect to the samplemobile phase with respect to the sample

15. Reverse phaseReverse phase
 In this column the packing material is relativelyIn this column the packing material is relatively
nonpolar and the solvent is polar with respect tononpolar and the solvent is polar with respect to
the sample. Retention is the result of thethe sample. Retention is the result of the
interaction of the nonpolar components of theinteraction of the nonpolar components of the
solutes and the nonpolar stationary phase.solutes and the nonpolar stationary phase.
Typical stationary phases are nonpolarTypical stationary phases are nonpolar
hydrocarbons, waxy liquids, or bondedhydrocarbons, waxy liquids, or bonded
hydrocarbons (such as C18, C8, etc.) and thehydrocarbons (such as C18, C8, etc.) and the
solvents are polar aqueous-organic mixturessolvents are polar aqueous-organic mixtures
such as methanol-water or acetonitrile-water.such as methanol-water or acetonitrile-water.

16. Size exclusionSize exclusion
 In size exclusion the HPLC column isIn size exclusion the HPLC column is
consisted of substances which haveconsisted of substances which have
controlled pore sizes and is able to becontrolled pore sizes and is able to be
filtered in an ordinarily phase according tofiltered in an ordinarily phase according to
its molecular size. Small moleculesits molecular size. Small molecules
penetrate into the pores within the packingpenetrate into the pores within the packing
while larger molecules only partiallywhile larger molecules only partially
penetrate the pores. The large moleculespenetrate the pores. The large molecules
elute before the smaller molecules.elute before the smaller molecules.

17. Ion exchangeIon exchange
 In this column type the sampleIn this column type the sample
components are separated based uponcomponents are separated based upon
attractive ionic forces between moleculesattractive ionic forces between molecules
carrying charged groups of oppositecarrying charged groups of opposite
charge to those charges on the stationarycharge to those charges on the stationary
phase. Separations are made between aphase. Separations are made between a
polar mobile liquid, usually waterpolar mobile liquid, usually water
containing salts or small amounts ofcontaining salts or small amounts of
alcohols, and a stationary phasealcohols, and a stationary phase
containing either acidic or basic fixedcontaining either acidic or basic fixed
sites.sites.

18. Selectivity FactorSelectivity Factor
 K’ values tell us where bands elute relativeK’ values tell us where bands elute relative
to the void volume. These values areto the void volume. These values are
unaffected by such variables as flow rateunaffected by such variables as flow rate
and column dimensions. The value tell usand column dimensions. The value tell us
where two peaks elute relative to eachwhere two peaks elute relative to each
other. This is referred to as the selectivityother. This is referred to as the selectivity
factor or separation factor (now and thenfactor or separation factor (now and then
as the chemistry factor).as the chemistry factor).

19. Types of Liquid ColumnTypes of Liquid Column
ChromatographyChromatography
(LCC)(LCC)
 LLC (Liquid Liquid)LLC (Liquid Liquid)
 LSCLSC (Liquid Solid -(Liquid Solid -
adsorption)adsorption)
 SEC (SEC (Size Exclusion)Size Exclusion)
 GLC GSCGLC GSC
 SFC (SupercriticalSFC (Supercritical
Fluid)Fluid)

20. Types of DetectorsTypes of Detectors
 Absorbance (UVAbsorbance (UV
with Filters, UV withwith Filters, UV with
Monochromators)Monochromators)
 IR AbsorbanceIR Absorbance
 FluorescenceFluorescence
 Refractive-IndexRefractive-Index
 Evaporative LightEvaporative Light
Scattering DetectorScattering Detector
(ELSD)(ELSD)
 ElectrochemicalElectrochemical
 Mass-Mass-
SpectrometricSpectrometric
 Photo-Diode ArrayPhoto-Diode Array

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

22. 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/opamhttp://www.chem.usu.edu/~sbialk/Classes/565/opamps/opam
ps.htmlps.html
 Skoog, Holler, and Neiman.Skoog, Holler, and Neiman. Principles of InstrumentalPrinciples of Instrumental
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 http://weather.nmsu.edu/Teaching_Material/SOIL698/Studenthttp://weather.nmsu.edu/Teaching_Material/SOIL698/Student
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 http://www.chemistry.adelaide.edu.au/external/soc-http://www.chemistry.adelaide.edu.au/external/soc-
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