hplc-high pressure/performance liquid chromatography
DESCRIPTION
ALL Chromatographic of HPLC in DetailsTRANSCRIPT
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HPLCHPLCWaste
InjectionSystem
IntroductionIntroduction
High Pressure Liquid Chromatography: The name “HPLC” originally referred to the fact that high The name “HPLC” originally referred to the fact that high
pressure was needed to generate the flow required for liquid pressure was needed to generate the flow required for liquid chromatography in packed columns.chromatography in packed columns.
The early 1970’s saw a tremendous leap in technology. The early 1970’s saw a tremendous leap in technology. These new “HPLC” instruments could develop up to 6,000psi These new “HPLC” instruments could develop up to 6,000psi (400 bar) of pressure, and included improved detectors and (400 bar) of pressure, and included improved detectors and columns.columns.
HPLC really began to take hold in the mid to late 1970’s. With HPLC really began to take hold in the mid to late 1970’s. With continued advances in performance, the name was changed continued advances in performance, the name was changed to High Performance Liquid Chromatography (HPLC). High to High Performance Liquid Chromatography (HPLC). High Performance Liquid Chromatography (HPLC) is now one of Performance Liquid Chromatography (HPLC) is now one of the most powerful tools in analytical chemistry, with the ability the most powerful tools in analytical chemistry, with the ability to separate, identify and quantitate the compounds that are to separate, identify and quantitate the compounds that are present in any sample that can be dissolved in a liquid. present in any sample that can be dissolved in a liquid.
Basic Principle Of HPLCBasic Principle Of HPLC
HPLC System Provide
High sensitivity High Accuracy
Find out purity / impurity profile
For the non volatile compound / high Mol. Wt.
Basic Principle Of HPLCBasic Principle Of HPLC
HPLC
Pressure Performance
High Pressure Pump High Accuracy High Sensitivity
Copyright © 2008, Advance Analytical Research & Training Institute
HPLCHPLC
Principles Of SeparationPrinciples Of Separation
Adsorption Chromatography• Normal Phase• Reversed Phase• Ion exchange• Affinity• Hydrophobic Interaction Partition Chromatography• Liquid Liquid chromatography• Gas chromatography Size exclusion Chromatography• Gel filtration• Gel permeation• Gel chromatography
Principles Of SeparationPrinciples Of Separation
Principles Of SeparationPrinciples Of Separation
Adsorption Chromatography
• The principle of separation is adsorption. Separation of
components takes place because of the difference in affinity
of compounds towards stationary phase. This principle is
seen in normal phase as well as reversed phase mode,
where adsorption takes place.
Adsorption ChromatographyAdsorption Chromatography
Principles Of SeparationPrinciples Of Separation
Ion exchange chromatography
• The principle of separation is ion exchange, which is
reversible exchange of functional groups. In ion exchange
chromatography, an ion exchange resin is used to separate a
mixture of similar charged ions. For cations, a cation
exchange resin is used. For anions, an anion exchange resin
is used.
Principles Of SeparationPrinciples Of Separation
Principles Of SeparationPrinciples Of Separation
Principles Of SeparationPrinciples Of Separation
Affinity Chromatography
• Affinity chromatography uses the affinity of the sample with
specific stationary phases. This technique is mostly used in
the field of biotechnology, microbiology, biochemistry etc.
Principles Of SeparationPrinciples Of Separation
Size exclusion chromatography
• In this type of chromatography, a mixture of components with
different molecular sizes are separated by using gels. The gel used
acts as molecular sieve and hence a mixture of substances with
different molecular sizes are separated. Soft gels like dextran,
agarose or polyacrylamide are used. Semi rigid gels like
polystyrene, alkyl dextran in non aqueous medium are also used.
The mechanism of separation is by steric and diffusion effects.
Principles Of SeparationPrinciples Of Separation
Partition ChromatographyPartition Chromatography
InstrumentationInstrumentation
InstrumentationInstrumentation
InstrumentationInstrumentation
RESERVOIRRESERVOIR To hold solvent or Mobile Phase (Because it moves).To hold solvent or Mobile Phase (Because it moves).
PUMPPUMP
used to generate and control the flow at a specified flow rate, used to generate and control the flow at a specified flow rate,
typically in milliliters per minute. Sometimes termed as “Solvent typically in milliliters per minute. Sometimes termed as “Solvent
delivery system” or “Solvent Manager”.delivery system” or “Solvent Manager”.
INJECTORINJECTOR
To introduce (“inject”) the SAMPLE into the flowing mobile phase To introduce (“inject”) the SAMPLE into the flowing mobile phase
stream, which carries the sample into the HPLC COLUMN. stream, which carries the sample into the HPLC COLUMN.
Sometimes termed as “Sample Manager” or “Auto Sampler”.Sometimes termed as “Sample Manager” or “Auto Sampler”.
COLUMNSCOLUMNScontains the chromatographic packing material needed to create contains the chromatographic packing material needed to create
the separation. This is called as Stationary Phase.the separation. This is called as Stationary Phase.
DETECTORDETECTOR
needed to “see” the separated “compound bands” as they elute needed to “see” the separated “compound bands” as they elute
from the HPLC column. Mobile Phase exits the detector in to the from the HPLC column. Mobile Phase exits the detector in to the
waste.waste.
Mobile PhaseMobile Phase
A modern HPLC apparatus is equipped with one or more glass or stainless steel reservoirs, each of which contain 500 ml or more of solvent.
Mobile phase preparation and use:• Sample must be soluble in mobile phase• Solvent filtration.• Solvent degassing. Sample must be soluble in mobile phase. If it is not soluble
then sample may clot in the column and damage it. Different grades are available. AR/ LR/ GR/ HPLC/
GRADIENT/ SPECTROSCOPIC etc. Clean, high purity HPLC grade solvents and reagents should be used while preparing mobile phase.
Mobile PhaseMobile Phase
Solvent filtration:
• Solvents should be filtered through a 0.45 micron filter. This is
especially critical with salts and buffers. Also ensure that the
filter material is compatible with the solvent used. The benefit
of solvent filtering includes:
1. Improved pump performance (including seals, check valves
and plunger)
2. Increase in injector life
• After filtering, store the solvent in a covered reservoir to
prevent dust and debris from entering the solvent.
Solvent filtrationSolvent filtration
Solvent filtrationSolvent filtration
Sample filtrationSample filtration
Sample filtersSample filters
Sample filtrationSample filtration
Mobile PhaseMobile Phase
SonicationSonication
Mainly used for premixed mobile phase.
If the mobile phase reservoir is placed in an ultrasonic bath,
the sound waves remove small bubbles which can escape
more easily.
not recommended for on-line mixing systems
often used in conjunction with vacuum degassing.
Vacuum DegasVacuum Degas
Vacuum DegasVacuum Degas
He DegasHe Degas
A stream of helium bubbles will sweep dissolved air out of
liquids.
Helium sparging is very effective; it can reduce the dissolved
air.
This makes helium sparging especially suitable for use with
on-line mixing systems.
In-line DegasIn-line Degas
This is the most convenient approach to degassing.
There is in built facility for degassing.
This only degas the solvent, not filter.
Mobile PhaseMobile Phase
Improper solvent preparation leads to:• Out gassing in pump head• Air bubbles or particles trapped in the detector flow
cell• Mobile phase contamination• Damage to pump, check valves and seals.• Plugged in-line filters, frits, check valves or connecting
tubing.• Poor injection precision.• High system back pressure.• Flow related base line noise.• Shifting retention times.• Abnormal peak shapes.• In correct qualitative/ quantitative results.
High Pressure PumpHigh Pressure Pump
Pumps are used to pass mobile phase through the column at
high pressure and at controlled flow rate.
Ideal pump:
• Generation of pressures up to 6000 psi
• Flow rate ranging from 0.1 to 10 ml/min
• Flow control and flow reproducibility of ± 0.5%
• It should be composition resistant and give a pulse free
output.
• Mobile phase change should be easy.
High Pressure PumpHigh Pressure Pump
Pumps are categorized into
1. Single head reciprocating piston pump (Dual Piston Pump)
2. Syringe pump
3. Diaphragm pump
Depending upon elution technique, they are categorized into
1. ISOCRATIC ELUTION
2. GRADIENT ELUTION
Parts Of PumpParts Of Pump
Pressure gauge (Dampener)• To reduce pulsations Piston• For suction of mobile phase from solvent reservoir and its
withdrawal to column Check valves• Piston in co ordination with check valve maintain flow rate Purge valve• For purging of system. If there is any bubble in mobile phase
and it is passed into the system then there are chances of damage of piston, check valves, column etc. so purging is done in which some of the mobile phase is bypassed and then it is allowed to pass into the system.
PistonPiston
This is the picture of piston along with seal. Piston is made up of This is the picture of piston along with seal. Piston is made up of
stainless steel, borosilicate or Safire glass. Motor driven piston stainless steel, borosilicate or Safire glass. Motor driven piston
moves through this seal and gives accurate flow. moves through this seal and gives accurate flow.
PistonPiston
If mobile phase is not filtered/ contaminated, particle gets If mobile phase is not filtered/ contaminated, particle gets
trapped between seal and plunger resulting in scratches on to trapped between seal and plunger resulting in scratches on to
the surface of plunger and gives non reproducible flow rate.the surface of plunger and gives non reproducible flow rate.
Check valveCheck valve
This is the picture of check valve. Depending upon the design of This is the picture of check valve. Depending upon the design of
pump, there are two or more than two check valves are present. pump, there are two or more than two check valves are present.
In check valve, there is a ball and seat arrangement which is In check valve, there is a ball and seat arrangement which is
explained in next slide.explained in next slide.
Check valveCheck valve
Ball
Seat
Holder
Check valveCheck valve
Single Head Reciprocating Piston Single Head Reciprocating Piston PumpPump
Single head reciprocating Single head reciprocating
piston pumppiston pump
•The twin piston pumps with shortThe twin piston pumps with short
stroke are among the most stroke are among the most
commonlycommonly used pumps for HPLC.used pumps for HPLC.
• BBoth pump heads are switched inoth pump heads are switched in
series, whereby the piston in the firstseries, whereby the piston in the first
pump head delivers a specific pump head delivers a specific
volumevolume per strokeper stroke..
Single Head Reciprocating Piston Single Head Reciprocating Piston PumpPump
Single Head Reciprocating Piston Single Head Reciprocating Piston PumpPump
Single Head Reciprocating Piston Single Head Reciprocating Piston PumpPump
Advantages:
• Modest cost
• Pressure up to 8000 psi
• External reservoir
• Simple mechanical design
Limitation:
• pulse noise
• Seal, valve maintenance
Dual Piston PumpDual Piston Pump
Syringe PumpSyringe Pump
Syringe PumpSyringe Pump
Advantages:
• Pulse free
• High pressure up to 10000 psi
• Convenient flow control
Limitation:
• Expensive
• Difficult solvent change refill
Diaphragm PumpDiaphragm Pump
High Pressure PumpHigh Pressure Pump
Types of HPLC pumps based on elution techniques:1. ISOCRATIC: A separation that employs a single solvent of
constant composition is termed as isocratic elution.2. GRADIENT: A separation that employs two (or more) solvent
systems that differ significantly in polarity is termed as gradient elution.
• Frequently separation technique is greatly enhanced by gradient elution. Here the proportion of the solvent is varied in programmed way, sometimes continuously and sometimes in a series of steps. Modern HPLC equipment is often equipped with devices that introduce solvents from two or more reservoirs into a mixing chamber at rates that vary continuously.
IsocraticIsocratic
GradientGradient
GradientGradient
GradientGradient
Elution TechniquesElution Techniques
Comparison between Isocratic & Comparison between Isocratic & GradientGradient
Comparison between Isocratic & Comparison between Isocratic & GradientGradient
IsocraticIsocratic GradientGradient
Least ComplexLeast Complex More complexityMore complexity
Composition remains constantComposition remains constant40% Water/60%Methanol40% Water/60%MethanolPremixed SolventsPremixed SolventsAutomated Solvent BlendingAutomated Solvent Blending
Composition varies with timeComposition varies with timeBottle A: 100% WaterBottle A: 100% WaterBottle B: 100 MethanolBottle B: 100 Methanol0 % B to 100% B over 10 minutes0 % B to 100% B over 10 minutes
Must wash column with high Must wash column with high strength solvent after prolonged strength solvent after prolonged useuse
Must re-equilibrate column before Must re-equilibrate column before re-injectingre-injecting
Compound co-elution more Compound co-elution more frequent frequent No compound co-elutionNo compound co-elution
difficult to get resolution of difficult to get resolution of compoundscompounds
Dwell volume effectsDwell volume effects
InjectorsInjectors
InjectorsInjectors
Manual Injectors
• Samples are loaded by the analyst
Auto samplers
• Loads and inject samples; limited pre-injection treatment of
samples (e.g., dilution or addition of aliquots of a standard)
Sample Managers
• Integrates sample pre-treatment and injection with other
system parameters such as the selection of the column,
analytical conditions and processing method.
Manual InjectorsManual Injectors
Rotor SealRotor Seal
LoopLoop
Manual InjectorsManual Injectors
Auto samplerAuto sampler
ColumnsColumns
ColumnsColumns
Column HardwareColumn Hardware
Column HardwareColumn Hardware
Column HardwareColumn Hardware
Column HardwareColumn Hardware
Analytical ColumnsAnalytical Columns
Analytical / Prep ColumnsAnalytical / Prep Columns
COA Of ColumnCOA Of Column
Pre ColumnsPre Columns
Protects the analytical column.
It is located between the pump and the sample injector.
Used when mobile phase ph is greater than 8.
Particle size is larger than analytical column.
It saturates the mobile phase so high PH mobile phase does
not dissolve the silica inside the analytical column.
Guard ColumnsGuard Columns
It is a short column.
Located between sample injector and analytical column.
Used when dirty samples are to be injected like blood, urine,
soil samples etc.
Contain same particle size and packing material as that of
analytical column.
It is cheap and can be refilled.
Pre columns & guard columnsPre columns & guard columns
Preparatory columns & Micro bore Preparatory columns & Micro bore columnscolumns
Preparatory Columns
• Used in bulk drugs.
• Mainly for synthesis purpose
• Preparatory column has a large column diameter to facilitate
large volume injections.
Micro bore Columns
• used for analytical and small volume assays.
• Diameter is 1-2 mm.
• Increase in sensitivity without loss in resolution.
Microbore & Analytical Microbore & Analytical ColumnColumn
Retention MechanismRetention Mechanism
Three primary characteristics of chemicals can be used to Three primary characteristics of chemicals can be used to
create HPLC separations. They are: create HPLC separations. They are:
• Polarity – (Normal Phase or Reversed Phase)Polarity – (Normal Phase or Reversed Phase)
• Electrical Charge – (Ion Exchange)Electrical Charge – (Ion Exchange)
• Molecular Size – (GPC or SEC Type)Molecular Size – (GPC or SEC Type)
Retention MechanismRetention Mechanism
Normal and reversed phase chromatography is mainly based Normal and reversed phase chromatography is mainly based
on POLARITY.on POLARITY.
In ion exchange chromatography, separation is achieved on In ion exchange chromatography, separation is achieved on
the basis of ionic properties of sample.the basis of ionic properties of sample.
In size exclusion chromatography, sample mixture is In size exclusion chromatography, sample mixture is
separated based on size of the molecules of the sample.separated based on size of the molecules of the sample.
Polarity RulesPolarity Rules
A simple rule describes this behavior for polarity-based A simple rule describes this behavior for polarity-based
retention mechanisms:retention mechanisms:
““Like Attracts Like, and Opposites are Not Attracted Like Attracts Like, and Opposites are Not Attracted
When We Use Polarity”When We Use Polarity”. – Just of opposite to magnetism.. – Just of opposite to magnetism.
• Polar will attract Polar (like), and repel Non-Polar Polar will attract Polar (like), and repel Non-Polar
(opposites). (opposites).
• Non-Polar will attract Non-Polar (like), and repel Polar Non-Polar will attract Non-Polar (like), and repel Polar
(opposites).(opposites).
• Same Principle is applied while selecting column stationary Same Principle is applied while selecting column stationary
phase & mobile phase for compound retention.phase & mobile phase for compound retention.
PolarityPolarity
Polarity Of Stationary & Mobile Polarity Of Stationary & Mobile PhasePhase
Polarity Of Sample AnalytePolarity Of Sample Analyte
Principles Of SeparationPrinciples Of Separation
Principles Of SeparationPrinciples Of Separation
Retention Mechanism In NPCRetention Mechanism In NPC
Competitive polar interactionsCompetitive polar interactions• The more polar component, the more stronger, it will The more polar component, the more stronger, it will
be adsorbed and so retained longer.be adsorbed and so retained longer. Physical adsorption is a dynamic reversible process.Physical adsorption is a dynamic reversible process. Polar (specific but nonionic) interactions of analyte with polar Polar (specific but nonionic) interactions of analyte with polar
adsorption sites (SiOH, -NHadsorption sites (SiOH, -NH22, -CN, Diol) cause its retention, -CN, Diol) cause its retention Different sorption affinities between analytes result in their Different sorption affinities between analytes result in their
separationseparation
• More polar analytes retained longerMore polar analytes retained longer
• Analytes with larger number of polar functional group Analytes with larger number of polar functional group are retained longerare retained longer
• Structural isomers are often separatedStructural isomers are often separated
Principles Of SeparationPrinciples Of Separation
Principles Of SeparationPrinciples Of Separation
Retention Mechanism In RPCRetention Mechanism In RPC
Non polar (nonspecific) interactions of analyte with Non polar (nonspecific) interactions of analyte with hydrophobic adsorbent surface (-C18, C8, Phenyl, C4)hydrophobic adsorbent surface (-C18, C8, Phenyl, C4)
Different sorption affinities between analytes results in their Different sorption affinities between analytes results in their separationseparation
• More polar analytes retained lessMore polar analytes retained less
• Analytes with larger hydrophobic part are retained longerAnalytes with larger hydrophobic part are retained longer
• Almost no separation of structural isomersAlmost no separation of structural isomers Nonspecific (hydrophobic) interactions are at least ten times Nonspecific (hydrophobic) interactions are at least ten times
weaker than polarweaker than polar• Small differences in component molecular structure could Small differences in component molecular structure could
have a significant effect in their retentionhave a significant effect in their retention
Principles Of SeparationPrinciples Of Separation
Principles Of SeparationPrinciples Of Separation
Normal Phase Chromatography
• Retention by interaction between polar stationary phase and
polar sample molecules using non polar mobile phase.
Principles Of SeparationPrinciples Of Separation
Reversed Phase Chromatography
• Retention by interaction between non polar hydrocarbon
chain of stationary phase and non polar part of sample
molecules using polar mobile phase.
Principles Of SeparationPrinciples Of Separation
Summary Of Separation ModesSummary Of Separation Modes
Properties Of Normal & Reversed Properties Of Normal & Reversed PhasePhase
PropertiesProperties Normal PhaseNormal Phase Reversed PhaseReversed Phase
Polarity Of Stationary Polarity Of Stationary PhasePhase
highhigh lowlow
Polarity Of mobile Polarity Of mobile PhasePhase
lowlow highhigh
Sample elution orderSample elution order Least polar firstLeast polar first Most polar firstMost polar first
Retention can be Retention can be increased byincreased by
Increasing surface of Increasing surface of stationary phasestationary phase
Increasing surface of Increasing surface of stationary phasestationary phase
Increasing of n-alkyl Increasing of n-alkyl chain length of chain length of stationary phasestationary phase
Reducing polarity of Reducing polarity of mobile phasemobile phase
Increasing polarity of Increasing polarity of mobile phasemobile phase
Reducing water Reducing water content of mobile content of mobile phase (with non polar phase (with non polar eluent)eluent)
Increasing polarity of Increasing polarity of sample moleculessample molecules
Reducing polarity of Reducing polarity of sample moleculessample molecules
Calculation For Polarity Calculation For Polarity IndexIndex
Q : Find out polarity index of Methanol : Water : ACN 60: 20: 20
Polarity index of methanol= 5.1 Polarity index of water= 9.0 Polarity index of ACN= 5.8
Polarity index of mobile phase= PaPb + PcPd + PePf Pa= Polarity Index of methanol
Pb= Ratio Of Methanol Pc= Polarity Index of water
Pd= Ratio Of Water Pe= Polarity Index of ACN
Pf= Ratio Of ACN
Calculation For Polarity Calculation For Polarity IndexIndex
Ans : Polarity Index Of Mobile Phase = (5.1)(0.6) + (9.0)(0.2) + (5.8)(0.2)
= 3.06 + 1.8 + 1.16 = 6.02
Q : Prepare two mobile phases which polarity index is less than 6.02 and more than 6.02.
ColumnsColumns
HPLC Scales (Analytical / HPLC Scales (Analytical / Preparative)Preparative)
Analytical Scale:• High Performance Liquid Chromatography (HPLC)
provides analytical data as to what compounds were present in a sample, and their concentration.
Preparative Scale:• HPLC can also supply a purified quantity of each
compound that is collected in a “ Fraction ” of the flow output from the Detector.
• The instrument component that performs this function is called a “Fraction Collector”.
• This process is called “Preparative Chromatography”.
Preparative HPLCPreparative HPLC
DetectorsDetectors
Mainly used to measure the concentration of components Mainly used to measure the concentration of components present in sample. Detectors used depends upon the present in sample. Detectors used depends upon the property of the compounds to be separated. Different property of the compounds to be separated. Different detectors available for HPLC are:detectors available for HPLC are:
• UV/VISUV/VIS Fixed wavelengthFixed wavelength Variable wavelength (including PDA)Variable wavelength (including PDA)• Refractive Index (RI)Refractive Index (RI)• FluorescenceFluorescence• Electrochemical (ECD) Electrochemical (ECD) • Others (Conductivity, Mass spectrometer, ELSD)Others (Conductivity, Mass spectrometer, ELSD)
Criteria For DetectorsCriteria For Detectors
High sensitivityHigh sensitivity Negligible baseline noiseNegligible baseline noise Large linear dynamic rangeLarge linear dynamic range Response independent of variations in operating parameters Response independent of variations in operating parameters
(pressures, temperature, flow-rate…etc.)(pressures, temperature, flow-rate…etc.) Response independent of mobile phaseResponse independent of mobile phase Low dead volumeLow dead volume Non-destructive Non-destructive Stable over long periods of operations.Stable over long periods of operations. SelectiveSelective
DetectorsDetectors
UV/Visible detectorUV/Visible detector
Selective detection minimizing effects from other
components.
High sensitivity detection at maximum absorption wavelength.
Most frequently used detector in HPLC analysis
Compounds must contain a UV absorbing chromophore
Must work in the linear range of Beer’s Law.
It can be operated at fixed wavelength and variable
wavelength.
Detector Flow CellDetector Flow Cell
Basically detector consists of a flow-cell through which the Basically detector consists of a flow-cell through which the
mobile phase and resolved sample moves. optics shine through mobile phase and resolved sample moves. optics shine through
the detector cell and variation in optical properties are detected.the detector cell and variation in optical properties are detected.
Detector Flow CellDetector Flow Cell
Radiation Source Radiation Source
UV / Visible UV / Visible SpectrophotometerSpectrophotometer
Effect of Wavelength On Effect of Wavelength On ChromatogramChromatogram
Effect of Wavelength On Effect of Wavelength On ChromatogramChromatogram
Photo Diode ArrayPhoto Diode Array
Reasons For Using PDAReasons For Using PDA
Analyte absorb at different wavelength (flexibility of detection)Analyte absorb at different wavelength (flexibility of detection)
Need to verify peak homogeneity ("peak purity")Need to verify peak homogeneity ("peak purity")
Need of UV spectroscopy data Need of UV spectroscopy data
• for library identificationfor library identification
• For faster method developmentFor faster method development
RI detectorRI detector
First HPLC detector developedFirst HPLC detector developed Typically referred to as Universal detectors.Typically referred to as Universal detectors. Detects all dissolved solutes- “non-specific”.Detects all dissolved solutes- “non-specific”. RI response depends on the difference in RI between mobile RI response depends on the difference in RI between mobile
phase and solute(s).phase and solute(s). Sensitivity reaches maximum when RI differences are Sensitivity reaches maximum when RI differences are
greatest.greatest. Can detect Can detect g level but only for isocratic runs.g level but only for isocratic runs. Commonly used for: Sugars, Polymers and Fatty Acids Commonly used for: Sugars, Polymers and Fatty Acids
RI detectorRI detector
Comparison b/w UV & RI DetectorComparison b/w UV & RI Detector
Fluorescence DetectorFluorescence Detector
Increased Selectivity and Sensitivity Increased Selectivity and Sensitivity
Analytes not only absorbs UV/Vis radiation but also releases Analytes not only absorbs UV/Vis radiation but also releases
the energy in the form of light of longer wavelength.the energy in the form of light of longer wavelength.
Sensitivity is in the pg region (up to 1000x UV)Sensitivity is in the pg region (up to 1000x UV)
detector is very sensitive, but its response is only linear over a detector is very sensitive, but its response is only linear over a
relatively limited concentration range.relatively limited concentration range.
Mainly used in food industry. Mainly used in food industry.
Fluorescence DetectorFluorescence Detector
Conductivity DetectorConductivity Detector
Generally used for Ion ChromatographyGenerally used for Ion Chromatography
Detects the ability of analyte to carry a chargeDetects the ability of analyte to carry a charge
• solutes are ionic (i.e. acid and bases)solutes are ionic (i.e. acid and bases)
• Inorganic anions and cationsInorganic anions and cations
Electrochemical DetectorElectrochemical Detector
““Destructive” detection modeDestructive” detection mode
Sample is either oxidized or reduced in the cell.Sample is either oxidized or reduced in the cell.
High sensitivity with picogram (10High sensitivity with picogram (10-12-12 grams) to high grams) to high
femtogram (10femtogram (10-15 -15 grams) range.grams) range.
Proper selection of buffer, electrode and voltage is critical to Proper selection of buffer, electrode and voltage is critical to
success as well as conditioning the mobile phase.success as well as conditioning the mobile phase.
Electrochemical DetectorElectrochemical Detector
Reference Reference ElectrodElectrodee
Working Working ElectrodElectrodee
Electrolyte Electrolyte (mobile phase)(mobile phase)
AuxiliarAuxiliaryyElectrodElectrodee
Analyte is oxidized Analyte is oxidized or or reducedreduced
DetectorsDetectors
RI RI UV/VIS UV/VIS Fluor. Fluor. ECD ECD Cond. Cond.
Response Response Universal Universal SelectiveSelective
(Chromop(Chromophore) hore)
SelectiveSelective
(Fluoroph(Fluorophore) ore)
SelectiveSelective
(Oxidation (Oxidation or or ReductionReduction) )
SelectiveSelective
(Ions) (Ions)
Sensitivity Sensitivity gram gram nanogram nanogram picogram picogram picogram picogram picogram picogram
Linear Linear Range Range
101044 101055 101033 101066 101055
Flow Flow Sensitivity Sensitivity
Yes Yes No No No No Yes Yes Yes Yes
Temp.Temp.
Sensitive Sensitive
Yes Yes No No No No Yes Yes Yes Yes
DetectorsDetectors
Data SystemsData Systems
Process the detector output and integrate it to form a Process the detector output and integrate it to form a
meaningful chromatogram.meaningful chromatogram.
Modem Integration system do more than just that.Modem Integration system do more than just that.
They do processing of the chromatogram, calculations, They do processing of the chromatogram, calculations,
statistical analysis, data back-up and storage.statistical analysis, data back-up and storage.
Data Systems also control various parameters of the system.Data Systems also control various parameters of the system.
Thanks to these advanced systems that we no longer have to Thanks to these advanced systems that we no longer have to
cut chromatogram peaks and weigh them on analytical cut chromatogram peaks and weigh them on analytical
balances for interpretation!balances for interpretation!
Thank YouThank You
Mechanical Separation Power:
Mechanical Separation Power:
Common Column Problems and RemediesCommon Column Problems and Remedies
Variability in retention and resolution Band tailing Short column lifetime
Variability in retention and resolution Band tailing Short column lifetime
Three most important problems in HPLC method development:
Three most important problems in HPLC method development:
Retention and resolution irreproducibility• Sample retention should be repeatable from run to run• It is important to check column retention during method development at least daily, using a particular set of conditions• Values of K and should not change by more than 2-3% over time.
Retention and resolution irreproducibility• Sample retention should be repeatable from run to run• It is important to check column retention during method development at least daily, using a particular set of conditions• Values of K and should not change by more than 2-3% over time.
Retention and Resolution Variations in HPLCRetention and Resolution Variations in HPLC
Effect Cause
Column-column differences
Variation in support, bonding
Column changes during use
Disturbance in bed, loss of bonded phase, dissolution of silica support, buildup of non-eluted material
Extra-column effects
From system to system: large injn. Volume, large tubing volume between. injection valve and column
Poor control of separation
Changes in mobile phase composition, flow rate and temp.
Column overload Too large a sample mass
Solutions to the Irreproducibility Problems
Solutions to the Irreproducibility Problems
Initially select a good column of less-acidic highly
purified support (if silica based) and maintain other
column parameters same throughout the application Eliminate chemical or silanol effects for silica-based
columns by using favourable mobile phase conditions. Equilibrate the column with proper mobile phase Use proper laboratory techniques that ensure stable
day-to- day operation. Ensure continuing supply of the same column.
Initially select a good column of less-acidic highly
purified support (if silica based) and maintain other
column parameters same throughout the application Eliminate chemical or silanol effects for silica-based
columns by using favourable mobile phase conditions. Equilibrate the column with proper mobile phase Use proper laboratory techniques that ensure stable
day-to- day operation. Ensure continuing supply of the same column.
Causes of Tailing (Asymmetrical) Peaks
Causes of Tailing (Asymmetrical) Peaks
Bad column: An initial bad column (poorly packed).Plugged frit or void.Sample overloadWrong solvent for sampleExtra column effectsChemical or secondary retention (silanol) effectsInadequate bufferingContaminating heavy metals
Bad column: An initial bad column (poorly packed).Plugged frit or void.Sample overloadWrong solvent for sampleExtra column effectsChemical or secondary retention (silanol) effectsInadequate bufferingContaminating heavy metals
Short column lifetimeShort column lifetime
Partially blocked (plugged) frit or column bedAdsorbed sample impurities (garbage).Initially poorly packed columnsMechanical or thermal shock creating voidsChemical attack on the stationary phase
Partially blocked (plugged) frit or column bedAdsorbed sample impurities (garbage).Initially poorly packed columnsMechanical or thermal shock creating voidsChemical attack on the stationary phase
Columns degrade for several reasons:Columns degrade for several reasons:
Symptoms of impending column death:Symptoms of impending column death:
Column backpressure increaseTailing bandsLoss in plate numberLoss of selectivity
Column backpressure increaseTailing bandsLoss in plate numberLoss of selectivity
Copyright © 2008, Advance Analytical Research & Training Institute