HPLC-HIgh Pressure/Performance Liquid Chromatography

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


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


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.


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.


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.

Partition ChromatographyPartition Chromatography

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.


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

Sample filtrationSample filtration

Sample filtersSample filters

Sample filtrationSample filtration

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

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.


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.


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.


Ball

Seat

Holder

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..


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


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

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

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

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

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


Normal Phase Chromatography

• Retention by interaction between polar stationary phase and

polar sample molecules using non polar mobile phase.


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.

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

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:

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

HPLC-HIgh Pressure/Performance Liquid Chromatography

Education

Transcript of HPLC-HIgh Pressure/Performance Liquid Chromatography