appendix 12b

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High Performance Liquid Chromatography (HPLC) OUTLINE o Background - What is HPLC? o TLC vs HPLC o Schematics / Instrumentation o Mobile Phases and Their Role in Separations o Types of Separation o Normal vs Reversed phase o Optimization of Separation o Mobile Phase Isocratic vs Gradient Elution

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Page 1: Appendix 12b

High Performance Liquid Chromatography (HPLC)

OUTLINEo Background - What is HPLC?o TLC vs HPLCo Schematics / Instrumentationo Mobile Phases and Their Role in

Separationso Types of Separationo Normal vs Reversed phase o Optimization of Separationo Mobile Phase Isocratic vs Gradient

Elution

Page 2: Appendix 12b

What is HPLC?

o HPLC is really the automation of traditional liquid chromatography under conditions which provide for enhanced separations during shorter periods of time!

o Probably the most widely practiced form of quantitative, analytical chromatography practiced today due to the wide range of molecule types and sizes which can be separated using HPLC or variants of HPLC!!

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High Performance Liquid Chromatography (HPLC)

o High pressure liquid chromatography

o High priced liquid chromatography

o Hewlett-Packard liquid chromatography

o High performance liquid chromatography

o Hocus pocus liquid chromatography

o High patience liquid chromatography

Page 4: Appendix 12b

What is HPLC?o High Performance Liquid

Chromatography

o High Pressure Liquid Chromatography (usually true]

o Hewlett Packard Liquid Chromatography (a joke)

o High Priced Liquid Chromatography (no joke)

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High Performance Liquid Chromatography

HPLC is characterized by the use of high pressure to push a mobile phase solution through a column of stationary phase allowing separation of complex mixtures with high resolution.

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High Performance Liquid Chromatography (HPLC)

o HPLC utilizes a liquid mobile phase to separate the components of a mixture.

o These components (or analytes) are first dissolved in a solvent, and then forced to flow through a chromatographic column under a high pressure.

o In the column, the mixture is resolved into its components.

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High Performance Liquid Chromatography (HPLC)

o The interaction of the solute with mobile and stationary phases can be manipulated through different choices of both solvents and stationary phases.

o As a result, HPLC acquires a high degree of versatility not found in other chromatographic systems and it has the ability to easily separate a wide variety of chemical mixtures.

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TLC vs. HPLC

Type of Analysis qualitative only qualitative &quantitative

Stationary Phase 2-dimensionalthin layer plate

3-dimensionalcolumn

Instrumentation minimal! much! with manyadjustable parameters

Sample Application spotting(capillary)

injection(Rheodyne injector)

Mobile Phase Movement capillary action(during development)

high pressure(solvent delivery)

Visualization of Results UV lightbox “on-line” detection(variable UV/Vis)

Form of Results spots, Rf’s(retention factors)

peaks, Rt’s(retention times)

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HPLC Instrumentation

HPLC instrumentation includes:o Reservoir;o pump;o injector;o column;o detector;o recorder or data system.

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HPLC InstrumentationMobile phase reservoir, filtering.o The most common type of solvent reservoir is a

glass bottle. o Most of the manufacturers supply these bottles

with the special caps, Teflon tubing and filters to connect to the pump inlet and to the purge gas (helium) used to remove dissolved air.

o Helium purging and storage of the solvent under helium was found not to be sufficient for degassing of aqueous solvents.

o It is useful to apply a vacuum for 5-10 min. and then keep the solvent under a helium atmosphere.

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Varian HPLC System

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HPLC Instrumentation

Pumps.o High pressure pumps are needed

to force solvents through packed stationary phase beds.

o However, many separation problems can be resolved with larger particle packing that require less pressure.

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HPLC Instrumentation

Pumps.o The HPLC pump is considered to be

one of the most important components in a liquid chromatography system which has to provide a continuous constant flow of the eluent through the HPLC injector, column, and detector

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HPLC Instrumentation

Pumps.o The two basic classifications are the

constant-pressure and the constant-flow pump.

o The constant-pressure pump is used only for column packing.

o The constant-flow pump is the most widely used in all common HPLC applications.

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HPLC Instrumentation

Pumps.Standard HPLC pump requirements are:o Flow rate range: from 0.01 to 10

ml/mino Pressure range: from 1 to 5000 psi

(340 atm)o Pressure pulsations: less than 1% for

normal and reversed phase mode less than 0.2% for size exclusion mode.

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HPLC Instrumentation

Injectorso Sample introduction can be

accomplished in various ways. o The simplest method is to use an

injection valve. o In more sophisticated LC systems,

automatic sampling devices are incorporated where sample introduction is done with the help of autosamplers and microprocessors.

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HPLC InstrumentationInjectoro Injectors should provide the possibility of

injecting the liquid sample within the range of 0.1 to 100 ml of volume with high reproducibility and under high pressure (up to the 4000 psi).

o They should also produce minimum band broadening and minimize possible flow disturbances.

o The most useful and widely used sampling device for modern LC is the microsampling injector valve.

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HPLC InstrumentationInjectors

o Usually 5 to 1000 L volumes, all directly onto the column • not much worry about capacity since the

columns have a large volume (packed).o Injector is the last component before the

column(s)o A source of poor precision in HPLC• errors of 2-3 %RSD are due just to injection• other errors are added to this due to

capillary action and the small dimensions/cavities inside the injector

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HPLC InstrumentationInjectors

o 6-PORT Rotary Valve is the standard manual injector

o Automatic injectors are availableo Two positions, load and inject in

the typical injectoro Injection loop internal volume

determines injection volume.

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HPLC Instrumentationo Injectors – Six-port Rheodyne valve in

which the sample fills an external loop

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HPLC InstrumentationInjectors – Micro-sampling injector Valves

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HPLC InstrumentationInjectors

Automatic Injectorso With commercially available automatic

sampling devices, large numbers of samples can be routinely analyzed by LC without operator intervention.

o Such equipment is popular for the analysis of routine samples (e.g., quality control of drugs), particularly when coupled with automatic data-handling systems.

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HPLC InstrumentationInjectors

Automatic Injectorso Automatic injectors are indispensable in

unattended searching (e.g., overnight) for chromatographic parameters such as solvent selectivity, flowrate, and temperature optimization.

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HPLC InstrumentationInjectors - Automatic Injectors

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HPLC Instrumentation

Columns

o HPLC is largely the domain of packed columnso some research into microbore/capillary

columns is going on.o Molecules move too slowly to be able to

reach and therefore “spend time in” the stationary phase of an open tubular column in HPLC. o In solution, not the gas phaseoLarger molecules in HPLC vs. GC

(generally)

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HPLC Instrumentation

Columns

o Stationary phases are particles which are usually about 1 to 20 m in average diameter (often irregularly shaped)o In Adsorption chromatography, there is

no additional phase on the stationary phase particles (silica, alumina, Fluorosil).

o In Partition chromatography, the stationary phase is coated on to (often bonded) a solid support (silica, alumina, divinylbenzene resin)

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HPLC Instrumentation

Detectorso Optical detectors are most frequently used.o These detectors pass a beam of light

through the flowing column effluent as it passes through a low volume ( ~ 10 ml) flowcell.

o The most commonly used detector in LC is the ultraviolet absorption detector.

o A variable wavelength detector of this type, capable of monitoring from 190 to 460-600 nm, will be found suitable for the detection of the majority samples.

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HPLC Instrumentation

Detectors

Current LC detectors have wide dynamic range normally allowing both analytical and preparative scale runs on the same instrument.

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HPLC InstrumentationDetectorsOn-line detectors:o Refractive indexo UV/Vis Fixed wavelengtho UV/Vis Variable wavelengtho UV/Vis Diode arrayo Fluorescenceo Conductivityo Mass-spectrometric (LC/MS)o Evaporative light scattering

MS as an on-line HPLC detector is the most sensitive, selective and most universal detector. But

it is still the most expensive one.

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HPLC Instrumentation

DetectorsOther types of detectors:o FD – Fluorescence. • Excitation wavelength generates

fluorescence emission at a higher wavelength.

• Analytes must have fluorophore group. • Can react analyte with fluorophore

reagent. • Very sensitive and selective. • More difficult methods transfer. • Results very dependent upon separation

conditions.

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HPLC Instrumentation

DetectorsOther types of detectors:o RI – Refractive Index. • Universal analyte detector.

Solvent must remain the same throughout separation. • VERY temperature sensitive. • Sometimes difficult to

stabilize baseline.

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HPLC InstrumentationDetectorsOther types of detectors:

MS – Mass Spec. • Mass to charge ratio (m/z). • Allows specific compound ID. • Several types of ionization techniques:

electrospray, atmospheric pressure chemical ionization, electron impact.

• The detector usually contains low volume cell through which the mobile phase passes carrying the sample components.

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HPLC Instrumentation

Detectors o Numerous Types (some obscure)o Original HPLC Detectors were

common laboratory instruments such as spectrophotometers, etc.oyou can even use a SPEC 20!oUsually a narrow linear range

(1E3, usually)o Must be solvent -compatible,

stable, etc.

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HPLC Instrumentation

Detectors

o Universal• respond to all analytes

o Analyte Specific• respond to specific properties of

analyteso Non-destructive• most

o Destructive• ELSD, MS and a few others.

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HPLC InstrumentationBasic detector requirements.An ideal LC detector should have the

following properties:o Low drift and noise level (particularly

crucial in trace analysis).o High sensitivity.o Fast response.o Wide linear dynamic range (this

simplifies quantitation).o Low dead volume (minimal peak

broadening).

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HPLC InstrumentationBasic detector requirements.o Cell design which eliminates remixing

of the separated bands.o Insensitivity to changes in type of

solvent, flow rate, and temperature.o Operational simplicity and reliability.o It should be tuneable so that detection

can be optimized for different compounds.

o It should be non-destructive.

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HPLC Instrumentation

DetectorsStandard Absorbance Detector….o Single Beam UV-VIS instrument with a flow-

through cell (cuvette)o Can use any UV-VIS with a special flow cell

o Extra connections lead to band-broadening if UV-VIS is far from HPLC column exit.

o Usually utilize typical UV-VIS lamps and 254 nm default wavelentho Can be set to other wavelengths (most)o Simple filter detectors no longer widely used

oadjustable wavelength units are cost-effective

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HPLC Instrumentation

DetectorsStandard Absorbance Detector….

o Non-destructive, not-universalo not all compounds absorb lighto can pass sample through several cells

at several different wavelenghtso Usually zeroed at the start of each run

using an electronic software command. You can have real-time zeroing with a reference cell.

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HPLC Instrumentation

DetectorsDiode Array Detector (DAD)The more common tool for research-grade HPLC

instrumentso quite versatile...

o Advances in computer technology since ~1985 or so have lead to the development of Diode Array instruments

o Non-destructive, non-universalo DAD scans a range of wavelengths every second

or few seconds. At each point in the chromatogram one gets a complete UV-VIS spectrum!o Huge volumes of datao Detailed spectra for each peak and each

region of each peak

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HPLC Instrumentation

DetectorsRefractive Index Detectoro One of a very few Universal HPLC

detectors. Non-destructiveo Responds to analytes changing

the RI of the mobile phaseorequires a separate reference

flow of mobile phase

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HPLC InstrumentationDetectorsRefractive Index Detectoro Extremely temperature sensitive,

usually heatedosensitive to temp changes of

+/- 0.001 °Co No longer really widely used

oAbsorbance detectors are relatively cheap.

o Useful for process work, on-line monitoring, etc.

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DetectorsRefractive Index Detector

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DetectorsRefractive Index Detector

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HPLC Instrumentation

DetectorsELSD (Evaporative Light Scattering Detector)o Universal, destructiveo Useful for very large molecules, and a wide

linear rangeo Analytes are de-solvated in the detectoro Molecules pass through what is essentially a

large cuvette for a UV-VIS instrumento The reduction in light intensity detected (due

to scattering by the analytes) is measuredo The larger and more concentrated a

particular molecule is, the greater the scattering.

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HPLC Instrumentation

Recorder / Data Systemo The main goal in using electronic data

systems is to increase analysis accuracy and precision, while reducing operator attention.

o In routine analysis, where no automation (in terms of data management or process control) is needed, a pre-programmed computing integrator may be sufficient.

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HPLC Instrumentation

Recorder / Data Systemo For higher control levels, a more

intelligent device is necessary, such as a data station or minicomputer.

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HPLC Instrumentation

Recorder / Data SystemThe advantages of intelligent

processors in chromatographs:o additional automation options

become easier to implement;o complex data analysis becomes more

feasible;o software safeguards can be designed

to reduce accidental misuse of the system.

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Types of HPLC Separations (partial list)

o Normal Phase: Separation of polar analytes by partitioning onto a polar, bonded stationary phase.

o Reversed Phase: Separation of non-polar analytes by partitioning onto a non-polar, bonded stationary phase.

o Adsorption: In Between Normal and Reversed. Separation of moderately polar analytes using adsorption onto a pure stationary phase (e.g. alumina or silica)

o Ion Chromatography: Separation of organic and inorganic ions by their partitioning onto ionic stationary phases bonded to a solid support.

o Size Exclusion Chromatography: Separation of large molecules based in the paths they take through a “maze” of tunnels in the stationary phase.

Page 55: Appendix 12b

HPLC - Normal vs. Reversed Phase

Normal Phase Reversed Phase

Stationary phase Polar (silica gel) Non-polar (C18)

Mobile phase Non-polar (organic solvents)

Polar (aqueous/organic)

Sample movement

Non-polar fastest Polar fastest

Separation based

on

Different polarities

(functionality)

Different hydrocarbon

content

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HPLC - Normal vs. Reversed Phase

o In normal-phase chromatography, the stationary bed is strongly polar in nature (e.g., silica gel), and the mobile phase is nonpolar (such as n-hexane or tetrahydrofuran).

o Polar samples are thus retained on the polar surface of the column packing longer than less polar materials.

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HPLC - Normal vs. Reversed Phase

o Reversed-phase chromatography is the inverse of this.

o The stationary bed is nonpolar (hydrophobic) in nature, while the mobile phase is a polar liquid, such as mixtures of water and methanol or acetonitrile.

o Here the more nonpolar the material is, the longer it will be retained.

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HPLC - Optimization of Separations

o Correct choice of column so the above equilibrium has some meaningful (non-infinity, non-zero) equilibrium constants.

o Correct choice of mobile phaseo Decision on the type of mobile phase

compositiono constant composition = isocratico varying composition = gradient elution

o Determination if flow rate should be constanto usually it is

o Decision on heating the columno heating HPLC columns can influence the

above equilibrium….

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HPLC - The Mobile Phaseo Must do the following:

o solvate the analyte molecules and the solvent they are in

o be suitable for the analyte to transfer “back and forth” between during the separation process

o Must be:o compatible with the instrument (pumps, seals,

fittings, detector, etc)o compatible with the stationary phaseo readily available (often use liters/day)o of adequate purity

o spectroscopic and trace-composition usually!o Not too compressible (causes pump/flow problems)

o Free of gases (which cause compressability problems)

Page 60: Appendix 12b

HPLC - Isocratic versus Gradient Elution

There are two elution types: isocratic and gradient.

o In the first type constant eluent composition is pumped through the column during the whole analysis. This is ISOCRATIC ELUTION.

o In the second type, eluent composition (and strength) is steadily changed during the run. This is GRADIENT ELUTION.

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HPLC - Isocratic versus Gradient Elution

o Isocratic elution has a constant mobile phase compositiono Can often use one pump!o Mix solvents together ahead of time!o Simpler, no mixing chamber

requiredo Limited flexibility, not used much in

researchomostly process chemistry or

routine analysis.

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HPLC - Isocratic versus Gradient Elution

o Gradient elution has a varying mobile phase compositionoUses multiple pumps whose

output is mixed togetherooften 2-4 pumps (binary to quaternary systems)

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HPLC - Isocratic versus Gradient Elution

o Changing mobile phase components changes the polarity indexocan be used to subsequently elute

compounds that were previously (intentionally) “stuck” on the column

oSome additional wear on the stationary phase

oColumn has to re-equiluibrate to original conditions after each run (takes additional time).

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ANY QUESTIONS ?