appendix 12b
TRANSCRIPT
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
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!!
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
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)
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.
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.
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.
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)
HPLC Instrumentation
HPLC instrumentation includes:o Reservoir;o pump;o injector;o column;o detector;o recorder or data system.
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.
Varian HPLC System
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.
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
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.
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.
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.
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.
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
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.
HPLC Instrumentationo Injectors – Six-port Rheodyne valve in
which the sample fills an external loop
HPLC InstrumentationInjectors – Micro-sampling injector Valves
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.
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.
HPLC InstrumentationInjectors - Automatic Injectors
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)
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)
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.
HPLC Instrumentation
Detectors
Current LC detectors have wide dynamic range normally allowing both analytical and preparative scale runs on the same instrument.
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.
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.
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.
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.
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.
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.
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).
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.
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
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.
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
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
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.
DetectorsRefractive Index Detector
DetectorsRefractive Index Detector
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.
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.
HPLC Instrumentation
Recorder / Data Systemo For higher control levels, a more
intelligent device is necessary, such as a data station or minicomputer.
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.
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.
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
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.
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.
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….
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)
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.
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.
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)
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).
ANY QUESTIONS ?