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 phaseo 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 chromatographyo High priced liquid chromatographyo Hewlett-Packard liquid chromatographyo High performance liquid chromatographyo Hocus pocus liquid chromatographyo High patience liquid chromatography
What is HPLC?o High Performance Liquid Chromatographyo 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-dimensional 3-dimensional thin layer plate column Instrumentation minimal! much! with many adjustable parameters Sample Application spotting injection (capillary) (Rheodyne injector)Mobile Phase Movement capillary action high pressure (during development) (solvent delivery)Visualization of Results UV lightbox “on-line” detection (variable UV/Vis) Form of Results spots, Rf’s peaks, Rt’s (retention factors) (retention times)
HPLC InstrumentationHPLC 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.
HPLC InstrumentationPumps.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 InstrumentationPumps.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 InstrumentationPumps.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 InstrumentationPumps.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 InstrumentationInjectorso 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 InstrumentationInjectorso 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 InstrumentationInjectorso 6-PORT Rotary Valve is the standard manual injectoro 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 InstrumentationInjectorsAutomatic 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 InstrumentationInjectorsAutomatic Injectorso Automatic injectors are indispensable in unattended searching (e.g., overnight) for chromatographic parameters such as solvent selectivity, flowrate, and temperature optimization.
HPLC InstrumentationColumnso HPLC is largely the domain of packed columns o 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 phase o Larger molecules in HPLC vs. GC (generally)
HPLC InstrumentationColumnso 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 InstrumentationDetectorso 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 InstrumentationDetectorsCurrent LC detectors have widedynamic range normally allowing bothanalytical and preparative scale runson 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 InstrumentationDetectorsOther 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 InstrumentationDetectorsOther 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 InstrumentationDetectorso Numerous Types (some obscure)o Original HPLC Detectors were common laboratory instruments such as spectrophotometers, etc. o you can even use a SPEC 20! o Usually a narrow linear range (1E3, usually)o Must be solvent -compatible, stable, etc.
HPLC InstrumentationDetectorso Universal • respond to all analyteso Analyte Specific • respond to specific properties of analyteso Non-destructive • mosto 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 InstrumentationDetectorsStandard 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 wavelenth o Can be set to other wavelengths (most) o Simple filter detectors no longer widely used o adjustable wavelength units are cost- effective
HPLC InstrumentationDetectorsStandard Absorbance Detector….o Non-destructive, not-universal o not all compounds absorb light o 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 InstrumentationDetectorsDiode Array Detector (DAD)The more common tool for research-grade HPLC instruments o quite versatile...o Advances in computer technology since ~1985 or so have lead to the development of Diode Array instrumentso 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 data o Detailed spectra for each peak and each region of each peak
HPLC InstrumentationDetectorsRefractive Index Detectoro One of a very few Universal HPLC detectors. Non-destructiveo Responds to analytes changing the RI of the mobile phase o requires a separate reference flow of mobile phase
HPLC InstrumentationDetectorsRefractive Index Detectoro Extremely temperature sensitive, usually heated o sensitive to temp changes of +/- 0.001 °Co No longer really widely used o Absorbance detectors are relatively cheap.o Useful for process work, on-line monitoring, etc.
HPLC InstrumentationDetectorsELSD (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 InstrumentationRecorder / 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 InstrumentationRecorder / Data Systemo For higher control levels, a more intelligent device is necessary, such as a data station or minicomputer.
HPLC InstrumentationRecorder / 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 Phaseo 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 Phaseo 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 Separationso 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 composition o constant composition = isocratic o varying composition = gradient elutiono Determination if flow rate should be constant o usually it iso Decision on heating the column o 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 processo Must be: o compatible with the instrument (pumps, seals, fittings, detector, etc) o compatible with the stationary phase o 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 ElutionThere 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 Elutiono Isocratic elution has a constant mobile phase composition o Can often use one pump! o Mix solvents together ahead of time! o Simpler, no mixing chamber required o Limited flexibility, not used much in research o mostly process chemistry or routine analysis.
HPLC - Isocratic versus Gradient Elutiono Gradient elution has a varying mobile phase composition o Uses multiple pumps whose output is mixed together ooften 2-4 pumps (binary to quaternary systems)
HPLC - Isocratic versus Gradient Elution o Changing mobile phase components changes the polarity index o can be used to subsequently elute compounds that were previously (intentionally) “stuck” on the column o Some additional wear on the stationary phase o Column has to re-equiluibrate to original conditions after each run (takes additional time).