high performance liquid chromatoghraphy (HPLC)
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  • Mobile Phases - Component solvents/mobile phases to make up gradient Gradient Controller - Sets up gradient - linearity, steps, ramps, number of solvents/mobile phases (binary, ternary, quaternary). Pump - Dual piston, Pulse free, Able to deliver 4000PSI, Precision flow rates of 0.001mL/min, Flow range 0.001-10.001 mL/min. Injector - How do you inject a sample in to a flowing sream at 4000PSI? If you tried you’d have the syringe plunger go thru a wall! The sample injector utilizes a set of valves in which a sample loop switchs in-and-out the flowing stream. You introduce the sample by injecting it into the sample loop which has a fixed volume. The fixed volume injected replaces the contents of the loop so therefore for manual injection you should have enough injected to completely replace the previous loop contents. The sample is automatically introduced into the flowing stream by valve switching.
  • 555- hydrolysis prior to LC; conc col in-line (C-18) (~SPE); gradient 25mM H 3 PO 4 :MeCN 90:10 > 10:90, PDA uses 2ºλ confirmed by response ratio. Typical cmpd -Cl 5 phenol has BP=310ºC -- not volatile. 605- Electrochemical - 4,4’-Diaminobiphenyl > Diphenoquinone. V=+0.8v 0.1M pH 4.7 OAc : MeCN. BP= 402ºC -- not volatile. Also true electrochemical VA determination. 610 - LC separates all 16. UV for Naphthalene, Acenaphthene, Acenaphthalene and Fluorene, rest Fluorescence; gradient MeCN:H 2 O . MeCN. Also GC/FID (4 sets of unresolved pairs) method. FL then UV, λ=254 8316 - Direct H 2 O inj. MP= H 2 O, UV=195nm. GC can run direct H 2 O inj but not recommended. GC run about 20 min. HPLC run about 12 min. 8330 - >4PPM Direct H 2 O inj. C-18 confirm with CN; MeOH: H 2 O. λ=254. TNT BP= 240ºC (explodes)! 8331 - Tetrazene- MP with ion-pairing (MeOH: H 2 O (C 10 -SO 3 H)(HOAc)), λ=280. 8332 - Nitroglycerine - MP= MeOH: H 2 O(3:2)-CN col. Confirm C-18 (1:1), λ=254. 8325 - LC/PB/MS - Benzidines and N-containing pests/herbs (Carbaryl, Siduron (Tupersan)). Also UV λ=230.
  • 2,4,5-T = trichlorophenoxyacetic acid Benzidine = 4.4’-Diaminobiphenyl Fluorene = p-Biphenylene methane
  • TNT - show numbering system Acrylonitrile = vinyl cyanide Naphthyl Methyl Carbamate
  • - “Like dissolves like”. In Normal Phase the analyte will be partitioned preferentially in the mobile phase and provide little interaction with the stationary phase. This is not desirable since selective retention on the column will be very hard to control. It can be controlled by modifying the stationary phase, a very time consuming and expensive proposition even if feasible. In Reverse Phase the opposite is true. The analyte will be partitioned preferentially in the stationary phase ( “Like dissolves like” ) . and by simply modifying the mobile phase, by adjusting the polarity, ionic strength or pH, selectivity can be virtually fully controlled.
  • It is common practice to make up these organic solvents as mixtures with water or, in a lot of cases, have each pure solvent mixed under instrument control and changed at a certain rate with time (gradient). Gradients can be simple or complex. Simple - as a linear gradient (ramp); Complex as steps (start and hold) and ramps together. You can have a solvent or several solvents being controlled at the same time with a changing modifier such as a pH buffer. Methanol - Most common solvent. Close to water in structure. Miscible in all proportions with H 2 O so that for less polar organics you can have the power of 90% Methanol with 10% H 2 O. Acetonitrile - Highly polar, very low UV absorbance. Also completely miscible with H 2 O but lacking in hydrogen bonding capability thus affording a different partitioning effect. Tetrahydrofuran - Molecule has high dipole moment. More soluble with non-polar compounds. Water - Also a very common solvent. Used to make up solvent modifiers to adjust pH (buffers) as well as ion-pairing reagents. Emphasize degassing (“bends”- bubble in detector) and for particle-free (dust could be up to 10X size of particles of solid support). 555- 25mM H 3 PO 4 :MeCN(90:10)->10:90 605 - 0.1M pH4.7 OAc: MeCN (1:1) isocratic. 610 - MeCN:H 2 O -> MeCN 8316 - 100% H 2 O 8330 - MeOH:H 2 O isocratic 8331 - MeOH:H 2 O(HOAc-C 10 SO 3 H) 8332- MeOH:H 2 O (3:2-CN), (1:1-C-18) 8325-A=MeCN:0.01M OAc (75:25), B= MeCN -> 60%MeCN total. λ= 190 for MeCN, λ= 205 for MeOH, λ= 190 for H 2 O, λ~ 290 for THF, λ~ 255 for 1% HOAc and λ~ 260 NH 4 OAc (1M)
  • 555- λ=210-310nm, main λ = 230nm. 605- V= +0.8v 610- UV=254nm, Fluor=280/389nm. (Fl then UV) 8330- UV=254nm. 8331(Tetrazene)- UV=280nm. 8332 (Nitroglycerine)- UV=214nm 8316 - UV=195nm. 8325 - PB/MS and UV @230nm
  • Here is a perfect example of why we should have a confirmatory column. Look at peaks 8, 9,10 and 11 on the C-18 chromatogram and notice the poor resolution of the group primarily due to peak 11: 2,6-dinitrotoluene. Now notice the CN chromatogram. See how 2,6-Dinitrotoluene is separated from the two amino-dinitrotoluene compounds and the 2,4-dinitrotoluene allowing baseline resolution for all four. Also note peak 14, HMX. It takes almost 50 min to get out while it was the first (< 2.5 min) to come out on C-18.
  • Here is a perfect example of why we should have a confirmatory column. Look at peaks 8, 9,10 and 11 on the C-18 chromatogram and notice the poor resolution of the group primarily due to peak 11: 2,6-dinitrotoluene. Now notice the CN chromatogram. See how 2,6-Dinitrotoluene is separated from the two amino-dinitrotoluene compounds and the 2,4-dinitrotoluene allowing baseline resolution for all four. Also note peak 14, HMX. It takes almost 50 min to get out while it was the first (< 2.5 min) to come out on C-18.
  • Here are two chromatograms which very dramatically show the effect of column particle size and flow rate. Note that resolution has, for the most part, (peaks 3 and 4) NOT deteriorated. You would think that the pressure needed to run chromatogram B would be extrememly high. However, the pressure needed to run chromatogram B, due to new porous particles making up the solid support, was only 2000 PSI (about 50% higher than for A).
  • Here are two chromatograms which very dramatically show the effect of column particle size and flow rate. Note that resolution has, for the most part, (peaks 3 and 4) NOT deteriorated. You would think that the pressure needed to run chromatogram B would be extrememly high. However, the pressure needed to run chromatogram B, due to new porous particles making up the solid support, was only 2000 PSI (about 50% higher than for A).

high performance liquid chromatoghraphy (HPLC) high performance liquid chromatoghraphy (HPLC) Presentation Transcript

  • High Performance Liquid Chromatography (HPLC)S What is HPLC?S Types of SeparationsS Columns and Stationary PhasesS Mobile Phases and Their Role in SeparationsS Injection in HPLCS Detection in HPLCS Variations on Traditional HPLC S Ion Chromatography S Size Exclusion Chromatography
  • What is HPLC?HPLC is really the automation of traditionalliquid chromatography under conditions whichprovide for enhanced separations duringshorter periods of time!Probably the most widely practiced form ofquantitative, analytical chromatographypracticed today due to the wide rangeof molecule types and sizes which canbe separated using HPLC orvariants of HPLC!!
  • H ighPerformanceL iquidC hromatography
  • H ighP ressureL iquidC hromatography
  • H ighP ricedL iquidC hromatography
  • PartitioningS Separation is based on the analyte’s relative solubility between two liquid phases Mobile Phase Stationary Phase Solvent Bonded Phase
  • Instrumentation Gradient Controller • Pump Column Detector Injector Mobile Phases
  • What does the analyst do?S Select the correct type of separation for the analyte(s) of interest, based on the sample type (among other factors).S Select an appropriate column (stationary phase) and mobile phaseS Select an appropriate detector based on whether universal or compound-specific detection is required or availableS Optimize the separation using standard mixturesS Analyze the standards and sample
  • What does the analyst do?S Select the correct type of separation for the analyte(s) of interest, based on the sample type (among other factors).
  • HPLC MethodsS Parameter Group Method Compounds • SDW05.23000’s EPA 555 Cl-PhenoxyAcids• WPP05.06000’s EPA 605 Benzidines• WPP05.13000’s EPA 610 PAHs• SHW06.26000’s SW-846 8316 Acrylics• SHW06.28000’s SW-846 8330’s Explosives• SHW07.06000’s SW-846 8325 Benzidines and N- Pesticides
  • Compounds 2,4,5-T Benzidine Fluorene
  • Compounds TNT (2,4,6-Trinitrotoluene)H2C=CH-CN Acrylonitrile Carbaryl
  • HPLC - ModesS Normal Phase. - Polar stationary phase and non-polar solvent.• Reverse Phase. - Non-polar stationary phase and a polar solvent.
  • Common Reverse Phase SolventsMethanol CH3OHAcetonitrile CH3CNTetrahydrofuranWater H2O
  • What does the analyst do? Select an appropriate column (stationary phase) and mobile phase
  • Columns and Stationary PhasesHPLC is largely the domain of packed columns: S some research into microbore/capillary columns is going on. S Molecules move too slowly to be able to reach and therefore “spend time in” the stationary phase of an open tubular column in HPLC. SIn solution, not the gas phase SLarger molecules in HPLC vs. GC (generally)
  • Columns and Stationary Phases Stationary phases are particles which are usually about 1 to 20 µm in average diameter (often irregularly shaped): S In Adsorption chromatography, there is no additional phase on the stationary phase particles (silica, alumina, Fluorosil). S In Partition chromatography, the stationary phase is coated on to (often bonded) a solid support (silica, alumina, divinylbenzene resin)
  • ColumnsS Solid Support - Backbone for bonded phases. S Usually 10µ, 5µ or 3µ silica or polymeric particles.S Bonded Phases - Functional groups firmly linked (chemically bound) to the solid support. S Extremely stable S ReproducibleS Guard - Protects the analytical column: S Particles S Interferences S Prolongs the life of the analytical column
  • Bonded Phases • C-2 Ethyl Silyl -Si-CH2-CH3 •C-8 Octyl Silyl -Si-(CH2)7-CH3• C-18 Octadecyl Silyl -Si-(CH2)17-CH3 •CN Cyanopropyl Silyl -Si-(CH2)3-CN
  • Stationary PhasesS Polar (“Normal” Phase): S Silica, alumina S Cyano, amino or diol terminations on the bonded phaseS Non-Polar (“Reversed Phase”) S C18 to about C8 terminations on the bonded phase S Phenyl and cyano terminations on the bonded phaseS Mixtures of functional groups can be used!!S Packed particles in a column require: S Frits at the ends of the column to keep the particles in S Filtering of samples to prevent clogging with debris S High pressure pumps and check-valves S Often a “Guard Column” to protect the analytical column
  • The Mobile Phase in HPLC...S Must do the following: S solvate the analyte molecules and the solvent they are in S be suitable for the analyte to transfer “back and forth” between during the separation processS Must be: S compatible with the instrument (pumps, seals, fittings, detector, etc) S compatible with the stationary phase S readily available (often use liters/day) S of adequate purity S spectroscopic and trace-composition usually! S Not too compressible (causes pump/flow problems) S Free of gases (which cause compressability problems)
  • Typical HPLC Pump (runs to 4,000+ psi)
  • Polarity Index for Mobile Phases…..S The polarity index is a measure of the relative polarity of a solvent. It is used for identifying suitable mobile phase solvents. S The more polar your solvent is, the higher the index. S You want to try to choose a polarity index for your solvent (or solvent mixture) that optimizes the separation of analytes S usually the index is a starting point S the polarity of any mixture of solvents to make a mobile phase can be modeled to give a theoretical chromatogram S Usually, optimization of solvent composition is experimentalS A similar number is the Eluent Strength (Eo]S Increasing eluent strength or polarity index values mean increasing solvent polarity.S Remember, the analyte(s) and samples must be mobile phase and stationary phase compatible!
  • Optimization of Mobile Phase Polarity…Changing the mobile phase composition alters theseparation.
  • Isocratic versus Gradient ElutionS Isocratic elution has a constant mobile phase composition S Can often use one pump! S Mix solvents together ahead of time! S Simpler, no mixing chamber required S Limited flexibility, not used much in research S mostly process chemistry or routine analysis.S Gradient elution has a varying mobile phase composition S Uses multiple pumps whose output is mixed together S often 2-4 pumps (binary to quarternary systems) S Changing mobile phase components changes the polarity index S can be used to subsequently elute compounds that were previously (intentionally) “stuck” on the column S Some additional wear on the stationary phase S Column has to re-equiluibrate to original conditions after each run (takes additional time).
  • Injection in HPLCS Usually 5 to 1000 µL volumes, all directly onto the column S not much worry about capacity since the columns have a large volume (packed).S Injector is the last component before the column(s)S A source of poor precision in HPLC S errors of 2-3 %RSD are due just to injection S other errors are added to this S due to capillary action and the small dimensions/cavities inside the injectorS 6-PORT Rotary Valve is the standard manual injectorS Automatic injectors are availableS Two positions, load and inject in the typical injectorS Injection loop internal volume determines injection volume.
  • LOAD (the sample loop)Inject (move the sampleloop into the mobilephase flow)
  • What does the analyst do?S Select an appropriate detector based on whether universal or compound- specific detection is required or available
  • DetectorsS UV SSingle wavelength (filter) [610, 8330] S Variable wavelength (monochromator) [8316, 8325] SMultiple wavelengths (PDA) [555]S Fluorescence [610]S Electrochemical [605]S Mass Spectrometric [8325]
  • ChromatogramsRestek® ULTRA C-18 Column (250mm x 4.6mm, 5µ),Mobile Phase: (1:1 Methanol:Water), 1.5 mL/min.
  • ChromatogramsRestek® CN Column (250mm x 4.6mm, 5µ),Mobile Phase: (1:1 Methanol:Water), 1.5 mL/min.
  • Chromatograms Supelcosil LC-PAH Columns. Conditions: A: 150mm x 4.6mm, 5µ. Flow Rate: 1.5 mL/min
  • Chromatograms Supelcosil LC-PAH Columns. Conditions: B: 50mm x 4.6mm, 3µ. Flow Rate: 3.0 mL/min
  • Detection in HPLCS Numerous Types (some obscure)S Original HPLC Detectors were common laboratory instruments such as spectrophotometers, etc. S you can even use a SPEC 20! S Usually a narrow linear range (1E3, usually)S Must be solvent -compatible, stable, etc.S Universal S respond to all analytesS Analyte Specific S respond to specific properties of analytesS Non-destructive S mostS Destructive S ELSD, MS and a few others.
  • Standard Absorbance Detector….S Single Beam UV-VIS instrument with a flow-through cell (cuvette)S Can use any UV-VIS with a special flow cell S Extra connections lead to band-broadening if UV-VIS is far from HPLC column exit.S Usually utilize typical UV-VIS lamps and 254 nm default wavelenth S Can be set to other wavelengths (most) S Simple filter detectors no longer widely used S adjustable wavelength units are cost-effectiveS Non-destructive, not-universal S not all compounds absorb light S can pass sample through several cells at several different wavelenghtsS Usually zeroed at the start of each run using an electronic software command. You can have real-time zeroing with a reference cell.
  • Diode Array Detector (DAD)S The more common tool for research-grade HPLC instruments S quite versatile...S Advances in computer technology since ~1985 or so have lead to the development of Diode Array instrumentsS Non-destructive, non-universalS DAD scans a range of wavelengths every second or few seconds. At each point in the chromatogram one gets a complete UV-VIS spectrum! S Huge volumes of data S Detailed spectra for each peak and each region of each peak
  • Refractive Index DetectorS One of a very few Universal HPLC detectors. Non-destructiveS Responds to analytes changing the RI of the mobile phase S requires a separate reference flow of mobile phaseS Extremely temperature sensitive, usually heated S sensitive to temp changes of +/- 0.001 °CS No longer really widely used S Absorbance detectors are relatively cheap.S Useful for process work, on-line monitoring, etc.
  • ELSD (Evaporative Light ScatteringDetector)S Universal, destructiveS Useful for very large molecules, and a wide linear rangeS Analytes are de-solvated in the detectorS Molecules pass through what is essentially a large cuvette for a UV-VIS instrumentS The reduction in light intensity detected (due to scattering by the analytes) is measuredS The larger and more concentrated a particular molecule is, the greater the scattering.
  • What does the analyst do?S Optimize the separation using standard mixtures and then for samples.
  • Optimization of Separations in HPLCS Correct choice of column so the above equilibrium has some meaningful (non-infinity, non-zero) equilibrium constants.S Correct choice of mobile phaseS Decision on the type of mobile phase composition S constant composition = isocratic S varying composition = gradient elutionS Determination if flow rate should be constant S usually it isS Decision on heating the column S heating HPLC columns can influence the above equilibrium….
  • Types of HPLC Separations (partial list) S Normal Phase: Separation of polar analytes by partitioning onto a polar, bonded stationary phase. S Reversed Phase: Separation of non-polar analytes by partitioning onto a non-polar, bonded stationary phase. S Adsorption: In Between Normal and Reversed. Separation of moderately polar analytes using adsorption onto a pure stationary phase (e.g. alumina or silica)
  • Types of HPLC Separations (partial list) S Ion Chromatography: Separation of organic and inorganic ions by their partitioning onto ionic stationary phases bonded to a solid support. S Size Exclusion Chromatography: Separation of large molecules based in the paths they take through a “maze” of tunnels in the stationary phase.
  • Advantages of LC compared to GC: 1.) LC can be applied to the separation of any compound that is soluble in a liquid phase. ‚oLC more useful in the separation of biological compounds, synthetic or natural polymers, and inorganic compounds 2.) Liquid mobile phase allows LC to be used at lower temperatures than required by GC ‚ LC better suited than GC for separating compounds that may be thermally labile
  • 3.) Retention of solutes in LC depend on their interaction with both the mobile phase and stationary phase. ‚ GC retention based on volatility and interaction with stationary phase ‚ LC is more flexible in optimizing separations  change either stationary or mobile phase 4.) Most LC detectors are non-destructive ‚ most GC detectors are destructive ‚ LC is better suited for preparative or process-scale separationsDisadvantage of LC compared to GC: 1.) LC is subject to greater peak or band-broadening. ‚ much larger diffusion coefficients of solutes in gases vs. liquids