7. HPLC Adv. Chrom - PowerPoint Presentation

7. HPLC Adv. Chrom

Revision 1. The P in HPLC is often used as an abbreviation for pressure. Explain why .pump uses high pressure to form liquid through column2. Draw a block diagram of the components of a typical HPLC.  3. How does separation by HPLC differ from that by GLC?temperature not involved polarity attraction to both phasesliquid mobile phase 4. What general rules apply to the selection of the two phases in HPLC?SP similar to analytesMP different but not too different

7.1 Injection systems operate at pump pressures of 2 MPa and higher (10 x atmospheric pressure)how to inject the sample against such a pressurehow to maintain constant pressure without surges and drop-offsneed pressure isolation: rheodyne 1 2 3 6 5 4 load run 1 2 3 6 5 4

7.2 Pumps desirable properties stable pulse-free flowavoids baseline fluctuationsreproducible flow ratesconsistency between runsa range of flow rates from 0.5-5 mL/minflexibility in separationsagainst very high pressures (up to 100 atm)needed to get liquid through column in reasonable timewithout being affected by different solvents (aqueous and non-aqueous)allows range of mobile phases to be used

Reciprocating piston pump (a) Do you think this pump by itself would create pulse-free flow? no (b) What could be done to fix the problem? two pumpsworking 180o out of phase

Mobile phase must be filtered and degassed to remove: fine particles which would block the check valves in the pump or the columndissolved gases, which may produce bubbles within the column or detectorreservoirs should be covered to avoid dust falloutshould be HPLC gradeguaranteed not to contain stabilisers added to other “pure” solventsmay not be as pure as AR grade, e.g. HPLC grade hexane may contain some heptane

Columns made from stainless steel to withstand the internal pressure15-30 cm in lengthnot coiled because there is no need to save spacepressure problems created by coilsinternal diameter of the column is typically 4-5 mmsmaller bore packed columns and capillary columns are now available for HPLCpacking has decreased in size to < 10 umstationary phase chemically bonded to Si-OH on packing surface (or capillary walls)

Stationary phases guard column - a short version (1-2 cm) of the main column between the injection system and the main columnintended to be “disposable” – catches any fine particles resulting from precipitation between sample and mobile phaseNameAttached GroupPolarity Reverse phase C8 to C18 Non-polar Normal phase -(CH 2 ) 3 CN Slightly polar Normal phase -(CH 2)3NH2 PolarAnion exchange -NR3+Ionic Cation exchange-SO3 -IonicSize exclusion  No polarity

Detectors most HPLC detectors based on familiar lab instruments Ex. 7.3 - Why? because HPLC uses solutionsuniversal response (or selective in a predictable way)high sensitivitylow noisewide linear rangeresponse independent of instrumental variationssmall internal volume #flow-through design #non-destructivestable responserelatively inexpensiveUV/VISrefractive indexconductivitymass spectrometryevaporative light scattering (ELS) fluorescence infrared flame emission potentiometric polarographic

Selectivity some adjustment that can be made to pick out specified analytes and ignore otherssome UV-VIS detectors are fixed wavelength (universal), others allow choice of wavelengths Selectivity Sensitivity Temp. Sensitivity Gradient elution compatibility UV/VIS Selective Medium high Low Yes ELS Universal High Low Yes RI Universal Low MediumNo ConductivityUniversal Medium High No MS Universal or Selective Medium high No No sugars in grape juice RI fluoride in toothpaste conductivity determination of FW range of polystyrene plastic ELS steroids in blood MS

Flow through design the flow from the column is continuousthe “sample” cell in which the detector measurement is made, must be flow-throughfresh eluant comes in, pushing out that which is already measuredthe simplest cell has two openings, one for the inlet, and one for the outletwhere cell pathlength is important for sensitivity (eg UV/VIS, fluorescence, conductivity)inlet and outlet must as far apart as possiblewithout increasing the cell volume UV beam quartz windows to waste from column DETECTOR

Internal volume a dead volume where no separation occursimpacts on the ability to the ability to resolve close-together peaksExampleflow rate of 1 mL/minutea detector with an internal volume of 1 mLwill take 1 minute to fill or to change over the contentsany reading from the detector is an average of the eluant from the last minutea changeover time of 1 secondmaximum flow rate of 3 mL/minutevolume required will be no more than 50 µLdetector cell volumes of less than 10  L are not uncommon

Solvent response correction mobile phase response at the detector must be zeroed like a reference beam/cell in spectroscopysolvent response will mask small variations due to the low levels of analyte eluting from the columnType 1 – split before columnAdv – copes with changes in solvent response during gradient elutionDisadv – two detectors may respond differently, half the flow rate lostType 2 – diversion after columnAdv – no dead time or flow rate problemsDisadv – two detectors respond differently, can’t cope with changes in response by detector during gradient elution Type 3 – no diversion Adv – no detector variation, dead time or flow rate problems Disadv – can only be zeroed initially, can’t cope with changes in response by detector during gradient elution COLUMN D D COLUMN D D COLUMN D Which do our instruments use?

Ion chromatography used to separate mixtures of ions either anions or cations, but not both at the same timemobile phase is aqueous with ionic or polar organic compounds dissolved (modifiers)these provide competition for the SP sites so that the analyte ions don’t stick to the SPhigher modifier conc = lower RTcation columns have negatively charged sites, vice versa for anion columnsanion chromatography is very important because of the difficulty in analysing anion mixtures at < 100 mg/Lcation chromatography less important because of all the various ways of analysing metals

Retention factors size - the smaller the ion, the less the attractionretention time for I- > Br- > Cl- > F-charge - the smaller the charge, the less the attractionretention time for Al3+ > Ca2+ > Na+

Detection most commonly used detector is conductivitypresence of modifiers in MP causes a background conductance makes it difficult to detect small increases due to the analytes (loss of sensitivity)unsuppressed – low modifier concentrations => long RTssuppressed – treatment of eluent between column & detector to remove background conductivitymobile phase is a sodium carbonate/hydrogen carbonate solutionsuppressor column is a cation exchange resin, loaded with hydrogen ions on the exchange sites Supp-SO3H + Na+ (aq)  Supp-SO3- Na + + H + ( aq ) H+ + HCO3-  H 2CO3

Size exclusion separates molecules by their molecular size by ability (or lack of it) to pass into the porous structure of the stationary phaseunusual in that there is no polarity attraction to the stationary phasethe mobile phase simply transports the mixtureonly works with large and very large molecules (FW > 2000)typical analytes are carbohydrates, proteins, plasticsrelationship between size/mass and retention volume (measured instead of time) can be determinedWhich comes out first?Larger molecules

Choice of phases Exercise 7.7 sugars in grape juice normalfluoride in toothpasteanionFW of plasticssize exclusionsteroids in bloodreversemobile must have some attraction to analyteslikely to be medium polarity FW < 2000 > 2000 Size exclusion Hexane Ethanol Water Solubility Reverse phase Normal phase Ion exchange Non-ionic Ionic

Elution modes in HPLC isocratic – same mobile phase throughout gradient elution – gradual change in MP (equivalent of temp programming in GC) 2nd MP means 2nd pumpClass Exercise 7.8column: reverse phase MP: ethanoltwo well-resolved peaks at 2 & 3 minutes & a broad peak at 10 minutesHow could this be improved, using gradient elution?the 10 min peak must be strongly attracted to the SP => it is non-polarmust make the MP less polarbegin introducing less polar solvent after 1st peak emerges, i.e. 2.5 mins