1. High Performance Liquid Chromatography (HPLC) b What is HPLC? b Types of Separations b Columns and Stationary Phases b Mobile Phases and Their Role in Separations b Injection in HPLC b Detection in HPLC b Variations on Traditional HPLC Ion ChromatographyIon Chromatography Size Exclusion ChromatographySize Exclusion Chromatography

2. What is HPLC? b High Performance Liquid Chromatography b Hewlett Packard Liquid Chromatography (a joke) b High Priced Liquid Chromatography (no joke) b HPLC is really the automation of traditional liquid chromatography under conditions which provide for enhanced separations during shorter periods of time! b 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!!

4. Types of HPLC Separations (partial list) b Normal Phase: Separation of polar analytes by partitioning onto a polar, bonded stationary phase. b Reversed Phase: Separation of non-polar analytes by partitioning onto a non-polar, bonded stationary phase. b Adsorption: In Between Normal and Reversed. Separation of moderately polar analytes using adsorption onto a pure stationary phase (e.g. alumina or silica) b Ion Chromatography: Separation of organic and inorganic ions by their partitioning onto ionic stationary phases bonded to a solid support. b Size Exclusion Chromatography: Separation of large molecules based in the paths they take through a “maze” of tunnels in the stationary phase.

6. What does the analyst do? b Select the correct type of separation for the analyte(s) of interest, based on the sample type (among other factors). b Select an appropriate column (stationary phase) and mobile phase b Select an appropriate detector based on whether universal or compound-specific detection is required or available b Optimize the separation using standard mixtures b Analyze the sample

7. Columns and Stationary Phases…... b HPLC is largely the domain of packed columns some research into microbore/capillary columns is going on.some research into microbore/capillary columns is going on. Molecules move too slowly to be able to reach and therefore “spend time in” the stationary phase of an open tubular column in HPLC.Molecules move too slowly to be able to reach and therefore “spend time in” the stationary phase of an open tubular column in HPLC. –In solution, not the gas phase –Larger molecules in HPLC vs. GC (generally) b Stationary phases are particles which are usually about 1 to 20  m in average diameter (often irregularly shaped) In Adsorption chromatography, there is no additional phase on the stationary phase particles (silica, alumina, Fluorosil).In Adsorption chromatography, there is no additional phase on the stationary phase particles (silica, alumina, Fluorosil). In Partition chromatography, the stationary phase is coated on to (often bonded) a solid support (silica, alumina, divinylbenzene resin)In Partition chromatography, the stationary phase is coated on to (often bonded) a solid support (silica, alumina, divinylbenzene resin)

9. Stationary Phases b Polar (“Normal Phase): Silica, aluminaSilica, alumina Cyano, amino or diol terminations on the bonded phaseCyano, amino or diol terminations on the bonded phase b Non-Polar (“Reversed Phase”) C18 to about C8 terminations on the bonded phaseC18 to about C8 terminations on the bonded phase Phenyl and cyano terminations on the bonded phasePhenyl and cyano terminations on the bonded phase b Mixtures of functional groups can be used!! b Packed particles in a column require: Frits at the ends of the column to keep the particles inFrits at the ends of the column to keep the particles in Filtering of samples to prevent clogging with debrisFiltering of samples to prevent clogging with debris High pressure pumps and check-valvesHigh pressure pumps and check-valves Often a “Guard Column” to protect the analytical columnOften a “Guard Column” to protect the analytical column

10. Optimization of Separations in HPLC b Correct choice of column so the above equilibrium has some meaningful (non-infinity, non-zero) K. b Correct choice of mobile phase b Decision on the type of mobile phase composition constant composition = isocraticconstant composition = isocratic varying composition = gradient elutionvarying composition = gradient elution b Determination if flow rate should be constant usually it isusually it is b Decision on heating the column or not usually we do NOT heat HPLC columns….usually we do NOT heat HPLC columns….

11. The Mobile Phase in HPLC... b Must do the following: solvate the analyte molecules and the solvent they are insolvate the analyte molecules and the solvent they are in be suitable for the analyte to transfer “back and forth” between during the separation processbe suitable for the analyte to transfer “back and forth” between during the separation process b Must be: compatible with the instrument (pumps, seals, fittings, detector, etc)compatible with the instrument (pumps, seals, fittings, detector, etc) compatible with the stationary phasecompatible with the stationary phase readily available (often use liters/day)readily available (often use liters/day) of adequate purityof adequate purity –spectroscopic and trace-composition usually! Not too compressible (causes pump/flow problems)Not too compressible (causes pump/flow problems)

12. Typical HPLC Pump (runs to 4,000+ psi) Polymer Piston, often made of industrial sapphire or ruby

13. Polarity Index for Mobile Phases….. b The polarity index is a measure of the relative polarity of a solvent. It is used for identifying suitable mobile phase solvents. The more polar your solvent is, the higher the index.The more polar your solvent is, the higher the index. You want to try to choose a polarity index for your solvent (or solvent mixture) that optimizes the separation of analytesYou want to try to choose a polarity index for your solvent (or solvent mixture) that optimizes the separation of analytes –usually the index is a starting point –the polarity of any mixture of solvents to make a mobile phase can be modeled to give a theoretical chromatogram –Usually, optimization of solvent composition is experimental

15. Optimization of Mobile Phase Polarity...

16. Isocratic versus Gradient Elution b Isocratic elution has a constant mobile phase composition Can often use one pump!Can often use one pump! Mix solvents together ahead of time!Mix solvents together ahead of time! Simpler, no mixing chamber requiredSimpler, no mixing chamber required Limited flexibility, not used much in researchLimited flexibility, not used much in research –mostly process chemistry or routine analysis. b Gradient elution has a varying mobile phase composition Uses multiple pumps whose output is mixed togetherUses multiple pumps whose output is mixed together –often 2-4 pumps (binary to quarternary systems) Changing mobile phase components changes the polarity indexChanging mobile phase components changes the polarity index –can be used to subsequently elute compounds that were previously (intentionally) “stuck” on the column –Some additional wear on the stationary phase –Column has to re-equiluibrate to original conditions after each run (takes additional time).

19. Injection in HPLC b Usually 5 to 1000  L volumes, all directly onto the column not much worry about capacity since the columns have a large volume (packed).not much worry about capacity since the columns have a large volume (packed). b Injector is the last component before the column(s) b A source of poor precision in HPLC errors of 2-3 %RSD are due just to injectionerrors of 2-3 %RSD are due just to injection other errors are added to thisother errors are added to this due to capillary action and the small dimensions/cavities inside the injectordue to capillary action and the small dimensions/cavities inside the injector b 6-PORT Rotary Valve is the standard manual injector b Automatic injectors are available b Two positions, load and inject in the typical injector b Injection loop internal volume determines injection volume.

20. LOAD (the sample loop) Inject (move the sample loop into the mobile phase flow)

21. Detection in HPLC b Numerous Types (some obscure) b Original HPLC Detectors were common laboratory instruments such as spectrophotometers, etc. you can even use a SPEC 20!you can even use a SPEC 20! Usually a narrow linear range (1E3, approx)Usually a narrow linear range (1E3, approx) b Must be solvent -compatible, stable, etc. b Universal respond to all analytesrespond to all analytes b Analyte Specific respond to specific properties of analytesrespond to specific properties of analytes b Non-destructive mostmost b Destructive ELSD, MS and a few others.ELSD, MS and a few others.

23. Standard Absorbance Detector…. b Single Beam UV-VIS instrument with a flow-through cell (cuvette) b Can use any UV-VIS with a special flow cell Extra connections lead to band-broadening if UV-VIS is far from HPLC column exit.Extra connections lead to band-broadening if UV-VIS is far from HPLC column exit. b Usually utilize Hg vapor lamps and 254 nm line (generic) Can be set to other wavelengths (most)Can be set to other wavelengths (most) Simple filter detectors no longer widely usedSimple filter detectors no longer widely used –adjustable wavelength units are cost-effective b Non-destructive, not-universal not all compounds absorb lightnot all compounds absorb light can pass sample through several cells at several different wavelenghtscan pass sample through several cells at several different wavelenghts b Usually zeroed at the start of each run using an electronic software command. You can have real-time zeroing with a reference cell.

25. Diode Array Detector (DAD) b The more common tool for research-grade HPLC instruments quite versatile...quite versatile... b Advances in computer technology since ~1985 or so have lead to the development of Diode Array instruments b Non-destructive, non-universal b DAD scans a range of wavelengths every second or few seconds. At each point in the chromatogram one gets a complete UV-VIS spectrum! Huge volumes of dataHuge volumes of data Detailed spectra for each peak and each region of each peakDetailed spectra for each peak and each region of each peak

27. Refractive Index Detector b One of a very few Universal HPLC detectors. Non-destructive b Responds to analytes changing the RI of the mobile phase requires a separate reference flow of mobile phaserequires a separate reference flow of mobile phase b Extremely temperature sensitive, usually heated sensitive to temp changes of +/- 0.001 °Csensitive to temp changes of +/- 0.001 °C b No longer really widely used Absorbance detectors are relatively cheap.Absorbance detectors are relatively cheap. b Useful for process work, on-line monitoring, etc. b Do you remember using an Abbe Refractometer? Organic?Organic?

28. ELSD (Evaporative Light Scattering Detector) b Universal, destructive b Useful for very large molecules, and a wide linear range b Analytes are de-solvated in the detector b Molecules pass through what is essentially a large cuvette for a UV- VIS instrument b The reduction in light intensity detected (due to scattering by the analytes) is measured b The larger and more concentrated a particular molecule is, the greater the scattering.

30. Other Detectors... b Mass Spec problem is interfacing a liquid flow with a vacuum system!problem is interfacing a liquid flow with a vacuum system! b Electrochemical b Conductivity b Fluorescence b Lecture on Ion-Chromatography and Size Exclusion Chromatography will continue later...