Transcript High Performance Liquid Chromatography (HPLC)
High Performance Liquid Chromatography (HPLC)
What is HPLC?
Types of Separations Columns and Stationary Phases Mobile Phases and Their Role in Separations Injection in HPLC Detection in HPLC
Variations on Traditional HPLC
Ion Chromatography
Size Exclusion Chromatography
What is HPLC?
High Performance Liquid Chromatography
High Pressure Liquid Chromatography (usually true]
Hewlett Packard Liquid Chromatography (a joke)
High Priced Liquid Chromatography (no joke)
HPLC is really the automation of traditional liquid chromatography under conditions which provide for enhanced separations during shorter periods of time!
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!!
Types of HPLC Separations (partial list)
Normal Phase: Separation of polar analytes by partitioning onto a polar, bonded stationary phase.
Reversed Phase: Separation of non-polar analytes by partitioning onto a non-polar, bonded stationary phase.
Adsorption: In Between Normal and Reversed. Separation of moderately polar analytes using adsorption onto a pure stationary phase (e.g. alumina or silica)
Ion Chromatography: Separation of organic and inorganic ions by their partitioning onto ionic stationary phases bonded to a solid support.
Size Exclusion Chromatography: Separation of large molecules based in the paths they take through a “maze” of tunnels in the stationary phase.
What does the analyst do?
Select the correct type of separation for the analyte(s) of interest, based on the sample type (among other factors).
Select an appropriate column (stationary phase) and mobile phase Select an appropriate detector based on whether universal or compound-specific detection is required or available
Optimize the separation using standard mixtures
Analyze the standards and sample
Columns and Stationary Phases.
HPLC is largely the domain of packed columns
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.
In solution, not the gas phase
Larger molecules in HPLC vs. GC (generally) 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 Partition chromatography, the stationary phase is coated on to (often bonded) a solid support (silica, alumina, divinylbenzene resin)
Stationary Phases
Polar (“Normal” Phase):
Silica, alumina Cyano, amino or diol terminations on the bonded phase
Non-Polar (“Reversed Phase”)
C18 to about C8 terminations on the bonded phase
Phenyl and cyano terminations on the bonded phase
Mixtures of functional groups can be used!!
Packed particles in a column require:
Frits at the ends of the column to keep the particles in Filtering of samples to prevent clogging with debris
High pressure pumps and check-valves Often a “Guard Column” to protect the analytical column
Optimization of Separations in HPLC
Correct choice of column so the above equilibrium has some meaningful (non-infinity, non-zero) equilibrium constants.
Correct choice of mobile phase Decision on the type of mobile phase composition
constant composition = isocratic
varying composition = gradient elution Determination if flow rate should be constant
usually it is Decision on heating the column
heating HPLC columns can influence the above equilibrium….
The Mobile Phase in HPLC...
Must do the following:
solvate the analyte molecules and the solvent they are in be suitable for the analyte to transfer “back and forth” between during the separation process
Must be:
compatible with the instrument (pumps, seals, fittings, detector, etc)
compatible with the stationary phase
readily available (often use liters/day) of adequate purity
spectroscopic and trace-composition usually!
Not too compressible (causes pump/flow problems)
Free of gases (which cause compressability problems)
Typical HPLC Pump (runs to 4,000+ psi)
Polarity Index for Mobile Phases…..
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.
You 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 A similar number is the Eluent Strength (E o ] Increasing eluent strength or polarity index values mean increasing solvent polarity. 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 the separation.
Isocratic versus Gradient Elution
Isocratic elution has a constant mobile phase composition
Can often use one pump!
Mix solvents together ahead of time!
Simpler, no mixing chamber required Limited flexibility, not used much in research
mostly process chemistry or routine analysis.
Gradient elution has a varying mobile phase composition
Uses multiple pumps whose output is mixed together
often 2-4 pumps (binary to quarternary systems) Changing 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).
Injection in HPLC
Usually 5 to 1000
L volumes, all directly onto the column
not much worry about capacity since the columns have a large volume (packed).
Injector is the last component before the column(s) 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 6-PORT Rotary Valve is the standard manual injector Automatic injectors are available Two positions, load and inject in the typical injector Injection loop internal volume determines injection volume.
Inject (move the sample loop into the mobile phase flow)
LOAD (the sample loop)
Detection in HPLC
Numerous Types (some obscure) Original HPLC Detectors were common laboratory instruments such as spectrophotometers, etc.
you can even use a SPEC 20!
Usually a narrow linear range (1E3, usually) Must be solvent -compatible, stable, etc.
Universal
respond to all analytes Analyte Specific
respond to specific properties of analytes Non-destructive
most Destructive
ELSD, MS and a few others.
Standard Absorbance Detector….
Single Beam UV-VIS instrument with a flow-through cell (cuvette) 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.
Usually utilize typical UV-VIS lamps and 254 nm default wavelenth
Can be set to other wavelengths (most) Simple filter detectors no longer widely used
adjustable wavelength units are cost-effective Non-destructive, not-universal
not all compounds absorb light can pass sample through several cells at several different wavelenghts 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)
The more common tool for research-grade HPLC instruments
quite versatile...
Advances in computer technology since ~1985 or so have lead to the development of Diode Array instruments
Non-destructive, non-universal
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 data
Detailed spectra for each peak and each region of each peak
Refractive Index Detector
One of a very few Universal HPLC detectors. Non-destructive
Responds to analytes changing the RI of the mobile phase
requires a separate reference flow of mobile phase
Extremely temperature sensitive, usually heated
sensitive to temp changes of +/- 0.001 ° C
No longer really widely used
Absorbance detectors are relatively cheap.
Useful for process work, on-line monitoring, etc.
ELSD (Evaporative Light Scattering Detector)
Universal, destructive
Useful for very large molecules, and a wide linear range Analytes are de-solvated in the detector
Molecules pass through what is essentially a large cuvette for a UV-VIS instrument
The reduction in light intensity detected (due to scattering by the analytes) is measured
The larger and more concentrated a particular molecule is, the greater the scattering.