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
 Select the correct type of separation for
do?
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 (Eo]
 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.
LOAD (the sample loop)
Inject (move the sample

loop into the mobile
phase flow)
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
 The more common tool for research-grade HPLC
(DAD)
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.

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High Performance Liquid

 Variations on Traditional HPLC

 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

 Probably the most widely practiced form of quantitative,

 Reversed Phase: Separation of non-polar analytes by partitioning

 Adsorption: In Between Normal and Reversed. Separation of

 Ion Chromatography: Separation of organic and inorganic ions by

 Size Exclusion Chromatography: Separation of large molecules based

 Non-Polar (“Reversed Phase”)

 Mixtures of functional groups can be used!!

 Packed particles in a column require:

Changing the mobile phase composition alters the

 Gradient elution has a varying mobile phase composition

Inject (move the sample

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