HPLC

HPLC
Pump

Jacket for
controlling column
temperature

6-port
Mobile Sample HPLC
valve
Phases loop column
A and B

syringe
MP waste
Detector
 Components

Mobile phase reservoirs

HPLC Pump(s)
Mixing valves
Sample injector (manual or auto)
Column
Detector
Plumming
Mobile phase waste container
 Types of HPLC Separations
 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.
 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)
 Columns and Stationary Phases.
 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 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 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 compressibility problems)
Typical HPLC Pump (runs to 4,000+ psi)
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.
 Isocratic versus Gradient Elution
 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

Inject
 Detection in HPLC
 Numerous Types (some obscure)
 Original HPLC Detectors were common laboratory
instruments such as spectrophotometers, etc.
– 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.
A UV-Visible absorption flow cell for
HPLC
 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.
 Supercritical fluid
chromatography (SFC)

 SFC is a hybrid of gas and liquid

chromatography that combines some of the best
features of each.

 SFC is of importance because it permits the

separation and determination of a group of
compounds that are not conveniently handled by
either gas liquid or liquid chromatography.
 Supercritical fluid
 Above the critical temperature
no phase transition
regardless of the applied
pressure
Supercritical fluid has physical
and thermal properties that are
between those of the pure liquid
and gas
fluid density is a strong
function of the temperature and
pressure
diffusivity much higher than a
liquid readily penetrates porous
and fibrous solids
Low viscosity
Supercritical fluid
chromatography
Instruments and Operating Variables
The pressures and temperatures required for creating supercritical
fluids lie within the operating limits of ordinary HPLC equipment.
Thus, instruments for SPC are similar in most regards to the
instruments for HPLC.

Effects of Pressure
Pressure changes in supercritical chromatography have
a pronounced effect on the capacity factor. This effect is
a consequence of the increase in density of mobile
phase with increases in pressure.

Stationary Phases
Both open-tubular and packed columns are used for SFC although
currently the former are favored. Open-tubular columns are similar
to the fused-silica columns with internal coatings of bonded and
crossed-linked siloxanes of various types.
 Mobile Phases
The most widely used mobile phase for SPC is carbon dioxide. It is
an excellent solvent for a variety of organic molecules. In addition, it
transmits in the ultraviolet and is odorless, nontoxic, readily
available, and remarkably inexpensive when compared with other
chromatographic mobile phases.

Detectors
A major advantage of SFC over HPLC is that the flame ionization
detector of gas chromatography can be employed. Mass
spectrometers are also more easily adapted as detectors for SFC
than HPLC.

Applications
SFC has been applied to a wide variety of including natural
products, drugs, foods, pesticides and herbicides, surfactants,
polymers and polymer additives, fossil fuels and explosives and
propellants.
 SFC Separations
SFC is a hybrid of gas and liquid chromatography
that combines some of the best features of each
As in HPLC, variation of the mobile phase
composition affects separation
In SFC, mobile phase affinity for the analyte is a
function of mobile phase density
Density is controlled by controlling system
pressure
Highly polar samples are not easy to handle (high
critical parameters & high reactivity)
 SFC Advantages vs HPLC
Supercritical fluids have low viscosities
- faster analysis (5 to 10 X faster)
- less pressure drop across the column
- the use of open tubular columns is feasible
Column lengths from 10 to 20 m are used
Can be used with a wide range of sensitive detectors
Resolving power is ~5X that of HPLC
 SFC Advantages vs GC

Can analyze non-volatile, polar, or adsorptive

solutes without derivatization.
Can analyze thermally labile compounds.
Can analyze solutes of much higher molecular
weight.
 SFC and Retention
Retention dependent on temperature, pressure,
mobile phase density, and composition of the
stationary and mobile phase.
Complex interactions and not easily predictable.
For supercritical fluids
- solvating properties similar to liquids
- viscosity closer to gases
Solvating power  density
 Other SFC Solvents
Nitrous Oxide - Similar in solvating and separations
properties to CO2
Alkanes - less safe and not as detector compatible
than CO2
- better solvent characteristics for non-polar
solutes
Halocarbons, xenon, etc. - specialty applications
only
More polar solvents for highly polar & high
molecular weight compounds
 Solvent Modifiers

Add organic modifiers to > solvent strength

- methanol
- isopropanol
- dichloromethane
- THF
- acetonitrile