igh-performance liquid chromatography 

(sometimes referred to - perhaps erroneously - as high-pressure

liquid chromatography), HPLC, is a chromatographic technique that can separate a mixture of compounds and

is used inbiochemistry and analytical chemistry to identify, quantify and purify the individual components of the

mixture.

HPLC typically utilizes different types of stationary phases, a pump that moves the mobile phase(s) and analyte

through the column, and a detector to provide a characteristic retention time for the analyte. The detector may

also provide additional information related to the analyte, (i.e. UV/Vis spectroscopic data for analyte if so

equipped). Analyte retention time varies depending on the strength of its interactions with the stationary phase,

the ratio/composition of solvent(s) used, and the flow rate of the mobile phase. It is a form of liquid

chromatography that utilizes smaller column size, smaller media inside the column, and higher mobile phase

pressures.

With HPLC, a pump (rather than gravity) provides the higher pressure required to move the mobile phase and

analyte through the densely packed column. The increased density arises from smaller particle sizes. This allows

for a better separation on columns of shorter length when compared to ordinary column chromatography.

Research with thin-layer and column chromatography showed that separations are much more effective
when the stationary phase is a very thin layer on the surface of very small and very uniform spherical
beads. However, resistance to flow of the mobile phase is very much higher, and in order to get a useful
flow of a liquid mobile phase, e. g., 1 - 3 milliliters/minute, pressures of around 15 Mpa (about 2,000
psi) must be applied to the mobile phase. It is possible to apply such pressure from a cylinder of
compressed gas, but most systems use a reciprocating piston pump or diaphragm pump with some
means of damping the pressure fluctuations from the piston. The sample is usually dissolved in the
mobile phase before injection. Columns are typically 4.6 mm ID (6 mm OD) stainless steel tubing 250
mm long. A typical packing will have octadecylsilyl (C18-Si-) (ODS) groups bonded to 5 µm silica beads.
The packing is held inside the column by “frits”, discs with pores about 0.5 µm in diameter.

Liquid-liquid chromatography began with samples dissolved in organic solvents and a stationary
phase of water adsorbed on particles or fibers of the solid support. More generally, the stationary
phase was more “polar” than the mobile phase. That is the so-called "normal phase"
chromatography. But stationary phases such as ODS have been particularly useful for separating
samples dissolved in water (and most HPLC is now done with bonded phases). Liquid
chromatography with the stationary phase less polar than the mobile phase is called “reverse
phase”, but is now the common situation. The mobile phase is very often not just water but a
mixture of water with methanol (CH3OH) or acetonitrile (CH3CN). “Solvent programming”, a
stepwise or continuous change (gradient elution) of the mobile phase composition, is used to
speed up separations, like temperature programming in gas chromatography.

“Chiral”columns have been developed relatively recently to separate optical isomers. This
separation is important because many pharmaceuticals are active in only one chiral form. For
instance, natural Vitamin E is D-a-tocopherol, while half of synthetic Vitamin E is the less active L-
isomer.

As in gas chromatography, a few microliters of the solution are measured into a “sample loop” on
an “injector”. When the injector is operated, the sample loop is suddenly switched into the flow of
mobile phase just before it reaches the column. The mobile phase leaving the column passes
immediately into a “detector” which is used to determine the presence and concentration of a
solute.

Because the pressure on the mobile phase drops rapidly as it leaves the column, bubbles may
form from dissolved gases. Chromatographers “degas” the mobile phase before use by boiling it,
ultrasonic treatment, bubbling helium through it to flush out other gases, or applying a vacuum.
There are no relatively inexpensive HPLC detectors as sensitive and broadly useful as the flame
ionization and electron capture detectors used in gas chromatography. A refractive index (RI)
detector responds to most components, but is not very sensitive. An ultraviolet (UV) detector is
quite sensitive for molecules which absorb ultraviolet light, and a variable wavelength UV detector
can be set to the absorption maximum for a particular molecule of interest, or to a short
wavelength where most molecules absorb. A diode array detector (DAD) disperses the transmitted
light into a spectrum, providing an absorption spectrum of each component that absorbs ultraviolet
light. Still more sensitive, and still less general, is the fluorescence detector which measures
fluorescence emitted from components which have absorbed ultraviolet light. There are various
special-purpose detectors. Amperometric systems measure electron flow which oxidizes or reduces
certain components (sugars, for instance), and polarimetric detectors, generally not very sensitive,
detect components are optically active. Mass spectrometric detectors are now used, but were late
to arrive because it was difficult to separate the mobile phase molecules so as to maintain
adequate vacuum in the mass spectrometer.

Special case: Supercritical fluid chromatography (SFC). Liquids can vaporize, and gases can
condense to the liquid phase. Both changes depend on the pressure and temperature. Every gas
has a critical temperature above which it cannot be condensed at any pressure. The critical
pressure is the pressure required at the critical temperature. If a liquid (such as a condensed gas)
is warmed, under high pressure, to the critical temperature and beyond, strange things happen.
The boundary between gas and liquid vanishes, and it is best not to ask physical chemists too
many questions about the remaining phase, the “supercritical fluid”. Whatever their exact nature,
supercritical fluids can be very useful solvents for HPLC, which runs under high pressure anyway.
The most commonly used of these fluids is carbon dioxide. (Supercritical fluids are also useful for
extracting things from solids, and, when the solids are clothing, for dry cleaning.)

Reports and procedures will specify the mobile phase composition, flow rate and perhaps the
pressure; the column dimensions; the column packing (particle type and size; coating); the
detector and its operating conditions (e. g., wavelengths for UV and fluorescence detectors),
integrator settings, and, somewhere, retention times.

There are some kinds of liquid chromatography that do not fit the partition or adsorption models
very well. They are often done in simple vertical columns.

Gel chromatography: The mobile phase carries sample through a gel, such as agar or
polyacrylamide. Small molecules pass through quickly. The larger molecules cannot get through as
quickly, and are separated by size. Various kinds of gels are used for polar (aqueous) or nonpolar
mobile phases. Sometimes these gels are called “molecular sieves”, but they are not the Molecular
Sieves (tradename) used in gas-solid chromatography to separate gases.

Exclusion chromatography: The packing is made of porous beads. Small molecules diffuse into the
pores and so do not move as fast as the mobile phase. The largest molecules cannot fit into the
pores, and move with the solvent flow. If the beads are charged, ions of the same charge are
repelled while those of opposite charge are attracted in ion exclusion chromatography.

Ion-pair chromatography: In liquid chromatography, strongly ionized sample components which

would not partition into the liquid phase can be combined with large ions having the opposite
charge [such as an alkyl sulfonate (negative) or a tetrabutylammonium (positive)] to produce “ion
pairs” that act like a compound and will partition.

Affinity chromatography: This uses a specific “ligand” (such as an antigen) bonded to the
stationary phase to separate a specific substance such as the corresponding antibody.

There are some special procedures and terms:

Headspace analysis: The sample is held for a time in a closed container, with a gas phase. Volatile
molecules move from the sample into the gas phase, depending on their volatility. When the
system has reached equilibrium, a sample of the gas phase is injected into a gas chromatograph,
and the concentration in the gas phase (headspace) is used to determine the concentration in the
liquid phase. 
Purge and trap: A relatively large volume of the liquid sample (perhaps a liter) is held in a
container. A gas (e. g., helium) is bubbled through to sweep out the volatile molecules, and then
passed through a short trap column containing some of the column packing (“solid phase”) which
collects and concentrates the volatiles in a much smaller volume. Then the trap is heated rapidly
and the volatiles are driven into the gas chromatograph.

Solid phase extraction (SPE): A relatively large volume of liquid sample is exposed to a small
volume of a stationary phase that extracts and concentrates material from the sample. That
material can then be redissolved and chromatographed.

Response factor: Detectors typically respond more strongly to some molecules than to others, so
that peak size alone is not the best means of measuring quantities of individual components. Once
the components have been identified, the analyst can chromatograph known amounts of individual
materials to relate the peak size to quantity. The relationship is the “response factor”.

Area percent: The area of a specific peak divided by the total area of all the peaks. It is commonly
used to report analysis results. Users should remember that response factors differ, so that area
percent is not the same as weight percent, and that some components may not be detected at all
by the detector used. 

Introduction

High Pressure Liquid Chromatography (HPLC) is

used in analytical chemistry or biochemistry to separate chemical compounds in mixtures for

analysis or

purification.

Components in a mixture are separated on a column

packed with silica-based particles (referred to as stationary phase) by pumping a solvent (referred to
as

mobile phase) through the column. Depending on the

unique affinity of each component (referred to as the

analyte) between the mobile phase and the stationary

phase, each analyte migrates along the column at different speeds and emerges from the column at
different

times, thus establishing a separation of the mixture.

Analytes with higher affinity for the mobile phase

migrate faster down the column, whereas those with

higher affinity for the stationary phase migrate slower.

This migration time (referred to as retention time) is

unique for each analyte and can be used in its identification. With the appropriate use of a detection
method

after the column, each analyte can also be quantified for

analysis.

Smaller column particle size can improve chromatographic resolution, but increased solvent delivery

pressure is needed. Further reduction of column particle size can allow for higher solvent flow rates,

reducing analysis time without sacrificing resolution.

This is what gives Ultra High Pressure Liquid Chromatography its advantage over other LC
techniques.

Note

Refer to Syringe Pump Application Note AN4 Ultra

HPLC System Configuration for additional information

about Ultra High Pressure Liquid Chromatography

applications.

Basic Types of HPLC Column Chemistries:

● Normal Phase Chromatography, e.g. Silica particles

● Reverse Phase Chromatography, e.g. C-18 coated

Silica

Basic Types of Mobile-Phase Delivery Systems:

● Isocratic – constant solvent mixture, e.g. 10%

Water in Methanol throughout the entire separation run.

● Gradient – time-varying solvent mixture, e.g.

starting a run with pure Hexane followed by a

slow increase of Ethyl Acetate concentration

until pure Ethyl Acetate at the end.

Basic components of an HPLC system are a solvent

delivery pump, sample injection port, column, and

detector. HPLC performance requires instruments with

characteristics not found in typical LC systems. Solvent

delivery must be pulseless and accurately calibrated at

flow rates in the 1 mL/minute to 100 µL/minute range.

Syringe pumps are generally acknowledged to be superior to piston pumps for HPLC applications
where low,

stable flow rates are required.

Teledyne Isco Syringe Pumps are excellent HPLC

pumps for these high performance applications.

Figure 1: Isocratic Solvent Delivery System

Injection Port

Injection Valve

Manual

Valve

Solvent Syringe Pump

Waste

Data Acquisition PC

Detector

Waste

Column

Syringe Pump Application Note

AN6Syringe Pump Application Note AN6

Theory

The Van Deemter equation is the key to understanding the fundamentals of chromatography.

According to the Van Deemter equation:

H = A + B/µ + Cµ

where: H is the column’s plate height, and µ is the

mobile phase linear flow rate. A, B, and C are constants.

A smaller plate height H indicates higher separation

resolution.

The constant A is the eddy diffusion term. Eddy diffusion results from multiple flow paths in the
column and

is independent of mobile phase flow rate. Due to the

packing particles, analyte molecules can follow multiple

pathways of differing path lengths. These multiple pathways of differing length spread the analyte
molecules

apart and cause peak broadening. The A term depends

on the compactness of the stationary phase. Voids in

the stationary phase contribute to peak broadening due

to channeling. In contrast, smaller packing particles

offer smaller differences in path length, thus reducing

peak broadening.

B is the longitudinal diffusion coefficient. It depends

on the diffusion coefficient of the analyte molecules in

the mobile phase. Faster mobile phase flow rates reduce

resident time, and shorter resident time of the analyte

molecules in the column reduces the effects of longitudinal diffusion. This reduction contributes to
better

separation efficiencies since the analyte molecules have

less opportunity to spread out through diffusion, thus

explaining the 1/v factor.

C is the analyte mass transfer coefficient. It depends

on the time needed for the analyte molecules to equilibrate between the mobile and stationary
phases. If this

equilibration is too slow, then some of the analyte molecules, which did not have enough time to
bond to the

stationary phase, will flow down the column with the

mobile phase; whereas, the other molecules, which did

not have enough time to detach from the stationary

phase, are left behind. Higher mobile phase flow rates

will contribute to the spreading out of the analyte molecules, thus explaining the v factor. Smaller
stationary

phase particles are expected to have shorter equilibration time.

As stated above, smaller stationary phase particles

or beads should contribute to smaller A and C values.

The A term will contribute less to H and allow for higher

resolution. As the C term becomes less significant to the

value of H, increasing mobile phase flow rates will not

sacrifice separation performance as much. This would

allow for faster separations with the same resolution.

Even though smaller column beads may not directly

affect the B value, the higher flow rates reduce its contribution to H. Smaller column beads would
allow for

faster separations with higher resolution.

Smaller beads will pack with smaller interstitial

spaces, thus reducing the A value, but offer higher resistance to solvent flow. In turn, this requires
higher

solvent pressures. With higher optimal separation flow

rates, column back pressure increases as an inverse

cube of the stationary phase particle size; i.e., Pα1/d

Typically, pressures of 1,000 psi are required for HPLC

with 5µm column beads, whereas 20,000 psi or above is

required for Ultra-HPLC (UHPLC) with 1.7µm beads.

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