HPLC - What is it?

Hewlett Packard HPLC sale:

You know you have to use HPLC in
your laboratory...you haven't done it
yourself...so where do you start?.....

High Performance Liquid

Chromatography (HPLC) is an
analytical process utilizing special
instruments designed to separate,
quantify and analyze components of
a chemical mixture. Samples of
interest are introduced to a solvent
flow path; carried through a column
packed with specialized materials
for component separation; and
component data is obtained through
the combination of a detection
mechanism coupled with a data
recording system. All this occurs
under pressures which may reach or
exceed 6,000 psi!
The basic components of an HPLC
system include
solvent resevoir
sample injector
pump(s)
analytical column

Model 1050 HPLC System

Including: 1050 Quaternary HPLC
Pump 79852A, 1050 Variable
Wavelength UV-VIS Detector 79853,
1050 21 Position Auto sampler
79855A, Computer and Monitor,
ChemStation. Installation and
Training Options are Available.
Other Pump, Detector and
Autosampler Options are available.
HP 1050 autosampler
HP 1050 quaternary pumps
HP 1050 interface 35900-C
HP UV 79853 detector
Computer and new Chemstation
analysis and control software
HP 1046A fluorescent detectors
available
$ Inquire at 763-712-8717

detector(s)
data recorder
waste container (or fraction
collector)
Other important elements are an
inlet solvent filter, post-pump inline
filter, sample filter, precolumn filter,
guard column, back-pressure
regulator and/or solvent sparging
system. The function of each of these
components is briefly described
below.
An HPLC system begins with the
solvent reservoir, which contains the
solvent used to carry the sample
through the system. The solvent
should be filtered with an inlet
solvent filter to remove any particles
that could potentially damage the
system's sensitive components.
Solvent is propelled through the
system by the pump. This often
includes internal pump seals, which
slowly break down over time. As
these seals break down and release
particles into the flow path, an inline
solvent filter prevents any postpump component damage.
The next component in the system is
the sample injector, also known as
the injection valve. This valve,
equipped with a sample loop of the
appropriate size for the analysis
being performed, allows for the
reproducible introduction of sample
into the flow path. Because the
sample often contains particulate
matter, it is important to utilize
either a sample filter or a precolumn

Other HPLC arrivals:

HP 1090 series, with uv or pda
Beckman system Gold HPLC
Gilson (very affordable system)
Hitachi components
Shimadzu 10 series
Waters 600E system with uv vis
detector
Kontron HPLC pumps
Pharmacia FPLC
Perceptive Biosystems BioCAD
Water 600E systems
When molecules in a mixture are
very similar, direct quantification
becomes difficult. HPLC is a
separating technology used to
separate very similar molecules from
a mixture and then quantify them
accurately. It is a member of a group
of technologies known as
"CHROMATOGRAPHY".
In chromatography, the components
are separated on the basis of their
chemical composition and structure.
Any molecule present in the mixture
will have its own unique composition
and structure and therefore will
have different interaction with its
surrounding. These interactions
make them all move uniquely on the
support provided to it. This support
is known as "stationary phase" and
the energy to move is provided by a
liquid or a gas "mobile phase".
On the basis of the material being
used as either phase, the
chromatographic technique is titled

filter to prevent valve and column

damage. Some systems utilize an
automatic injector or autosampler to
accomplish this task.
Following the injector, an analytical
column allows the primary sample
separation to occur. This is based on
the differential attraction of the
sample components for the solvent
and the packing material within the
column. However, a sacrificial guard
column is often included just prior
to the analytical column to
chemically remove components of
the sample that would otherwise foul
the main column.
Following the analytical column, the
separated components pass through
a detector flow cell before they pass
into the waste reservoir. The sample
components' presence in the flow
cell prompts an electrical response
from the detector, which is digitized
and sent to a recorder. The recorder
helps analyze and interpret the data.
Recorders can be as simple as a pen
recording what happens in the
detector onto moving chart paper or
as sophisticated as a computer with
powerful software for hardware
control and data interpretation.
As a final system enhancement, a
back pressure regulator is often
installed immediately after the
detector. This device solvent bubble
formation until the solvent is
completely through the detector.
This is important because bubbles in
a flow cell can interfere with the
detection of sample components.

accordingly.
HPLC (High Performance Liquid
Chromatography) is a mode of
chromatography wherein the
stationary phase is used in form of
fine particles packed in a cylindrical
column made up of inert material
like stainless steel. Stationary phase
adsorbs the analytes and holds them
reversibly for particular time. All
the molecules in the mixture will
spend different time with different
stationary phase and therefore come
out at the end one by one.
Adsorption being the base of
separation, usage of fine particles
offers more surface area for the
molecules to interact with. The time
that a molecule takes to travel from
one end to other end of the
stationary phase is known as the
RETENTION TIME. Hence all
molecules will display their own
unique retention time for a
particular stationary-mobile phase
combination.
Selection of stationary phase and
mobile phase is done on the basis of
the molecule of interest to get
optimum retention time. As a result,
HPLC acquires a high degree of
versatility not found in other
chromatographic systems and it has
the ability to easily separate a wide
variety of chemical mixtures.
Silicagel is the most frequently used
adsorbent. The adsorbent is packed
in a column. The density of the

Alternatively, an inert gas sparging

system may be installed to force
dissolved gasses out of the solvent
being stored in the solvent reservoir.
Each of the components described
above requires fittings to couple it
into a system.
It is important to note that improper
selection or installation of these
fittings can lead to leaks or the
formation of dead volume, both of
which can result in poor HPLC
performance.
GMI can assist in component choice
or complete system configuration to
accomplish most lab and production
requirements. We spend extra time
in our reconditioning 'up front' so
that you, our customer, receive a
COMPLETE working system upon
arrival to your laboratory.
HPLC LINGO:
ELUTION: This term describes the
transport of a species through the
stationary phase by the continuous
flow (addition) of mobile phase.
ELUANT: Mobile phase that carries
the sample through the column.
ELUATE or EFFLUENT: Mobile
phase with separated components
after they emerge from the column.
ISOCRATIC ELUTION: A
separation in which the mobile
phase composition remains
unaltered. The mobile phase may
comprise of a single solvent or a premixed mixture of solvents.
GRADIENT ELUTION: HPLC is
frequently used for the separation of

packing directly affects the

resolution. Hence the HPLC
columns are densely packed with the
adsorbent to achieve significant
degree of resolution. But very high
density of packing demands for the
mobile phase to be introduced at
very high pressure to generate
effective flow. So the technique is
also sometimes named as HIGH
PRESSURE LIQUID
CHROMATOGRAPHY.
At the end of the column, a detector
is kept. As the molecules come out
from the end, the detector detects
and gives signal. Till the molecules
keeps traveling through the
detector's path it keeps detecting it
and gives a peak on
CHROMATOGRAM. The peak
area calculation quantifies the
molecule of interest on the basis of
standard values.
Remarkable interference can be
observed by minor changes in the
stationary and mobile phase
composition, purity and/or density.
Therefore it is must to get standard
grade reagents for HPLC. Such
quality control requirements makes
HPLC a little expensive technology
as compared to other similar
technologies.
GMI staff have performed
chromatography and can assist you
with your technology selection. You
never have to be embarrassed by
your level of knowledge with these

mixtures that contain compounds

with a wide range of polarities. In
such situations, isocratic conditions
may not provide an acceptable
separation (i.e., it is not possible to
obtain sufficient resolution or the
separation takes an unacceptably
long period of time). To solve these
problems, the composition of the
mobile phase is changed during the
separation. Two or three solvents
that differ in polarity are employed.
After sample introduction, the ratio
of these solvents is programmed to
vary either continuously or in steps,
resulting in enhanced separation
efficiency.
The terms binary gradient, ternary
gradient, and quaternary gradient
refer to the use of 2, 3, and 4
solvents, respectively, to make up the
mobile phase composition in a
gradient elution method.
CHROMATOGRAM: When a
detector that responds to solute
concentration is placed at the
column outlet, a plot of the
generated signal versus time (or
volume of mobile phase) is called a
chromatogram. Such a plot, which
usually comprises of one or more
peaks, may be used for qualitative
and quantitative purposes the
location of a peak on the time (or
volume) axis serves to identify the
component, and the area under the
peak provides a quantitative
measure of that component.
SENSITIVITY: Minimum limit of
detection of a given species.

technologies since it is our goal to

HELP you with intelligent and
educated decisions in procuring
equipment. We will never 'over sell'
you or talk you into unneeded
purchases unlike some 'dealers' with
the sole goal of unloading their
inventory.
GMI IS
on the lookout for your un-needed
instrumentation
especially your HP / Agilent HPLC's,
Waters,
and Perceptive Biosystems BioCADS

DISTRIBUTION CONSTANT: It
describes the equilibrium involving
the transfer of an analyte between
the mobile and stationary phases.
This constant, also called partition
ratio or partition coefficient, is
defined as K= Cs/Cm, a ratio of the
analyte molar concentration in the
stationary phase to that in the
mobile phase.
RETENTION TIME, t: The time
taken by the analyte peak to reach
the detector after sample
introduction is called the retention
time. A more accurate measure of
the retention time of an analyte is
obtained by subtracting from this
value the time taken for an
unretained solute to emerge from

Determined by the smallest ratio of

peak height-to-baseline noise
(signal-to-noise ratio) that allows
accurate and reproducible
determination of peak height or
area. This varies with detection
method, instrument used and species
being detected.
UNRETAINED COMPOUND:
Component of a mixture that moves
through a column at the same rate
as the mobile phase, i.e., migration is
not retarded by physical or chemical
interaction with the stationary
phase.
VOID VOLUME: Total volume of
mobile phase in a fully wet packed
column - the space between the
particles of the stationary phase
(interstitial volume) plus the volume
within the particles (pore volume). It
is also defined as the volume of
mobile phase required to carry an
unretained component through a
column.
RESOLUTION: Measure of the
degree of separation between two
successively eluting components in a
chromatographic run (two adjacent
peaks in a chromatogram).
The resolution between species A
and B may be expressed as:
R = 2 [tB tA]/ (WA + WB), where t
and W correspond to the retention
time and peak width at baseline,
respectively. A resolution value of 1.5
implies a complete separation of the
two species

the column (i.e., the dead time, t0),

resulting in the adjusted retention
time, t. The retention time is the
most important parameter for
component identification under set
experimental conditions.
CAPACITY FACTOR (or Retention
Factor) k : A measure of the
retention volume (or time) of a
particular component of the sample
with a given combination of
stationary phase and mobile phase.
It is defined for species A as k A =
(tA-t0)/t0, or, kA = tA/t0 where t0
is the retention time for an
unretained compound and tA =
adjusted retention time of species A.
ALPHA ( ) VALUE: A measure of
the separation of any two
components under a given set of
conditions. It is defined for two
components A and B as: = k
A/k B (where k is the respective
capacity factor). It is the ratio of the
retention volumes of the two
components; i.e., their relative
retention.
BAND (or Zone): Layer of sample or
component moving through the
stationary phase, carried by the
mobile phase. Represented on the
chromatogram as a peak when it
emerges from the column.
BAND-BROADENING (or Zonebroadening): Spreading of the
sample or component band. Arises
due to inefficiency of the column bed
or inappropriate choice of mobile
phase.
COLUMN EFFICIENCY: Degree to

which species flow through the

column as "bands", without being
spread; less band-broadening
implies less likely overlap of peaks in
the chromatogram. Efficiency is
expressed numerically as Plate
Count, N, or as a Height Equivalent
to Theoretical Plate (HETP), H. It is
a measure of the quality of a filled
column.
HETP, H = L/N
number of plates N = 16(t/W)2
where L = column length, t =
retention time, and W = peak width
at baseline.
The origin of these terms is from the
treatment of the chromatographic
column as made up of a number of
discrete narrow bands called
theoretical plates, similar to a
distillation column.