Basic Principles of HPLC

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Basic Principles of HPLC
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H
igh

• P

erformance
L
iquid
C
hromatography
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Introduction

• HPLC is a form of liquid chromatography used to
  separate compounds that are dissolved in
  solution. HPLC instruments consist of a reservoir
  of mobile phase, a pump, an injector, a
  separation column, and a detector.
• Compounds are separated by injecting a sample
  mixture onto the column. The different component
  in the mixture pass through the column at
  differentiates due to differences in their
  partition behavior between the mobile phase and
  the stationary phase. The mobile phase must be
  degassed to eliminate the formation of air
  bubbles.

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HPLC system
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This unit is equipped with two pump units and a
UV/Vis detector. The gradient is controlled via
the pump controllers.
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LC methods are not as sensitive
to temperature. Columns are commonly mounted
outside the instrument.
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Solvents All solvents should be HPLC grade.
This is a type of reagent grade material. It
has been filtered using a 0.2 µm filter.
Filtered solvent helps extend pump life by
preventing scoring. It also reduces the chances
of a column plugging.
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Solvent degassing All solvents should be degassed
prior to use. This reduces the chances of bubbles
being formed in the column or detector. Oxygen
present at high pressure can also cause a
problem. Methods that can be used ! Displacement
with a less soluble gas ! Applying a vacuum !
Heating the solvent.
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HPLC Injectors
In order to introduce a sample onto the column
for analysis, a special valve called the injector
must be used to transfer the sample into the
pressurized system
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Injectors may look different from the outside, but internally, most are 6-port rotary valves. These valves consist of a fixed body (the "stator") plus an internal seal that rotates (the "rotor"). Three internal passages connect alternate pairs of external ports. The valves can switch between two positions, referred to as the "inject" and "load" positions, respectively. In the load position, the pump is connected to the column, and the sample inlet is connected to one end of a piece of tubing, called the sample loop. The other end of the sample loop is connected to the waste port. Rotation results in reconnecting the various lines that enter the valve, so that a sample volume can be inserted into the mobile phase that flows from the pump to the column inlet. Injectors may look different from the outside, but internally, most are 6-port rotary valves. These valves consist of a fixed body (the "stator") plus an internal seal that rotates (the "rotor"). Three internal passages connect alternate pairs of external ports. The valves can switch between two positions, referred to as the "inject" and "load" positions, respectively. In the load position, the pump is connected to the column, and the sample inlet is connected to one end of a piece of tubing, called the sample loop. The other end of the sample loop is connected to the waste port. Rotation results in reconnecting the various lines that enter the valve, so that a sample volume can be inserted into the mobile phase that flows from the pump to the column inlet.
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The column HPLC has seen significant improvement
over the last 20 years primarily due to
improved column technology. Packings are more
uniform and smaller. Phases are commonly
chemically bound to the packing. Packing methods
have improved.
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Pump types Isocratic pump - delivers constant
mobile phase composition solvent must be
pre-mixed lowest cost pump Gradient pump
-delivers variable mobile phase composition can
be used to mix and deliver an isocratic mobile
phase or a gradient mobile phase
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?? Reciprocation pumps --- advantages small
internal volume, high output pressures, ready
adaptability to gradient elution and constant
flow rates --- disadvantages produce a pulsed
flow, cause baseline noise. ?? Displacement
pumps --- advantages output is pulse free ---
disadvantages limited solvent capacity (lt250
mL) inconvenience when change solvents ??
Pneumatic pumps --- advantages inexpensive,
output is pulse free --- disadvantages limited
solvent capacity, not amenable to gradient
elution and limit pressures lt2000 psi
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Packings Originally, these were irregular silica
and alumina. A range of synthetic,
regularly shaped packings are now available. !
Porous - channels through packing !! Superficially
porous - rough surface !! Smooth - bead like.
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Packing size As packing size is decreased,
efficiency and pressure requirements are
increased. ! Common diameters for analytical
work diameter ! plates "" 10 µm "" 5000 "" 5 µm
"" 9000 "" 3 µm" " 15,000 All are for a 15 cm x
4.6 mm column
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Column body Typically consist of stainless steel
with a high precision internal bore. Some
manufacturers offer column inserts - " dont need
to repurchase the column " fittings. Others offer
columns where the external body can be compressed
to improve packing efficiency.
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HPLC column examples
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Column stationary phases Today, most packing fall
into four classes. Silica or alumina Bound phases
on either alumina or silica. Gels Controlled-pore
glass or silica
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Absorption phases - alumina ! common mobile
phases ! hexane, chloroform, 2-propanol. !
example application - amines. silica ! common
mobile phases ! hexane, chloroform, 2-propanol. !
example applications - ethers, esters, !
porphyrins, fat-soluble vitamins.
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Partition phases Can be broken down into ! Normal
phase - polar materials bound to the support. !
Reverse phase - non-polar materials bound to the
support. ! Mixed phase - ! may have some of each.
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Partition phases Normal ! Amino (-NH2) ! Cyano
(-CN) ! Diol (glycidoxy-ethylmethoxysilane) Rever
se ! C-2 or RP-2 (-Si-CH2CH3) ! C-8 or RP-8
(-Si-(CH2)7CH3) ! C-18 or RP-18
(-Si-(CH2)17CH3) Increasing the C number results
in a thicker, more retentive phase
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Ion exchange phases Strong cation ! - sulfonic
acid group Strong anionic !- quarternary
amine Weak anion ! - primary amine Weak cation !
- COOH
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Size exclusion phases Gels - organic or aqueous
based Controlled-pore - silica or glass Must be
selected based on pressure requirements and size
range required for your application.
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Capillary and Microbore columns. Several
companies have begun offering columns with
smaller ID. Microbore column - 1 mm ID,
packed column. Capillary column - lt 1 mm ID,
internal bound phase. These columns require
smaller solvent flows, reduced sample size and
improved detector design.
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Capillary and Microbore columns. Capillary and
Microbore columns. Aromatic Compounds mobile
phase!2 ethylacetate in hexane flow rate " 4
µl/min column ""Fusica II, 300µm I.D. x ! ! ! 25
cm silica sample ! 1. toluene ! 2. nitrobenzene !
3. acetophenone ! 4. 2,6-dinitrobenzene injection!
! 60 nl detection! ! UV 254 nm
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Silica based columns
Silica is the ideal support for HPLC columns. It
offers a large mechanical stability,
excellent physicochemical surface properties, a
wide range of bonding chemistries and is
compatible with a broad range of organic solvents.
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pH stability In general, HPLC columns are stable
within a pH range of 2 to 8. If you are measuring
a pH value, the measurement must be done in the
aqueous media before mixing the eluent with
organic solvents. Modern HPLC columns can be
used outside that pH range. The new bonding
chemistries allow use down to pH 1 for some
stationary phases. However, please check vendors
product information before using silica based
column outside the pH range of 2 to 8. However,
best lifetimes are obtained between pH 2.0 and pH
6.8.
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Mechanical stability Stationary phases based on
silica are mechanically very stable. The packed
columns show no pressure limit and can be used at
more than 40 MPa (6000 psi) without any problem.
However, please avoid pressure shocks on the
column. Pressure shocks lead to channelling in
the column, which results in peak splitting in
the corresponding chromatogram.
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• Mobile phases (Eluents)
• Silica based stationary phases are compatible
  with all organic solvents in the above mentioned
  pH range. .
• Filter all prepared buffer through a 0.5 µm
  filter before using them in your HPLC system

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The use of non pure solvents in HPLC causes
irreversible adsorption of impurities on the
column head. These impurities block adsorption
sites, change the selectivity of the column and
lead to peak splitting in the chromatogram. In
gradient elution, impurities cause so called
Ghost Peaks. Ghost peaks are peaks that always
appear in the same position on the chromatogram.
Their origin is not the sample, but the
impurities from the solvents or solvent
additives. Therefore, it is highly recommended to
run a gradientwithout injection in the beginning
of each method to determine the ghost peaks
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To avoid irreversible adsorption at the column
head, you should always use a pre-column. The use
of a pre-column increases the life time of a
column dramatically. In addition, a pre-column
can filter solid parts stemming from pump seals
or injection rotors. An alternative to a
pre-column is an in-line filter. These filters
are attached directly to the column. These
filters get rid of solid parts in the eluent but
will not avoid irreversible adsorption of organic
impurities
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Detector Systems Virtually every chemical and
physical property that can be measured
in solution has been look at. Detectors fall
roughly into two classes Bulk property - measures
an overall change in the mobile phase. Solute
property - measures a solute specific property.
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• Properties of a good detector
• A detector must provide
• high sensitivity, low detection limits,
• linearity,
• Reproducibility.
• This is true for any detector.
• Each detector will have specific advantages and
  will vary as to peak shape and spread, noise and
  flow/temperature dependence they have.

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• UV/Vis detector
• A solute property detector.
• Sample must exhibit absorption in UV/Vis range.
  Solvent must not absorb significantly at the
  measured wavelength.
• Types " Filter photometer - single
• Variable wavelength
• Multiwavelength.

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Refractive index detector Bulk property detector
- general purpose. Based on refraction of light
as it passes from one media to another. Presence
of a solute changes the refractive index of the
solvent.
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Heat of absorption detector A small amount of
heat is released when a sample absorbs on a
suitable surface. This detector can measure this.
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Electrochemical detectors A number of properties
have been evaluated Detector types ! ! Dielectic
constant ! ! Amperometric ! ! Conductometric ! !
Polarographic ! ! Potentiometric
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Dielectric constant detector Bulk property
detector. Measures changes in polarity of the
liquid phase passing through the
cell. Conductometric detector Measures
conductivity of the solvent. Useful for solutions
of ions.
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Amperometric detectors Most frequently applied
type of electrochemical detector. A known
potential is applied across a set of electrodes -
typically a glassy carbon type. Ability to
oxidize or reduce a species can
be measured. Typically limited to working with a
specific class of materials per analysis
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Partitioning

• Separation is based on the analytes relative
  solubility between two liquid phases

Stationary Phase
Mobile Phase
Solvent
Bonded Phase
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HPLC - Modes

• Normal Phase.- Polar stationary phase and
  non-polar solvent.

Reverse Phase. - Non-polar stationary phase
and a polar solvent.
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Common Reverse Phase Solvents

• Methan0l

CH3OH
Acetonitrile
CH3CN
Tetrahydrofuran
Water
H2O
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Columns

• Solid Support - Backbone for bonded phases.
• Usually 10µ, 5µ or 3µ silica or polymeric
  particles.
• Bonded Phases - Functional groups firmly linked
  (chemically bound) to the solid support.
• Extremely stable
• Reproducible
• Guard - Protects the analytical column
• Particles
• Interferences
• Prolongs the life of the analytical column

Analytical - Performs the separation.
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Bonded Phases

• C-2 Ethyl Silyl -Si-CH2-CH3

• C-8 Octyl Silyl -Si-(CH2)7-CH3

• C-18 Octadecyl Silyl -Si-(CH2)17-CH3

• CN Cyanopropyl Silyl -Si-(CH2)3-CN

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Instrumentation
Gradient Controller

Column
Pump
Detector
Injector
Mobile Phases
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Detectors

• UV
• Single wavelength (filter) 610, 8330
• Variable wavelength (monochromator) 8316, 8325
• Multiple wavelengths (PDA) 555
• Fluorescence 610
• Electrochemical 605
• Mass Spectrometric 8325

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Chromatograms
Restek ULTRA C-18 and CN Columns (250mm x 4.6mm,
5µ), Mobile Phase (11 MethanolWater), 1.5
mL/min.
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Chromatograms
A
B
Supelcosil LC-PAH Columns.
Conditions A 150mm x 4.6mm, 5µ. Flow Rate 1.5
mL/min
Conditions B 50mm x 4.6mm, 3µ. Flow Rate 3.0
mL/min