PCG Matthias

HPLC
Liquid chromatography
1.Adsorption- competition between liquid mobile phase and
solid absorbent
2.Partition - competition between liquid and stationary phase
3.Size reduction
4.Affinity
5.Ion exchange
What is HPLC?
The most widely used analytical separations

technique - Utilizes a liquid mobile phase & packed
column to separate components of mixture
uses high pressure to push solvent

through the column
Popularity:
sensitivity
-ready adaptability to accurate quantitative

determination
-suitability for separating nonvolatile species or

thermally
fragile ones
History
Early LC carried out in glass columns

diameters: 1-5.cm
-lengths: 50-500cm
Size of solid stationary phase
-diameters: 150-200 um Flow rates still low 1

Separation times long!
Decrease particle size of packing causes increase in

comm
efficiency! diameters 3-10 um
This technology required sophisticated instruments

-new method called HPLCPopularity:
-widespread applicability to substances that are of
prime interest to industry, to many fields of
science, and to the public
Ideally suited for separation and identification of

amuno acids, proteins, nucleic acids, hydrocarbons,
carbohydrates, pharmaceuticals, pesticides, pent
antibiotics, sterolds, and a variety of other inorgan
substances
Advantages of HPLC
1.Higher resolution and speed of analysis

2.Greater reproducibility due to close control of the
parameters
1.affecting the efficiency of separation
2.Easy automation of instrument operation and
data analysis
3.Adaptability to large-scale, preparative
procedures
Advantages of HPLC are result of 2 major advances:

-stationary supports with very small particle sizes
and large
surface areas
-appliance of high pressure to solvent flow
Parameters used in HPLC

1.Retention parameters
2.Column efficiency parameters
Retention: When a component in a sample

interacts with the stationary phase in the column
and a delay in elution occurs
Column efficiency: Goodness of a colum

Composition of HPLC System
1.Solvent
2.Solvent Delivery System (Pump)
3.Injector
4.Sample
5.Column
6.Detectors
7.Waste Collector
Recorder (Data Collection)
Mobile Phase Degassing
Dissolved gases in the mobile

phase can come out of
solution and form bubbles as the pressure changes
from the column entrance to the exit
- May block flow through the system Sparging is

used to remove any dissolved gas from the mobile
phase
An inert and virtually insoluble gas, such as helium,

is forced into the mobile phase solution and drives
out any dissolved gas.
Degassing may also be achieved by filtering the

mobile phase under a
vacuum
Solver
Are used to store Mobile-Phase. The solvent

reservoir must be made of inert material such as
glass and must be smooth so as to avoid growth of
microorganisms on its walls. It can be transparent
or can be amber colored. A graduated bottle gives
a rough estimate of mobile-phase volume in the
bottle. Solvent reservoirs are placed above HPLC
system (at higher level) in a tray. They should never
be kept directly above the system as any spillage of
solvent on the system may damage electronic parts
of
HPLC.
Isocratic elution:
Use of a constant-composition mobile phase in

liquid chromatography
Gradient elution:
Vary the mobile phase composition with time
-If there is a wide polarity range of components to

be eluted.
- Allows for faster runs.
- Ex: mobile phase composition

can be programmed to vary from 75% water: 25%
acetonitrile at time zero to 25% water: 75%
acetonitrile at the end of the run.
- More polar components will tend to elute first.
• More non-polar components will elute later in

the gradient
Common Reverse Phase Solvents
1.Methanol CH,OH
2.Acetonitrile CH,CN
3.Tetrahydrofuran
4. Water
Tubing
1.Very small inner diameter

2.Consistent i.d.
3.Very strong
4.Easy to cut
5.Fittings available
Auto samplers
"Are fully automatic injection

systems enabling greater productivity and the
highest level of precision.
"The HPLC Autosampler incorporates an elegant

swivel head
that allows a manual injection and a special

Rheodyne injection
valve to give accurate full or partial loop fill

injections.
Columns
1.Solid Support - Backbone for bonded phases.
Usually 10p, 5u or 3u silica or polymeric particles.
2.Bonded Phases - Functional groups firmly linked
(chemically bound) to the solid support.
-Extremely stable
- Reproducible
3. Guard column - Protects the analytical column:
Prolongs the life of the analytical column
-Analytical column- Performs the separation.
Column Packing
-Usually spherical silica particles of uniform
diameter (2-10μm)
-The smaller particles yield higher separation

efficiencies.
-The silica particles are very porous
-Allows for greater surface area for

Interactions between the stationary phase and the
analytes.
Other packing materials may also be used:
- Zirconia (ZrO,)
Particle Diameter
1.Has a greater effect on resolution than column

length
1.Short columns with small particles ideal
2.5μm is standard size
3.3μm better, but restricted range of packings
available
4.Downside is high back pressure and issues with
retention of small particles inside the column,
blockages
Detector
*Detect various compounds as they elute out from

column. The detector gives response in terms of a
milivolt signal that is then processed by the
computer (integrator) to give a chromatogram.
Basically detector consists of a flow-cell through
which the mobile phase and resolved sample
moves optics shine through the detector cell and
variation in optical properties are detected.
-A Ultra violet or UV detector detects absorbance

of UV light by chromophores in the analyte
compound.
-A refractive index detector will sense variation in

refractive index of mobile phase stream passing
through flow-cell
- Similarly Fluorescence Detectors

checks for Florescence.
VOIN WANTE
Various Detetectors are Listed below
1.Ultraviolet (UV)
2.Fixed wavelength detector
3.Variable wavelength detector
4.Diode Array
5.Fluorescence
6.Electrical Conductivity Index
7.Electrochemical
8.Light scattering
9.IR Absorbance
10. Mass-Spectrometric
APPLICATIONS OF HPLC
1.Assay of cephalosporins
2.Assay of theophylline of barbiturates,
phenothiazines, benzodiazepine derivatives,
rauwolfia alkaloids etc. by C-18 reversed phases
3.Quantitative analysis of several analgesics like
aspirin, caffeine, paracetamol, phenacetin, etc.
4.Analysis of urine and serum samples
5.Separation of antipyrine and benzocaine in ear
drops
6.Separation of steroids like cortisone and cortisol
which are used in the treatment of
1.Rheumatoid arthritis.
2.Assay of hydro cartisone, fluocinolone
acetonide,triamcinolone acetonide in tablets and
other formulations
3.Analysis of oestrogen in female urine.
4.Analysis of lipids.
5.Separation of tropane akaloids, ergot alkaloids,
indole alkaloids, cinchona alkaloids, etc.
6.Analysis and separation of amino acids and
proteins
7.Analysis of carbohydrates.

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HPLC

The most widely used analytical separations

uses high pressure to push solvent

-ready adaptability to accurate quantitative

-suitability for separating nonvolatile species or

Early LC carried out in glass columns

Size of solid stationary phase

-diameters: 150-200 um Flow rates still low 1

Decrease particle size of packing causes increase in

efficiency! diameters 3-10 um

This technology required sophisticated instruments

Ideally suited for separation and identification of

1.Higher resolution and speed of analysis

Advantages of HPLC are result of 2 major advances:

-appliance of high pressure to solvent flow

Parameters used in HPLC

Retention: When a component in a sample

Column efficiency: Goodness of a colum

Mobile Phase Degassing

Dissolved gases in the mobile

solution and form bubbles as the pressure changes

from the column entrance to the exit

- May block flow through the system Sparging is

An inert and virtually insoluble gas, such as helium,

Degassing may also be achieved by filtering the

Are used to store Mobile-Phase. The solvent

Use of a constant-composition mobile phase in

Vary the mobile phase composition with time

-If there is a wide polarity range of components to

- Allows for faster runs.

- Ex: mobile phase composition

- More polar components will tend to elute first.

• More non-polar components will elute later in

Common Reverse Phase Solvents

1.Very small inner diameter

"Are fully automatic injection

"The HPLC Autosampler incorporates an elegant

that allows a manual injection and a special

valve to give accurate full or partial loop fill

-Analytical column- Performs the separation.

-The smaller particles yield higher separation

-The silica particles are very porous

-Allows for greater surface area for

Other packing materials may also be used:

1.Has a greater effect on resolution than column

*Detect various compounds as they elute out from

-A Ultra violet or UV detector detects absorbance

-A refractive index detector will sense variation in

- Similarly Fluorescence Detectors

Various Detetectors are Listed below

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