High performance liquid chromatography (HPLC), also known as high pressure liquid chromatography, is essentially a form of column chromatography in which the stationary phase consists of small particle packings (3-50 µm) contained in a column with a small bore (2-5 mm), one end of which is attached to a source of pressurised liquid eluant (mobile phase)
3. 3
High performance liquid chromatography (HPLC), also
known as high pressure liquid chromatography, is
essentially a form of column chromatography in which
the stationary phase consists of small particle packings
(3-50 µm) contained in a column with a small bore (2-5
mm), one end of which is attached to a source of
pressurised liquid eluant (mobile phase).
The four forms of high performance liquid
chromatography most often used are Partition,
Adsorption, ion-exchange and Size exclusion,.
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The origins of Liquid
Chromatography began
in the early 1900’s with
the work of the Russian
botanist, Mikhail S.
Tswett. His famous
studies focused on
separating compounds
(leaf pigments), which
were extracted from
plants using a solvent.
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▪ Prior to the 1970's, few reliable chromatographic
methods were commercially available to the
laboratory scientist. Like open-column
chromatography, paper chromatography, and
thin-layer chromatography.
▪ High pressure liquid chromatography was
developed in the mid-1970's and quickly improved
with the development of column packing materials
and the additional convenience of on-line
detectors.
9. Disadvantages of
Conventional column Chromatography
▪ Mobile phase moves under gravity feed or low pressure
pumping
▪ Slow flow rates.
▪Separation times are higher , even in hrs.
▪ Sample is to be collected separately & then passed on to
the detector.
▪ No good efficiency.
▪ Not very reproducible.
10. Advantages of HPLC over
Conventional column Chromatography
▪ Provides a specific sensitive and precise
method for analysis of complicated samples.
▪ Ease of sample preparation & introduction.
▪ Speed of analysis
▪ Analysis is specific, accurate & precise.
▪ Substances that are thermolabile, or have low
volatility could be analysed by HPLC, unlike GC.
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Isocratic elution & Gradient elution
✓There are 2 basic types of chromatographic
mobile phase operating modes which can be
used in HPLC. The first type is called “Isocratic”.
In this mode, the mobile phase composition
remains the SAME throughout the run.
✓The second type is called “Gradient”
chromatography. This type is used for very
complex samples with many compounds. In this
mode, the mobile phase composition is
CHANGED during the separation. As the
separation proceeds, the elution strength of the
mobile phase is strengthened..
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Gradient elution can be carried out by two ways
▪ Use a system where there are two bottles of solvents
and two pumps. The speed of each pump is controlled
(Gradient Controller) over time to deliver more or less of
each solvent.
The two streams are mixed (Mixer) to create the mobile
phase gradient over time. At the beginning, the mobile
phase is made up of mostly the weaker solvent. As the
time progresses, the proportion of the stronger solvent
goes up
▪ Another system design uses a single pump and multiple
solvent bottles. The flow from each bottle is controlled by
a gradient proportioning valve which creates the desired
solvent strength
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i] Mobile Phase Reservoir &
solvent treatment system
▪ Reservoirs are made up of stainless steel.
▪ Solvent treatment involves degassing & mixing
them in proper proportions if required.
▪ Degassing can be done by following different
methods
1. Spraying (on-line)
2. Heat (off-line)
3. Vacuum (off-line)
4. Sonication (off-line)
5. Distillation (off-line)
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ii] Pumps
▪ Required to deliver a constant flow of mobile phase at a
pressure ranging from 1 to 550 bar ( 0.1-55 MPa;
14.6-8000 psi)
▪ Those with very high pressure provide wide range of flow
rate
Low flow rates(10-100 microlit/min.) microbore
columns,
Intermediate flow(0.5-2 ml/min.) conventional analytical
HPLC columns,
High flow rates preparative or Semi preparative
columns & for slurry packing techniques.
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• Criteria for selection of pump
1. Reproducible mobile phase flow rate.
2. Baseline pulsation should be minimum To
minimize detector noise for trace analysis.
3. Should be suitable for operation at various
pressures (3000-6000 psi).
4. Provide constant flow rate.
5. Made up of materials resistant or chemically
inert to the chemical & solvents commonly
used.
6. Adaptable to gradient analysis.
7. Suitable for use of small volumes of solvents.
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8. Protect from Evaporation.
9. Low maintainance cost.
10.Suitable for operation at variable flow rates
Analytical : 0.5 to 2ml/min.
Preparatie : 2 to 10ml/min.
Mcrobore columns: microlit/min.
11. Should have small hold up volume.
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Types of pumps
1) Syringe type or screw driven pumps
e.g. Displacement pump.
2) Reciprocating pumps
a. Single piston pump
b. Dual piston pump
c. Reciprocating diaphragm pump.
3) Pneumatic pumps
a. Direct pressure pumps
b. Amplifier pumps
4) Constant pressure pumps
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1. Syringe type / Displacement pump.
Mobile phase
Seal
Piston
MOTOR
Lead screw
Outlet
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ADVANTAGES DISADVANTAGES
i. Pulse free delivery
at high pressure
ii. Independent of
viscosity of solvent
i. Limited solvent
capacity
ii. Change of solvent is
inconvenient
iii. Suitable for batch
operation & no
continuous
operation possible.
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ADVANTAGES DISADVANTAGES
i. Constant flow rates
ii. Independent of viscosity
of solvent
iii. Suitable for continuous
operation.
iv. Gradient elution
possible
v. Relatively inexpensive
vi. Wide range of flow
rates achieved.
i. Pulsating output.
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3. Pneumatic pumps
ADVANTAGES
i. Inexpensive
ii. Easy to operate
iii. Pulse free
DISADVANTAGES
i. Flow rate dependant
on viscosity of
solvent.
ii. Gradient operation
is difficult.
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4. Constant pressure pumps
ADVANTAGES
i. High liquid pressure
achieved
ii. Valving
arrangement
provide rapid refill of
solvent chamber
(capacity 70ml)
iii. Pulse less &
continuous pumping
DISADVANTAGES
i. Inconvenient for
gradient analysis.
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iii] Injectors
▪ The samples should be introduced with
high degree of reproducibility for better
efficiency.
▪ Overloading band broadning
▪ Sample injection should not disturb the
pressure system.
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▪ Sample injectors/ Injection ports are of 3 basic types
1. Syringe injection:
- sample is injected directly into the column through a self
sealing septum with a syringe that can withstand pressure
up to 1500 psi.
2. Stop flow injectors:
- injected with syringe only but by removing upper head of
the column after stopping the solvent flow momentarily.
3. Solvent flow injectors:
- sample is deposited before the column inlet & then
swept by a valving action into the column by the mobile
phase. i.e. injection valves. This does not disturb
pressure system at all.
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iv]Columns
▪ The columns are made up of high quality
stainless steel polished internally to
mirror finish.
▪ Smaller diameter & longer length of
column ensures better efficiency, as it
increases no. of theoretical plate but
shorter length provides faster separation.
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▪ Types of columns
1. Analytical columns
(length 10-25 cm in & internal diameter 2-6 mm)
2. Preparative columns
(length 25-100 cm in & internal diameter 6 mm or more )
3. Guard columns
protective, have large diameter packings.
▪ Column thermostat:- For certain applications, close control of column
temperature is required. Better chromatograms are by maintaining
column temp. constant to a few tenths degree centigrade. Generally
columns have heaters that maintain temperature between 100-150ْ C.
Columns may also be fitted with water jackets fed from a constant temp.
bath to give precise temp. control.
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▪ Types of column packings:-
I. Pellicular
II. Porous particle
The material varies with the type of interaction involved in
separation.
a. Adsorption HPLC e.g. HPLC grade silica
b. Partition HPLC
i] Normal phase HPLC e.g., ethane 1-2 diole (polar) &
ii] Reversed phase HPLC e.g., Octadecylsilane (non-polar)
c. Ion exchange HPLC e.g., ion exchange residues chemically
bonded with silica.
d. Size exclusion HPLC e.g., sephadex
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v]Detectors
✓ Classification
1. Photometric detectors
a) Single wavelength detectors
b) Multiwavelength detector
c) Variable wavelength detector
d) Programmable detector
e) Diode array detector
f) Fluorescence detector
2. Refractive index detectors
3. Mass spectrometry (MS)
4. Other detectors
▪ Electrochemical (ECD), conductivity, infrared, mass
(evaporative), radioactivity and post-column reaction system
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Characteristics of a UV/Vis Detector
UV/Vis absorbance detector typically consists of :
⚫ A deuterium source (190 - 700 nm)
⚫ A monochromator involving a moveable grating controlled by
stepper motor to select a certain wavelength through the exit
slit to a small flow cell (about 10 mL)
⚫ Two photodiodes to measure the light intensity of the sample
and reference beam
Principle for absorbance detector is the Beer’s Law
Absorbance = molar absorptivity x pathlength x concentration
A = e b c = - Log I / I0 where I0 = Initial light intensity
It is the most common detector for HPLC, capable of ng detection
⚫ Noise/drift characteristics important for sensitivity
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The most powerful UV/Vis absorbance detectors in use today are
photodiode-array (PDA) based instruments
It permit that very rapid collection of data over a selected spectral
range. Thus, spectral data for each chromatographic peak can be
collected and stored.
This stored data may then be compared with the spectrum of a
pure standard from a library - a spectral analysis study of peak
purity.
The PDA detector is very useful for the identification of
components that are difficult to resolve (overlapping peaks) since the
characteristic spectrum for each of the unresolved components is
likely to be different.
PDA Detectors
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Photodiode Array Detector: Principles
Principles and components similar to that of UV/Vis detector
A flow cell is placed before a stationary grating and passes the
entire light spectrum of the light source
A diode array with many elements measures the spectral intensity
of each wavelength
Records spectra to assist the determination of peak identity and
purity
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Sterile exclusion , aqueous
mobile phase
Sterile exclusion , non-
aqueous mobile phase
Water soluble
MW>2000
Sample
MW<2000
Nonionic
Ionic
Sterile exclusion ,
aqueous mobile phase
Sterile exclusion ,
aqueous mobile phase
Anion exchange or ion pair
Cataion exchange or ion pair
Acidic
Basic
Water insoluble
Low
polarity
Medium high
polarity
