liquid chromatography

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LIQUID CHROMATOGRAPHY
M.PRASAD NAIDU Msc Medical Biochemistry, Ph.D
Research scholar.
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LIQUID CHROMATOGRAPHY
1. Introduction
2. Retention Mechanisms in Liquid Chromatography
3. Method Development
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Liquid Chromatography
Liquid Chromatography (LC) is a chromatographic
technique in which the mobile phase is a
liquid. LC is a much older technique than GC,
but was overshadowed by the rapid development of
GC in the 1950s and 1960s. LC is currently
the dominate type of chromatography and is even
replacing GC in its more traditional
applications. Advantages of LC compared to
GC LC can be applied to the separation of any
compound that is soluble in a liquid phase. LC
more useful in the separation of biological
compounds, synthetic or natural polymers, and
inorganic compounds Liquid mobile phase allows
LC to be used at lower temperatures than required
by GC LC better suited than GC for separating
compounds that may be thermally labile
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Advantages of LC compared to GC
(continued) Retention of solutes in LC depend
on their interaction with both the mobile phase
and stationary phase. GC retention based on
volatility and interaction with stationary
phase LC is more flexible in optimizing
separations ? change either stationary or mobile
phase Most LC detectors are
non-destructive most GC detectors are
destructive LC is better suited for preparative
or process-scale separations Disadvantage of LC
compared to GC LC is subject to greater
peak or band-broadening. RESOLUTION!!!! much
larger diffusion coefficients of solutes in gases
vs. liquids
Low- and High-performance Liquid
Chromatography Many types of liquid
chromatography are available, based on different
stationary phase and mobile phase
combinations. - each type may be further
characterized based on its overall efficiency or
performance
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Low-performance liquid chromatography LC
methods that use large, non-rigid support
material particles gt 40 mm in diameter
poor system efficiencies and large plate
heights such systems have the following
characteristics broad peaks poor limits of
detection long separation times columns can
only tolerate low operating pressures
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Column chromatography an example of the
equipment used in low-performance liquid
chromatography
Solvent reservoir
Column head
Column
Column packing
Porous glass plate
Sample is usually applied directly to the top of
the column. Detection is by fraction collection
with later analysis of each fraction
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High-performance liquid chromatography (HPLC)
LC methods that use small, uniform, rigid
support material particles lt 40 mm in
diameter usually 3-10 mm in practice good
system efficiencies and small plate heights
(H!) such systems have the following
characteristics narrow peaks shorter
separation times
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A typical HPLC system

• Higher operating pressures
• need for mobile phase
• delivery requires special
• pumps and other system
• components
• Sample applied using
• closed system (i.e.,
• injection valve)
• detection uses a flow-through detector

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Similar to GC, solutes can be eluted from a
column by using either a constant column
conditions or gradient elution Isocratic
elution use of a constant mobile phase
composition to elute solutes simple,
inexpensive difficult to elute all solutes with
good resolution in a reasonable amount of time
? general elution problem Gradient elution
changing the composition of the mobile phase with
time ? solvent programming going
from a weak mobile phase to a strong one. weak
mobile phase ? solvent A strong mobile phase ?
solvent B solvent change can be stepwise, linear
or non-linear
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Gradient elution of mixture of 30 amino-acids
In choosing a mobile phase for LC, several
factors need to be considered type of
stationary phase used determines what will be
a strong or weak mobile phase solubility of
the solutes viscosity of the mobile phase
type of detector used and solvent's background
signal purity of the solvents miscibility
of the solvents (for gradient elution)
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LIQUID CHROMATOGRAPHY
1. Introduction
2. Retention Mechanisms in Liquid Chromatography
3. Method Development
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Types of Liquid Chromatography (Retention
Mechanisms ) Techniques in LC are classified
according to the method of solute
separation Adsorption chromatography Affinity
chromatography Partition chromatography
Size-exclusion chromatography Ion-exchange
chromatography
1. Adsorption chromatography (Liquid-solid
Chromatography)

• A LC technique which separates solutes based on
  their adsorption to an underivatized solid
  particles is known as adsorption chromatography,
  or liquid-solid chromatography.
• This technique is suited for non-polar
  small compounds (MWlt5000).

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b. One advantages of adsorption chromatography,
as is also true for GSC, is that it is able to
retain and separate some compounds that can not
be separated by other methods. One such
application is in the separation of geometrical
isomers.
c. Mechanism (a) Retention of solute in
adsorption chromatography can be viewed as solute
A displacing n moles of solvent M from a surface.
Amp nMsp Asp nMmp
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2. Partition chromatography (Liquid-liquid
Chromatography)
a. Partition chromatography, or liquid-liquid
chromatography is a Chromatographic technique in
which solute are separated based on their
partition between a liquid mobile phase and a
liquid stationary phase coated on a solid
support.
b. The support material used in partition
chromatography is usually silica. Un-bonded and
banded stationary phase.
c. Mechanism
The retention of solute in partition
chromatography is given by
k KD (Vs/Vm)
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d. Applications of partition Chromatography
Normal Phase LC and Reversed-phase LC
Stationary phase polar
non-polar
How can ions be separated by using partition
Chromatography?
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e. Ion-pair Chromatography
(a) Ion-pair chromatography is used for the
separation of ionic and ionizable compounds and
mixtures of neutral and ionic compounds.
(b) In this method, counterions (species of
opposite charge to the solutes) thereby regulate
the retention. Typically alkyl amines or tetra
alkyl amines are added to ion pair with acids
whereas alkyl sulfates, sulfonates, or phosphates
are used to ion pair with bases
A- B A-B
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3. Ion-Exchange Chromatography
a. Ion-exchange chromatography is a liquid
chromatography technique in which solutes are
separated by their adsorption onto a support
containing fixed charge onto a support containing
fixed charges on its surface.
b. Ion-exchange is a fairly common technique used
in water softeners and in the industrial removal
or replacement of ionic compounds for products.
Ion-exchange is used in chromatography for
separation of a wide variety of charged
compounds, including inorganic ions, organic
ions, and biological compounds (such as amino
acids, proteins and nucleic acids)
c. Mechanism
n(supportA-B) Cn
(supportA-)nCn nB
K (BnCn/BnCn)
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4. Affinity chromatography
For biomolecule separation
Antibody-antigen
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5. Size-exclusion Chromatography for polymer and
bio-polymer
Retention of a solute is dependent on Standard
entropy effect. Diameter of the pore is very
important for solute selectivity. Multiple pore
sizes should be used for separate solutes with
different sizes.
GPC Gel permeation Chromatography (polymer
scientists) GFC Gel filtration Chromatography
(biochemists)
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LIQUID CHROMATOGRAPHY
1. Introduction
2. Retention Mechanisms in Liquid Chromatography
3. Method Development
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LC Method Development
Problem Definition
Mode selection
What type of LC should be used?
Stationary phase and mobile phase, Temperature
Selectivity Optimization
Column length, particle size, flow rate,
instrument configuration, sample injection
System optimization
Method Validation
Accuracy, sensitivity, specificity,
detection Limit, quantification limit, linearity
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Selection of a mobile phase for a particular LC
application can be done by using various tables
that summarize properties for common LC
solvents LIKE DISSOLVES LIKE!
Solvent Refractive Index Viscosity (cP) Boiling Point (oC) Polarity Index (P) Eluent Strength (eo)
Fluoroalkanes 1.27-1.29 0.4-2.6 50-174 lt-2 -0.25
cyclohexane 1.423 0.90 81 0.04 -0.2
N-hexane 1.327 0.30 69 0.1 0.01
1-chlorobutane 1.400 0.42 78 1.0 0.26
Carbon tetrachloride 1.457 0.90 77 1.6 0.18
i-propyl ether 1.365 0.38 68 2.4 0.28
toluene 1.494 0.55 110 2.4 0.29
Diethyl ether 1.350 0.24 35 2.8 0.38
tetrahydrofuran 1.405 0.46 66 4.0 0.57
chloroform 1.443 0.53 61 4.1 0.40
ethanol 1.359 1.08 78 4.3 0.88
Ethyl acetate 1.370 0.43 77 4.4 0.58
dioxane 1.420 1.2 101 4.8 0.56
methanol 1.326 0.54 65 5.1 0.95
acetonitrile 1.341 0.34 82 5.8 0.65
nitromethane 1.380 0.61 101 6.0 0.64
Ethylene glycol 1.431 16.5 182 6.9 1.11
water 1.333 0.89 100 10.2 large
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Mobile-phase selection in partition Chromatography


(P1 P2)/2
k2/k1 10
For NPLC (capacity factors)
(P2 P1)/2


k2/k1 10
For RPLC (capacity factors)
Example 28-1 In a reversed-phase column, a
solution was found to have A retention time of
31.3 min, while an un-retained species required
0.48 min For elution when the mobile phase was
30 (by volume) methanol and 70 water. Calculate
(a) k , (b) a water/methanol composition that
should bring k to a value of about 5, and (c)
the retention time of this solution under this
new mobile phase (assuming that the retention
time of the un-retained species is not changed).
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