LIQUID CHROMATOGRAPHY

Group No:14
110119029-CHAPALA CHATRAPATHI KARTHIK
110119031- CHINTAPALLI SIVA MANIKANTA
110119053 -KUNDRAPU LOHIT
 Liquid Chromatography 

 Liquid chromatography extracts the molecules in the liquid mobile phase using a

stationary phase that is solid or an immiscible liquid.

 Liquid chromatography is used for analytical or separature applications

 Chromatography with a liquid mobile phase, works very well with liquid samples

(especially food testing, environmental samples, and biotechnology applications)

 Can be both qualitative (retention time or volume) or quantitative (peak area or peak

volume—standards)

 LC chromatograms must show good resolution (difference in retention) and be

based on a column set up with good efficiency (narrow peaks).

 LC: Analyte Requirements

 Must be able to place analyte into a liquid that can be injected into the column

 Since it is usually possible to find some solvent for the analyte, LC has

become very popular (especially for analytes that cause problems in GC, such

as polymers and biological compounds).

 Since volatilization is not required, LC can be run at lower temperatures (less

likely to disrupt analyte).

 Analyte interaction will be with both the stationary and mobile liquid phases

(better resolution than GC with more flexibility in controlling retention times).

Block Diagram of Liquid Chromatography
 Components of liquid Chromatographic technique

Injection Site:
The sample or the mixture here does not have mobility. Hence it required a
mobile phase to carry it forward. The injection site is where this sample
mixture is introduced in the column.

Packed column:

 The columns are commonly made from the stainless steel and thick glass
polymers such as polyether ether ketone, a combination of stainless-steel and
glass or an amalgamation of stainless-steel and polymers are also used.
  Typically LC analytical columns range from 3 to 25 centimetres long with
the diameter of 1 to 5 mm. these columns are commonly straight unlike that
of a gas chromatography columns.
 Particles used for packing columns have a characteristic diameter ranging
from 5 to 10 millimetre

Detectors:

 It detects the constituents of the mixture that are being eluted of the
chromatography column
 The first effective affected in line liquid chromatography detector was
refractive index detector.
 The other detectors used are UV detectors fluorescence detectors mass
spectrometers and electro chemical detector.
 LC: Role of the Mobile Phase 

  Strong Mobile Phase: a solvent or solution that quickly elutes

analyte (analyte is more soluble in the mobile phase)

 Weak Mobile Phase: a liquid that slowly elutes analyte (analyte has

better solubility in the stationary phase) Isocratic Elution: using

constant composition in the mobile phase (recall the “general elution

problem”)

 Solvent Programming: Begin with a weak mobile phase (A), then

switch over time to stronger mobile phase (B).

Types of Liquid Chromatography
 Adsorption chromatography
• Liquid-Solid chromatography

• Solutes are separated based on their different

abilities to adsorb to the support's surface

• Uses an underivatized solid support

(stationary phase = solid support)

• Oldest type of chromatography

 Partition chromatography

 Liquid-Liquid chromatography

 Solutes are separated based on their

different abilities to partition between the

stationary phase and mobile phase

 Uses a solid support coated or chemically

derivatized with a polar or non-polar layer

 Most common type of liquid

chromatography at present
 Gel-permeation chromatography
 Size exclusion chromatography
 Separates large and small solute based on
their different abilities to enter the pores of
the support large solutes elutes faster than
smaller ones
 Uses a porous support that does not adsorb
solutes
 Commonly used to separate biological
molecules or polymers which differ by size
(MW)
 Ion exchange chromatography
 Separates ions based on their different
abilities to interact with the fixed exchange
sites
 Uses ion exchange resin (with fixed charges)
on its surface
 Cation-exchange: support with negative
groups
 Anion-Exchange: support with positive
groups
 Affinity chromatography

 Separates molecules based on their

different abilities to bind to the affinity
ligand
 Uses a support that contains an
immobilized biological molecule
(affinity ligand)
 Commonly used to purify and analyze
biological molecules
 Most Selective type of Chromatography
 Paper chromatography
 Paper-partition chromatography is a
simplified version of column
chromatography, which makes use of strips
or hollow cylinders of filter paper to hold
both the solid and liquid phases

 The strips of paper are placed in a

chromatography chamber with a saturating
and equilibrating vapour and hung from a
solvent reservoir
 Thin-layer chromatography
 Thin Layer Chromatography is a technique used to
isolate non-volatile mixtures. The experiment is
conducted on a sheet of aluminium foil, plastic, or
glass which is coated with a thin layer of adsorbent
material. The material usually used is aluminium
oxide cellulose, or silica gel.

 This technique is useful for separating organic

compounds. Because of the simplicity and rapidity of
TLC, it is often used to monitor the progress of
organic reactions and to check the purity of products.
 Liquid chromatography Detectors
 The most common liquid chromatography detectors are the ultraviolet
detector, the fluorescence detector, and the refractometer.
Liquid chromatography detectors have less sensitivity (defined as the
minimum detectable concentration) than gas chromatography detectors (in
fact several orders of magnitude less).

 The fluorescence detector is probably one of the most sensitive detectors

and the refractive index detector the least sensitive.

 In special applications (e.g., carbohydrate analysis), the light scattering

detector is growing in popularity but, unfortunately, although universal in
response, has a sensitivity (as defined previously) that is only slightly
better than that of the refractive index detector.
 UV Absorbance Detector (UV/Vis)

It is the most common type of detector in liquid chromatography,

Absorbance detector measures the ability of solutes to absorb light at a
particular wavelength range. This absorbance is described by the Beer-
Lambert Law.

A = εl c
Where:
A= Absorbance of light at a given wavelength
ε = Molar absorption coefficient of the solute
l = path length of the flow-cell
c = concentration of solute
 Fluorescence Detector
 Fluorescence spectroscopy (also known
as fluorimetry or spectrofluorometry) is a type of
electromagnetic spectroscopy that analyzes
fluorescence from a sample.

 It involves using a beam of light,

usually ultraviolet light, that excites the electrons in
molecules of certain compounds and causes them to
emit light; typically, but not necessarily, visible
light .
 Fluorescence is primarily concerned with electronic and
vibrational states. Generally, the species being examined has
a ground electronic state, and an excited electronic state of
higher energy. Within each of these electronic states there are
various vibrational states.

 In fluorescence, the species is first excited, by absorbing a

photon, from its ground electronic state to one of the various
vibrational states in the excited electronic state. Collisions
with other molecules cause the excited molecule to lose
vibrational energy until it reaches the lowest vibrational state
from the excited electronic state.

 The molecule then drops down to one of the various

vibrational levels of the ground electronic state again,
emitting a photon in the process. As molecules may drop
down into any of several vibrational levels in the ground
state, the emitted photons will have different energies, and
thus frequencies. Therefore, by analyzing the different
frequencies of light emitted in fluorescent, along with their
relative intensities, the structure of the different vibrational
levels can be determined.
 Differential refractometer

 A differential refractometer (DRI), or refractive index

detector (RI or RID) is a detector that measures
the refractive index of an analyte relative to the solvent.

 They are often used as detectors for high-performance

liquid chromatography and size exclusion
chromatography.

 They are universal detectors because they can detect

anything with a refractive index different from the
solvent, but they have low sensitivity.
 Light is created by a source and passed through flow cells containing
mobile phase eluting from the column (sample stream) and a reference
stream (usually mobile phase with no solute in it). The light passing
through these flow cells is passed through a second time using a mirror
and passed to a detector where its intensity is measured. When the
refractive index of liquid in the sample and reference flow cell are the
same, little or no bending of light occurs at the interface between the
low-cells. This allows the largest amount of light possible to reach the
detector.
 Electrochemical detector
This detector measure the ability of a solute to undergo either oxidation (i.e., loss of
electrons) or reduction (i.e., gain of electrons)

One way in which such a reaction can be monitored is by measuring the change in
current under a constant electric field. Another way is to measure the change in the
electric field produced when a constant current is present

Conductivity Detector
This detector measures the ability of a solution to conduct a current when placed
in an electrical field. This ability depends on the number of ions or ionic
compounds present in the solution
The relationship between the current, electric field and conductivity of the solution
is shown as follows: I = C E
 References:

 Handbook Of Analytical Instruments by R. S. KHANDPUR

 Instrumental Methods and Analysis by D.U

 https://www.slideserve.com/raja/liquid-chromatography

 https://www.slideserve.com/mercer/liquid-chromatography

 https://en.wikipedia.org/wiki/Chromatography_detector