Search for notes by fellow students, in your own course and all over the country.
Browse our notes for titles which look like what you need, you can preview any of the notes via a sample of the contents. After you're happy these are the notes you're after simply pop them into your shopping cart.
Title: Chromatography: Principle, Types and Applications
Description: Chromatography is a physical separation method in which the components of a mixture are separated on the basis of their differential distribution between two immiscible phases. It is further divided into various types on the basis of the type of interaction that is taking place between the analytes in the mixture and the separator. This document provides a brief overview of the principle of chromatography, the types of chromatography, how to read a chromatogram and the applications of chromatography. The diagrams and the flowchart will help in summarizing the concepts and could be used to make presentations for the academic course as well.
Description: Chromatography is a physical separation method in which the components of a mixture are separated on the basis of their differential distribution between two immiscible phases. It is further divided into various types on the basis of the type of interaction that is taking place between the analytes in the mixture and the separator. This document provides a brief overview of the principle of chromatography, the types of chromatography, how to read a chromatogram and the applications of chromatography. The diagrams and the flowchart will help in summarizing the concepts and could be used to make presentations for the academic course as well.
Document Preview
Extracts from the notes are below, to see the PDF you'll receive please use the links above
CHROMATOGRAPHY
CHROMATOGRAPHY
Greek word:
Colour
To write
Carotene
Mikhail Tswett invented
chromatography in
1901 to separate plant
pigments such as Chlorophyll,
carotene and xanthophyll
...
Stationary phase :
Mobile phase :
May be a solid, gel,
liquid or a solid/liquid
mixture that is
immobilized
...
HOW DOES CHROMATOGRAPHY WORKS?
Because of
differential
partition of
component of
samples in the
two phases
separation
occurs
...
The value of K depends on the solubility of the analytes in the condensed phase
...
Chromatographic
Performance
Parameters
The successful chromatographic separation
of analytes in a mixture depends upon the
➢ Selection of the most appropriate
process of chromatography
➢ Optimization of the experimental
conditions associated with the
separation
...
It consists of a series of peaks or bands
...
Qualitative data- what is
present? Through retention time
...
Quantitative data- how much
analyte is present?– Area under
the peak
READING A CHROMATOGRAM
Retention time
tR = tR’ + tM
tM : Time taken by analyte molecule to pass through
free spaces between the Stationary phase
...
Time
Importance of Retention factor:
1
...
2
...
Resolution:
Peak should start and finish at baseline
...
CHROMATOGRAPHIC EFFICIENCY
Chromatography columns are considered to consist of a number of adjacent zones, each zone is
called a theoretical plate
...
Width of the analyte peak, expressed
in terms of its standard deviation
Distance travelled by analyte within the
column
Small the value of H
↓
More the number of N
↓
More the efficiency of column
...
Multiple Pathways: smaller the particle
size of matrix, less variable pathways
and thus less elution time will be taken
...
VAN DEEMTER PLOT:
Eluent flow rate
1
α
𝐿𝑜𝑛𝑔𝑖𝑡𝑢𝑑𝑖𝑛𝑎𝑙 𝑑𝑖𝑓𝑓𝑢𝑠𝑖𝑜𝑛
α 𝐸𝑞𝑢𝑖𝑙𝑖𝑏𝑟𝑎𝑡𝑖𝑜𝑛 𝑡𝑖𝑚𝑒
OPTIMIZATION DEPENDS ON :
•
•
•
•
•
Stationary Phase and mobile phase
Detector: more sensitivity
Flow rate: affects retention time and diffusion and thus performance
Amount of sample
Temperature: improve performance (must avoid decomposition of sample) by
increasing solubility and reducing viscosity
...
• Flat and horizontal baseline
• No artefactual peaks
• Shortest possible analysis times
...
Sample is dissolved in solvent which is
allowed to flow continuously over
stationary phase leading to
Progressive separation and elution
of sample
...
Analytes binds to stationary phase with a
strength determined by their affinity which
are then selectively eluted by using a
mobile phase that has higher affinity for
stationary phase than them
...
Solvent kept at top of
chamber and moves by
chromatography capillary action and
gravitational force –
more rapid flow
Descending
paper
Solvent moves upward
Ascending
against gravitational flow
paper
chromatography by capillary force – slow
speed
Solvent front
Mobile phase: solvent (can
be polar or non polar)
Analysis- Spots corresponding to different compounds may be located by their
color, UV light, Ninhydrin or by treatment with iodine vapors
...
Stationary phase – a thin layer of adsorbent like
silica gel (polar substance) over a flat inert
surface of glass / plastic plate/ aluminium
Mobile phase – non polar solvent
Eg
...
Analysis Of Results Of Planar Chromatography
Retention factor:
𝑫𝒊𝒔𝒕𝒂𝒏𝒄𝒆 𝒕𝒓𝒂𝒗𝒆𝒍𝒍𝒆𝒅 𝒃𝒚 𝒔𝒐𝒍𝒖𝒕𝒆
𝑫𝒊𝒔𝒕𝒂𝒏𝒄𝒆 𝒕𝒓𝒂𝒗𝒆𝒍𝒍𝒆𝒅 𝒃𝒚 𝒔𝒐𝒍𝒗𝒆𝒏𝒕
0 ≤ Rf ≤ 𝟏
Rf = 0 → Immobile solute
High affinity to stationary phase
Rf = 1 → Solute has no affinity for stationary phase
High solubility in mobile phase and therefore moves with solvent
COLUMN
CHROMATOGRAPHY
Stationary phase: packed in
glass or metal column
Mobile phase: gas or liquid
that pass over it
...
Eluate: analyte separates through
the column and emerges
individually in the eluate as it
leaves column
...
Polar compounds need to be retained in column
...
2
Removal of O2, or
hydrocarbons and
needs to maintain a
constant pressure and
temperature
Solvent used: Acetone, Hexane or
methanol
Non polar or low polar analytes are
directly inserted
...
Mobile phase:
Inert gas used
...
Capillary columns further divided into:
Wall-coated Open
Tubular (WCOT)
Support-coated
Open Tubular
(SCOT)
Porous layer
Open Tubular
(PLOT)
Partition
coefficient of
analytes are
sensitive to
temperature
...
Detectors can be of different types and chose on the basis of type of analyte :
Flame ionisation
detector(FID)
For all organic compound
Nitrogen phosphorus detector N and S containing analytes
(NPD)
Analytes are ionized in the flame generated from
the mixture of H2 and air
...
Flame photometric detector
P and S containing analytes
These analytes emit light when burned in FID- type
detector, light is detected and quantified
...
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
Mobile phase passes through columns under 10–400 atmospheric pressure, and with a
high (0
...
Use of small particles in the stationary phase, and
application of high pressure increases separation power
...
Small particles: higher N and
hence higher pressure needed
Mobile phase needs pretreatment, such as filtering or
sparging (degassing)
...
Capable of outputs of at least 50 MPa
and there must be no pulses (i
...
cyclical
variations in pressure)
...
So sample is flushed
without interruption of the flow
of eluents
...
Most commonly used detectors are:
Variable wavelength detectors
based upon ultraviolet–visible spectrophotometry
Scanning wavelength
detectors:
record the complete absorption spectrum of each analyte, thus
aiding identification
...
Mass spectrometer detectors
enable the analyte to be detected and its structure determined
simultaneously
...
Are commonly used in the analysis of
carbohydrates
...
UV detector is used for identification
...
2
...
This difference in the binding strength of analytes leads to their separation
...
Adsorbent: generally used silica (Si-OH), Alumina, carbon
Hydrophobic interaction chromatography
Stationary phase: hydrophobic group containing compounds
Analyte: proteins ( via exploiting their hydrophobicity)
Exposed hydrophobic group ( extracted by addition of salt ions such a
Ammonium Sulphate) interacts with the hydrophobic matrix and forms
protein matrix interaction
Hydroxylapatite chromatography
Adsorbent: Crystalline hydroxylapatite
(Ca10(PO4)6(OH)2)
Anatytes: protein or Nucleic acid
Advantage: can separate ssDNA and dsDNA
Both DNAs can bind at low phosphate buffer
conc
...
Increase, first ssDNA
get desorbed
...
PARTITION CHROMATOGRAPHY
Separation of analyte depends on : Retention factor and partition coefficient
Stationary phase: Liquid; attached either by physical means or covalently
...
Normal phase liquid chromatography
Reverse phase liquid chromatography
Stationary phase: Polar and solid
–Alkylamine bonded to silica
Mobile phase: Non polar (organic solvent)
–Hexane, heptane, dichloromethane or ethyl
acetate
tR increases with polarity → most polar analyte
component will elute last
...
Polarity of organic compound in
increasing order is:
REVERSE PHASE
NORMAL PHASE
Polar
stationary
phase
Non Polar
stationary
phase
4
...
Distribution of analyte in the column → determined by the mobile phase trapped in stationary phase and
available to the analyte→ depends on porosity and size of analyte
Distribution coefficient Kd
between inner and outer mobile
Small molecules: distributed
phase: Function of molecular
between mobile phase that is
inside and outside of particles,
size
so have slower speed and will
Kd vary between 0 to 1
be eluted later
Kd = 1 completely in the pores
Kd=0 complete exclusion
Large molecules: do not enter
the pores, pass through the
interstitial space and elutes
first
5
...
Separation for enzymes, nucleotides, nucleic acid, Immunoglobin etc
...
ION EXCHANGE CHROMATOGRAPHY
Principle: attraction between opposite charged stationary phase (ion exchanger) and analyte
Two types based on the charge of exchanger
Cation exchanger chromatography
High affinity towards
Cationic analytes (+ve)
Anion exchanger chromatography
High affinity towards
Anionic analytes (-ve)
Stationary phase has
negatively charge (-ve)
Stationary phase has
Positively charge (+ve)
Negatively charged (-ve)
components elutes first
Positively charged (+ve)
components elutes first
The exchangers can be Strong (ionizable at all working pH) or Weak exchanger( ionized at narrow pH)
...
Factors Affecting Ion Exchanger Chromatography:
Elution:
pH: can affect both the ionization of exchanger and the analyte
Analyte – more stable at pH
Ionic strength:
↑se in ionic strength permits binding of selected samples
↓se in ionic strength causes elution
...
Initial conditions – all test analytes bind to the
exchanger
...
Separation of amino acids,
glycated hemoglobin,
isolation of nucleic acid
from blood
The elution volumes of
globular proteins are
determined largely by their
relative molecular mass
(Mr)
Purification of enzymes and other proteins, including receptor proteins and
immunoglobulins
...
Material that can be
either liquid or gas
...
Advantage of using supercritical fluids → Combines advantages of GC and HPLC:
• SF are less viscous than liquids, and has more diffusion coefficient → high separation efficiency
...
• Columns packed with a wide spectrum of stationary phases
...
WHICH LIQUID CHROMATOGRAPHY TO USE:
WHAT TYPE OF CHROMATOGRAPHY DO I NEED?
NON VOLATILE
VOLATILE AND STABLE
Liquid
chromatography
Gas
chromatography
Surface techniques
Neutral
Adsorption chromatography
Partition chromatography
Ionic /charged
Ion exchange chromatography
+ve charge:
Cation Exchange
-ve charge:
Anion Exchange
Size exclusion
chromatography
APPLICATIONS OF CHROMATOGRAPHY:
Drug testing: Bodily fluids including blood and urine can undergo chromatography to separate the
naturally occurring compounds therein from any metabolic byproducts produced by the consumption of
drugs
...
May also be used to purify intermediate products throughout the stages of synthesis
...
BIOANALYTICAL CHROMATOGRAPHY IN CLINICAL ANALYSIS
The three main types of chromatographic techniques widely used
are: HPLC, GC and SCF
• Targeted analysis of drugs
and clinical biomarkers
• Non targeted multi analyte
analysis for identifying drug
of abuse
...
• Proteomics, metabolomics,
lipidomics
...
• Non targeted screening in
metabolomics and lipidomics
• Endogenous compounds
analysis
• Targeted analysis of many
volatile compounds
determined could be drugs
or narcotics
• Biomarker for Tuberculosis,
neurodegenerative and
metabolic syndrome
GC
• Chiral separation such as
racemic cetirizine and citalopram
in human plasma or ketamine
metabolites in human urine
...
, , Pilařová, V
...
, , Plíšek, J
...
, , & Nováková, L
...
Current state of bioanalytical
chromatography in clinical analysis
...
https://doi
...
1039/c7an01807j
Liquid chromatography is the most widely used
chromatographic technique in routine clinical
analysis
...
Relative representation of applications using various LC approaches in clinical analysis
...
, , Pilařová, V
...
, , Plíšek, J
...
, , & Nováková, L
...
Current state of
bioanalytical chromatography in clinical analysis
...
https://doi
...
1039/c7an01807j
Thankyou
Title: Chromatography: Principle, Types and Applications
Description: Chromatography is a physical separation method in which the components of a mixture are separated on the basis of their differential distribution between two immiscible phases. It is further divided into various types on the basis of the type of interaction that is taking place between the analytes in the mixture and the separator. This document provides a brief overview of the principle of chromatography, the types of chromatography, how to read a chromatogram and the applications of chromatography. The diagrams and the flowchart will help in summarizing the concepts and could be used to make presentations for the academic course as well.
Description: Chromatography is a physical separation method in which the components of a mixture are separated on the basis of their differential distribution between two immiscible phases. It is further divided into various types on the basis of the type of interaction that is taking place between the analytes in the mixture and the separator. This document provides a brief overview of the principle of chromatography, the types of chromatography, how to read a chromatogram and the applications of chromatography. The diagrams and the flowchart will help in summarizing the concepts and could be used to make presentations for the academic course as well.