Introduction To Chromatographic Techniques

Introduction to the Science of
Chromatography
Dr. Vijay J. Jadhav
Professor
Department of Veterinary Public Health and Epidemiology, LUVAS
National Workshop on
Application of chromatographic methods to detect
contaminants in Food and Environmental samples
(5-7 March, 2024)
Introduction
The term "chromatography“
1890 Russian–Italian botanist M. S. Tswett- First
application of column chromatography for
separation of plant pigments
Science of seperation
Major applications:
Purification of reaction mixtures in chemical synthesis
Purification of bio-molecules such as proteins for
pharmaceutical research
Analysis of complex sample mixtures of forensic and
environmental sciences
Chromatography at Work
• Forensic Testing- GC for explosives detection
– RDX (1,3,5-Trinitroperhydro- 1,3,5-triazine),
– TNT (2-Methyl-1,3,5-trinitrobenzene)
– PETN ([3-Nitrooxy-2,2-bis(nitrooxymethyl) propyl] nitrate)
• Doping tests- Drug abuse by sports person-

– Synthetic hormone intake
• Ebola Immunization- Finding out effective antibody

• Food flavours- Basmati rice, wine flavors etc.
Theory of Chromatography
• Separation of sample components on the basis of their difference
in their distribution between two non-miscible phases
• Stationary phase, either liquid or solid, is fixed in the system,
whereas, mobile phase flows through the chromatographic system
• The distribution equilibrium (DE) determines its effective
migration velocity of each analyte
• Difference amongst analytes w.r.t. DE decide the mean residence
time in the stationary phase for each analyte which is unique.
Stationary Phase, s
Mobile Phase, m with Velocity, v
Stationary Phase, s
Classification of Chromatographic Methods
• Column Chromatography - The stationary
phase is held in a narrow tube through which the
mobile phase is forced either by pressure or by
gravity.
– Simple column chromatography
– High pressure liquid chromatography (HPLC)
– Gas chromatography (GC).
• Planar Chromatography - The stationary phase
is supported on a flat plate or in the fibres of a
paper. Mobile phase moves through the stationary
phase by capillary action or by gravity.
– Paper chromatography
– Thin layer chromatography (TLC).
General Specific Stationary Type of
Classification Method Name Phase Equilibrium
Liquid-liquid Liquid adsorbed Partition between
partitioning on a solid immiscible
Liquid Chromatography (LC)
liquids
Liquid-bonded Organic species Partition between
phase bonded to a solid liquid and a
surface bonded surface
Liquid-solid or Solid Adsorption
adsorption
Ion exchange Ion-exchange Ion exchange
resin
Size exclusion Liquid in Partition/sieving
interstices of a
polymeric solid
General Specific Stationary Type of
Classification Method Name Phase Equilibrium
Gas Gas-liquid Liquid adsorbed Partition between
Chromatograp on a solid gas and liquid
hy (GC) Gas-bonded Organic species Partition between
phase bonded to a solid liquid and
surface bonded surface
Gas-solid Solid Adsorption
(Source: Skoog and Leary, 1992)

Applications of Chromatography
• Purification of Biological molecules

– (More than 100 Daltons)
• Detection of Chemical molecules
– (Less than 100 Daltons)
Purification of Biomolecules
• Separation and purification of large
molecules especially protein is challenging
– Removal of non protein substances
– Separation of desired protein from mixture of
variety of proteins
– Preventing inactivation of desired protein
– Maintaining its stability
• Ion Exchange
Chromatography
• Chromatographic Methods
Based on Hydrophobicity
• Affinity Chromatography
• Immobilized Metal Affinity
Chromatography
• Size Exclusion
Chromatography (GPC)
Detection of chemicals
• Medicinal drugs, toxins, pesticides and
industrial contaminants into tissues of
organisms
• Intentional or unintentional exposure
• Pharmocokinetic studies, food safety
analysis and forensic investigations.
• Extraction and Cleanup methods
– Liquid-liquid extraction
– Elimination of water: Freezing, lyophilisation, use of
unhydrous salt
– Elimination of fat: open column chromatography with
alumina, gel permeation chromatography, liquid liquid
partitioning, chilling
– Elimination of proteins: precipitation in the presence of
strong acides and ammonium sulphate, enzymic digestion
using proteases,
– Solvent removal: rotary vaccum evaporation
– Use of sorbents: alumina, florisil, silica, silica gel, activated
charcoal, C18
– Matrix solid phase dispersion: blending of solid phase
extraction sorbents directly with solid samples
• Instrumental method
– High performance/pressure thin layer
chromatography (HPTLC)
– Gas chromatography with electron capture
detector, nitrogen-phosphorous (thermionic)
detector, thermal Conductivity detector, flame
ionisation detector, flame photometric detector
and mass spectrometer etc.
– High performance/pressure liquid
chromatography with ultraviolet and visible
detector, photodiode detector, fluorescence
detector, electrochemical detector, mass
spectrometer
Standardization and Validation of
Chromatographic methods
• Standardization (Optimization): Finding out
best combination that gives maximum
output with respect to performance of the
method
• Validation: Examination of stability of
method under different conditions
Validation parameters
• Accuracy
• Precision
– Repeatability
– Intermediate Precision
– Reproducibility
• Specificity
• Detection Limit
• Quantitation Limit
• Linearity
• Range
• Robustness
• Specificity:
– Ability to assess unequivocally the analyte in the presence of
components which may be expected to be present
– Typically these might include impurities, degradants, matrix,
etc.
• Accuracy:
– Closeness of agreement between an accepted reference value
and the value found
• Precision
– Expresses the closeness of agreement (degree of scatter)
between a series of measurements obtained from multiple
sampling of the same homogeneous sample under the
prescribed conditions
– Repeatability, Intermediate Precision and Reproducibility.
Precision
• Repeatability: Expresses the precision
under the same operating conditions over a
short interval of time.
• Intermediate precision: Expresses within-
laboratories variations i.e. different days,
different analysts, different equipment, etc.
• Reproducibility: Expresses the precision
between laboratories (collaborative studies)
• Detection Limit: Lowest amount of analyte
in a sample which can be detected but not
necessarily quantitated as an exact value.
• Quantitation Limit: Lowest amount of
analyte in a sample which can be
quantitatively determined with suitable
precision and accuracy.
• Linearity: Ability (within a given range) to
obtain test results which are directly
proportional to the concentration (amount)
of analyte in the sample.
• Range: Interval between the upper and
lower concentration (amounts) of analyte in
the sample (including these concentrations)
for which it has been demonstrated that the
analytical procedure has a suitable level of
precision, accuracy and linearity.
• Robustness Measure of its capacity to
remain unaffected by small, but deliberate
variations in method parameters and
provides an indication of its reliability
during normal usage.
Thank You All !!

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Introduction to the Science of

• Doping tests- Drug abuse by sports person-

• Ebola Immunization- Finding out effective antibody

Mobile Phase, m with Velocity, v

(Source: Skoog and Leary, 1992)

• Purification of Biological molecules

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