Download Chromatography

Chromatography:
HPLC, HPSEC, MS,
LC-MS/MS, GC
Isariya Techatanawat, PhD
Director of Bioequivalence Study Group,
Research and Development Institute,
The Government Pharmaceutical
Organization
Chromatography
• Chromatography is a technique for
separating and/or identifying the
components in a mixture.
• Components in a mixture have different
tendencies to adsorb onto a surface or
dissolve in a solvent.
• All chromatographic methods require one
static part (stationary phase) and one
moving part (mobile phase).
Type of Chromatography
1.
2.
3.
4.
Adsorption Chromatography
Partition Chromatography
Ion Exchange Chromatography
Size Exclusion Chromatography
1. Adsorption Chromatography
• Solid stationary phase
• Liquid or gaseous mobile phase
• Each solute has its own equilibrium
between adsorption onto the surface of the
solid and solubility in the solvent, the least
soluble or best adsorbed ones travel more
slowly.
• The result is a separation into bands
containing different solutes.
1. Adsorption Chromatography
2. Partition Chromatography
• Stationary phase is a non-volatile liquid.
• Mixture to be separated is carried by gas
or liquid as mobile phase.
• Solutes distribute themselves between the
moving and the stationary phases, with
more soluble component in mobile phase
reaching the end of chromatography
column first.
2. Partition Chromatography
3. Ion-Exchange
Chromatography (IEC)
• Ion-exchange chromatography based
upon electrical charge.
• Likes may repel, while opposites are
attracted to each other.
• Stationary phases are characterized by
nature and strength of acidic or basic
functions on their surfaces and the types
of ions that they attract and retain.
3. Ion-Exchange
Chromatography (IEC)
• Opposite charge are electrostatically bound
to the surface.
• When the mobile phase is eluted through
resin, electrostatically bound ions are
released as other ions are bonded
preferentially
Ion-Exchange
Chromatography (IEC)
• Cation exchange is used to retain and
separate positively charged ions on a
negative surface.
• Anion exchange is used to retain and
separate negatively charged ions on a
positive surface.
3. Ion-Exchange
Chromatography (IEC)
3. Ion-Exchange
Chromatography (IEC)
• Adsorption based on binding of opposite
charges.
• Different components (virus, proteins,
DNA) have different charges, which
means strength of binding varies from
component to component.
4. Size Exclusion Chromatography
• Mixture passes as a gas or a liquid through a
porous gel.
• Pore size is designed to allow the large
solute particles to pass through uninhibited.
• Small particles permeate gel and are slowed
down. The smaller the particles, the longer it
takes for them to get through column.
• Separation is according to particle size.
4. Size Exclusion Chromatography
HIGH
PERFORMANCE
LIQUID
CHROMATOGRAPHY
(HPLC)
Principles of
Liquid Chromatography
HPLC
• HPLC is improved form of column
chromatography.
• Smaller particle size for column packing
material
• Provide greater surface area for interactions
between stationary phase and the molecules.
• Allow better separation of mixture component
HPLC
• Instead of solvent being allowed to drip
through a column under gravity, it is forced
through under high pressures.
• To separate, identify, quantitate
compounds.
• Compounds in trace concentrations as low
as parts per trillion [ppt] may also easily be
identified.
HPLC
• Normal Phase HPLC
• Reversed Phase HPLC
Normal Phase HPLC
• Stationary phase is polar (eg silica particles).
• Mobile phase is non-polar solvent (eg. Hexane).
Normal Phase HPLC
• Stationary phase is polar and retains polar
yellow dye most strongly.
• Non-polar blue dye is won in the retention
competition by the mobile phase and
elutes quickly.
Reversed Phase HPLC
• Stationary phase is non-polar. Silica is modified
to make it non-polar by attaching long
hydrocarbon chains to its surface (eg. C8 or C18
carbon atoms).
• Mobile phase is polar solvent (eg. mixture of
water and methanol).
Reversed Phase HPLC
• The most strongly retained compound is
non-polar blue dye, as its attraction to nonpolar stationary phase.
• Polar yellow dye is won in competition by
the polar, aqueous mobile phase, moves
the fastest, and elutes earliest.
HPLC
HPLC Separation
What is Chromatogram?
HPLC: Qualitative Analysis
HPLC: Quantitative Analysis
HPLC Chromatogram
Isocratic LC System
Gradient LC System
Gradient LC System
Preparative Chromatography
High Performance
Size Exclusion
Chromatography
(HPSEC)
Size Exclusion
Chromatography (SEC)
• Smaller molecules penetrate more of the
pores on their passage through the bed.
• Larger molecules may penetrate pores
above a certain size so they spend less
time in the bed.
• The larger molecules elute first, while the
smaller molecules travel slower [because
they move into and out of more of the
pores] and elute later.
Size Exclusion
Chromatography (SEC)
Size Exclusion
Chromatography (SEC)
• Biomolecules could be separated based
on their size, by passing, or filtering, them
through a controlled-porosity packing.
• Stationary phases are synthesized with a
pore-size distribution.
• Mobile phases are good solvents for
analytes and may prevent any interactions
[based on polarity or charge] between
analytes and stationary phase surface.
Size Exclusion
Chromatography (SEC)
• Large components such as virus typically
elute just after void volume.
• Contaminating proteins and nucleic acids
elute at volume greater than void volume.
SEC: Exclusion Limit
• SEC resin are often characterized by their
exclusion limit.
• Exclusion limit is the molecular weight of
the smallest molecule which cannot enter
the pores of the matrix.
• All molecules bigger than the exclusion
limit elute in the void volume.
Typical steps in SEC
• Equilibration – prepare column for binding
target biomolecule/virus
• Load – apply biomolecule/virus to column
• Elution – collect purified biomolecule/virus
from column
• Cleansing/Sanitization – ensure no viable
microorganisms present
• Storage – fill column with solution that
minimizes biological growth and maintains
integrity of resin
Separation by SEC
HPSEC
HPSEC
HPSEC
• Chromatogram shows how much material
exited the column at any one time, with the
higher molecular weight, larger polymer
coils eluting first, followed by successively
lower molecular weight (and therefore
smaller) chains emerging later.
• The primary separation is according to
elution volume.
HPSEC
• Chromatogram is compared to calibration
that shows the elution behavior of a series
of polymers for which the molecular weight
is known.
• Molecular weight distribution of the sample
is calculated.
Calibration graph used to determine
polymer MW from its retention time
Average molecular weights of
polymer nearly symmetrical
Process flow for the preparation
of clinical flu vaccine
Expansion of Vero cells
Infection
Harvest and Clearance
Benzonase
TFF
AIEX
SEC
Purified Vaccine Bulk
Mass Spectrometry
(MS)
Mass Spectrometry
• Measures mass-to-charge ratio of ions to
identify and quantify molecules.
Mass spectrometer
1. Ion source: Sample molecules are ionised.
2. Mass analyzer: Sample molecules are
separated according to their mass/charge
ratio (m/z).
3. Ion detector: Separated ions are detected
and sent to data system. m/z ratios and their
relative abundance is presented as m/z
spectrum .
Ionisation methods
• Electrospray Ionisation (ESI)
• Matrix Assisted Laser Desorption Ionisation
(MALDI)
• Atmospheric Pressure Chemical Ionisation (APCI)
• Chemical Ionisation (CI)
• Electron Impact (EI)
• Fast Atom Bombardment (FAB)
• Field Desorption / Field Ionisation (FD/FI)
• Thermospray Ionisation (TSP)
Mass Analyser
• To separate ions formed in ionisation
source according to their mass-to-charge
(m/z) ratios.
• Mass analysers: quadrupoles, time-offlight (TOF) analysers, magnetic sectors ,
and both Fourier transform and
quadrupole ion traps.
Ion Detector
• To monitor ion current and amplify it.
• Signal is transmitted to data system where
it is recorded as mass spectra .
• m/z values of ions are plotted against their
intensities to show number of components
in sample, molecular mass of each
component, and relative abundance of
various components in sample.
Mass Spectrometer
Mass Spectrum
Mass Spectrum
Mass Spectrometry
• m/z spectrum shows dominant ions at m/z
556.1, which are consistent with the
expected protonated molecular ions,
(M+H+).
• Measured molecular weight is 555.1 Da.
Mass spectrum of sulfamethazine acquired
without collision-induced dissociation
exhibits little fragmentation
Mass spectrum of sulfamethazine acquired with
collision-induced dissociation exhibits more
fragmentation and more structural information
Mass
Spectrum
Mass Spectrometry
• Samples (M) with molecular weights
greater than 1200 Da give rise to multiply
charged molecular-related ions such as
(M+nH)n+ in positive ionisation mode and
(M-nH)n- in negative ionisation mode.
Mass Spectrometry
• Proteins have many suitable sites for
protonation as all of the backbone amide
nitrogen atoms could be protonated
theoretically, as well as certain amino acid
side chains such as lysine and arginine
which contain primary amine
functionalities.
Applications for
Mass Spectrometry
Field of
Study
Applications
Proteomics
•Determine protein structure, function, folding and interactions
•Identify a protein from the mass of its peptide fragments
•Detect specific post-translational modifications throughout complex
biological mixtures
•Quantitate (relative or absolute) proteins in a given sample
•Monitor enzyme reactions, chemical modifications and protein digestion
Drug
Discovery
•Determine structures of drugs and metabolites
•Screen for metabolites in biological systems
Clinical
Testing
•Perform forensic analyses such as confirmation of drug abuse
•Detect disease biomarkers (newborns screened for metabolic diseases)
Genomics
•Sequence oligonucleotides
Environment •Test water quality or food contamination
Geology
•Measure petroleum composition
•Perform carbon dating
Tandem (MS/MS)
mass spectrometers
• More than one analyser.
– quadrupole-quadrupole
– magnetic sector-quadrupole
– quadrupole-time-of-flight geometries.
LC-MS
LC-MS/MS
LC-MS vs LC-MS/MS
LC-MS/MS
Full scan mass spectrum of ginsenoside Rb1
showing primarily sodium adduct ions
Full scan product ion (MS/MS) spectrum from
the sodium adduct at m/z 1131.7
Subsequent full scan product ion spectrum
(MS3) from the ion at m/z 789.7
LC-MS/MS
Identification of proteins
Identification of proteins
Biochemical Applications
• Rapid protein identification using capillary
LC/MS/MS and database searching
Molecular Weight Determination
Determining molecular weight
of green fluorescent protein
• 27,000 Dalton with 238 amino acids.
Rapid protein identification
using capillary LC/MS/MS and
database searching
Protein identification
Full scan MS/MS spectra from doubly
charged parent ion m/z 807.2 and
matching theoretical sequence
identified by database searching
Identification of
structurally similar aflatoxins
Analysis of peptides using
CE/MS/MS
Gas
Chromatography
(GC)
Gas Chromatography
• Mobile phase is gas. Stationary phase can
either be solid or non-volatile liquid.
• GC involves a sample being vapourised
and injected onto the chromatographic
column.
• Sample is transported through the column
by the flow of inert gaseous mobile phase.
Gas Chromatography
GC: Instrumental components
• Carrier gas
– Carrier gas must be chemically inert.
– Commonly used gases: nitrogen, helium,
argon, carbon dioxide.
• Sample injection port
– Temperature of the sample port is usually
about 50°C higher than the boiling point of the
least volatile component of the sample.
GC: Instrumental components
• Columns
– Packed columns
– Capillary columns
• Column temperature
– Oven temperature is kept constant for a
straightforward separation
GC: Instrumental components
• Detectors
– Concentration dependant detectors:
• Signal is related to concentration of solute
• Not destroy the sample
• Dilution of with make-up gas will lower response
– Mass flow dependant detectors
• Destroy sample
• Signal is related to rate at which solute molecules
enter the detector.
• Response is unaffected by make-up gas
Support
Selectivity
gases
Flame
Hydrogen
Mass flow
Most organic cpds.
ionization (FID)
and air
Thermal
Concentrati
conductivity
Reference Universal
on
(TCD)
Halides, nitrates, nitriles,
Electron
Concentrati
Make-up peroxides, anhydrides,
capture (ECD) on
organometallics
NitrogenHydrogen
Mass flow
Nitrogen, phosphorus
phosphorus
and air
Hydrogen
Flame
Sulphur, phosphorus, tin,
and air
photometric
Mass flow
boron, arsenic, germanium,
possibly
(FPD)
selenium, chromium
oxygen
Aliphatics, aromatics, ketones,
Photo-ionization Concentrati
esters, aldehydes, amines,
Make-up
(PID)
on
heterocyclics, organosulphurs,
some organometallics
Hall electrolytic
Hydrogen, Halide, nitrogen, nitrosamine,
Mass flow
conductivity
oxygen
sulphur
Detector
Type
Detect Dynami
ability c range
100 pg 107
1 ng
107
50 fg
105
10 pg 106
100 pg 103
2 pg
107
Gas Chromatography
References
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http://www.waters.com/
http://www.chemguide.co.uk
https://https://www.thermofisher.com
Chromatography. The Royal Society of Chemistry.
An Introduction to Mass Spectrometry. The University of Leeds.
Mass Spectrometry in Biotechnology
Gary Siuzdak , Academic Press 1996 SiuzdakBiotechnology”
An Introduction to Gel Permeation Chromatography and Size Exclusion
Chromatography. Agilent Technologies.
Size-exclusion Chromatography of Polymers. Encyclopedia of Analytical
Chemistryilent
Chromatography for influenza vaccine manufacture: Principles,
Equipment and Design. NC State University
Basics of LC/MS. Agilent Technologies.
Gas chromatography. Sheffield Hallam University.
http://teaching.shu.ac.uk/hwb/chemistry/tutorials/chrom/gaschrm.htm