affinity chromatography 1 (1)
TRANSCRIPT
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Affinity Chromatography
andIon Exchange Chromatography
Shannon E. Spence
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What on Earth did scientist do before
Chromatography?
-
Extractionis based on the difference in solubility materialis grounded, placed with a solvent which
dissolves soluble compounds. A second
extract solvent . The mixture is placed in a
separatory funnel
-Crystallizationalso based on the difference of solubility. Thesolubility is solved in a fixed volume of solvent.
The purified compound crystallizes as solution
cools, evaporates or diffuses
-Distillilation
separates components based on their volatility
typically via vaporization-condensationmethod
Filtrationseparate components of a mixture based on
their particle size. Used most often to
separate a liquid from a solid
www.chemguide.co.uk/.../idealfract.html
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What entices the scientists to
Chromatography?Just like the previous techniques
chromatography is a way to separate
two components based on a specific
characteristic
What makes chromatography so
useful...The results are reproducible with
better accuracy than the before
mentioned separation techniques
Chromatography can separate more
complex mixtures than the previoustechniques
Chromatography is less time
consuming and cheaper
http://www.residues.com/ion_chromatography.html
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Brief History of Chromatography
1903 Tswett, a Russian botanist
coined the term chromatography. He
passed plant tissue extracts through a
chalk column to separate pigments by
differential adsorption chromatogrpahy
1915 R.M Willstatter, German Chemistwin Nobel Prize for similar
experiement
1922 L.S Palmer, American scientist
used Tswetts techniques on various
natural products
1931 Richard Kuhn used
chromatography to separate isomersoh polyene pigments; this is the first
known acceptance of chromatographic
methods
http://www.chemgeo.uni -hd.de/texte/kuhn.html
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History of the Main techniques
1938 Thin Layer chromatography by
Russian scientist N.A Izamailov and
M.S Shraiber
1941 Liquid-Liquid partition
chromatography developed by Archer
John, Porter Martin and RichardLaurence Millington Synge
1944 Paper Chromatography one of
the most important methods in the
development of biotechnology
1945 Gas Chromatography 1st
analytical gas-solid (adsorption)
chromatography developed by FritzPrior
1950 Gas Liquid Chromatography by
Martin and Anthony James; Martin
won the Nobel Prize in 1952
British chemist ArcherJohn Porter Martin, co-
recipient, with Richard L. M. Synge, of the 1952
Nobel Prize in chemistry, "for their invention ofpartition chromatography."
http://www.chemistryexplained.com/Ma-Na/Martin-Archer-John-Porter.html
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History of the Main Techniques
1966 HPLC named by Csaba Horvath,
but didnt become a popular method
until 1970s
1950s Ion-Exchange chromatography
declassified this technique
1970s Ion Chromatography wasdeveloped by Hamish Small and co-
workers at the Dow Chemical
company
1930s Affinity Chromatography was
developed for the study of enzymes
and other proteins
library.thinkquest.org
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Chromatography
Applies the principles of the fractional separation procedures
Non-instrumental analysis which partitions components between two
phases usually a mobile phase and a stationary phase, based onthe difference in the components physical properties
Can separate complex mixtures composed of many very similar
components.
Chromatography is often coupled with analytical instruments to
complete analysis. A single chromatographic analysis can isolate, identify , and
quantitate multiple components of mixtures
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Principles of Chromatography
Chromatography is used when there is a difference in the retention times of
different components
Two types of phases
1) Stationary phase
2) mobile phases Properties of Chromatographic Properties
1) immiscible stationary and mobile phases
2) an arrangement where a mixture is depositied at one end of the
stationary phase
3) flow of the mobile phase towards the other end of the stationaryphase
4) different rates of partitioning for each component
5) means for visualizing the separation of each component
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Techniques
Ion Exchange Chromatography (IEC)separates biomolecules based on the their net surface charge
Ion Chromatography (IC)more general form of IEC allows separation of ions and polar molecules
based on the charge properties of the molecules
Affinity Chromatography (AC)
is the purification of a biomolecule with respect to the specific bindingof that biomolecule due to the chemical structure
Gas Chromatography (GC)is a technique used to separate organic molecules that are volatile
Gas-Liquid Chromatography (GLC)another name for GC
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More Detailed History of
IE Chromatography 1850 H. Thompson and J.T way treated various clas with ammonium sulfateor carbonate in solution to extract the ammonia and release the calcium
1927 Zeolite (sodium aluminum silicates) mineral columns were used toremove interfering calcium and magnesium ions from solution to determinesulfate content of water
1940s Modern Ion-Exchange Chromatography was developed during thewartime Manhattan Project
- this technique was used to separate and concentrate the radioactiveelements needed to make an atomic bomb. The adsorbents would latchonto charged transuranium elements differentially eluting them
1970s Hamish Small and co-workers ofDow Chemical Company developed
ion-chromatography usable for automated analysis
- IC uses weaker ionic resins for the stationary phase and a neutralizing
stripper column to remove background eluent ions
- used for determining low concentrations of ions in water and other
environmental studies
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Terminology
Elution
- washing of the mixture
Eluent- additional solvents used for
elution
Effluent- exiting fluid stream
Residency
- time spent on column
Stationary Phase
-
Mobile Phase
- fluid carrying the mixture of
analytes
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Ion-Exchange Chromatography
Usually employed with HPLC
Ions are charged molecules
- cation positively charged ion
- anion negatively charged ion
These ions do not separate smoothly under the traditional methods of theliquid and mobile phases of chromatography
Requires alteration methods of either the mobile phase or stationary phase
are required
- mobile phase suppresses the ionic nature of the analyte
- stationary phase incorporate ions of the opposite charge to attract and
retain analyte
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How do you get those columns
to work There is a glass columncoated with a resin
polymer
The resin is eitherpositively charged (an
acid) or negatively
charged (a base)
An analyte will have ions
opposite of the resins
charge eluting off the ion
of interest
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Resin
Common resins are copolymerized styrenes
vinylic and aromatic functional groups
styrene derivative and divinylbenzene
Creates better stability due to crosslinking of the benzene rings Creates a swelling within the polymer affecting the porosity while taking in
the mobile phase liquid
Aromatic substitution reaction makes these polymers ideal for charged
functional groups
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Cation Exchange resins
The functional group in cation exchange resins are usually acids
Sulfonic acids SO3H (strong acid resin) are added to the resin by
sulfonation reactions
Res-(SO3H) + M+
Res-(SO3M) + H+
Carboxylic acid COOH (weak acid resin)
Res-COOH + M+ Res-COOM + H+
With both the strong and the weak acid exchange sites an acidic Hydrogen
is attached to a functional group chemically bound to the resin
Cation exchange is good for removing metal ions from an aqueous solution
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Anion Exchange Resins
The functional groups added to the resin is similar to cation resins but are
basic instead of acidic
Quaternary ammonium a strong base -- CH2N(CH3)3+OH-
CH2N(CH3)3+OH- + B- Res-CH2N(CH3)3
+Cl- + OH-
Polyalky amine a weak base -- NH(-R)2+OH-
NH(-R)2+OH- + B- Res-NH(-R)2
+B- +OH-
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To Affinity and Beyond
The rate of ion exchange is controlled by the law of
mass action. At equal concentration the greater affinity
molecule will control the cation exchange resins in the
acid form.
However if a much higher concentration of strong acid
passed through the greater affinity molecule, such as
sodium, will form the resin, reversing equilibrium and
convert the resin back to an acidic form.
Generally it is possible to return either ion exchangeresin column to a desired starting form by passing a
large excess of the desired ion at very high
concentration through the resin
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What makes it unique
Careful selection of the ionic
composition of the eluent, and the
gradual adjustment of its strength
during elution using a controlled
gradient
The components of a mixture of
ions can be induced to separate
just as the components of a
mixture separated by partitionion
chromatography
The parameters controlling the
relative residence of the analyte or
other eluent ions is the resin
stationary phase or the ionic
solution mobile phase
1) both the relative selectivity of
the resin for the ions and their
relative concentrations in each
phase
2) In ion exchange, selectivity
resides in relative ion-pairing
interaction strengths only in the
stationary phase
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Relative Affinity of Ions
The higher the charge the higher the
affinityNa+ < Ca2+ < Al3+ and Cl- < SO4
2-
The Ion with the greatest size and charge
has the highest affinity
Li+ < Na+ < K+ < Cs+ < Be2+ < Mg2+ < Cu2+ and F- < Cl- < Br- < I-
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Applications of IEC
In cell and molecular biology, ion
exchange chromatography is used to
separate different proteins out of an
eluant.
areas of research such as the
environment, industry, commercial
products of organic molecules without UV-vis absorption
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Ion Chromatography
The analysis of ionic analytes by
separation on ion exchange stationary
phases with eluent suppression of excess
eluent ions
ex) when cations are being exchanged to effect aseparation, variable concentrations of HCl are used as
an eluent passing through the analytical anion columnwithout being retained forming largely undissociated
species such as water, carbonic acid and bicarbonate
ions
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AFFINITYCHROMATOGRAPHY
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Affinity History
1930s, first developed by Arne Wilhelm
Tiselius, won the Nobel Prize in 1948
Used to study enzymes and other proteins Relies on the affinity of various
biochemical compounds with specific
properties
ex) enzymes for their substrates
antibodies for their antigens
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How do they get those iddy bitty
molecules in there?
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So now what.
The Sample is injected into the equilibrated
affinity chromatography column
Only the substance with affinity for the ligand are
retained on the column
The substance with no affinity to the ligand will
elute off
The substances retained in the column can beeluted off by changing the pH of salt or organic
solvent concentration of the eluent
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Specificity of Affinity
Chromatography Specificity is based on three aspect of affinity
1) the matrix
2) the ligand
3) the attachment of the ligands to the matrix
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Matrix
The matrix simply provides a posre structure to increase
the surface area to which the molecule can bind
This has been what kept the Affinity Chromatographyfrom being developed earlier and useful to the scientific
community
The matrix must be activated for the ligand to bind to itbut still able to retain its own activation towards the
target molecule
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Matrix
Amino, hydroxyl, carbonyl and thio groups located with
the matrix serve as ligand binding sites
Matrix are made up of agarose and otherpolysaccharides
The matrix also must be able to withstand the
decontamination process of rinsing with sodiumhydroxide or urea
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Ligand
The Ligand binds only to the desired molecule within the solution
The ligand attaches to the matrix which is made up of an inert
substance
The ligand should only interact with the desired molecule and form a
temporary bond
The ligand/molecule complex will remain in the column, eluting
everything else off
The ligand/molecule complex dissociates by changing the pH
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So many ligands so little time
The chosen ligand must bind strongly to the molecule of
interest
If the ligand can bind to more than onel molecule in thesample a technique, negative affinity is performed
- this is the removal of all ligands, leaving the
molecule of interest in the column
-done by adding different ligands to bind to theligands within the column
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Applications
Used in Genetic Engineering
Production of Vaccines
And Basic Metabolic Research
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Gas Chromatography
Gas-Liquid Chromatography
AndGas-Solid Chromatography
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History
1945
technique developed by Fritz Prior out of post-WWII
Europe
Fritz Prior was a only a graduate student at the time 1947
Prior succeeded in separating O2 and CO2 on a
charcoal column
1950ArcherJ.P Martin and Anthony James developed Gas-
Liquid Partition Chromatography (GLPC)
this has become the method of choice
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Gas Chromatography
Gas- Solid Chromatography (GSC)
is a process of repeated adsorption/desorption of
sample from the carrier gas to the solid adsorbent
Gas-Liquid Partioning Chromatography (GLPC)involves a sample being vaporized and injected onto the
head of the chromatographic column. The sample is
transported through the column by the flow of inert,
gaseous mobile phase. The column itself contains a
liquid stationary phase which is adsorbed onto the
surface of an inert solid.
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Instrumentation
Carrier Gas
Flow controller
Injector port
Column oven
Column
Detector
Recorder
http://teaching.shu.ac.uk/hwb/chemistry/tutorials/chrom/gaschrm.htm
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Carrier Gas
The carrier gas must be chemically inert.
Commonly used gases include nitrogen, helium,
argon, and carbon dioxide. The choice of carrier gas is often dependant
upon the type of detector which is used.
The carrier gas system also contains a
molecular sieve to remove water and other
impurities.
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Sample injection port
For optimum column efficiency, the sample should not be too large, and
should be introduced onto the column as a "plug" of vapour
- slow injection of large samples causes band broadening and loss of
resolution.
The most common injection method is where a microsyringe is used to
inject sample through a rubber septum into a flash vapouriser port at the
head of the column.
The temperature of the sample port is usually about 50C higher than the
boiling point of the least volatile component of the sample.
For packed columns, sample size ranges from tenths of a microliter up to 20
microliters. Capillary columns, on the other hand, need much less sample, typically
around 10-3 mL. For capillary GC, split/splitless injection is used.
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Sample injection port
The injector can be used in one of
two modes; split or split less.
The injector contains a heated
chamber containing a glass liner
into which the sample is injected
through the septum.
The carrier gas enters the
chamber and can leave by three
routes (when the injector is in split
mode).
The sample vaporizes to form a
mixture of carrier gas, vaporized
solvent and vaporized solutes
A proportion of this mixture passes
onto the column, but most exits through
the split outlet.
The septum purge outlet prevents
septum bleed components from
entering the column
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Columns
There are two general types of column,
1) packed
contain a finely divided, inert, solid support material (commonly based on
diatomaceous earth) coated with liquid stationary phase. Most packed columns are
1.5 - 10m in length and have an internal diameter of 2 - 4mm.
2) capillary(also known as open tubularCapillary columns have an internal diameter of a few tenths of a millimeter. They
can be one of two types;
a) wall-coated open tubular(WCOT)
consist of a capillary tube whose walls are coated with liquid stationary
phase
b) support-coated open tubular(SCOT).the inner wall of the capillary is lined with a thin layer of support material
such as diatomaceous earth, which the
stationary phase has been adsorbed.
SCOT columns are generally less efficient than WCOT columns. Both types of
capillary column are more efficient than packed columns.
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Column
In 1979, a new type of WCOT column was devised - the Fused Silica Open
Tubular(FSOT) column
These have much thinner walls than the glass capillary columns, and are
given strength by the polyimide coating. These columns are flexible and can
be wound into coils. They have the advantages of physical strength,
flexibility and low reactivity.
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Column Temperature
For precise work, column temperature must be controlled to within
tenths of a degree.
The optimum column temperature is dependant upon the boiling
point of the sample.
As a rule of thumb, a temperature slightly above the average boilingpoint of the sample results in an elution time of 2 - 30 minutes.
Minimal temperatures give good resolution, but increase elution
times.
If a sample has a wide boiling range, then temperature programming
can be useful. The column temperature is increased (either continuously or in
steps) as separation proceeds
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Detectors There are many detectors which can be used in gas chromatography. Different detectors will give different types of selectivity.
A non-selective detector responds to all compounds except the carrier gas
a selective detectorresponds to a range of compounds with a common
physical or chemical property and a specific detectorresponds to a single
chemical compound.
Detectors can also be grouped into concentration dependant detectors and
mass flow dependant detectors.
The signal from a concentration dependant detector is related to the
concentration of solute in the detector, and does not usually destroy the
sample
D
ilution of with make-up gas will lower the detectors response.
Mass flow dependant detectors usually destroy the sample, and the signal
is related to the rate at which solute molecules enter the detector.
The response of a mass flow dependant detector is unaffected by make-up
gas.
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Flame Ionization Detector (FID) The effluent from the column is mixed with
hydrogen and air, and ignited.
Organic compounds burning in the flame
produce ions and electrons which can
conduct electricity through the flame.
A large electrical potential is applied at the
burner tip, and a collector electrode is
located above the flame.
The current resulting from the pyrolysis ofany organic compounds is measured.
FIDs are mass sensitive rather than
concentration sensitive; this gives the
advantage that changes in mobile phase
flow rate do not affect the detector's
response.
The FID
is a useful general detector for theanalysis of organic compounds; it has high
sensitivity, a large linear response range,
and low noise.
It is also robust and easy to use, but
unfortunately, it destroys the sample.
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Applications
Main purpose is to
separate and analyze
multiple component
mixtures: Essential oils
Hydrocarbons
solvent
Biomedical
Biochemical
Physics
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Mass Spectrometry
Creates ions from molecules
It analyzes those ions, providing
information about its molecular weight and
chemical structure based on thefragmentation patterns
http://www.chem.arizona.edu/massspec/intro_html/intro.html
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Instrumentation
Sample introduction/separation
Ionization method
- Electron Impact ionization Ion separation method
- Low (unit) resolution 1 Dalton
- High resolution 0.0001 Dalton Ion Detector
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History
1886 Eugene Goldstein
observed rays which travelled through channels of a perforated
cathode. These rays would travel towards an anode
1899 William Wien
discovered the rays could be deflected by either a strongelectrical field or a strong magnetic field
constructed a device which could separate the positive rays by
their mass to charge ratio (m/z)
1918 and 1919 ArthurJeffrey Dempster and F.W Aston
(respectively)created the modern day Mass spectrometer
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History of Modern Day MS
1929 WalkerBleakney
developed Electron Impact mass spectrometry
hard impact technique
1987 Franz Hillenchamp and Michael Karas
developed Matrix Assisted Laser
Desorption/Ionization
used in the identification of biomolecules
2002 John Bennett Fenn
developed ESI
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Mass Spectrometry
Mass spectrometry is an analytical tool used for measuring the molecular mass of a
sample.
For large samples such as macromolecules,molecular masses can be measured to
within an accuracy of 0.01% of the total molecular mass of the sample
within a 4 Daltons (Da) or atomic mass units (amu).
This is sufficient to allow minor mass changes to be detectedthe substitution of one amino acid for another, or a post-translational
modification.
For small organic molecules the molecular mass can be measured to within an
accuracy of5 ppm or less, which is often sufficient to confirm the molecular formula
of a compound, and is also a standard requirement for publication in a chemical
journal.
Structural information can be generated using certain types of mass spectrometers,
usually those with multiple analyzers which are known as tandem mass
spectrometers. This is achieved by fragmenting the sample inside the instrument and
analyzing the products generated.
This procedure is useful for the structural elucidation of organic compounds and for
peptide or oligonucleotide sequencing.
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Applications
Biotechnology: the analysis of proteins, peptides,
oligonucleotides
Pharmaceutical: drug discovery, combinatorial
chemistry, pharmacokinetics, drug metabolism Clinical: neonatal screening, haemoglobin analysis,
drug testing
Environmental: PAHs, PCBs, water quality, food
contamination Geological: oil composition
Physics: identification of space particles
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Great Scott, it can be used in
Biochemistry too! Accurate molecular weight measurements:
sample confirmation, to determine the purity of a sample, to verify amino
acid substitutions, to detect post-translational modifications, to calculate the
number of disulphide bridges
Reaction monitoring:
to monitor enzyme reactions, chemical modification, protein digestion Amino acid sequencing:
sequence confirmation, de novo characterization of peptides, identification
of proteins by database searching with a sequence "tag" from a proteolytic
fragment
Oligonucleotide sequencing:
the characterization or quality control of oligonucleotides
Protein structure:
protein folding monitored byH/D exchange, protein-ligand complex
formation under physiological conditions, macromolecular structure
determination
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Mass spectrometers can be
divided into three fundamentalparts.
Ionization source
Analyzer
Detector
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Ionization Source
The sample has to be introduced into the ionization source of the
instrument.
Once inside the ionization source, the sample molecules are
ionized, because ions are easier to manipulate than neutral
molecules. These ions are extracted into the analyzer region of the mass
spectrometer where they are separated according to their mass (m)
-to-charge (z) ratios (m/z) .
The separated ions are detected and this signal sent to a data
system where the m/z ratios are stored together with their relative
abundance for presentation in the format of a m/z spectrum .
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Analyzer
The analyzer and detector of the mass spectrometer, and often the
ionization source too, are maintained under high vacuum to give the ions a
reasonable chance of travelling from one end of the instrument to the other
without any hindrance from air molecules.
The entire operation of the mass spectrometer, and often the sample
introduction process also, is under complete data system control on modernmass spectrometers.
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Sample Introduction
The method of sample introduction to the ionization source often
depends on the ionization method being used, as well as the type
and complexity of the sample.
The sample can be inserted directly into the ionization source, or
can undergo some type of chromatography in route to the ionizationsource.
This method of sample introduction usually involves the mass
spectrometer being coupled directly to a high pressure liquid
chromatography (HPLC), gas chromatography (GC) or capillary
electrophoresis (CE) separation column.
The sample is separated into a series of components which then
enter the mass spectrometer sequentially for individual analysis.
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Ionization Methods Many ionization methods are available and each has its own advantages and
disadvantages
The ionization method to be used should depend on the type of sample under
investigation and the mass spectrometer available.
Ionization methods include the following:
1. Atmospheric Pressure Chemical Ionization (APCI)
2. Chemical Ionization (CI)3. Electron Impact (EI)
4. Electrospray Ionization (ESI)
5, Fast Atom Bombardment (FAB)
6. Field Desorption / Field Ionization (FD/FI)
7. Matrix Assisted LaserDesorption Ionization (MALDI)
8. Thermospray Ionization (TSP)
The ionization methods used for the majority of biochemical analyses are
Electrospray Ionization (ESI) and , and Matrix Assisted LaserDesorption Ionization
With most ionization methods there is the possibility of creating both positively and
negatively charged sample ions, depending on the proton affinity of the sample,
therefore beginning an analysis, the user would need to determine if the ions are
cations or anions.
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Ionization MethodsIonization
method
Typical
Analytes
Sample
Introduction
Mass
Range
Method
Highlights
Electron Impact (EI) Relativelysmallvolatile
GC orliquid/solidprobe
to1,000Daltons
Hard methodversatileprovidesstructure info
Chemical Ionization(CI)
Relativelysmall
volatile
GC orliquid/solid
probe
to1,000D
altons
Soft methodmolecular ion
peak [M+H]+
Electrospray (ESI) PeptidesProteinsnonvolatile
LiquidChromatographyor syringe
to200,000Daltons
Soft methodions oftenmultiplycharged
Fast AtomBombardment (FAB)
CarbohydratesOrganometallicsPeptides
nonvolatile
Sample mixedin viscousmatrix
to6,000Daltons
Soft methodbut harderthan ESI or
MALDI
Matrix Assisted LaserDesorption
(MALDI)
PeptidesProteins
Nucleotides
Sample mixedin solid
matrix
to500,000Daltons
Soft methodvery high
mass
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Electrospray Ionization (ESI)
A high voltage of 3 or 4 kV is applied to the tip of the capillary,
which is situated within the ionization source of the mass
spectrometer, and as a consequence of this strong electric
field, the sample emerging from the tip is dispersed into an
aerosol of highly charged droplets, a process that is aided bya co-axially introduced nebulizing gas flowing around the
outside of the capillary.
This gas, usually nitrogen, helps to direct the spray emerging
from the capillary tip towards the mass spectrometer. The
charged droplets diminish in size by solvent evaporation,assisted by a warm flow of nitrogen known as the drying gas
which passes across the front of the ionization source.
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Electrospray Ionization (ESI)
Eventually charged sample ions, free from solvent, are released from the
droplets, some of which pass through a sampling cone or orifice into an
intermediate vacuum region, and from there through a small aperture into
the analyzer of the mass spectrometer, which is held under high vacuum.
The lens voltages are optimized individually for each sample.
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Electron Impact (EI)
The gas molecules exiting the GC are
bombarded by a high-energy electron beam
(70eV)
An electron will strike the molecule, supplyingenough energy to remove an electron from that
molecule
Will produce a singly charges ion containing one
unpaired electron
The instability on this molecule causes it to
fragment into smaller pieces
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Analyzers
Analysis and Separation of Sample Ions
The main function of the mass analyzer is to separate
the ions formed in the ionization source of the mass
spectrometer according to their mass-to-charge (m/z)
ratios.
There are a number of mass analyzers, the more
common known mass analyzers are quadrupoles , time-of-flight (TOF) analyzers, magnetic sectors , Fourier
transform and quadrupole ion traps .
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Analyzers
These mass analyzers have different features
Including the m/z range that can be covered,
the mass accuracy, and the achievable resolution.
The compatibility of different analyzers with different ionization
methods varies.
For example, all of the analyzers listed above can be used in
conjunction with electrospray ionization, whereas MALDI is not
usually coupled to a quadrupole analyzer.
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Analyzers
Tandem (MS-MS) mass spectrometers are instruments that have
more than one analyzer and so can be used for structural and
sequencing studies.
Two, three and four analyzers have all been incorporated intocommercially available tandem instruments, and the analyzers do
not necessarily have to be of the same type, in which case the
instrument is a hybrid one.
More popular tandem mass spectrometers include those of thequadrupole-quadrupole, magnetic sector-quadrupole , and more
recently, the quadrupole-time-of-flight geometries.
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Detectors
The detector monitors the ion current, amplifies it and
the signal is then transmitted to the data system where it
is recorded in the form of mass spectra .
The m/z values of the ions are plotted against their
intensities to show the number of components in the
sample the molecular mass of each component, and the
relative abundance of the various components in the
sample.
The type of detector is supplied to suit the type ofanalyzer; the more common ones are the
photomultiplier, the electron multiplier and the micro-
channel plate detectors.
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Key Terminology
Molecular Ion (M.+)is the charged molecule which remains intact, usually is the
molecular weight of molecule
Reference Spectramass spectral patterns which are reproducible
Base peak
100% abundance
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Interpreting Spectra
Ex) Methanol
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Samples (M) with molecular masses up to 1200
Da give rise to singly charged molecular-related
ions, usually protonated molecular ions of the
formula (M+H)+
in positive ionization mode, anddeprotonated molecular ions of the formula (M-
H)- in negative ionization mode.
Protonated molecular ions are expected when
the sample is analyzed under positive ionizationconditions.
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References
Robinson, Skelly Frame, Frame II, Undergraduate Instrumental Analysis, Chromatography pg,
721-851
Robinson, Skelly Frame, Frame II, 6th Edition, Undergraduate Instrumental Analysis. Mass
Spectrometry, pg. 613-721
Matthews, PhD, Fred, Organic Spectroscopy, Spring 2007 Lecture notes, Mass Spectroscopy and
GLC
B
rennan, PhD
, Carrie, Instrumental Analysis, Spring 2007 Lecture Notes, Chromatography Brennan, PhD, Carrie, Quantitative Analysis, Fall 2006 Lecture Notes, Chromatography
Silberberg, Chemistry 3rd Edition, pg 75
Silverstrin, Webster, Kiemle, Spectrometric Identification of Organic Compounds, 7th Edition,
Chapter 1 Mass Spectrometry
McMurry, Organic Chemistry, 6th Edition, Chapter 12
http://pubs.acs.org/hotartcl/tcaw/98/sep/creat.html, accessed June 16, 2007
www.chem.arizona.edu/massspec/inter_html/inter.html accessed July 3,2007 www.astbury.leeds.ac accessed July 3, 2007
www.chemistry.wustl.edu accessed July 3, 2007