Advanced Affinity Chromatography for postgraduate students

Principle behind affinity chromatography

1. Affinity Chromatography
and
Ion Exchange Chromatography
Shannon E. Spence

2. What on Earth did scientist do before
Chromatography?
-Extraction
is based on the difference in solubility material
is grounded, placed with a solvent which
dissolves soluble compounds. A second
extract solvent . The mixture is placed in a
separatory funnel
-Crystallization
also based on the difference of solubility. The
solubility 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-condensation
method
Filtration
separate 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

3. 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 previous
techniques
Chromatography is less time
consuming and cheaper
http://www.residues.com/ion_chromatography.html

4. 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 Chemist
win Nobel Prize for similar
experiement
•1922 L.S Palmer, American scientist
used Tswett’s techniques on various
natural products
•1931 Richard Kuhn used
chromatography to separate isomers
oh polyene pigments; this is the first
known acceptance of chromatographic
methods
http://www.chemgeo.uni-hd.de/texte/kuhn.html

5. 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 Richard
Laurence Millington Synge
•1944 Paper Chromatography one of
the most important methods in the
development of biotechnology
•1945 Gas Chromatography 1
st
analytical gas-solid (adsorption)
chromatography developed by Fritz
Prior
•1950 Gas Liquid Chromatography by
Martin and Anthony James; Martin
won the Nobel Prize in 1952
British chemist Archer John Porter Martin, co-
recipient, with Richard L. M. Synge, of the 1952
Nobel Prize in chemistry, "for their invention of
partition chromatography."
http://www.chemistryexplained.com/Ma-Na/Martin-
Archer-John-Porter.html

6. History of the Main Techniques
•1966 HPLC named by Csaba Horvath,
but didn’t become a popular method
until 1970s
•1950s Ion-Exchange chromatography
declassified this technique
•1970s Ion Chromatography was
developed 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

7. 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 on
the 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

8. 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 stationary
phase
4) different rates of partitioning for each component
5) means for visualizing the separation of each component

9. 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 binding
of 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

10. More Detailed History of
IE Chromatography
•1850 H. Thompson and J.T way treated various clas with ammonium sulfate
or carbonate in solution to extract the ammonia and release the calcium
•1927 Zeolite (sodium aluminum silicates) mineral columns were used to
remove interfering calcium and magnesium ions from solution to determine
sulfate content of water
•1940s Modern Ion-Exchange Chromatography was developed during the
wartime Manhattan Project
-this technique was used to separate and concentrate the radioactive
elements needed to make an atomic bomb. The adsorbents would latch
onto charged transuranium elements differentially eluting them
•1970s Hamish Small and co-workers of Dow 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

11. 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

12. 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 the
liquid 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

13. How do you get those columns
to work
•There is a glass column
coated with a resin
polymer
•The resin is either
positively charged (an
acid) or negatively
charged (a base)
•An analyte will have ions
opposite of the resins
charge eluting off the ion
of interest

14. 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

15. Cation Exchange resins
•The functional group in cation exchange resins are usually acids
•Sulfonic acids –SO
3H (strong acid resin) are added to the resin by
sulfonation reactions
Res-(SO
3H) + M
+
Res-(SO
3M) + 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

16. 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 --CH
2N(CH
3)
3+OH
-
–CH
2N(CH
3)
3+OH
-
+ B-Res-CH
2N(CH
3)
3
+
Cl
-
+ OH
-
•Polyalky amine a weak base --NH(-R)
2+OH
-
NH(-R)
2+OH
-
+ B
-
Res-NH(-R)
2
+
B-+OH
-

17. 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 exchange
resin column to a desired starting form by passing a
large excess of the desired ion at very high
concentration through the resin

18. 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

19. Relative Affinity of Ions
•The higher the charge the higher the
affinity
Na
+
< Ca
2+
< Al
3+
and Cl
-
< SO
4
2-
•The Ion with the greatest size and charge
has the highest affinity
Li
+
< Na
+
< K
+
< Cs
+
< Be
2+
< Mg
2+
< Cu
2+
and F
-
< Cl
-
< Br
-
< I
-

20. Ion Exchange Chromatography
The parameters controlling the residence
times of analyte or other eluent ions in the
resin stationary phase or ionic solution
mobile phase are
1) both the relative selectivity of the resin
for the ions
2) their relative concentrations in each
phase

21. 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

22. 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 a
separation, variable concentrations of HCl are used as
an eluent passing through the analytical anion column
without being retained forming largely undissociated
species such as water, carbonic acid and bicarbonate
ions

23. AFFINITY
CHROMATOGRAPHY

24. 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

25. How do they get those iddy bitty
molecules in there?

26. 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 be
eluted off by changing the pH of salt or organic
solvent concentration of the eluent

27. 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

28. 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 Chromatography
from being developed earlier and useful to the scientific
community
•The matrix must be activated for the ligand to bind to it
but still able to retain it’s own activation towards the
target molecule

29. Matrix
•Amino, hydroxyl, carbonyl and thio groups located with
the matrix serve as ligand binding sites
•Matrix are made up of agarose and other
polysaccharides
•The matrix also must be able to withstand the
decontamination process of rinsing with sodium
hydroxide or urea

30. 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

31. 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 the
sample 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 the
ligands within the column

32. Applications
•Used in Genetic Engineering
•Production of Vaccines
•And Basic Metabolic Research

33. Gas Chromatography
Gas-Liquid Chromatography
And
Gas-Solid Chromatography

34. 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
•1950
Archer J.P Martin and Anthony James developed Gas-
Liquid Partition Chromatography (GLPC)
this has become the method of choice

35. 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.

36. Instrumentation
•Carrier Gas
•Flow controller
•Injector port
•Column oven
•Column
•Detector
•Recorder
http://teaching.shu.ac.uk/hwb/chemistry/tutorials/chrom/gasc
hrm.htm

37. 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.

38. 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 50°C 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.

39. 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

40. 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 tubular
Capillary 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.

41. 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.

42. 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 boiling
point 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

43. Detectors
•There are many detectors which can be used in gas chromatography.
•Different detectors will give different types of selectivity.
•A non-selectivedetector 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 detectorsand
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
•Dilution 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.

44. 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 of
any 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 the
analysis 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.

45. Applications
•Main purpose is to
separate and analyze
multiple component
mixtures:
•Essential oils
•Hydrocarbons
•solvent
•Biomedical
•Biochemical
•Physics

46. Mass Spectrometry
•Creates ions from molecules
•It analyzes those ions, providing
information about it’s molecular weight and
chemical structure based on the
fragmentation patterns
http://www.chem.arizona.edu/massspec/intro_html/intro.html

47. Instrumentation
•Sample introduction/separation
•Ionization method
-Electron Impact ionization
•Ion separation method
-Low (unit) resolution –1 Dalton
-High resolution –0.0001 Dalton
•Ion Detector

48. Operation of Instrument
•Computer System
•Sample Introduction
•Ionization Method
•Ion-Separation
•Detector

49. 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 strong
electrical 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 Arthur Jeffrey Dempster and F.W Aston
(respectively)
created the modern day Mass spectrometer

50. History of Modern Day MS
•1929 Walker Bleakney
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

51. 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 detected
the 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 of 5 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
peptideor oligonucleotidesequencing.

52. 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

53. 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 by H/D exchange, protein-ligand complex
formation under physiological conditions, macromolecular structure
determination

54. Mass spectrometers can be
divided into three fundamental
parts.
•Ionization source
•Analyzer
•Detector

55. 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 .

56. 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 modern
mass spectrometers.

57. 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 ionization
source.
•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.

58. 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 Laser Desorption Ionization (MALDI)
8. Thermospray Ionization (TSP)
•The ionization methods used for the majority of biochemical analyses are
Electrospray Ionization (ESI) and , and Matrix Assisted Laser Desorption 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.

59. Ionization Methods
Ionization
method
Typical
Analytes
Sample
Introduction
Mass
Range
Method
Highlights
ElectronImpact (EI) Relatively
small
volatile
GC or
liquid/solid
probe
to
1,000
Daltons
Hard method
versatile
provides
structure info
Chemical Ionization
(CI)
Relatively
small
volatile
GC or
liquid/solid
probe
to
1,000
Daltons
Soft method
molecular ion
peak [M+H]+
Electrospray (ESI) Peptides
Proteins
nonvolatile
Liquid
Chromatography
or syringe
to
200,000
Daltons
Soft method
ions often
multiply
charged
Fast Atom
Bombardment (FAB)
Carbohydrates
Organometallics
Peptides
nonvolatile
Sample mixed
in viscous
matrix
to
6,000
Daltons
Soft method
but harder
than ESI or
MALDI
Matrix Assisted Laser
Desorption
(MALDI)
Peptides
Proteins
Nucleotides
Sample mixed
in solid
matrix
to
500,000
Daltons
Soft method
very high
mass

60. Electrospray Ionization (ESI)
•ESI is one of the Atmospheric Pressure Ionization (API)
techniques and is well-suited to the analysis of polar
molecules ranging from less than 100 Da to more than
1,000,000 Da in molecular mass.
•During standard electrospray ionization the sample is
dissolved in a polar, volatile solvent and pumped through a
narrow, stainless steel capillary at a flow rate of between 1
µL/min and 1 mL/min.

61. 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 by
a 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.

62. 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.

63. Electron Impact (EI)
•The gas molecules exiting the GC are
bombarded by a high-energy electron beam
(70eV)
•An electron will strike the molecule, supplying
enough 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

64. 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 .

65. 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.

66. 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 into
commercially 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 the
quadrupole-quadrupole, magnetic sector-quadrupole , and more
recently, the quadrupole-time-of-flight geometries.

67. Detectors
•The detectormonitors 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/zvalues of the ions are plotted against their
intensitiesto 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 of
analyzer; the more common ones are the
photomultiplier, the electron multiplier and the micro-
channel plate detectors.

68. Key Terminology
•Molecular Ion (M.+)
is the charged molecule which remains intact, usually is the
molecular weight of molecule
•Reference Spectra
mass spectral patterns which are reproducible
•Base peak
100% abundance

69. Interpreting Spectra
•Ex) Methanol

70. •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, and
deprotonated molecular ions of the formula (M-
H)
-
in negative ionization mode.
•Protonated molecular ions are expected when
the sample is analyzed under positive ionization
conditions.

71. References
•Robinson, Skelly Frame, Frame II, Undergraduate Instrumental Analysis, Chromatography pg,
721-851
•Robinson, Skelly Frame, Frame II, 6
th
Edition, Undergraduate Instrumental Analysis. Mass
Spectrometry, pg. 613-721
•Matthews, PhD, Fred, Organic Spectroscopy, Spring 2007 Lecture notes, Mass Spectroscopy and
GLC
•Brennan, PhD, Carrie, Instrumental Analysis, Spring 2007 Lecture Notes, Chromatography
•Brennan, PhD, Carrie, Quantitative Analysis, Fall 2006 Lecture Notes, Chromatography
•Silberberg, Chemistry 3
rd
Edition, pg 75
•Silverstrin, Webster, Kiemle, Spectrometric Identification of Organic Compounds, 7
th
Edition,
Chapter 1 Mass Spectrometry
•McMurry, Organic Chemistry, 6
th
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.acaccessed July 3, 2007
•www.chemistry.wustl.eduaccessed July 3, 2007