14/01/2011
Introduction to Chemical
Analysis Using Spectroscopy
Rob Keyzers
School of Chemical & Physical Sciences
Victoria University of Wellington
November 2010
How do we identify a molecule?
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How do we identify a molecule?
• That’s fine if we have 10 g of a simple
chemical…
• What if we have less than 10 mg?
• What if it’s very (VERY) complex?
How do we identify a molecule?
• We need instruments capable of analysis:
• Sensitive (sub-ng – mg level)
• Non-destructive (we may have the total world’s
supply!)
• Informative (get as much info per experiment)
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What do we do?!?
USE SPECTROSCOPY!!!
What is spectroscopy?
• Interaction of matter with electromagnetic
energy to probe molecular structure
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What is spectroscopy?
• Radio
• Gives information about nuclei and their chemical environment
(Nuclear Magnetic Resonance; NMR)
• Infrared
• Gives information about chemical functionality
• UV/Visible
• Gives information about double bonds and colours
• X-Ray
• Gives information about whole molecules (IN CRYSTALS ONLY!)
• Odd one out
– Mass spectrometry
• Gives information about molecular weight and formulae
Mass Spectrometry (MS)
• Recall
• All matter composed of:
– Electrons (e-)
– Protons (P+)
– Neutrons (No)
• P+ and No have essentially equal masses
• Defined as 1/12th of weight of 12C
• Called 1 Atomic Mass Unit (AMU) or Dalton (Da)
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Mass Spectrometry (MS)
• Each P+ weighs 1.6726231 x 10-27 kg
• Each No weighs 1.6749286 x 10-27 kg
• Each e- weighs 9.1093897 x 10-31 kg
• 1 AMU weighs 1.6605402 x 10-27 kg
Mass Spectrometry (MS)
• Each e- weighs proportionally only 5 x 10-4 th
the weight of 1 AMU
• Therefore e-’s do not contribute significantly to
molecular mass!
• ALL THE SIGNIFICANT MASS OF AN
ATOM IS BASED ON P+ AND No
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Mass Spectrometry (MS)
•
Element’s can be found as mixtures with differing numbers of P+ and
No
• Same element and general properties, called different ISOTOPES
• Hydrogen
– 1H (1 P+, 0 No), 99.98% of naturally occurring H (Proton)
– 2H (1 P+, 1 No), 0.02% of naturally occurring H (Deuterium)
– 3H (1 P+, 2 No), trace amount of naturally occurring H (Tritium)
– Relative atomic mass of naturally occurring H is
(0.9998 x 1.0078)+(0.002 x 2.0141) = 1.01 AMU
• Carbon
– 12C (6 P+, 6 No), 98.9% of naturally occurring C
– 13C (6 P+, 7 No), 1.1% of naturally occurring C
– 14C (6 P+, 8 No), trace amount of naturally occurring C
– Relative atomic mass of naturally occurring C is
(0.989 x 12.0000)+(0.011 x 13.0033) = 12.01 AMU
Mass Spectrometry (MS)
• Nitrogen
– 14N (7 P+, 7 No), 99.6% of naturally occurring N
– 15N (7 P+, 8 No), 0.4% of naturally occurring N
– Relative atomic mass of naturally occurring N is
(0.996 x 14.0031)+(0.004 x 15.0001) = 14.01 AMU
• Chlorine
– 35Cl (17 P+, 18 No), 75.5% of naturally occurring Cl
– 37Cl (17 P+, 20 No), 24.5% of naturally occurring Cl
– Relative atomic mass of naturally occurring Cl is
(0.755 x 34.9689)+(0.245 x 36.9659) = 35.46 AMU
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Mass Spectrometry (MS)
• Atoms therefore come in different types
• A type
– No heavier isotope that contributes significantly to atomic
weight
» e.g. Hydrogen, fluorine, phosphorus, iodine
• A+1 type
– Have a significant isotope 1 AMU heavier
» e.g. Carbon, nitrogen
• A+2 type
– Have a significant isotope 2 AMU heavier
» e.g. Bromine, chlorine, sulfur, oxygen
• etc
Mass Spectrometry (MS)
• A mass spectrometer…
• Detects molecular masses
• Detects a different mass for EVERY combination of
isotopes
• Can be used to ID molecular formula of a compound
• Very sensitive (can detect 10-9 g)
• All compounds analysed MUST be
electrically charged
• +ve or –ve IONS are detected
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Mass Spectrometry (MS)
• e.g. Ethanol, C2H6O
46.04
• When all 12C, 1H, 16O (called MONOISOTOPIC composition, M
peak) weighs 46.04186 AMU
• Note small peaks at 47.04186, 48.04186
• 47 from molecule where 1 x C=13C, 48 from when 2 x C=13C
100.0
80.0
60.0
40.0
20.0
0.0
46.00
46.50
47.00
47.50
48.00
Mass Spectrometry (MS)
• e.g. Ethanol, C2H6O
• Why are the peaks at 47 and 48 smaller?
• How do we get a peak at 47?
• One of the C’s must be 12C and the other 13C.
• The probability of getting a C as 13C (remembering 13C
accounts for 1.1% of all naturally occurring C) is 1.1% + 1.1% =
2.2% (2 C’s therefore 2 chances of 1.1% each). The peak at 47
is therefore 2.2% of the height (area) of the peak at 46.
• What about 48?
• BOTH C’s must be 13C. The probability of this is 1.1% x 1.1% =
0.012%!!! The peak at 48 is therefore 0.012% the height (area)
of the peak at 46.
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Mass Spectrometry (MS)
• e.g. Ethanol, C2H6O
• Why are the peaks at 47 and 48 smaller?
• How do we get a peak at 47?
• One of the C’s must be 12C and the other 13C.
• The probability of getting a C as 13C (remembering 13C
accounts for 1.1% of all naturally occurring C) is 1.1% + 1.1% =
2.2% (2 C’s therefore 2 chances of 1.1% each). The peak at 47
is therefore 2.2% of the height (area) of the peak at 46.
• What about 48?
• BOTH C’s must be 13C. The probability of this is 1.1% x 1.1% =
0.012%!!! The peak at 48 is therefore 0.012% the height (area)
of the peak at 46.
Mass Spectrometry (MS)
• What about something with more significant isotopes?
123.97
120.0
121.97
• e.g. Bromopropane (C3H7Br)
• Br has two isotopes, 79Br and 81Br in 50.5% and 49.5% abundance,
respectively, therefore get M:M+2 peaks in almost 1:1 ratio
• M+1 and M+3 for 13C versions of the 79Br and 81Br molecules.
• The groups of peaks for different isotopes in a molecule is called a
“Molecular Cluster”
100.0
80.0
60.0
40.0
20.0
0.0
121.00
122.00
123.00
124.00
125.00
126.00
127.00
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Mass Spectrometry (MS)
• So, how does MS work?
Sample
Ion source
Mass
discriminator
Detector
Data
analysis
• Highly modular instrument
Ionization source
• To be detected, a sample must be ionized
and introduced to the discriminator in the
gas phase.
• Gaseous samples only need to be ionized
• Solid/liquid samples must be vaporised and ionized
• Certain sources suitable for gases
• Electron impact (EI)
• Other sources good for liquids
• Atmospheric Pressure Ionization sources (API’s)
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Ionization source
e-
2 eM+
M
M + HA
[M+H]++ A-
Mass discriminator
• Once ionized…
• Ions accelerated into a mass discriminator
• Separates charged masses on some basis
• Related to mass to charge ratio (electrical current per atomic
mass)
• Multiple methods
•
•
•
•
•
Magnetic sector
Quadrupole
Time of Flight
Orbitrap
Ion-Cyclotron Resonance...
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Mass discriminator
Mass discriminator
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Detectors
• The part that does the business!
• Also generally the same between instruments
• Electron multiplier
• Some instruments use Fourier transform
electronics
• The ICR and the Orbitrap
• Discriminator and detector the same entity!
Detectors
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So, what do we get?
• Two types of information
• Low Resolution (0.1 Da accuracy)
– Good for fragmentation patterns (“fingerprinting”)
– Library matching of known compounds
So, what do we get?
C3H10Cl7N5O2
C8H6BrCl2NO8
C7BrCl2N8O3
CH3Br2N10O5
C2H9Br2N3O10
C3H2Cl3N3O13
H137Br2ClNO3
C8H120Cl2N3O4
CH117BrN12O
C2H123BrN5O6
CH130Cl4NO6
H124Cl4N8O
C16H121Br
C3H137BrCl4
C5H14Br2Cl3N4
H116N6O12
C10H111ClN9
C11H117ClN2O5
C10Cl5N7
C11H6Cl5O5
H5Cl4N4O12
H10BrCl4N6O5
C4H3BrCl3N9O2
C5H9BrCl3N2O7
C6H7Cl6N4O3
H2BrN4O16
C10H13Br2Cl2O2
C15H110N5O2
H13Cl8N6O
C11H3BrClO9
C3H134Br2O4
C4H16Br3N2O4
C5H123Cl3N4O3
C3H121Cl3N7O2
C4H127Cl3O7
C4H130Br2N4
C2H14Br3N5O3
C4H21BrCl7
C8H11Br2Cl2N3O
C13H108N8O
C14H114NO6
C2H4BrNO17
C8H9Cl6NO4
C2HBrCl3N12O
C3H7BrCl3N5O6
C17H3Br2N2
CH6BrCl4N10O
C2H12BrCl4N3O6
C9H4Cl5N3O4
C12H113ClN6O
CH112N10O8
C2H118N3O13
C7H16Br2Cl3NO
H106N17O3
H28Br4ClN
CH19Br3ClN3O3
C2H126Cl4N5O2
CH127BrNO10
H121BrN8O5
CH21Br2Cl4NO3
MW=392.865417
MW=392.8653836
MW=392.865379
MW=392.8654608
MW=392.8654654
MW=392.8653252
MW=392.8653142
MW=392.86554
MW=392.8656172
MW=392.8656218
MW=392.865174
MW=392.8651694
MW=392.8651126
MW=392.8657182
MW=392.8650714
MW=392.8650776
MW=392.8650496
MW=392.8650542
MW=392.865783
MW=392.8657876
MW=392.865811
MW=392.865013
MW=392.8658648
MW=392.8658694
MW=392.8649312
MW=392.8649212
MW=392.8659278
MW=392.865906
MW=392.8659028
MW=392.8648978
MW=392.8648284
MW=392.8660096
MW=392.8660258
MW=392.8646836
MW=392.8646882
MW=392.866166
MW=392.8646674
MW=392.8646236
MW=392.8645856
MW=392.8645638
MW=392.8645684
MW=392.8662634
MW=392.8662734
MW=392.8645226
MW=392.8645272
MW=392.8662938
MW=392.8663506
MW=392.8663552
MW=392.8644454
MW=392.8663918
MW=392.8664152
MW=392.8664198
MW=392.8664136
MW=392.8664106
MW=392.8643598
MW=392.8664954
MW=392.8665116
MW=392.8642842
MW=392.8642796
MW=392.8642196
dm=0.0 ppm
dm=0.0 ppm
dm=-0.1 ppm
dm=0.2 ppm
dm=0.2 ppm
dm=-0.2 ppm
dm=-0.2 ppm
dm=0.4 ppm
dm=0.6 ppm
dm=0.6 ppm
dm=-0.6 ppm
dm=-0.6 ppm
dm=-0.7 ppm
dm=0.8 ppm
dm=-0.8 ppm
dm=-0.8 ppm
dm=-0.9 ppm
dm=-0.9 ppm
dm=1.0 ppm
dm=1.0 ppm
dm=1.0 ppm
dm=-1.0 ppm
dm=1.2 ppm
dm=1.2 ppm
dm=-1.2 ppm
dm=-1.2 ppm
dm=1.3 ppm
dm=1.3 ppm
dm=1.3 ppm
dm=-1.3 ppm
dm=-1.5 ppm
dm=1.6 ppm
dm=1.6 ppm
dm=-1.8 ppm
dm=-1.8 ppm
dm=1.9 ppm
dm=-1.9 ppm
dm=-2.0 ppm
dm=-2.1 ppm
dm=-2.1 ppm
dm=-2.1 ppm
dm=2.2 ppm
dm=2.2 ppm
dm=-2.2 ppm
dm=-2.2 ppm
dm=2.3 ppm
dm=2.4 ppm
dm=2.4 ppm
dm=-2.4 ppm
dm=2.5 ppm
dm=2.6 ppm
dm=2.6 ppm
dm=2.6 ppm
dm=2.6 ppm
dm=-2.6 ppm
dm=2.8 ppm
dm=2.8 ppm
dm=-2.8 ppm
dm=-2.9 ppm
dm=-3.0 ppm
• Two types of information
• High Resolution (>0.0001 Da accuracy)
60.0
40.0
20.0
0.0
390.00
392.86
80.0
395.00
398.86
100.0
395.86
394.86
120.0
396.86
– Gives molecular formulae
– Still may need more info
400.00
405.00
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Now we have an idea of
molecular formula, what now?...
• Check for functional groups!
• Can be used to differentiate different isomers
• How?
• Use IR spectroscopy!
Infrared (IR) spectroscopy
• Bonds in a molecule act like a spring
• Vibrate in and out
• The frequency of vibration depends on the weights at the end
the spring (the atoms) and the length of the spring (bond
length)
• The atoms and bond length are dependent on FUNCTIONAL
GROUP identity!
• Energy required is in IR range
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Infrared (IR) spectroscopy
• Bonds in a molecule act like a spring
• What types of vibration are possible?!?
– Diatomic
– Polyatomic
Infrared (IR) spectroscopy
• Absorption of different wavelengths ID’s different
functional groups
• Measured in wave-numbers (cm -1)
e.g.
H-O stretch = 3300 - 3650 cm-1
C-O stretch = 1000 – 1300 cm-1
H-N Stretch = 3300 – 3500 cm-1
Carbonyls (ester) = 1735 – 1750 cm-1
Carbonyls (acid) = 1700 – 1730 cm-1
Carbonyls (amide) = 1630 – 1680 cm-1
etc etc...
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Infrared (IR) spectroscopy
Now we have our molecular formula
and an idea of functionality….
• Well, now what?
• What if you had possible isomers with the same
functionality/formula?
e.g.
• How do we tell the difference?
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Nuclear Magnetic Resonance
(NMR)
• Certain types of nucleus (e.g. H, C, N, F,
P) can be probed individually
• Tells us what type of nucleus is present
• Tells us what “chemical environment” each
nucleus is in
• Same principle as MRI
• Only certain isotopes work
• 1H, 2H, 13C, 15N, 18O…
• Called “spin active” nuclei
• VERY poor sensitivity (< 10-4 g)
Nuclear Magnetic Resonance
(NMR)
• How does it work?
• Each spin active nucleus acts like a magnet
• In an external magnetic field, the nuclei try to
align with the external field
Apply magnetic field
(BO)
Normal
Aligned
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Nuclear Magnetic Resonance
(NMR)
• You can “flip” each magnet from aligned to
anti-aligned.
• When you do, it will flip back and give off radio energy
• You can measure the energy and this tells you about
the “chemical environment”
• The frequency of radio energy changes because of
what’s around each nucleus
Apply magnetic field
(BO)
Normal
Aligned
Nuclear Magnetic Resonance
(NMR)
• How do we do it?
• All measured inside a super-conducting magnet
• Produces extremely high, consistent magnetic field
• Extremely expensive!
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Nuclear Magnetic Resonance
(NMR)
• “Chemical environment”
• Each nucleus in a different chemical environment will give one
signal in the NMR spectrum
• Based on symmetry
• Each signal different because of what’s around it
– Other spin active nuclei
– Electrons (which are also spin active)
Nuclear Magnetic Resonance
(NMR)
• Many different types of NMR experiment
• The two most basic are 1H and 13C
•
13C
is most simple to understand
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13C
NMR Spectrum
• The scale
• Measured in ppm (frequency) from 0 at right hand side
(backwards)!
• Goes up to around 220 ppm
• The position on the scale is called the “chemical shift”
• Value indicative of carbon functionality and type (alkane,
alkene, carbonyl, acid, amide etc)
13C
NMR Spectrum
e.g. 1-Propanol and 2-propanol
• If we know molecular formula from MS then can
determine structure
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13C
NMR Spectrum
13C
NMR Spectrum
e.g.
e.g.
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1H
NMR
• A bit more complex than 13C
• Get different chemical shifts for 1H’s
• Also get integration data (tells you how many 1H’s
in a chemical environment)
• Get “coupling patterns” (tells you how many next
door neighbour 1H’s an individual 1H has)
• Similar kind of chemical shift chart for 1H
1H
NMR
The “n+1” rule
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1H
NMR
NMR
• What I have shown you is BASIC NMR
• Only “1D” NMR spectra
• Many other experiments
–
–
–
–
Tell you which 1H is next door to another
Tell you which 1H is attached to which 13C
Tell you which 1H is within two to three bonds of a 13C
etc etc…
NMR is the most used analytical technique for
solving chemical structures, in conjunction with MS
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NMR and MS
• The combination of IR, MS and NMR has been
used to solve structures like:
• Try solving those by paper-chase!
NMR and MS
• All three techniques now also instrumental for
•
•
•
•
protein identification
understanding biological processes and interactions
probing fluid motion and hydrodynamics
The list goes on….
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