Introduction to Chemical Analysis Using Spectroscopy14/01/2011 4 What is spectroscopy? •Radio...

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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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• 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?


• 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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• 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


• 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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• 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


• 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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• 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


• 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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• e.g. Ethanol, C2H6O• 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

46.00 46.50 47.00 47.50 48.00

0.0

20.0

40.0

60.0

80.0

100.0

46

.04


• 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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• 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.


• What about something with more significant isotopes?• 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”

121.00 122.00 123.00 124.00 125.00 126.00 127.00

0.0

20.0

40.0

60.0

80.0

100.0

120.0

12

3.9

7

12

1.9

7

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• So, how does MS work?

• Highly modular instrument

Sample

Ion sourceMass

discriminatorDetector

Data

analysis

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

M M+

e- 2 e-

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

http://upload.wikimedia.org/wikipedia/commons/1/12/Orbitrap_Patent.gif

http://upload.wikimedia.org/wikipedia/commons/8/8a/Orbitrappe.png

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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?

• Two types of information• High Resolution (>0.0001 Da accuracy)

– Gives molecular formulae

– Still may need more info

390.00 395.00 400.00 405.00

0.0

20.0

40.0

60.0

80.0

100.0

120.0

39

5.8

6

39

8.8

6

39

2.8

6

39

6.8

6

39

4.8

6

C3H10Cl7N5O2 MW=392.865417 dm=0.0 ppm

C8H6BrCl2NO8 MW=392.8653836 dm=0.0 ppm

C7BrCl2N8O3 MW=392.865379 dm=-0.1 ppm

CH3Br2N10O5 MW=392.8654608 dm=0.2 ppm

C2H9Br2N3O10 MW=392.8654654 dm=0.2 ppm

C3H2Cl3N3O13 MW=392.8653252 dm=-0.2 ppm

H137Br2ClNO3 MW=392.8653142 dm=-0.2 ppm

C8H120Cl2N3O4 MW=392.86554 dm=0.4 ppm

CH117BrN12O MW=392.8656172 dm=0.6 ppm

C2H123BrN5O6 MW=392.8656218 dm=0.6 ppm

CH130Cl4NO6 MW=392.865174 dm=-0.6 ppm

H124Cl4N8O MW=392.8651694 dm=-0.6 ppm

C16H121Br MW=392.8651126 dm=-0.7 ppm

C3H137BrCl4 MW=392.8657182 dm=0.8 ppm

C5H14Br2Cl3N4 MW=392.8650714 dm=-0.8 ppm

H116N6O12 MW=392.8650776 dm=-0.8 ppm

C10H111ClN9 MW=392.8650496 dm=-0.9 ppm

C11H117ClN2O5 MW=392.8650542 dm=-0.9 ppm

C10Cl5N7 MW=392.865783 dm=1.0 ppm

C11H6Cl5O5 MW=392.8657876 dm=1.0 ppm

H5Cl4N4O12 MW=392.865811 dm=1.0 ppm

H10BrCl4N6O5 MW=392.865013 dm=-1.0 ppm

C4H3BrCl3N9O2 MW=392.8658648 dm=1.2 ppm


C6H7Cl6N4O3 MW=392.8649312 dm=-1.2 ppm

H2BrN4O16 MW=392.8649212 dm=-1.2 ppm

C10H13Br2Cl2O2 MW=392.8659278 dm=1.3 ppm

C15H110N5O2 MW=392.865906 dm=1.3 ppm

H13Cl8N6O MW=392.8659028 dm=1.3 ppm

C11H3BrClO9 MW=392.8648978 dm=-1.3 ppm

C3H134Br2O4 MW=392.8648284 dm=-1.5 ppm

C4H16Br3N2O4 MW=392.8660096 dm=1.6 ppm

C5H123Cl3N4O3 MW=392.8660258 dm=1.6 ppm

C3H121Cl3N7O2 MW=392.8646836 dm=-1.8 ppm

C4H127Cl3O7 MW=392.8646882 dm=-1.8 ppm

C4H130Br2N4 MW=392.866166 dm=1.9 ppm

C2H14Br3N5O3 MW=392.8646674 dm=-1.9 ppm

C4H21BrCl7 MW=392.8646236 dm=-2.0 ppm

C8H11Br2Cl2N3O MW=392.8645856 dm=-2.1 ppm

C13H108N8O MW=392.8645638 dm=-2.1 ppm

C14H114NO6 MW=392.8645684 dm=-2.1 ppm

C2H4BrNO17 MW=392.8662634 dm=2.2 ppm

C8H9Cl6NO4 MW=392.8662734 dm=2.2 ppm

C2HBrCl3N12O MW=392.8645226 dm=-2.2 ppm

C3H7BrCl3N5O6 MW=392.8645272 dm=-2.2 ppm

C17H3Br2N2 MW=392.8662938 dm=2.3 ppm

CH6BrCl4N10O MW=392.8663506 dm=2.4 ppm


C9H4Cl5N3O4 MW=392.8644454 dm=-2.4 ppm

C12H113ClN6O MW=392.8663918 dm=2.5 ppm

CH112N10O8 MW=392.8664152 dm=2.6 ppm

C2H118N3O13 MW=392.8664198 dm=2.6 ppm

C7H16Br2Cl3NO MW=392.8664136 dm=2.6 ppm

H106N17O3 MW=392.8664106 dm=2.6 ppm

H28Br4ClN MW=392.8643598 dm=-2.6 ppm

CH19Br3ClN3O3 MW=392.8664954 dm=2.8 ppm

C2H126Cl4N5O2 MW=392.8665116 dm=2.8 ppm

CH127BrNO10 MW=392.8642842 dm=-2.8 ppm

H121BrN8O5 MW=392.8642796 dm=-2.9 ppm

CH21Br2Cl4NO3 MW=392.8642196 dm=-3.0 ppm

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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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• Bonds in a molecule act like a spring• What types of vibration are possible?!?

– Diatomic

– Polyatomic


• 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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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)


(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

Normal

Apply magnetic field

(BO)

Aligned

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(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

Normal

Apply magnetic field

(BO)

Aligned


(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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(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)


(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

e.g.

13C NMR Spectrum

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….

http://en.wikipedia.org/wiki/File:Bruker_Avance1000.jpg

Introduction to Chemical Analysis Using Spectroscopy14/01/2011 4 What is spectroscopy? •Radio...

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