HANDOUT

SPECTROSCOPY ANALYSIS OF CHEMISTRY

bY:

Drs. Tri Santoso ,M.Si

PROGRAM STUDI PENDIDIKAN KIMIAJURUSAN MATEMATIKA DAN ILMU PENGETAHUAN ALAM

FAKULTAS KEGURUAN DAN ILMU PENDIDIKANMARCH, 2010

I. Introduction:  Modern spectroscopic methods have largely replaced chemical tests as the standard means of identifying chemical structures, and for a practising practical organic chemist 1H-NMR has become a routine tool for identifying the products of reactions.

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 Spectroscopic techniques are based on the absorption of specific forms of energy by a molecule and monitoring the affect that this has on the molecule.

Spectroscopy:

Theoretical Background:

Since spectroscopy is based on the interaction of electromagnetic radiation (EMR) with a molecule, an understanding of electromagnetic radiation is a must.

Spectroscopy monitors the changes in energy states of a molecule, so one must be familiar with the important energy states and concept of quantisation of energy within a molecule.

Electromagnetic Radiation (EMR):

The part of the electromagnetic radiation spectrum that you are most familar with is "visible light" but this is just a small portion of all the possible types. Can you think of common applications of other regions of the electromagnetic spectrum ?    

Electromagnetic radiation has both particle and wave properties.  Can you think of an

example of each ? 

Wave-like properties: It is important to understand wavelength and frequency and how they relate to one another.

Particle-like properties: A particle of energy is called a photon.  Each photon has a discrete amount of energy : a quantum.

Energy States: There are many energy 'states' in a molecule.  Of particular interest to the organic chemist will be those relating to the energy associated with the nuclear spin state, the vibration of a bond or an electronic energy levels (orbitals)

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Quantisation of Energy:  

The absorption of energy causes an atom or molecule to go from an initial energy state (the ground state) to another higher enrgy state (an excited state)   The energy changes are frequently described using an energy level diagram.

The energy states are said to be quantised because there are only certain values that are possible, there is not a continuous spread of energy levels available.  

Spectroscopic methods: Ultra-Violet (UV-VIS) Infra red (IR) Nuclear Magnetic Resonance (1H-NMR and 13C-NMR) Mass Spectrometry (MS) Getting Structures fromSpectra

II. Ultraviolet-Visible (uv-vis) Spectroscopy

Basics Ultraviolet-visible spectropscopy (uv = 200-400 nm, visible = 400-800 nm) corresponds to electronic excitations between the energy levels that correspond to the molecular orbitals of the systems.  In particular, transitions involving orbitals and lone pairs (n = non-bonding) are important and so uv-vis spectroscopy is of most use for identifying conjugated systems which tend to have stronger absorptions.The lowest energy transition is that between the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) in the ground state.  The absorption of the EM radiation excites an electron to the LUMO and creates an excited state. The more highly conjugated the system, the smaller the HOMO-LUMO gap, E, and therefore the lower the frequency and longer the wavelength, .  The colours we see in inks, dyes, flowers etc. are typically due to highly conjugated organic molecules. The unit of the molecule that is responsible for the absorption is called the chromophore, of which the most common are C=C ( to *) and C=O (n to *) systems.

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The following table contains some data for polyenes and demonstrates how the wavelength of the absorbance increases as the conjugated system becomes more extended.  

H(CH=CH)nHmax /

nm max 

1 170 15,0002 217 21,0003 258 35,000

Terminology

Absorbance A, a measure of the amount of radiation that is absorbedBand Term to describe a uv-vis absorption which are typically broad.Chromophore Structural unit responsible for the absorption.Molar absorptivity , absorbance of a sample of molar concentration in 1 cm cell.Extinction coefficicent An alternative term for the molar absorptivity.Path length l, the length of the sample cell in cm.Beer-Lambert Law A = c.l    (c = concentration in moles / litre)max The wavelength at maximum absorbancemax The molar absorbance at max

HOMO Highest Occupied Molecular OrbitalLUMO Lowest Unoccupied Molecular Orbital

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III. Infra Red SpectrophometryBasics: Infra red (IR) spectroscopy deals with the interaction between a molecule and radiation from the IR region of the EM spectrum (IR region = 4000 - 400 cm-1). The cm-1 unit is the wave number scale and is given by 1 / (wavelength in cm).

IR radiation causes the excitation of the vibrations of covalent bonds within that molecule. These vibrations include the stretching and bending modes.

An IR spectrum show the energy absorptions as one 'scans' the IR region of the EM spectrum.  As an example, the IR spectrum of butanal is shown below.

In general terms it is convienient to split an IR spectrum into two approximate regions:

4000-1000 cm-1 known as the functional group region, and < 1000 cm-1 known as the fingerprint region

Most of the information that is used to interpret an IR spectrum is obtained from the functional group region.

In practice, it is the polar covalent bonds than are IR "active" and whose excitation can be observed in an IR spectrum.

In organic molecules these polar covalent bonds represent the functional groups. Hence, the most useful information obtained from an IR spectrum is what

functional groups are present within the molecule (NMR spectroscopy typically gives the hydrocarbon fragments).

Remember that some functional groups can be "viewed" as combinations of different bond types.  For example, an ester, CO2R contains both C=O and C-O bonds, and both are typically seen in an IR spectrum of an ester.

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In the fingerprint region, the spectra tend to be more complex and much harder to assign.

MOST IMPORTANT THING TO REMEMBER.....

When analysing an IR spectrum avoid the temptation to try to assign every peak. The fingerprint region, however, can be useful for helping to confirm a structure by direct comparison with a known spectra.

Infra-Red (IR) Spectroscopy

 Hookes' Law

To help understand IR, it is useful to compare a vibrating bond to the physical model of a vibrating spring system.  The spring system can be described by Hooke's Law, as shown in the equation given on the left. 

Consider a bond and the connected atoms to be a spring with two masses attached.

Using the force constant k (which reflects the stiffness of the spring) and the two masses m1 and m2, then the equation indicates how the frequency, , of the absorption should change as the properties of the system change.

Consider the following trends: 1. for a stronger bond (larger k value), increases.

As examples of this, in order of increasing bond strength compare: CC bonds: C-C (1000 cm-1), C=C (1600 cm-1) and CC (2200 cm-1), CH bonds: C-C-H (2900 cm-1), C=C-H (3100 cm-1) and CC-H (3300 cm-1),(n.b. make sure that you understand the bond strengths order)2.  for heavier atoms attached (larger value), decreases.As examples of this, in order of increasing reduced mass compare: C-H  (3000 cm-1) C-C  (1000 cm-1) C-Cl (800 cm-1) C-Br (550 cm-1) C-I   (about 500 cm-1)

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The following diagram reflects some of the trends that can be accounted for using Hookes' Law.  It also gives an approximate outline of where specific types of bond stretches may be found.

Infra-Red (IR) Spectroscopy

Important absorptions:

The more important absorptions that you should probably learn to recognise, in order of importance are:  

Bond Base Value

Strength / Shape Comments

1 C=O 1715 s, "finger" Exact position depends on type of carbonyl2 O-H 3600 s, brd Broad due to H bonding3 N-H 3500 m Can tell primary (two peaks) from secondary (one peak)4 C-O 1100 s Also check for OH and C=O

5 C=C 1650 w alkene  m-s aromatic 

Alkene weak due to low polarity  Aromatic usually in pairs

6 CC 2150 w, sharp Most obvious in terminal alkynes

7 C-H 3000 s As hybridisation of C changes sp3-sp2-sp, the frequency increases

8 CN 2250 m, sharp Characteristic since little else around itabbreviations :  "s" = strong, "m" = medium and "w" = weak

If you know these, then you can identify most of the functional groups of interest. Note that it is rarely useful to look for C-C since the large majority organic molecules will have them.

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You should also be aware that the exact substitution pattern of a particular bond causes shifts in the position of the absorption and, therefore, ranges of values are typically given in most tables.

It is possible to rationalise the shifts of absorbances based on electronic effects due to proximal groups, conjugation and / or ring strain.

In general, when you are trying to work out what a molecule is, you will not just have the IR spectrum, but you will have other information as well, such as the formula or most likely the NMR. Always cross check between these sets of information.  For example, if the molecular formula indicates only one O, then you can not have an ester (CO2R) or a carboxylic acid (CO2H) !

Sample IR Spectra :  By looking at IR spectra that contain known functional groups and comparing and contrasting them with other IR spectra, one can develop the skills required to be able to "interpret" an "unknown" IR spectra.  Remember that for an organic chemist, the primary role of IR is to identify the functional groups that are present. A few examples reflecting some of the more important functional groups are provided below. Compare them to try to appreciate the subtle differences, comparing frequency, intensity and shape.  In the first example, of the aromatic hydrocarbon, toluene, we can see both the aromatic and aliphatic CH stretches, and two absorptions for the aromatic C=C stretches.

Acetone (2-propanone) is the "classic" carbonyl containing compound with the obvious C=O stretch in the middle of the spectra. Note that the peak is a very strong absorption. Compare it with the C=C in the previous case which are weaker and sharper.

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The characteristic absorption of the alcohol, 2-propanol, is the broad band due to the hydrogen bonded -OH group.

Carboxylic acids contain both C=O and OH groups. Note the broadness of both absorptions due to the hydrogen bonding and that the C=O is typically at slightly lower frequency than that of a ketone.

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An ester has the following key absorptions, the C=O and typically two bands for the C-O (not always easy to identify) since there are both sp3 C-O and sp2 C-O bonds.

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IV. Nuclear Magnetic Resonance (NMR) Spectroscopy

Basics: Nuclei with an odd mass or odd atomic number have "nuclear spin" (in a similar fashion to the spin of electrons). This includes 1H and 13C (but not 12C). The spins of nuclei are sufficiently different that NMR experiments can be sensitive for only one particular isotope of one particular element.  The NMR behaviour of 1H and 13C nuclei has been exploited by organic chemist since they provide valuable information that can be used to deduce the structure of organic compounds. These will be the focus of our attention.

Since a nucleus is a charged particle in motion, it will develop a magnetic field.  1H and 13C  have nuclear spins of 1/2 and so they behave in a similar fashion to a simple, tiny bar magnet.  In the absence of a magnetic field, these are randomly oriented but when a field is applied they line up parallel to the applied field, either spin aligned or spin opposed.  The  more highly populated state is the lower energy spin state spin aligned situation.  Two schematic  representations of these arrangements are shown below:  

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In NMR, EM radiation is used to "flip" the alignment of nuclear spins from the low energy spin aligned state to the higher energy spin opposed state. The energy required for this transition depends on the strength of the applied magnetic field (see below) but in is small and corresponds to the radio frequency range of the EM spectrum.

As this diagram shows, the energy required for the spin-flip depends on the magnetic field strength at the nucleus. With no applied field, there is no energy difference between the spin states, but as the field increases so does the separation of energies of the spin states and therefore so does the frequency required to cause the spin-flip, referred to as resonance.

  The basic arrangement of an NMR spectrometer is shown to the left.  The sample is positioned in the magnetic field and excited via pulsations in the radio frequency input circuit. The realigned magnetic fields induce a radio signal in the output circuit which is used to generate the output signal.  Fourier analysis of the complex output produces the actual spectrum. The pulse is repeated as many times as necessary to allow the signals to be identified from the background noise.

Chemical Shift

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An NMR spectrum is a plot of the radio frequency applied against absorption. A signal in the spectrum is referred to as a resonance. The frequency of a signal is known as its chemical shift.

The chemical shift in absolute terms is defined by the frequency of the resonance expressed with reference to a standard compound which is defined to be at 0 ppm. The scale is made more manageable by expressing it in parts per million (ppm) and is indepedent of the spectrometer frequency.

It is often convienient to describe the relative positions of the resonances in an NMR spectrum.  For example, a peak at a chemical shift, , of 10 ppm is said to be downfield or deshielded with respect to a peak at 5 ppm, or if you prefer, the peak at 5 ppm is upfield or shielded  with respect to the peak at 10 ppm.

 Typically for a field strength of 4.7T the resonance frequency of a proton will occur around 200MHz and for a carbon, around 50.4MHz.  The reference compound is the same for both, tetramethysilane (Si(CH3)4 often just refered to as TMS).

What would be the chemical shift of a peak that occurs 655.2 Hz downfield of TMS on

a spectrum recorded using a 90 MHz spectrometer ? 

At what frequency would the chemical shift of chloroform (CHCl3, =7.28 ppm) occur

relative to TMS on a spectrum recorded on a 300 MHz spectrometer ? 

A 1 GHz (1000 MHz) NMR spectrometer is being developed, at what frequency and

chemical shift would chloroform occur ? 

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Shielding in H-NMR

The magnetic field experienced by a proton is influenced by various structural factors. Since the magnetic field strength dictates the energy separation of the spin states and hence the radio frequency of the resonance, the structural factors mean that different types of proton will occur at different chemical shifts. This is what makes NMR so useful for structure determination, otherwise all protons would have the same chemical shift.

The various factors include:

inductive effects by electronegative groups magnetic anisotropy hydrogen bonding

Electronegativity

The electrons around the proton create a magnetic field that opposes the applied field. Since this reduces the field experienced at the nucleus, the electrons are said to shield the proton. It can be useful to think of this in terms of vectors....

Since the field experienced by the proton defines the energy difference between the two spin states, the frequency and hence the chemical shift, /ppm, will change depending on the electron density around the proton. Electronegative groups attached to the C-H system decrease the electron density around the protons, and there is less shielding (i.e. deshielding) so the chemical shift increases. This is reflected by the plot shown in the graph to the left which is based on the data shown below.

 

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Compound, CH3X CH3F CH3OH CH3Cl CH3Br CH3I CH4 (CH3)4Si

X F O Cl Br I H SiElectronegativity

of X 4.0 3.5 3.1 2.8 2.5 2.1 1.8

Chemical shift, / ppm 4.26 3.4 3.05 2.68 2.16 0.23 0

These effects are cumulative, so the presence of  more electronegative groups produce more deshielding and therefore, larger chemical shifts.

Compound CH4 CH3Cl CH2Cl2 CHCl3

/ ppm 0.23 3.05 5.30 7.27

These inductive effects at not just felt by the immediately adjacent protons as the disruption of electron density has an influence further down the chain.  However, the effect does fade rapidly as you move away from the electronegative group.  As an example, look at the chemical shifts for part of a primary bromide

H signal -CH2-CH2-CH2Br / ppm  1.25  1.69  3.30

Magnetic Anisotropy

The word "anisotropic" means "non-uniform".  Magnetic anisotropy means that there is a "non-uniform magnetic field". Electrons in systems (e.g. aromatics, alkenes, alkynes, carbonyls etc.) interact with the applied field which induces a magnetic field that causes the anisotropy.  As a result, the nearby protons will experience 3 fields: the applied field, the shielding field of the valence electrons and the field due to the system. Depending on the position of the proton in this third field, it can be either shielded (smaller ) or deshielded (larger ), which implies that the energy required for, and the frequency of the absorption will change.

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Hydrogen Bonding

Protons that are involved in hydrogen bonding (this usually means -OH or -NH) are typically observed over a large range of chemical shift values.  The more hydrogen bonding there is, the more the proton is deshielded and the higher its chemical shift will be. However, since the amount of hydrogen bonding is susceptible to factors such as solvation, acidity, concentration and temperature, it can often be difficult to predict.

HINT : It is often a good idea to leave assigning  -OH or -NH resonances until other assignments have been made.

Experimentally, -OH and -NH protons can be identified by carrying out a simple D2O (deuterium oxide, also known as heavy water) exchange experiment.

Run the regular H-NMR experiment Add a few drops of D2O Re-run H-NMR experiment Compare the two spectra a look for peaks that have "disappeared"

Why would a peak disappear ?

Consider the alcohol case for example:   R-OH  +  D2O   <=>   R-OD   +   HOD During the hydrogen bonding, the alcohol and heavy water can "exchange" -H and -D each other, so the alcohol becomes R-OD. Although D is NMR active, it's signals are of different energy and are not seen in the H-

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NMR, hence the peak due to the -OH disappears.  (Note that the HOD will appear...)

Suggest a reason why the acidic protons in a carboxylic acid appear so far downfield (about

12 ppm) ? 

H-NMR Chemical shifts

The chemical shift is the position on the scale (in ppm) where the peak occurs. Typical /ppm values for protons in different chemical environments are shown in

the figure below. There are two major factors that influence chemical shifts

(a) deshielding due to reduced electron density (due electronegative atoms) and (b) anisotropy (due to magnetic fields generated by bonds).

Note that the figure shows the typical chemical shifts for protons being influenced by a single group.

In cases where a proton is influenced by more than one group, the effects are essentially cumulative.

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H-NMR Spectra I

The time has arrived to look at a few H-NMR spectra.....

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Coupling in H-NMR

So far the H-NMR spectra that we have looked at have all had different types of protons that are seen as singlets in the spectra.  This is not the normal case.... spectra usually have peaks that appear as groups of peaks due to coupling with neighbouring protons, for example, see the spectra of 1,1-dichloroethane shown below.

 

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Before we look at the coupling, lets review the assignment of the peaks first:

= 5.9 ppm, integration = 1H deshielded : agrees with the -CHCl2 unit = 2.1 ppm, integration = 3H, agrees with -CH3 unit.

Now, what about the coupling patterns ?

 Coupling arises because the magnetic field of vicinal (adjacent) protons influences the field that the proton experiences.

To understand the implications of this we should first consider the effect the -CH group has on the adjacent -CH3.

The methine -CH can adopt two alignments with respect to the applied field. As a result, the signal for the adjacent methyl -CH3 is split in two lines, of equal intensity, a doublet.

Now consider the effect of the -CH3 group has on the adjacent -CH .The methyl -CH3 protons give rise to 8 possible combinations with respect to the applied field. However, some combinations are equivalent and there are four magnetically different effects. As a result, the signal for the adjacent  methine -CH is split into four lines, of intensity ratio 1:3:3:1, a quartet.

The proximity of  "n" equivalent H on neighbouring carbon atoms, causes the signals to be split into "n+1" lines.

This is known as the multiplicity or splittingorcoupling pattern of each signal. Equivalent protons (or those with the same chemical shift) do not couple to each

other. If the neighbours are not all equivalent, more complex patterns arise. To a first approximation, protons on adjacent sp3 C tend to behave as if they are

equivalent.

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Now we can do more a complete analysis, including the application of the "n+1" rule to 1,1-dichloroethane:

= 5.9 ppm, quartet, integration = 1H, deshielded

grees with the -CHCl2 unit next to a -CH3 unit (n = 3, so n + 1 = 4 lines)

= 2.1 ppm, doublet, integration = 3H

grees with -CH3 unit, next to a -CH- (n = 1, so n + 1 = 2 lines)

Coupling Constant, J  

The coupling constant, J (usually in frequency units, Hz) is a measure of the interaction between a pair of protons. In a vicinal system of the general type, Ha-C-C-Hbthen the coupling of Ha with Hb, Jab, MUST BE EQUAL to the coupling of Hb with Ha, Jba, therefore Jab = Jba.  The implications are that the spacing between the lines in the coupling patterns are the same as can be seen in the coupling patterns from the H-NMR spectra of 1,1-dichloroethane (see left).

Pascal's Triangle   The relative intensitites of the lines in a coupling pattern is given by a binomial expansion or more conviently by Pascal's triangle. To derive Pascal's triangle, start at the apex, and generate each lower row by creating each number by adding the two numbers above and to either side in the row above together.  The first six rows are shown to the right. So for H-NMR a proton with zero neighbours, n = 0,  appears as a single line, a proton with one neighbours, n =1 as two lines of equal intensity, a proton with two neighbours, n = 2, as three lines of intensities 1 : 2 : 1,   etc.What would the multiplicity and the relative intensitites be for the secondary H in propane ? 

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Summary

Individual resonances are split due to coupling with "n" adjacent protons Number of lines in coupling pattern, L = n + 1

Complex Coupling Patterns

In reality, coupling patterns are often more complex than the simple n+1 rule since the neighbouring protons are often not equivlalent to each other (i.e. there are different types of neighbours) and therefore couple differently.  In these cases, the "n+1" rule has to be refined so that each type of neighbour causes n+1 lines. For example for a proton with two types of neighbour, number of lines, L = (n1 + 1)(n2 + 1). However, in many cases the lines overlap with each other and the result is further distortion from the "ideal" pattern. Coupling patterns invloving aromatic or alkene protons are often complex.

H-NMR Spectra II

Now for a few more spectra, this time where there are coupling patterns.....

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Interpretting 1H-NMR Spectra

Let's summarise what can be obtained from a 1H NMR spectrum:  

How many types of H ? Indicated by how many groups of signals there are in the spectra

What types of H ?  Indicated by the chemical shift of each group

How many H of each type are there? Indicated by the integration (relative area) of the signal for each group.

What is the connectivity ? Look at the coupling patterns. This tells you what is next to each group

Chemical shift

The chemical shift is the position on the scale (in ppm) where the peak occurs. Typical /ppm values for protons in different chemical environments are shown in

the figure below. There are two major factors that influence chemical shifts (a) deshielding due to

reduced electron density (due electronegative atoms) and (b) anisotropy (due to magnetic fields generated by bonds).

Note that the figure shows the typical chemical shifts for protons being influenced by a single group. In cases where a proton is influenced by more than one group, the effects are essentially cumulative.

Integration

The area of a peak is proportional to the number of H that the peak represents The integral measures the area of the peak The integral gives the relative ratio of the number of H for each peak

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Coupling

The proximity of other "n" H atoms on neighbouring carbon atoms, causes the signals to be split into "n+1" lines.

This is also known as the multiplicity or splitting of each signal.

Be aware that the exact substitution pattern around a particular H causes changes in the chemical shift and therefore ranges of values are given in the tables and the above figure. Having a good "feel" for the typical chemical shifts will save yourself lots of time in examinations, and avoid confusion.

An example of an H NMR is shown below.

Based on the outline given above the four sets of information we get are:

5 basic types of H present in the ratio of 5 : 2 : 2 : 2 : 3. These are seen as a 5H "singlet" (ArH), two 2H triplets, a 2H quartet and a 3H triplet. Each triplet tells us that there are 2H in the adjacent position, and a quartet tells us that there are 3H adjacent. (Think of it as the lines you see, L = n + 1, where n = number of equivalent adjacent H) This tells us we that the peaks at 4.4 and 2.8 ppm must be connected as a CH2CH2 unit. The peaks at 2.1 and 0.9 ppm as a CH2CH3 unit. Using the chemical shift charts, the H can be assigned to the peaks as below:

7.2ppm (5H) = ArH ; 4.4ppm (2H) = CH2O;

2.8ppm (2H) = Ar-CH2; 2.1ppm (2H) = O=CCH2CH3 and

0.9ppm (3H) = CH2CH3

C-NMR Spectroscopy

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It is useful to compare and contrast H-NMR and C-NMR as there are certain differences and similarities:

13C has only about 1.1% natural abundance (of carbon atoms) 12C does not exhibit NMR behaviour 13C nucleus is about 400 times less sensitive than H nucleus to the NMR phenomena Due to low abundance, we do not usually see 13C-13C coupling Chemical shift range is normally 0 to 220 ppm Similar factors affect the chemical shifts in 13C as seen for H NMR Long relaxation times (excited state to ground state) mean no integrations "Normal" 13C spectra are "broadband, proton decoupled" so the peaks show as single

lines Number of peaks indicates the number of types of C

Here is the simple correlation table of 13C chemical shifts:

The following information is to be gained from a typical  broadband decoupled 13C NMR spectrum:  

Interpretting C-NMR Spectra

The following information is to be gained from a typical  broadband decoupled 13C NMR spectrum:  How many types of C ? Indicated by how many signals there are in the spectra

What types of C ? Indicated by the chemical shift of each signal

Here are some examples of 13C-NMR spectra.  

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C-NMR Spectroscopy

It is useful to compare and contrast H-NMR and C-NMR as there are certain differences

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and similarities:

13C has only about 1.1% natural abundance (of carbon atoms) 12C does not exhibit NMR behaviour (nuclear spin, I = 0) As a result, C is about 400 times less sensitive than H nucleus to the NMR

phenomena Due to low abundance, we do not usually see 13C-13C coupling Chemical shift range is normally 0 to 220 ppm Chemical shifts measured with respect to tetramethylsilane, (CH3)4Si (i.e. TMS) Similar factors affect the chemical shifts in 13C as seen for H NMR Long relaxation times (excited state to ground state) mean no integrations "Normal" 13C spectra are "broadband, proton decoupled" so the peaks show as

single lines Number of peaks indicates the number of types of C

The general implications of these points are that 13C take longer to acquire, though they tend to look simpler. Overlap of peaks is much less common than for H-NMR which makes it easier to determine how many types of C are present.

Here is the simple correlation table of 13C chemical shifts:

C=O indicates aldehydes and ketones O=C-X indicates carbpxylic acids and derivatives such as esters and amides

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V. Mass Spectroscopy (MS)

Basics   Mass spectrometry is based on slightly different principles to the other spectroscopic methods. 

The physics behind mass spectrometry is that a charged particle passing through a magnetic field is deflected along a circular path on a radius that is proportional to the mass to charge ratio, m/e.  In an electron impact mass spectrometer, a high energy beam of electrons is used to displace an electron from the organic molecule to form a radical cation known as the molecular ion. If the molecular ion is too unstable then it can fragment to give other smaller ions. 

The collection of ions is then focused into a beam and accelerated into the magnetic field and deflected along circular paths according to the masses of the ions. By adjusting the magnetic field, the ions can be focused on the detector and recorded.

Probably the most useful information you should be able to obtain from a MS spectrum is the molecular weight of the sample.

This will often be the heaviest ion observed from the sample provided this ion is stable enough to be observed.

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Terminology  

Molecular ion The ion obtained by the loss of an electron from the moleculeBase peak The most intense peak in the MS, assigned 100% intensityM+ Symbol often given to the molecular ionRadical cation +ve charged species with an odd number of electrons

Fragment ions Lighter cations formed by the decomposition of the molecular ion.  These often correspond to stable carbcations.

Spectra

The MS of a typical hydrocarbon, n-decane is shown below. The molecular ion is seen as a small peak at m/z = 142.  Notice the series ions detected that correspond to fragments that differ by 14 mass units, formed by the cleave of bonds at successive -CH2- units

The MS of benzyl alcohol is shown below. The molecular ion is seen at m/z = 108.  Fragmentation via loss of 17 (-OH) gives a common fragment seen for alkyl benzenes at m/z = 91.  Loss of 31 (-CH2OH) from the molecular ion gives 77 corresponding to the phenyl cation. Note the small peaks at 109 and 110 which correspond to the presence of small amounts of 13C in the sample (which has about 1% natural abundance).

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Isotope patterns

Mass spectrometers are capable of separating and detecting individual ions even those that only differ by a single atomic mass unit.

As a  result molecules containing different isotopes can be distinguished. This is most apparent when atoms such as bromine or chlorine are present (79Br : 81Br,

intensity 1:1 and 35Cl : 37Cl, intensity 3:1) where peaks at "M" and "M+2" are obtained. The intensity ratios in the isotope patterns are due to the natural abundance of the

isotopes. "M+1" peaks are seen due the the presence of 13C in the sample.

The following two mass spectra show examples of haloalkanes with characteristic isotope patterns.

The first MS is of 2-chloropropane. Note the isotope pattern at 78 and 80 that represent the M amd M+2 in a 3:1 ratio. Loss of 35Cl from 78 or 37Cl from 80 gives the base peak a m/z = 43, corresponding to the secondary propyl cation. Note that the peaks at m/z = 63 and 65 still contain Cl and therefore also show the 3:1 isotope pattern.    

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The second MS is of 1-bromopropane. Note the isotope pattern at 122 and 124 that represent the M amd M+2 in a 1:1 ratio. Loss of 79Br from 122 or 81Br from 124 gives the base peak a m/z = 43, corresponding to the propyl cation. Note that other peaks, such as those at m/z = 107 and 109 still contain Br and therefore also show the 1:1 isotope pattern.    

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Getting Structures from Spectra

Good problem solving skills are often based on a good method of attack. Here is some general advice of how to go about determining structures from spectral data, be it from real spectra of an experimental sample, or in the context of a question.  The order of events here works towards using the H-NMR at the last stage since it is potentially the most useful for "assembling" the structure. However, at each stage it is a good idea to use information in other spectra (if available) to seek support your interpretation or address any doubts. Try to get a molecular formula at the earliest  possible opportunity and then calculate the index of hydrogen deficiency (IHD).

MSo determine the molecular weighto identify the presence of isotopes patterns for Cl or Br

UVo is the system conjugated ?

IRo identify the functional groups that are present

13C-NMRo how many types of carbon ?o what types of carbon ?

H-NMR

o how many types of hydrogen ?o how many of each type ?o what types of hydrogen ?o how are they connected ?

Having completed an analysis of the available spectra,

list the pieces that have been identifed check the pieces the MW and / or molecular formula and refine the pieces to fit assemble the pieces paying particular attention to H-NMR chemical shifts and

coupling patterns

Once you think you have the answer, check it with the H-NMR very carefully... it is probably the most critical test to pass.

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VI. Atomic Absorption SpectroscopyThe study of absorption spectra by means of passing electromagnetic radiation through an atomic medium that is selectively absorbing; this produces pure electronic transitions free from vibrational and rotational transitions

(Academic Press Dictionary of Science and Technology)

1. Introduction.

Figure1. Elements detectable by atomic absorption are highlighted in pink in this periodic table

Atomic absorption methods measure the amount of energy (in the form of photons of light, and thus a change in the wavelength) absorbed by the sample. Specifically, a detector measures the wavelengths of light transmitted by the sample (the "after" wavelengths), and compares them to the wavelengths, which originally passed through the sample (the "before" wavelengths). A signal processor then integrates the changes in wavelength, which appear in the readout as peaks of energy absorption at discrete wavelengths (see schematic of an atomic-absorption experiment).

Any atom has its own distinct pattern of wavelengths at which it will absorb energy, due to the unique configuration of electrons in its outer shell. This allows for the qualitative analysis of a pure sample. In order to tell how much of a known element is present in a sample, one must first establish a basis for comparison using known quantities. It can be done producing a calibration curve. For this process, a known wavelength is selected, and the detector will measure only the energy emitted at that wavelength. However, as the concentration of the target atom in the sample increases, absorption will also increase proportionally. Thus, one runs a series of known concentrations of some compound, and records the corresponding degree of absorbance, which is an inverse percentage of light transmitted. A straight line can then be drawn between all of the known points. From this line, one can then extrapolate the concentration of the substance under investigation from its absorbance. The use of special light sources and specific wavelength selection allows the quantitative determination of individual components of a multielement mixture.

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The phenomenon of atomic absorption (AA) was first observed in 1802 with the discovery of the Fraunhofer lines in the sun's spectrum. It was not until 1953 that Australian physicist Sir Alan Walsh demonstrated that atomic absorption could be used as a quantitative analtical tool. Atomic absorption analysis involves measuring the absorption of light by vaporized ground state atoms and relating the absorption to concentration. The incident light beam is attenuated by atomic vapor absorption according to Beer's law.The process of atomic absorption spectroscopy (AAS) involves two steps: 1.      Atomization of the sample 2.      The absorption of radiation from a light source by the free atoms     

The sample, either a liquid or a solid, is atomized in either a flame or a graphite furnace. Upon the absorption of ultraviolet or visible light, the free atoms undergo electronic transitions from the ground state to excited electronic states.

To obtain the best results in AA, the instrumental and chemical parameters of the system must be geared toward the production of neutral ground state atoms of the element of interest. A common method is to introduce a liquid sample into a flame. Upon introduction, the sample solution is dispersed into a fine spray, the spray is then desolvated into salt particles in the flame and the particles are subsequently vaporized into neutral atoms, ionic species and molecular species. All of these conversion processes occur in geometrically definable regions in the flame. It is therefore important to set the instrument parameters such that the light from the source (typically a hollow-cathode lamp) is directed through the region of the flame that contains the maximum number of neutral atoms. The light produced by the hollow-cathode lamp is emitted from excited atoms of the same element which is to be determined. Therefore the radiant energy corresponds directly to the wavelength which is absorbable by the atomized sample. This method provides both sensitivity and selectivity since other elements in the sample will not generally absorb the chosen wavelength and thus, will not interfere with the measurement. To reduce background interference, the wavelength of interest is isolated by a monochromator placed between the sample and the detector.

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2. Instrumentation

Figure 2. Perkin-Elmer Spectrophotometer Model 460In atomic absorption (see schematic of an atomic-absorption experiment), there are two methods of adding thermal energy to a sample. A graphite furnace AAS uses a graphite tube with a strong electric current to heat the sample. In flame AAS (see photo above), we aspirate a sample into a flame using a nebulizer. The flame is lined up in a beam of light of the appropriate wavelength. The flame (thermal energy) causes the atom to undergo a transition from the ground state to the first excited state. When the atoms make their transition, they absorb some of the light from the beam. The more concentrated the solution, the more light energy is absorbed!The light beam is generated by lamp that is specific for a target metal. The lamp must be perfectly aligned so the beam crosses the hottest part of the flame. The light passed through the flame is received by the monochromator, which is set to accept and transmit radiation at the specified wavelength and travels into the detector. The detector measures the intensity of the beam of light. When some of the light is absorbed by metal, the beam's intensity is reduced. The detector records that reduction as absorption. That absorption is shown on output device by the data system. We can find the concentrations of metals in a sample running a series of calibration standards through the instrument. The instrument will record the absorption generated by a given concentration. By plotting the absorption versus the concentrations of the standards, a calibration curve can be plotted. We can then look at the absorption for a sample solution and use the calibration curves to determine the concentration in that

3. Techniques of Measurement and EPA Methods Using FAAS

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Atomic absorption spectrometry is a fairly universal analytical method for determination of metallic elements when present in both trace and major concentrations. The EPA employs this technique for determining the metal concentration in samples from a variety of matrices.A) Sample preparationDepending on the information required, total recoverable metals, dissolved metals, suspended metals, and total metals could be obtained from a certain environmental matrix. Table 1 lists the EPA method number for sample processing in terms of the environmental matrices and information required. For more detail information, readers could refer to EPA document SW-846 "Test methods for evaluating solid wastes".

Table 1 EPA sample processing method for metallic element analysis

Analysis Target Method Number Environmental Matrice

total recoverable metals 3005 ground water/surface water

dissolved metals 3005 ground water/surface watersuspended metals 3005 ground water/surface water

total metals 3010 aqueous samples, wastes that contain suspended solids and mobility-procedure extracts

total metals 3015 aqueous samples, wastes that contain suspended solids and mobility-procedure extracts

total metals 3020 aqueous samples, wastes that contain suspended solids and mobility-procedure extracts

total metals 3050 sediments, sludges and soil samplestotal metals 3051 sludges, sediment, soil and oil

Appropriate acid digestion is employed in these methods. Hydrochloric acid digestion is not suitable for samples, which will be analyzed by graphite furnace atomic absorption spectroscopy because it can cause interferences during furnace atomization.

B) Calibration and standard curvesAs with other analytical techniques, atomic absorption spectrometry requires careful calibration. EPA QA/QC demands calibration through several steps including interference check sample, calibration verification, calibration standards, bland control, and linear dynamic range.The idealized calibration or standard curve is stated by Beer's law that the absorbance of an absorbing analyte is proportional to its concentration.Unfortunately, deviations from linearity usually occur, especially as the concentration of metallic analytes increases due to various reasons, such as unabsorbed radiation, stray light, or disproportionate decomposition of molecules at high concentrations. Figure 3 shows an idealized and deviation of response curve. The curvature could be minimized, although it is impossible to be avoided completely. It is desirable to work in the linearity response range. The rule of thumb is that a minimum of five standards and a blank should be prepared in order to have sufficient information to fit the standard curve appropriately. Manufacturers should be consulted if a manual curvature correction function is available for a specific instrument.

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Figure 3. Idealized/deviation response curveIf the sample concentration is too high to permit accurate analysis in linearity response range, there are three alternatives that may help bring the absorbance into the optimum working range:

1) sample dilution2) using an alternative wavelength having a lower absorptivity3) reducing the path length by rotating the burner hand.

C) EPA method for metal analysisFlame atomic absorption methods are referred to as direct aspiration determinations. They are normally completed as single element analyses and are relatively free of interelement spectral interferences. For some elements, the temperature or type of flame used is critical. If flame and analytical conditions are not properly used, chemical and ionization interferences can occur.Graphite furnace atomic absorption spectrometry replaces the flame with an electrically heated graphite furnace. The major advantage of this technique is that the detection limit can be extremely low. It is applicable for relatively clean samples, however, interferences could be a real problem. It is important for the analyst to establish a set of analytical protocol which is appropriate for the sample to be analyzed and for the information required. Table 2 lists the available method for different metal analysis provided in EPA manual SW-846.

Table 2. EPA methods for determination of metals by direct aspiration

Analyte Method number Analyte Method

number Analyte Method number

aluminum 7020 antimony 7040 barium 7080Aberyllium 7090 cadmium 7130 calcium 7140chromium 7190 cobalt 7200 copper 7210

iron 7380 lead 7420 lithium 7430magnesium 7450 manganese 7460 molybdenum 7480

nickel 7520 osmium 7550 potassium 7610silver 7760A sodium 7770 strontium 7780

thallium 7840 tin 7870 vanadium 7910zinc 7951        

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D) InterferencesSince the concentration of the analyte element is considered to be proportional to the ground state atom population in the flame, any factor that affects the ground state population of the analyte element can be classified as interference. Factors that may affect the ability of the instrument to read this parameter can also be classified as interference. The following are the most common interferences: a) Spectral interferences are due to radiation overlapping that of the light source. The interference radiation may be an emission line of another element or compound, or general background radiation from the flame, solvent, or analytical sample. This usually occurs when using organic solvents, but can also happen when determining sodium with magnesium present, iron with copper or iron with nickel. b) Formation of compounds that do not dissociate in the flame. The most common example is the formation of calcium and strontium phosphates.c) Ionization of the analyte reduces the signal. This is commonly happens to barium, calcium, strontium, sodium and potassium.d) Matrix interferences due to differences between surface tension and viscosity of test solutions and standards.e) Broadening of a spectral line, which can occur due to a number of factors. The most common line width broadening effects are:

1. Doppler effectThis effect arises because atoms will have different components of velocity along the line of observation.2. Lorentz effectThis effect occurs as a result of the concentration of foreign atoms present in the environment of the emitting or absorbing atoms. The magnitude of the broadening varies with the pressure of the foreign gases and their physical properties.3. Quenching effectIn a low-pressure spectral source, quenching collision can occur in flames as the result of the presence of foreign gas molecules with vibration levels very close to the excited state of the resonance line.4. Self absorption or self-reversal effectThe atoms of the same kind as that emitting radiation will absorb maximum radiation at the center of the line than at the wings, resulting in the change of shape of the line as well as its intensity. This effect becomes serious if the vapor, which is absorbing radiation is considerably cooler than that which is emitting radiation.

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Atomic Absorption Resources.EPA document SW-846 "Test methods for evaluating solid wastes".

Textbooks:

Haswell, S.J., 1991. Atomic Absorption Spectrometry; Theory, Design and Applications. Elsevier, Amsterdam.

Reynolds, R.J. et al., 1970. Atomic Absorption Spectroscopy. Barnes & Noble Inc., New York.

Schrenk, W.G., 1975. Analytical Atomic Spectroscopy. Plenum Press, New York.

Varma, A., 1985. Handbook of Atomic Absorption Analysis. Vol. I. CRC Press, Boca Raton.

Scientific journals related to Atomic Absorption Spectroscopy:

Journal of Analytical Atomic SpectrometryPublished by: Royal Society of ChemistrySpectrochimica Acta Part B: Atomic Spectroscopy Published by: Elsevier Science

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