2.

3: Instrumentation

Instrumentation
A section of this laboratory manual details the operation and theory associated with the instruments that you will be using in 5.32.
Please study the instructions described in for each instrument before you embark on using them for the first time. Use of any
instrument requires reading this section, learning from one of the TAs how to operate this instrument, and demonstrating to the
TA's satisfaction that you are capable of handling the instrument properly.

Instrument Sign-Up Rules

Sign-up schedules are posted for all major instruments (NMR, IR, UV, and GCMS). Schedules for use of these instruments are very
tight; therefore, significant abuses of sign-up privileges cannot be tolerated. In each case, note the following Sign-Up Rules:
1. Sign up for time on instruments no more than two days in advance.
2. If you discover that you are not able to use the time that you have signed up for, then be sure to erase your name in advance so
that someone else can use it. The TA's will let you know if you violate this rule.
3. Show up promptly for time for which you have signed up.
4. For your first NMR spectrum, you will need a full 30 minute slot and should bring only one sample. Once you are proficient in
using the instrument, a normal NMR spectrum will require 15-25 minutes (one 30 minute slot should be more than sufficient for
one sample and probably two samples could be done). You are not allowed to sign up for more than two 30-minute slots on a
single day.

FTIR Spectrophotometer
The Instrument
In a conventional IR spectrophotomer, a sample IR beam is directed through the sample chamber and measured against a reference
beam at each wavelength of the spectrum. The entire spectral region must be scanned slowly to produce good quality spectrum. In
5.32, we will be using a Nicolet FTIR Spectrophotometer (Nicolet was heavily involved in the design of the Hubble telescope!). IR
spectroscopy has been dramatically improved by the development of the Fourier Transform method in much the same way as NMR
has been revolutionized by this method.

Figure 2.3.1: Diagram of the Michelson Interferometer used in an FTIR Spectrophotometer.

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The heart of an FTIR Spectrophotometer is a Michelson Interferometer built around the sample chamber. Radiation from an IR
source is directed through the sample cell to a beam splitter. Half of the radiation is reflected from a fixed mirror while the other
half is reflected from a mirror which moved continuously over a distance of about 2.5 micrometers. When the two beams are
recombined at the detector, an interference pattern is produced. A single scan of the entire distance takes about 2 seconds and is
stored in the computer. In order that several scans may be added, they must coincide exactly. Obviously, this would be impossible
considering the thermal fluctuations and vibrations in the laboratory. In order to solve this problem, a helium-neon laser is
simultaneously directed through the Michelson Interferometer and the interference pattern of the laser is used as a frequency
reference.
The performance of an FTIR is dramatically superior to that of conventional instruments. Generally, only a small amount of sample
will produce an excellent spectrum in a fraction of the time.

Preparation of the sample

Due to the sensitivity of the FTIR instrument, the most convenient and satisfactory method involves simple evaporation of a
solution of the sample (chloroform, ether, dichloromethane; or even a CDCl3 NMR sample may be used) onto a KBr salt plate and
acquisition of the spectrum from the thin film remaining. This method provides excellent spectra with flat baseline unless the thin
film is too powdery in which case excessive scattering of the light leads to an irregular baseline. The sample may alternatively be
prepared as a nujol mull (mull accessories: agate mortar and pestle, nujol and NaCl discs may be obtained from LS).

Preparation of Instrument

If the instrument has just been turned on, then it is necessary to run a TEST ( F10 ) to be sure that all components are ON. If the
instrument is not turned on or does not check out when the TEST is performed, then ask the instrument TA for help. In addition, it
is important that N2 is flowing through the chamber so that most of the CO2 and H2O are flushed from the chamber and from inside
the instrument.
F4 SCAN BACKGROUND is performed with a blank IR plate in the chamber.
F8 then F4 DISPLAY BACKGROUND will show the spectrum of CO2 and H2O that remain in the chamber. If the background

shows excessive CO2 and H2O, then be sure the N2 is flowing briskly, wait a minute or two and try again. Once a good background
has been obtained, several students in succession can use the same background.

Scanning the Sample

Place the sample plate in the FTIR and wait for N2 to purge out the air.
F5 SCAN SAMPLE. Wait until the scan and Fourier transform are completed.
F8 then F1 DISPLAY SPECTRUM will automatically subtract the stored background and display the spectrum.
F7 PRINT. Important: Make sure that the printer is on-line before pressing F7.
Type PEAKPICK S 4000 600 to find the peaks in the spectrum. This data is printed by pressing F7 . If no one else is using the
instrument next, please turn off the nitrogen purge.

Principles of IR Spectroscopy
The different regions of the electromagnetic spectrum will be used in this section to learn about the structure and reactions of
organic molecules. For each spectroscopic method, it is helpful to understand how much energy corresponds to each wavelength
and how this relates to the physical process after absorption of radiation. Organic molecules can absorb IR radiation between 4000
cm-1 and 400 cm-1 which corresponds to an absorption of energy between 11 kcal/mole and 1 kcal/mole. This amount of energy
initiates transitions between vibrational states of bonds contained within the molecule.

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IR spectroscopy is a very powerful method for the identification of functional groups. The most important regions of the IR
spectrum are >1650 cm-1, whereas the fingerprint region (600 - 1500 cm-1) of the spectrum cannot easily be used for identification
of unknown compounds. Many references exist which tabulate the IR frequencies for various functional groups and organic
compounds (a short table appears at the end of this section). However, the most valuable resource available to you for the
interpretation of IR spectra is understanding the five basic principles of IR spectroscopy. Transitions between vibrational energy
levels follow the same equation as for a classical harmonic oscillator:
−
−
1 k m1 m2
Equation for the Classical Harmonic Oscillator: ν̄ = √ μ =
2pc μ m1 + m2

1. k is the force constant.

1. k is proportional to bond strength or bond order. C=O vibrates at a higher frequency than C-O. Furthermore, the change in
the force constant of different carbonyl groups can be understood based on the contribution of resonance structures. The
base value for the stretching frequency of a carbonyl (e.g., acetone) is ν CO ˜ 1715 cm-1. Acid chlorides have bond order
slightly greater than 2 because an acylium ion resonance structure may be drawn (ν CO 1800 cm-1). Alternatively, Phenyl

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 ketones, vinyl ketones and amides have a CO bond order slightly less than 2 and display a lower energy ν CO.

2. μ is the reduced mass.

1. Heavier atoms slower vibration, lower energy. Compare C-O vs. H-O or H-O vs H-S.
3. Overtone Peaks.
1. Notice in the above spectrum that a small peak is found at 3450 cm-1, even though the compound does not contain any O-H
or C-H bonds. This peak is the overtone of the C=O vibration (at 1735 cm-1). It corresponds to the transition from the
ground vibrational state (n=0) to the second vibrationally excited state (n=2) rather than the first. Carbonyl overtones are
always small and are easily found at slightly less than twice the normal C=O frequency.
4. Dipole moment.
1. The strength of an IR peak is roughly dependent on the change in dipole moment during vibration. C=O bonds are very
polar because of the greater electronegativity of oxygen and so give very intense bands. Also note that if a molecule is so
symmetrical that the stretching of a bond does not produce any change in dipole moment, then no IR peak will be found in
the spectrum. Compare the spectra of 1-butyne and 3-hexyne. 1-butyne shows an alkyne C-H stretch at 3280 cm-1 and an
alkyne C≡C stretch at 2080 cm-1. -hexyne shows no C≡C stretching peak.

5. Vibrational Modes.
1. The vibrations of two neighboring bonds can be coupled into symmetric and antisymmetric vibrational modes. One example
is the vibration of CH2 groups within an alkane (or the NH2 group of a primary amine). The symmetric stretch requires
slightly more energy (2925 cm-1) for a transition while an antisymmetric stretch requires slightly less energy (2850 cm-1).
For acetic anhydride, notice that although the two C=O groups are identical by symmetry, two peaks are found in the C=O
region of the IR spectrum.

If a functional group's normal vibrational frequency happens to coincide in frequency with a weak overtone peak of a

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 neighboring bond, then the peak will be observed as a Fermi doublet. In the case of aliphatic aldehydes, the aldehydic C-H
stretching frequency at 2720 cm-1 couples with the overtone of the C-H bending transition at 1380 cm-1. Fermi coupling also
explains the observation of two peaks near 2300 cm-1 in the spectrum of CO2.

Characteristic IR Frequencies
XH Region (3600 cm-1 to 2400 cm-1)
cm-1 comments

3600 ν (free OH) Sharp peak Alcohol or Phenol free OH

Alcohol: 3400 to 3200 cm-1

3600-2800 ν (H-bonded OH) Very Broad peak: Phenol: 3600 to 3000 cm-1
Carboxylic Acid: 3600 to 2400 cm-1

Amines show broad peaks, Amides show sharp peaks

3500-3300 ν (NH)
Primary Amines display two peaks (ν and ν )
s as

CH Region (3300 cm-1 to 2700 cm-1)

cm-1 comments

3300 Alkyne ν (CH) strong, sharp

3150-3000 Alkene or Phenyl ν (CH) medium intensity

3050 Cyclopropane or Epoxide ν (CH) weak

νs(CH), ν (CH) observed for CH2 or CH3

as
2960,2870 Alkane ν (CH)
groups

2750 Aldehyde ν (CH) sharp, medium intensity

−C≡N, −C≡C−, > C=C=C < Region (2300 cm-1 to 2000 cm-1)
cm-1 comments

sharp, weak to med intens, almost always

2250 ν (−C≡N)
observed
sharp, weak to med intens, check for ν (C-H) at
2150 ν (RC≡CH)
3300
sharp, weak to med intens, obsd only for R,R'
2260-2190 ν (R−C≡C−R )
′

different

1950 ν (> C=C=C <) sharp, strong allene

>C=O Region (1800 cm-1 to 1650 cm-1)

cm-1 comments

1800 Acid Chloride

CO Bond Order >2

1820,1760 Anhydride two peaks are observed (ν \; ν )

s as

1735 Ester RCO2R'

1755 Carbonate ROCO2R'

1735 Urethane ROCONR'2

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 cm-1 comments

1720 Aldehyde/Ketone aldehyde has ν (CH) at 2750 cm-1

1650 Amide

CO Bond Order <2

1630 Urea R2NCONR'2

C=N, C=C, NO2 Region (1660 cm-1 to 1500 cm-1)

-1
cm comments

1690-1640 ν (C=N) weak to med intensity, sharp

1660-1640 ν (C=C) weak to med intensity, sharp

1590 ν (NO2) strong, sharp

Principles of NMR Spectroscopy

One of the first things to do after obtaining your 1H and 13C NMR spectra is to identify the resonances associated with the solvent
used for the NMR sample using the table below.

$$$ per sample 1H NMR 13C NMR

CDCl3 $ 0.20 7.26 (1 peak) 77.01 (3 peaks)

206.2 (1 peak)
CD3COCD3 $ 1.54 2.04 (5 peaks)
29.8 (7 peaks)

CD3SOCD3 $ 2.76 2.49 (5 peaks) 39.6 (7 peaks)

C6D6 $ 1.73 7.15 (broad) 128.0 (3 peaks)

Peaks in the 13C NMR spectra corresponding to the deuterated solvent molecules show unique or peculiar spin coupling patterns,
making these especially easy to identify. This is particularly obvious for the 13C NMR spectrum of CDCl3 - coupling to one
detuerium atom produces a 1:1:1 triplet. A deuterium atom has nuclear spin quantum number I=1 and so its possible spin states are
+1, 0, -1 each leading to one peak of the multiplet. The 13C NMR spectrum of CD3COCD3 displays a seven peak multiplet pattern
characteristic for the -CD3 group

The appearance of the solvent resonances in the 1H NMR spectrum arise from the residual solvent molecules which contain one H
atom. The cost of deuterated NMR solvents is proportional to their level of isotopic purity and inevitably, some percentage of
molecules will contain one H atom. The solvent CDCl3 has a small amount of CHCl3 present, so a singlet is found in the 1H NMR
spectrum at 7.26 ppm. The solvent CD3COCD3 contains a small amount of the contaminant CD2HCOCD3. The hydrogen of a -
CD2H group will show up at the same chemical shift as for a CH3 group in acetone, but it will be spin coupled to two deuterium
atoms (each spin 1). The result is the pattern above having five peaks.
For 5.32, all the organic compounds which we will work with will involve only the elements listed on the following page in their
natural abundances. The spin-spin coupling behavior of these elements in 1H and 13C NMR spectra are easily understood. The

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important determinant of this feature is the spin (I) of the element involved.
I = 0 No spin-spin coupling effect is observed. Examples: 12C, 16O, 28Si, 32S.
I = 1/2 Spin-spin coupling with neighboring 1H and 13C atoms is always operative. Examples: 1H, 31P and 19F. The magnitude of
the coupling depends on the separation between atoms. Spin coupling with I=1/2 nuclei is discussed in the next section.
I = 1 Quadrupolar Nuclei. Different effects are possible. Deuterium is a weak quadrupolar nucleus. I=1 multiplets observed. 14N is
a stronger quadrupolar nucleus. Broad peaks are observed for NH protons. Cl,Br,I are strong quadrupolar nuclei. No coupling effect
is observed.

Spin Coupling Effects of Elements found in Organic Compounds

Observed Effect in 1H and
Natural Abund. Nuclear Spin (I) 13
C Spectra
2
JHH (0-25 Hz), 3JHH (0-18
Hz), 4JHH (usually <2 Hz)
1
1 JCH = 115 to 250 Hz, but
H H 99.98% 1/2
is not observed in the
standard (1H decoupled)
13
C spectrum.
2
2 JHD (2-4 Hz)
H(D) 0.015% 1 1
JCD (15 to 35 Hz)
12
C C 98.9% 0 No Effect (I = 0)

13 very small 13C sattelites are

C 1.1% 1/2
observed in 1H NMR
14
N nucleus is strongly
quadrupolar (I>1/2). This
feature allows its spin state
to change rapidly for most
14
N N 100% 1 Ncontaining compounds.
Therefore, protons bonded
to Nitrogen appear as
broadened peaks rather
than triplets.
15
N 0.37% 1/2
16
O O 99.8% 0 No Effect (I = 0)
17
O 0.04% 5/2
18
O 0.20% 0
2
JHF (40-90 Hz), 3JHF (5-50
19
F F 100% 1/2 Hz)
1
JCF (200-300 Hz)
28
Si Si 92.2% 0 No Effect (I = 0)
29
Si 4.7% 1/2
30
Si 3.1% 0
2
JHP (10-20 Hz), 3JHP (5-10
31 Hz), 4JHP (0-3 Hz) 1JCP
P P 100% 1/2
(50-100 Hz), 2JCP (5-10
Hz), 3JCP, 4JCP
32
S S 95% 0 No Effect (I = 0)
33
S 0.8% 3/2

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 Observed Effect in 1H and
Natural Abund. Nuclear Spin (I) 13C Spectra

34
S 4.2% 0
36
S 0.02% 0
35 Strong quadrupolar nuclei
Cl Cl 76% 3/2
(I=1)
37
Cl 24% 3/2 Spin state of Cl, Br or I
79 changes so rapidly that the
Br Br 50.7% 3/2
effect on other nuclei is
81 averaged
Br 49.3% 3/2
(No coupling observed in
127
I I 100% 5/2 1 13
H C NMR)

Analysis of Molecular Symmetry Using 13C (and 1H) NMR Spectra

For most organic compounds, all peaks in the 13C NMR spectrum are usually singlets. Why are multiplets not produced by spin
coupling with 13C and 1H nuclei (each have I=1/2)?
1. The low natural abundance of 13C (only 1.1% compared to 98.9% 12C) means that most individual 13C atoms will have only 12C
atoms nearby which do not exhibit spin-spin coupling.
2. Although H atoms attached to each 13C atom would normally be expected to couple with 13C atoms, the acquisition of 13C
spectra is actually performed with deliberate decoupling of the 1H nuclei by the instrument.
For these reasons, the 13C NMR spectra of most ordinary organic compounds exhibit only singlet resonances for each carbon in
the 13C NMR spectrum. This offers an exceptional opportunity to learn about the symmetry of a molecule by analyzing the 13C
NMR spectrum. Only compounds which contain D, F or P atoms will show multiplet peaks in the 13C NMR. Due to the sharpness
of singlet resonances in the 13C NMR and the large chemical shift range (200 ppm), it is very unlikely that two carbons which are
in different environments will display one singlet. If a molecule contains symmetry, then atoms or groups related by that symmetry
element experience identical environments and one peak represents the two carbons. Unfortunately, the 13C NMR cannot be
integrated to tell how many carbon atoms are represented by each resonance. So you often have to figure out in a logical fashion
which lines are representing equivalent carbon atoms. Since the 1H NMR spectrum can be integrated to determine how many
protons are represented by each resonance, one can use the integration of the 1H NMR spectrum in a logical fashion to help
analysis of the 13C NMR. The examples which follow demonstrate different cases of molecular symmetry.

Case 1. Molecular Symmetry - Kemp's Triacid.

The structure of Kemp's triacid has three-fold rotational symmetry. Therefore, in the 13C NMR spectrum only four lines are
observed. In the 13C NMR spectrum the three methyl groups are equivalent and are represented by one singlet and each of the three
CO2H groups are identical by symmetry. Similarly, each of the three CH2 groups are identical to each other. But in the 1H NMR,
the three equatorial hydrogens (on the same side of the ring as the -CO2H groups) are in a different environment from the three
axial hydrogens (on the same side as the methyl groups). Therefore, one doublet represents the equatorial hydrogens and the other
doublet represents the axial hydrogens. Why are the CH2 protons represented by a pair of doublets? because each of these protons
is different from its geminal partner, it couples with it to form a doublet.

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Here are some other symmetrical molecules: In the first case, a plane of symmetry parallel to the page bisects the bicyclic
framework. Of the seven carbons of the bicyclic framework, C(1) and C(4) are identical environments. Similarly, C(2) and C(3) are
identical. C(5) and C(6) are identical. C(2) is on the same side of the ring system as the hydrogen so is not identical to C(5) or C(6).
Four 13C resonances are observed for these seven carbons. The tert-butyl substituent will be discussed below. Bromobenzene also
possess a plane of symmetry which renders the ortho carbons equivalent to one another and the meta carbons equivalent to one
another Four 13C peaks are found. Benzyl bromide possesses a plane of symmetry which renders the protons of the CH2 group
equivalent to one another in the 1H NMR.

Case 2. Freely Rotating Substituents.

The compound phenylglycine displays four aromatic carbon signals in its 13C NMR spectrum. This molecule contains no plane or
axis of symmetry, but the phenyl group gives a symmetrical pattern. The phenyl group freely rotates about the single bond by
which it is attached to the substituent. The equivalency of the two ortho carbons results because they experience identical
environments over the course of this free rotation. For ordinary organic compounds, phenyl groups will always display this
symmetry. In addition, tert-butyl groups can freely rotate and will render the three methyl groups equivalent in NMR spectra.

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Case 3. Diasterotopic Groups. Phenylalanine and acetal
Let's look at the molecule phenylalanine. From the above discussion, a monosubstitued phenyl group rotates freely and therefore
four aromatic peaks are expected in the 13C NMR. We might be tempted to suggest that the two protons of the CH2 group would be
equivalent in the 1H NMR spectrum. Both bonds attached to the CH2 group can rotate freely and we already learned that the two
protons of benzyl bromide are equivalent. In the spectrum of phenylalanine below, two resonances are observed for the two protons
of the CH2 group!
The two protons of phenylalanine are not equivalent even though free rotation is occurring. In order to understand this result, it is
helpful to examine carefully the Newman projections of each of three staggered rotamers. Ha is between the CO2H and NH2 groups
in rotamer A and Hb is between these groups in rotamer B. In rotamer A, Ha sees the phenyl group next to the NH2 group. This is
not the same environment that Hb sees when it is between the CO2H and NH2 groups in rotamer B (it sees the phenyl group next to
the CO2H group). Free rotation does not make Ha and Hb experience identical environments. The protons Ha and Hb are called
diastereotopic protons and they experience diastereomeric environments.

Benzyl bromide has equivalent protons for its CH2 group because the entire molecule contains a mirror plane - it is achiral.
Phenylalanine is a chiral molecule and so contains no mirror plane. As a rule, if a molecule is chiral, then all of its CR2 substituents
will be diastereotopic.
This can be seen for the amide compound shown below. Even though the carbon atom denoted by * is three bonds removed from
the C(CH3)2 unit of the isopropyl group, two doublets are observed in the 1H NMR spectrum for these two methyl groups.

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The last example of diastereotopic groups is acetal which is especially peculiar. Acetal is achiral. A mirror plane renders the two
ethoxy groups equivalent to one another. However, the CH2 protons of acetal are diastereotopic and two complex multiplets are
observed in the 1H NMR spectrum.

Analysis of 1H NMR Coupling Patterns

First Order Coupling Patterns
Given the values of the coupling constants, it is a straightforward task to predict the appearance of the multiplet (Figure 1). A
proton which couples to another proton with a 4.0 Hz coupling constant will be observed as a doublet with a 4.0 Hz separation of
the peaks. If the proton is coupled to two protons with J = 4.0 Hz, it will appear as a 1:2:1 triplet, with each outer line separated
from the taller center line by 4.0 Hz. (Similarly, coupling to three protons with J = 4.0 Hz is observed as a 1:3:3:1 quartet, see
Pascal's triangle below.) In the case that the two coupling constants are different (J = 4.0, 12.0 Hz), four lines are observed.
Although the appearance of the multiplets becomes more complicated as more spin 1/2 nuclei are added, the same procedure can be
used to simulate any first order multiplet.

Figure 2.3.1: The doublet, triplet and dd patterns.

Extracting Coupling Constants from Simple First Order Spectra

The more difficult task is to extract the coupling constants from the appearance of a multiplet found in a spectrum. For the doublet
of doublets (dd) just described, the smaller coupling constant J1 = (line A - line B) = (line C-line D) and the larger coupling
constant J2 = (line A - line C) = (line B-line D). However, line B - line C is not a coupling constant of this multiplet, it is the
difference between the two coupling constants, J2-J1 (similarly, line A - line D = J1+J2).
Note also that not all four line patterns can be analyzed as a doublet of doublets. Sometimes, such a pattern could be just two
separate doublets (or even four singlets). The possibilities may be easily distinguished by comparing the spectra obtained at
different magnetic fields and noting that coupling constants are constant in Hz, whereas chemical shifts are constant in ppm.

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Coupling to n Equivalent Protons: Pascal's Triangle
When a proton couples to several (n) other protons with equal coupling constant J, n+1 lines will be observed in relative intensities
as shown in Pascal's triangle. As more adjacent protons are attached, the relative intensity of the outer lines diminish exponentially
to the point where they may be lost in the baseline or mistaken for impurities. For example, (CH3)2CHCH2Br has one methine
proton which is coupled equally to each of the eight adjacent protons. Although the observed ratio is expected to be
1:8:28:56:70:56:28:8:1, if the outer lines were not detected, the ratio of the remaining seven lines would be 1:3.5:7:8.75:7:3.5:1 and
this might be mistaken for a simple quartet of quartets. Clearly, the outer lines of the multiplet are often the most important for
correct recognition and this highlights the necessity for obtaining a 1H NMR spectrum devoid of stray impurity peaks.

Coupling to Three Inequivalent Protons: The ddd Patterns

Several different ddd patterns are possible depending on the values of the coupling constants J1,J2 and J3 (Figure 2). The maximum
number of lines possible for coupling to three spin 1/2 nuclei is 8 (2n) if no lines coincide (all lines would be expected to have
equal intensity). Even when some of the lines coincide, the sum of the intensities of each line relative to that of the smallest (outer
lines) will equal 8. The two cases below demonstrate that in order to extract the coupling constants from a ddd pattern, it is
necessary to figure out if J3>J1+J2 or if J3<J1+J2 (J1 is smallest, J3 is largest).

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Figure 2.3.2: Possible appearances of the ddd pattern.

Case 1
J1 = (line A - line B) = 2.8 Hz , J2 = (line A - line C) = 4.8 Hz . In this case the portions of the multiplet are separate so that (line A
- line D) = 7.7 Hz is not a coupling constant, it is J1+J2. J3 is (line A - line E) = 12.9 Hz . As a check, J1 +J2 +J3 = (line A - line H)
= 20.6 Hz.
Case 2

J1 = (line A - line B) = 8.7 Hz , J2 = (line A - line C) = 11.1 Hz . In this case, the portions of the multiplet cross over one another so
that (line A - line D) = 12.6 Hz , this is J3. J1+J2 is given by the value of (line A - line E) = 19.9 Hz. As a check, J1 +J2 +J3 = (line A
- line H) = 31.8 Hz.

In analyzing a multiplet, keep in mind that (a) a multiplet which is not subject to second-order effects will be symmetrical and (b)
the sum of the coupling constants = the spread of the multiplet.

Minor Deviations from First Order Spectra

In many spectra, the recognition of a multiplet and correct analysis is rendered more difficult due to minor deviations from the first
order nature. As the multiplets representing two protons coupled to one another become closer to one another in frequency, the
multiplets "lean" toward one another meaning that the intensities are greater for the inside lines than for the outside lines. A special
nature to this leaning effect is that the multiplets lean toward each other with the coupling constants shared between them. This
helps to identify the multiplets which are coupled to one another. If the leaning is not too severe, the multiplet can still be treated as
first order.

Effects of Strong Coupling

When two multiplets which are coupled to each other overlap severely, they obviously can not be treated as first order patterns.
Unfortunately, the effects on other resonances can also be very peculiar. In the first spectrum (styrene), the notations HA,HM and
HX represent that the resonances are well separated. Each of the resonances appears as a doublet of doublets (four lines) - this
spectrum can be treated as first order. In the second spectrum (vinyl chloride), the notations HA, HB represent the two protons on
the double bond which are strongly overlapping. The HX resonance appears as a six line pattern - this spectrum cannot be treated as
first order (the two smaller lines are called combination lines).

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Although computer programs exist for simulation of such complex coupling patterns, the best remedies are to try different solvents
(e.g., d6-acetone or d6-benzene) using a spectrometer at a high enough MHz to remove the effects of strong coupling.

AA'XX' Second Order Patterns

First, consider the molecule cyclopropene. The two protons on the double bond (HA) are chemically equivalent by symmetry, and
the two CH2 protons (HX) are chemically equivalent by symmetry. Because the molecule has a high degree of symmetry, another
fact is true: the geometric relationship of a single HA proton to each of the HX protons is identical. When a molecule has the two
possible JAX values equal, each pair of protons is called magnetically equivalent. and the spectrum will be first order. In this case, it
is designated A2X2. Note also that no effect of coupling between the two HA protons is observed.

Most molecules of this type do not have sufficient symmetry to make the two possible JAX values equal. Consider the two aromatic
compounds shown below. For all p-disubstituted aromatic compounds (and therefore all monosubstituted benzene derivatives) the
ortho protons are rendered chemically equivalent by free rotation of the ring. However, the proton HA is ortho to one of the HX
protons and para to the other. Because the coupling constant JAX ? JAX', the more complicated AA'XX' pattern results. It is
important to recognize on sight that this pattern is not first order. Although it is tempting to blur your eyes and call each pattern a
doublet, the four smaller peaks of each resonance are too large to be overlooked. Alternatively, analysis of each pattern as a doublet
of triplets would also be erroneous because the four smaller peaks are too small to be consistent with the expected 1:2:1:1:2:1 ratio
of intensities (and such an analysis would clearly not be consistent with the known structure!).
In the case of ortho- substitution with equal substituents (o-dichlorobenzene), the proton HA is ortho to one of the HX protons and
meta to the other. Again two complex AA'XX' patterns result which should not be mistaken for first order dd patterns.

The appearance of AA'XX' patterns depends on the exact values of JAA' and JXX' as well as JAX and JAX'. Although computer
simulation can be used to help figure out the exact values, the most important skill to acquire is learn to recognize typical AA'XX'
patterns which show up for certain types of organic functional groups
Consider a second series of examples, the XCH2CH2Y molecules. For many molecules of this type (BrCH2CH2CN is a good
example), two triplets are observed which leads one to proceed with a first order analysis. However, in a rigorous sense, the
XCH2CH2Y subunit is a magnetically inequivalent (AA'XX') spin system. In the anti and eclipsed conformations shown below, it

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can be seen that JAX ? JAX'. However, for 3-bromoacetonitrile, free rotation averages the coupling constants JAX and JAX' over all
conformations and this leads to a very nearly first order spectrum (JAX ˜ JAX').

In the case of 3-trimethylsilylpropionic acid, rotation is severely restricted due to the size of the trimethylsilyl substituent. The anti-
conformation predominates in which it is apparent that JAX ? JAX'. Alternatively, N-methylmorpholine is locked in a cyclic
conformation for which JAX ? JAX'.

Tables of NMR Coupling Constants and Chemical Shifts

Once the multiplets of a 1H NMR spectrum are correctly analyzed, the coupling constants are very powerful for determining the
structure of the organic compound. Spin-spin coupling constants are constant in Hz and the mechanism of spin-spin coupling
involves entirely a through bond effect. So the magnitude of coupling constants depends on the number of bonds separating the
atoms and the geometry of the bonds relative to each other. The first can be observed in the following series:

GEMINAL COUPLING is only observed for diastereotopic CH2 groups. (If the protons of a CH2 group are equivalent, then no
coupling will be observed between them). For phenylalanine, we learned that the property of chirality renders the Ha and Hb
protons inequivalent and so they couple to each other. Geminal coupling constants are often quite large, and the exact value of the
geminal coupling constant can tell a lot about the groups attached. Reviewing the three compounds discussed in the previous
section, the geminal coupling constant of 12.4 Hz for Kemp's Triacid is representative for a CH2 group flanked by two sp3 carbons.
The phenyl group of phenylalanine is electron-rich and facilitates more efficient coupling (J = 14.2 Hz) between the hydrogens of
an attached CH2 group. Alternatively, an oxygen atom is electronegative and leads to a smaller coupling constant (J = 9.4 Hz)
between the hydrogens of an attached CH2 group.

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Some other values for geminal coupling constants are listed below. In each case, R* is an alkyl substiituent containing a chiral
carbon atom. For small rings, the coupling geminal coupling constant is small as is the case for terminal alkenes.

VICINAL COUPLING is sensitive to the dihedral angle between the two adjacent C-H bonds (shown by the Karplus curve at
right). The magnitude of vicinal coupling constants can be zero if the dihedral angle ˜ 90° or as large as 13 Hz for ϕ = 180°. This is
frequently the case for bicyclic systems. For cyclohexane rings which adopt a rigid chair conformation, the coupling constants for
axial and equatorial protons are easily understood. For freely rotating systems, the coupling constant is averaged over all angles
during rotation.

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Long-Range Coupling
Coupling of protons separated by four or five bonds will generally be no larger than 1-2 Hz and will often be 0 Hz. Two factors
which facilitate long-range coupling are (a) intervening p bonds and (b) a "W" arrangement of bonds.

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1
H NMR Chemical Shifts for CH3X Compounds (CDCl3) (add 0.2 to 0.4 ppm for CH3CH2-X)

13
C NMR Chemical Shifts for sp and sp3 Carbons

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13
C NMR Chemical Shifts for sp2 Carbons

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Hewlett Packard GC/Mass Spectrometer
The GC-Mass Spectrometer has become the most sensitive and most powerful instrument for the identification of organic
compounds. Analysis of trace quantities of organic compounds - especially organochlorine compounds - has advanced to a stage
that 1 part in 1015 can be identified unambiguously as a contaminant in food or water sources. For this purpose, thousands of
spectra of insecticides and industrial pollutants have been compiled in databases. A solution of the organic compound containing 1-
5 milligrams in 0.5 mL is prepared and a special syringe is used to inject 0.05 microliters of this solution into the heated injector
port of the GCMS. The volatilized compound is carried in the helium flow to a splitter where only 1% of the sample is diverted to
the GC column (the rest goes out the window). The GC column used in this instrument is 10-30 meters long and only 0.4 mm in
diameter! ( called a capillary column). The conditions above assure that no more than 10-9 gram of sample are applied to this
column.

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As the organic compounds elute from the GC, they are introduced immediately into a Quadrupole Mass Spectrometer and a series
of mass spectra are repeatedly recorded for each two-second interval. In the ionization chamber, the molecules are bombarded by
electrons travelling across a 70 eV potential. When a molecule is struck by one of these high energy electrons, it loses one of its
own electrons and become a radical cation M•+. Many of the molecular ions will be formed with enough excess energy to undergo
subsequent reactions in the ionization chamber. From the ionization chamber, the positively charged ions are attracted through the
magnetic field of a quadrupole ion chamber maintained under high vacuum. For a given strength of the magnetic field, only those
ions having the correct charge-mass ration (m/e) will be deflected through the magnetic field and reach the detector. The instrument
varies the magnetic field continuously to produce a spectrum of m/e peaks.
In the next section are detailed mechanisms for common fragmentation and ionization reactions. It is much more difficult to solve
the structure of an organic compound from the mass spectrum alone than it is to match the spectrum with that of the known
compound. But with the help of NMR and IR spectroscopy, mass spectrometry can play an important role in structure
determination.

Reactions Observed in Mass Spectroscopy

A FRAGMENTATION REACTION involves the cleavage of one bond of the radical cation M•+ to form a cation A+ and a
radical B• . The reaction is especially facilitated by substituents which stabilize the cation A+.
∙+ + ∙
M ∅A +B (2.3.1)

α -Cleavage Reaction. The α -cleavage reaction is driven by the formation of a stable carbocation as shown for the three examples
below. In the first case, ethers readily undergo the a-cleavage reaction forming a stable oxonium ion (also common for similar N, S
compounds). In the second example, alkenes undergo α -cleavage reaction to form a stable allylic cation (also common for aryl
compounds and alkynes). In the third example, ketones readily undergo α -cleavage to produce stable acylium ions.

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Inductive Cleavage Reaction. Alkyl halides often undergo the inductive cleavage reaction in which the halogen atom (e.g. Cl• )
simply breaks off the initial radical cation. This is most prevalent for bromides and iodides and for compounds which can produce a
stable carbocation.

In each case, fragmentation reactions above produce both a radical and a cation from the initial radical cation M•+. Also, a
consequence of the nitrogen rule: For molecules that do not contain nitrogen, fragmentation reactions of the even-massed radical
cations will produce oddmassed A+ ions. This can be a useful method for finding which ions are produced by one of the above
reactions.
A REARRANGEMENT REACTION involves the cleavage of more than one bond of the radical cation M•+ to ultimately form a
new radical cation A•+ and a neutral compound B. Often, rearrangement reactions may proceed by complementary pathways so that
the mass numbers for both parts of the molecule can be found in the mass spectrum.
∙+ + ∙+
M ∅A +B or A+B (2.3.2)

Rearrangement of Cyclic Structures. When a cyclohexene derivative is ionized in the mass spectrum, a particularly facile cleavage
of the ring is reminiscent of a retro-Diels-Alder reaction.

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Radical site rearrangements (McLafferty Rearrangement). Carbonyl derivatives readily undergo a rearrangement reaction in
which M•+ undergoes intramolecular H atom transfer to the carbonyl oxygen atom. The best geometry involves a six-membered
transition state and subsequent a-cleavage or inductive cleavage leads to the fragment ions of each piece.

For each example of rearrangement reactions, the observed ions are radical cations. molecule which does not contain nitrogen, the
McLafferty rearrangement of the even-massed M•+ ion produces an even-massed A•+ ion and so are easily distinguished from the
odd-massed ions from a simple fragmentation reaction. spectrum arising from a McLafferty + 1 rearrangement. above, the
mechanism involves a second H atom transfer.

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2.3: Instrumentation is shared under a CC BY-NC-SA 4.0 license and was authored, remixed, and/or curated by LibreTexts.

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