College of Science, Health and Engineering

DEPARTMENT OF CHEMISTRY

OUTREACH PROGRAMS
MELBOURNE CAMPUS

STUDENT NOTES 2016

VCE INSTRUMENTAL CHEMISTRY PROGRAM

UNIT 3: CHEMICAL PATHWAYS

Enquiries Jessica Kvansakul, College of Science, Health and Engineering, La Trobe University, 3083
T 03 9479 6516 E outreach@latrobe.edu.au W latrobe.edu.au/outreach

VCE Instrumental Chemistry Program 2016

Welcome to the Year 12 Instrumental

Chemistry Program for 2016
Thank you for attending La Trobe University to complete the Instrumental Chemistry
Program.
Today you will observe four methods of instrumental analysis which scientists can
use to determine the composition of raw materials, ensure that ingredients in
products comply with industry standards and health regulations and to assess
various environmental conditions.
You will participate in four analyses using AAS, GC, NMR and UV-VIS instruments to
determine respectively:

the amount of iron in supplied food sample(s).

number and identification of organic compounds in a mixture
the structure of chemical compounds
the amount of phosphate in water

The purpose of this workshop is to teach you about these instrumental methods and
to demonstrate with real-world examples, how these instruments can be utilized.
Safety in the Laboratory
Since we will be working in a Chemistry Department Teaching Laboratories, there
are several rules that must be followed for your own safe ty. Your teacher will have
covered laboratory safety and risk assessments in the earlier part of your
chemistry course where you have been trained to follow safe working procedures.

The experiments you are doing today do not include the use of dangerous
chemicals; however caution should still be employed with all chemicals.

A lab coat must be worn.

At times you will be provided with safety glasses which must be worn over
your eyes not propped on your forehead.
Disposable gloves are available for you to wear for specific experiments.
Closed in shoes must be worn no sandals or thongs.
Long hair must be tied back.
Be very careful when handling glassware.

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Atomic Absorption Spectroscopy

Atomic absorption spectroscopy is a very widely used analytical techniqu e, which can
detect 70 different elements. It is very sensitive, capable of detecting concentrations
elements at part per million (ppm) levels or, in some cases, part per billion (ppb) levels.
APPLICATIONS
AAS is used in environmental analysis, forensics, and metallurgy to name a few. The
following applications are examples where this instrumental technique is used:

Analysis of nutrient metals (Fe, Ca, Al, Mg, etc.) in soil samples at parts per million
(ppm) concentrations.
Analysis of trace metals (Hg, Pb, Se, As, etc.) in environmental water samples at
parts per billion (ppb) concentrations.
Calcium determination in food products.
Iron levels in blood samples.
Metals present in engine oil to predict the possibility of engine failure

HOW DOES IT WORK?

AAS is a technique based on the absorption of UV-Visible light by gaseous atoms.

The sample to be analysed is converted into atomic vapour by spraying the solution
into a flame through a nebuliser.
A hollow cathode lamp (HCL) containing the element to be determined is used as the
light source.
Atoms of the element in the flame absorb light energy at precisely the wavelength
emitted by the HCL.
Amount of light absorbed at a particular wavelength is a function of the concentration
of that a particular element.

Figure 1: Basic components of an atomic absorption spectrophotometer.

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The basic components of AAS are listed below- briefly describe the function of each
1. A hollow cathode lamp ______________________________________
2. A slot burner ______________________________________
3. A nebuliser ______________________________________
4. A monochromator ______________________________________
5. A detector ______________________________________

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Exp1: Quantitative determination of iron in food sample(s) by AAS.

In this experiment you will analyse samples of butter beans, weet-bix and oats by atomic
absorption. You will determine the concentration of iron (Fe) in the prepared samples,
calculate its percentage weight based on the mass of sample and compare this percentage
to the reported value on the products label.
Background
Iron is a mineral that is naturally present in many foods, added to some food products, and
available as a dietary supplement. Iron is an essential component of hemoglobin, an erythrocyte
protein that transfers oxygen from the lungs to the tissues. As a component of myoglobin, a
protein that provides oxygen to muscles, iron supports metabolism. Iron is also necessary for
growth, development, normal cellular functioning, and synthesis of some hormones and
connective tissue. Some food sources of iron are listed in the table below.
Milligrams

Food
Breakfast cereals, fortified with 100% of the DV for iron, 1 serving

per serving Percent DV*

18
100

Oysters, eastern, cooked with moist heat, 3 ounces

44

White beans, canned, 1 cup

44

Chocolate, dark, 45%69% cacao solids, 3 ounces

39

Beef liver, pan fried, 3 ounces

28

Lentils, boiled and drained, cup

17

Spinach, boiled and drained, cup

17

Tofu, firm, cup

17

Kidney beans, canned, cup

11

Sardines, Atlantic, canned in oil, drained solids with bone, 3 ounces

11

Chickpeas, boiled and drained, cup

11

Rice, white, long grain, enriched, parboiled, drained, cup

* DV = Daily Value (Source: https://ods.od.nih.gov/factsheets/Iron-HealthProfessional/)

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Summary of the analysis

A. Preparation of standard solutions of iron.
B. Sample preparation: Acid digestion and dissolution of a known mass of solid
C.
D.
E.
F.

sample(s).
Measurement of absorbance of each iron standard and construction of a calibration
curve (CC).
Measurement of absorbance of prepared sample solution(s).
Calculations: use the CC to determine the [Fe] in the fertilizer solution and ultimately
its percentage
Weight in the original sample(s).

Standard Iron Solutions

Four standard solutions of iron (Fe) having concentrations of 1, 2, 4 and 5mg/L are
used.
1. Sample Preparation.
A known mass of samples were digested in 5 M hydrochloric acid (HCl) and 6 M
nitric acid (HNO3) by boiling under reflux. Further dilution is required to obtain a
solution suitable for analysis.
2. Calibration Curve.
The first part of this analysis consists of constructing a calibration or working curve.
The net absorbance (sample or standard absorbance minus absorbance of the
blank) of each of the iron standards is determined and graphed against its respective
concentration to construct the calibration curve.
3. Sample Measurement. Measure the absorbance of the presented sample solution(s).
Your demonstrator will show you how steps C and D are performed on the AAS as
well as discussing the operation of AAS and applications of the technique.

Calculation. Use the results from the AAS measurement of standards and sample(s) and
either the sample solution dilution conditions provided above or your own sample dilution
conditions to determine the weight percentage of iron present.

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Results:
1. Record absorbance values of standards
Fe
Blank
Concentration 0.00 mg/L
mg/L

Fe
1.00 mg/L

Fe
2.00 mg/L

Fe
4.00 mg/L

Fe
5.00 mg/L

Absorbance
Net
Absorbance

2. Absorbance of supplied sample(s)

Food sample

Butter beans

Oats

Red meat

Weetbix

Absorbance
Net
Absorbance

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3. Construct a Calibration Curve (CC) of Net Absorbance verses Iron Concentration (mg/L).
Label axes.

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Calculations
Use the net AAS absorbance measurement for the sample and the calibration curve you
constructed to determine the iron concentration in the diluted sample. Remember to use the
appropriate sample dilution conditions to calculate the weight percentage of Fe.
Questions
A) From your analysis, which of the food sample(s) has got the highest percentage of iron
and how much of this particular food is needed to meet the recommended daily intake of
iron?

B) What two gases were used in the flame in this analysis?

C) When an element such as iron is introduced into the flame, atoms of iron are produced.
In what state are these atoms and what are they capable of doing while in this state?

D) Which of the following elements could be analysed using AAS?

Ca, Al, P, S, Cu, C, N, Zn

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E) If a sample of fish was suspected of containing Hg, Zn and Cu, and assuming an HCL
lamp for each metal was available, could AAS be used to analyse for each metal in a
single sample of the fish? Why wouldnt the presence of all three metals in the one
sample interfere with the analysis of any one metal?

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UV-VIS Spectrophotometry

A UVVisible spectrophotometer makes use of the fact that many substances absorb
light of characteristic wavelengths. When Ultraviolet UV radiation (wavelength range
of 200 380 nm) passes through a solution of an organic compound, radiation can
be absorbed and electrons in the compound are excited to a higher energy level.
The amounts of energy absorbed are discrete amounts, being just that amount
required to promote electrons from one energy level to another.
The UV spectrum can be used to assist in identifying a substance (qualitative
analysis) because the maximum absorption occurs at a wavelength specific to each
particular substance.
Visible spectroscopy involves the absorption of visible light (390nm-700nm) by
solutions of transition metal ions. These solutions can be coloured because the d
electrons within the metal atoms can be excited from one electronic state to another.
The colour of the metal ion solutions is strongly affected by the presence of certain
anions and ligands.
UVVisible spectroscopy is used also for determining the concentration of a
substance in a sample, i.e. quantitative analysis.
Applications
UV-Vis technique is used in clinical analysis, hospital pathology laboratories,
environmental analysis and plastics and food industry. Some specific examples are:
a) determining the levels of nutrients, additives and contaminants in water and
foods
b) measuring the concentrations of substances in body fluids such as urine or
blood
c) measuring haemoglobin content and sugar levels in blood
d) determining the amount of coloured dyes in plastics

The instrument

Figure 2: Basic components of a UV- Visible spectrophotometer. (Source: Wikipedia).

Figure 1 Schematic of UV- visible spectrophotometer (source: Wikipedia)

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The basic components of a UV-Vis spectrophotometer are listed below - briefly

describe the function of each
1. A lamp ______________________________________
2. A monochromator ______________________________________
3. A sample cell ______________________________________
4. A detector ______________________________________
5. A recording device ______________________________________
Exp 2: UV-VIS Determination of Phosphate in Water
In this analysis, an absorbance spectrum generated in the visible portion of the
electromagnetic spectrum will be used to determine the phosphate content of a water
sample or the phosphate content in a fertilizer. In both cases, the orthophosphate form
(PO43-) of phosphorous will be determined.
The phosphate concentration is determined by using the visible spectrum of the bluecoloured complex (called molybdenum blue) which forms after phosphate reacts with
molybdenum and stannous chloride reagents. The wavelength at which the complex shows
the strongest absorbance is a characteristic of the complex and the absorbance intensity at
this wavelength differs with the concentration of phosphate present.
Because the concentrations of phosphate being analysed are very low (mg/L) the
relationship between concentration and absorbance is described by Beers Law. As a result,
a straight-line calibration curve can be constructed from the absorbance values of a series
of phosphate standards and from this curve; the absorbance value of the water of fertilizer
sample is used to determine the phosphate concentration present.
SUMMARY OF THE ANALYSIS
A. Phosphate standards either existing prepared versions or those prepared by
students on the day.
B. Sample preparation: existing water or fertilizer sample or samples presented on the
day will be used.
C. Measure absorbance at 690-700nm of molybdenum blue.
D. Construct a calibration curve
E. Determine phosphate content of the water sample

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Your demonstrator will give you a brief overview of UV-VIS spectroscopy; highlight its
applications, and the operation of the instrument, and how the standards and sample (s) are
to be prepared.
If in-situ preparation is agreed to, each pair of students will be responsible for preparing a
standard or sample and cleaning up their workstation. Specific instructions for each
preparation will be provided.
Under direction students will operate the Shimadzu UV-VIS spectrophotometer.
YOUR INVOLVEMENT:
1. Participate in standard/ sample preparation and / or instrument operation.
2. Record absorbance of each phosphate standard and that of the sample(s).
3. Use the net absorbance values of the standards and their concentration to construct
a calibration curve.
4. Use the calibration curve to determine the phosphate concentration in the sample(s).
5. Provide written answers to questions covering the instrumental/ analytical technique
used, and the sources and possible impact of phosphate in water.
RESULTS
1. Record of absorbance values and net absorbance calculations.
NB. Net absorbance = (Absorbance of a standard or sample minus absorbance of blank)
STANDARD
SOLUTION

ABSORBANCE

NET
ABSORBANCE

PHOSPHATE
CONCENTRATION

Blank

0mg/L

Phosphate Std 1

1mg/L

Phosphate Std 2

2mg/L

Phosphate Std 3

4mg/L

Phosphate Std 4

5mg/L

Water Sample

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2. Calibration graph: Net absorbance versus Concentration (mg/L)

(Label the axes and draw a line of best fit through (0,0) and the data points)

From the curve, the phosphate concentration in the water sample is ___________mg/L.

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3. This quantitative analysis for phosphate relies on the relationship between absorbance
and concentration. What is the name of the law which describes this relationship? Under
what conditions does this relationship fail?

4. What is the purpose of the blank?

5. What are some possible sources of phosphate in the environment?

6. How do phosphates get into waterways/ lakes/ oceans and why is it a concern if they
do?

7. Briefly describe how a UV-Vis spectrophotometer works.

8. The complex formed between phosphate and molybdenum is blue in colour. What colour
of light is this complex transmitting? What colour (s) is it absorbing?

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Chromatography

Chromatography is a technique commonly used to separate and identify the components in

a mixture. There are two instrumental chromatographic techniques based on column
chromatography, high performance liquid chromatography (HPLC) and gas chromatography
(GC). These are commonly used for the separation
and identification of very complex mixtures of similar
compounds, such as drugs in blood and
hydrocarbons in oil samples.
High-performance liquid chromatography (HPLC) and
Gas Liquid chromatography (GLC) are both forms of
liquid chromatography.
The sample partitions itself between the mobile and
stationary phases. The mobile phase is a liquid in
HPLC, and a gas in GLC. The stationary phase in the
column in both cases is a high boiling point liquid on
a solid support.

Figure 3: Example of a column chromatography

The gravity-controlled column chromatography the sample mixture which initially appears as
one band, gradually separates, as in the example above (figure 3), into three bands. The
band emerging first, is the least strongly adsorbed on the column, while the last band to
come off is that most strongly adsorbed.
HPLC is widely used in pharmaceutical and industrial analyses. It can separate compounds
with very high relative molecular masses whilst GC which is the most sensitive of the
chromatographic techniques is limited to compounds that can be readily vaporised without
decomposing. These compounds usually have low relative molecular masses.
Both HPLC and gas liquid chromatography (GLC) make use of two parameters:

Retention Times by which the components of a mixture are identified, and

Peak Areas from which the percentage composition can be determined.

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GAS CHROMATOGRAPHY
There are two types of gas chromatography, gasliquid chromatography (GLC) and gas
solid chromatography (GSC). Both gas chromatographic techniques operate in a similar
way as outlined below.
Gas supply: The mobile phase is a gas, generally inert gases such as helium, argon,
nitrogen, called the carrier gas.
An Injection Port:
Consists of a rubber
septum through which a
syringe needle is inserted
to inject the sample.
Oven: The injection port is
maintained at a
temperature high enough
to vaporize the least
volatile component in the
sample mixture. Since the
partitioning behaviour is
Figure 4: Schematic diagram of a gas chromatograph
dependent on
temperature, the
separation column is usually contained in a thermostat-controlled oven. Most columns
contain a liquid stationary phase on a solid support.
Detector: There are several types available. The most common is the Flame-Ionization
Detector.

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Experiment 3: Separation of an organic solvent mixture by GLC

Summary of the analysis
GLC will be used to separate and identify components in a mixture of aliphatic compounds.
The mixture contains ethanol, ethyl acetate, hexane, acetone and methanol.
Your demonstrator will give you a brief overview of GLC, highlighting instrument operation
and its applications. Qualitative and quantitative analysis of the mixture will be
demonstrated.
You will need to do the following:
a)
b)
c)
d)
e)

Record GC operating conditions

Record retention times of each component in the mixture
Record retention times of individual components
Identify components in mixture
Observe the steps to quantify the acetone content in the solvent mixture and use the
data provided to calculate its percentage composition
f) Provide written answers to questions.

RESULTS
GC Details:

Column type:
Stationary Phase:
Injector
temperature:
Column
temperature:
Carrier gas:
Type of detector:
Sample volume:

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Retention times:
Sample

Retention Times (mins)

Solvent mixture

Peak 1
Peak 2
Peak 3
Peak 4
Peak 5

ethanol
ethyl acetate
hexane
acetone
methanol

Draw the structural formula of hexane, ethyl acetate, and ethanol

hexane (BP = 69 C)

ethyl acetate (BP = 77 C)

(ethyl ethanoate)

ethanol (BP = 78 C)

Comment on the order in which the components were eluted:

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QUANTITATIVE DETERMINATION OF ACETONE IN THE SOLVENT MIX.

A series of different concentrations of acetone in ethanol are used. The area of the acetone
peak is indicative of its concentration in any standard and in the solvent mix. Accuracy is
improved if the detector response (sensitivity) of each compound in the mix is established
first and included in concentration calculations. However, time restraints with this
demonstration prevent performing these extra steps here. Nevertheless, a reasonable
estimation of concentration can be determined without these extra steps.
A plot of peak area versus acetone concentration (v/v) gives a calibration curve, which is
then used to determine the concentration of acetone in the mix.
Here is a set of typical peak area and concentrations for acetone under the isothermal
conditions used in this analysis. Use this data to construct a calibration curve and label the
axes.
Acetone
Peak area
(integration units)
Concentration
%(v/v)

160x 103

317 x 103

667 x 103

10

20

40

The acetone peak area in the solvent mix is ___________________ integration units

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Construct a calibration curve (line of best fit) and determine the percentage (v/v) of acetone
in the solvent mix.

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QUESTIONS
1. What is chromatography?

2. Describe the basic principles of chromatography using the following words:

Separation, component mixture, partitioning, mobile and stationary phase,
adsorption, desorption

3. List four types of chromatographic separation.

4. All chromatography involves the separation of components depending on how long

each component spends in the mobile phase and/ or the stationary phase. In GC
applications, what is the mobile phase and where is the stationary phase?

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5. List four applications where gas chromatography (GC) is used in chemical analysis

6. In this demonstration the GC was operated under isothermal conditions. What does
this mean?

7. A guide to the amount of any one component in a mixture can be gained by dividing
its peak area by the total peak area of all the components in the mixture. Is this the
most accurate approach? Explain

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Experiment 4: Structure Identification Using Nuclear Magnetic Resonance

(NMR) Spectroscopy

Your demonstrator will explain:

1. Safety in the Nuclear Magnetic Resonance (NMR) laboratory
2. NMR Theory
3. Application of NMR Technique to Molecular Structure Elucidation
SAFETY IN THE NUCLEAR MAGNETIC RESONANCE (NMR) LABORATORY

STOP!!
OBSERVE THE HAZARD AND
WARNING SIGNS ON THE LAB
DOOR BEFORE ENTERING
THE SUPERCONDUCTING
MAGNETS OF A SPECTROMETER
ARE ALWAYS AT FIELD.

Strong magnetic fields are produced outside the magnet. The magnetic field,
however, drops rapidly with increasing distance from the centre of the magnet and
an important measurement is the distance from the centre of the magnet at which
the field drops to 5 gauss. This is the cut-off point for most safety issues.
The horizontal distance of the 5 gauss line from the centre of the 400 MHz magnet
is 1.7 metres.

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Most of the newer NMR magnet systems are actively shielded. The following
must be understood when installing or working with such a shielded magnet:

The active shielding of the superconducting coil reduces the stray magnetic
fields, and therefore its effects.

Nevertheless, the magnetic field gradient is much stronger compared to nonshielded magnets, hence the distance interval between various stray field
contour lines is much smaller, and caution must be taken to avoid bringing
ferromagnetic objects close to the magnet.

In spite of the active shielding, the stray magnetic field directly above and directly
below the magnet is very high and the attractive forces on ferromagnetic objects
are very strong!

Keep all heavy iron objects away from the magnets. Do not allow metal chairs
within the barriers.

OBSERVE THE FOLLOWING WARNINGS:

Individuals with medical devices (e.g. cardiac pacemakers and metal prostheses) must
remain outside the 5-gauss perimeter. The NMR spectrometers generate strong
magnetic fields that can affect the operation of some pacemakers and harm implanted
or attached devices, such as prosthetic parts and metal blood vessel clips. Persons
with these types of medical concerns should contact their physicians about the
possible health risks before entering the Facility.
Magnetic field sensitive devices such as memory sticks, cards with magnetic strips,
cellular phones, laptop computers and mechanical watches should remain outside the
5-gauss perimeter. Strong magnetic fields surrounding the NMR spectrometers can
damage the strip of magnetic media found on credit cards, ATM cards, driver's
licenses, and other kinds of cards. Mechanical wrist and pocket watches will also
malfunction and be permanently damaged when exposed to a strong magnetic field.
Metal objects must remain outside the 5-gauss perimeter. Strong magnetic fields
surrounding the NMR spectrometers attract objects containing steel, iron, and other
ferromagnetic materials. This includes most ordinary tools, electronic equipment,
compressed gas cylinders, steel chairs and steel carts. Unless restrained, such
objects can suddenly fly toward the magnet which can cause personal injury and
extensive damage to the probe, Dewar and superconducting solenoid. The greater the
mass of the object, the more strongly it is attracted by the magnet. Only nonferromagnetic materials should be used near the instruments.

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NMR THEORY
Nuclear Magnetic Resonance (NMR) spectroscopy uses energy in the radio
frequency range of the electromagnetic spectrum has become the dominant method
of analysis for organic compounds, because in many cases it provides a way to
determine an entire structure using one set of analytical tests. It is also increasingly
used in inorganic chemistry and biochemistry, where it also provides a lot of valuable
structural information. The medical technique of MRI (Magnetic Resonance Imaging)
uses the same principles.
Since MRI uses proton NMR, it images the
concentration of protons. Many of those protons
are the protons in water, so MRI is particularly
well suited for the imaging of soft tissue, like the
brain, eyes, and other soft tissue structures in
the head as shown at left. The bone of the skull
doesn't have many protons, so it shows up dark.
Also the sinus cavities image as a dark region.
Nuclear Magnetic Resonance is a property of
the nucleus of an atom, concerned with what is
known as nuclear spin (I). This is equivalent to
the nucleus acting like a miniature bar magnet.
Although isotopes can have a variety of values
Figure 5: Example of an MRI scan
for I (including zero), the most useful for
spectroscopy are those nuclei which have I = 1/2.
Fortunately, this includes hydrogen 1 (1H), carbon 13 (13C), fluorine 19 (19F) and
phosphorus 31 (31P), so that some of the commonest elements in organic chemistry
can be analysed using NMR.

a
Figure 6: a. Excitation of nuclei by radio waves. b. Schematic representation of an NMR analysis.

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The nuclei can act like little magnets and their orientation can be flipped by energy
(radio waves) supplied by the magnetic field.
The constant flipping gives rise to a resonance frequency and different chemical
environments around each proton will give rise to a different chemical shift in the
spectrum.
In a real molecule, the effective magnetic field "felt" by a particular nucleus includes
not only the applied field, but also the magnetic effect of nearby nuclei and electrons.
This causes the signal to absorb at a slightly different frequency than for a single
atom; it is convenient to reference this resonant frequency to a
reference standard (usually tetramethylsilane, TMS, defined as
zero).
TMS has a symmetrical tetrahedral structure and all of the H
atoms in the molecule are in an identical environment. It
produces a single peak well away from the peaks of most
molecules (assigned as 0 ppm). It is the reference standard of choice for 1H and
13C NMR analysis.
Figure 7: Structure of TMS

When we plot the output from this absorption, we obtain a series of peaks known as
an NMR spectrum (or "spectra" if you have more than one spectrum) such as the
typical example shown in Fig. 8. The difference (in parts per million, ppm) from the
zero point is referred to as the chemical shift (). A typical range for is around 12
ppm for 1H and around 220 ppm for 13C. It is customary to have the zero point at
the right hand end of the spectrum, with numbers increasing to the left ("downfield")
as shown in Fig.9.

Figure 8: NMR Spectrum of Paracetamol

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1H

13C

Figure 9: Proton and carbon resonances are distributed along the x-axis.

This axis represents a scale of chemical environment known as a chemical shift

measured in parts per million (ppm).

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Integration of Peak Areas:

A valuable aspect of 1H NMR is that the area under each peak is proportional to the
number of hydrogens that are giving rise to that peak. (This is somewhat analogous
to GC, where the area under each peak is proportional to the amount of substance
giving rise to that peak.) A convenient way of analysing these peak areas is to
electronically "integrate" the peak, to convert the area into a distance. This distance
is routinely printed onto a 1H NMR spectrum as a line, such that the vertical distance
of the integration line is proportional to the number of hydrogens. We need to
compare the ratios of these vertical distances, and from this we can find the ratio of
the numbers of hydrogens. This ratio can be very helpful in determining the structure
of an unknown substance using NMR, but be careful- integrations are only
approximate! The area under each peak gives the ratio of hydrogen atoms on
adjacent carbons.
Equivalence:
If two or more protons (or indeed with two or more carbons) are in an equivalent
environment, then they will have the same chemical shift and appear as one signal.
A common example of this is a CH3 group, where all three protons are always
equivalent. In a CH2 group, the two protons are also equivalent, unless adjacent to a
chiral centre. Look for any plane of symmetry in the molecule, which will render the
two halves equivalent.
Splitting Pattern:
Splitting of peaks into multiplets can happen when the magnetic field of a proton is
affected by the protons on adjacent carbons (carbons that are coupled). The
number of peaks caused by splitting equals n + 1, where n is the number of H atoms
on the neighbouring atom i.e.,
CH splits the signal from hydrogens attached to an adjacent atom into 2 peaks
CH2 splits the signal from hydrogens attached to an adjacent atom into 3 peaks
CH3 splits the signal from hydrogens attached to an adjacent atom into 4 peaks
13C NMR
Since 13C makes up only 1% of natural carbon, coupling between carbon atoms is
rarely observed. It is used in NMR spectroscopy to identify different carbon atom
environments within a molecule. However, coupling by nearby hydrogens would very
often make 13C spectra very hard to read, so we routinely use a technique called
decoupling to eliminate all coupling effects from all hydrogens. This is done by
blasting the sample with radio waves which excite in the 1H region (this scrambles
all of the hydrogens), while observing in the 13C region. There is a price to pay:
integration can no longer be done accurately. However, there is also a benefit: the
hydrogens transfer some of their energy to the carbons and this improves the
otherwise feeble absorption of energy by the 13C nuclei. A side effect of this is that

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carbons which have no hydrogens attached to them tend to be considerably smaller

than the other carbons and such carbons can easily be identified in a 13C spectrum.
Therefore in a decoupled 13C spectrum we see no coupling. This means that each
carbon gives rise to a single sharp peak, and in a clear 13C spectrum the total
number of such peaks (excluding TMS and solvent) is equal to the number of types
of carbon in the molecule.
PUTTING IT ALL TOGETHER: DETERMINING A STRUCTURE FROM AN NMR
SPECTRUM
Assume that you know a compound having the molecular formula C4H8O2.
Treating this as a low resolution spectrum to start with, there are three clusters of
peaks and so three different environments for the hydrogens. The hydrogens in
those three environments are in the ratio 2:3:3. Since there are 8 hydrogens
altogether, this represents a CH2 group and two CH3 groups.
What about the splitting?
The CH2 group at about 4.1 ppm is a quartet. That tells you that it is next door to a
carbon with three hydrogens attached i.e. a CH3 group.
The CH3 group at about 1.3 ppm is a triplet. That must be next door to a CH 2 group.
This combination of these two clusters of peaks - one a quartet and the other a triplet
- is typical of an ethyl group, -CH3CH2. It is very common. Get to recognise it!
Finally, the CH3 group at about 2.0 ppm is a singlet. That means that the carbon
next door doesn't have any hydrogens attached.
So what is this compound? You would also use chemical shift data to help to identify
the environment each group was in, and eventually you would come up with:

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VCE Instrumental Chemistry Program 2016

Questions:
1. What splitting pattern in the 1H NMR spectrum would you expect for the
hydrogen atom(s) coloured red in the compounds shown below?
Your choices are: s singlet d doublet t triplet q quartet m multiplet. Enter the
appropriate letter in the answer box to the right of each formula.

2. How many peaks would you see in a 13C spectrum of hexane?

FINAL SUMMARY FOR 1H NMR

1H NMR spectra provide the following information:
a. The Number of Signals: each chemically different proton in a structure is
also magnetically different.
b. The Relative Areas of Each Signal: the strength of the NMR signal is
proportional to the number of protons giving rise to that signal, or, the area
under the absorption curve is proportional to the number of protons.
c. The Position of the Signal with respect to an internal standard (chemical
shift):
tetramethylsilane, (CH3)4S or TMS, is often used as an internal standard
since almost all proton signals appear downfield from the TMS signal.

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