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EXPERIMENT 3
CHARACTERISATION OF AN UNKNOWN SAMPLE BY NMR
SPECTROSCOPY
The chemicals listed below will be used in this experiment. The likely hazards
associated with each of the chemicals are noted and recommended procedures for
handling are given. You must read this page and the experimental description
carefully before starting the experiment and before coming into the laboratory. Note
any potential hazards and adopt precautions as your safe lab practice. When you are
satisfied that you understand any possible difficulties that might arise and the
recommended procedures for dealing with them, sign the declaration and have it
initialled by a demonstrator. This must be done prior commencing lab work. At the
beginning of the lab session demonstrators will quiz you about the safety information
and experimental procedure in order to identify your ability to work safely and
efficiently. If you fail to prove ability for safe and efficient work you will not be
allowed to start lab practical. Please note, that it is your own responsibility to
complete the lab practical during time that is allocated to you. Be sure to request
information or help if you are in doubt on any point.
| Chemical | Hazard | Precautions |
| Unknown compound A | Harmful, skin, eye and respiratory irritant. |
Do not ingest, avoid skin/eye contact, wear gloves |
| Unknown compound B | Harmful | Do not ingest, avoid skin/eye contact, wear gloves |
| Unknown compound C | Harmful, skin, eye and respiratory irritant. |
Do not ingest, avoid skin/eye contact, wear gloves |
| Unknown compound D | Toxic, May cause cancer, Harmful if swallowed |
Do not ingest, avoid skin/eye contact, wear gloves |
| Dimethyl Sulphoxide d6 | Harmful | Do not ingest, avoid skin/eye contact, wear gloves |
| Tetramethyl silane TMS | Highly flammable and harmful if inhaled |
Do not ingest, avoid skin/eye contact, wear gloves, use a fume hood. . Keep away from source of ignition, keep container tightly closed |
AssignmentTutorOnline
Declaration – I have read and understood the contents of the safety information sheet
and the script for the experiment
Signed (student): ……………………………………………………..
Checked (demonstrator): ………………………………………….. Date:………………………
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EXPERIMENT 3
SPECTROSCOPIC CHARACTERISATION OF AN UNKNOWN SAMPLE.
NMR SPECTROSCOPY
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OVERVIEW
A supplier has made an error in the labelling of 4 different chemical compounds. The
chemicals are Phenacetin, Aspirin, Ibuprofen Na salt and trans cinnamic acid. The
samples have now been labelled as A, B, C and D. Your task will be to indentify one
of the unknown samples.
Please work in pairs throughout the following experiments.
SUPPLEMENTARY INFORMATION
| Phenacetin: Molecular weight = 179.2 g/mol λmax in methanol= 248.6 nm Molecular structure |
Aspirin: Molecular weight = 180.2 g/mol λmax in methanol= 271.8 and 224.0 nm Molecular structure |
trans Cinnamic acid: Ibuprofen Na salt
Molecular weight = 148.2 g/mol Molecular weight = 228.3 g/mol
λmax in methanol= 267.0 and 214.2 nm λmax in methanol= 221.4 nm
Molecular structure Molecular structure
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EXPERIMENT 3
NUCLEAR MAGNETIC RESONANCE (NMR) SPECTROSCOPY
LEARNING AIMS
To gain familiarity with the use of laboratory NMR equipment
To gain understanding of the interpretation of NMR spectra
To extend data manipulation and presentational skills
To continue developing GLP skills
LEARNING OUTCOMES
To critically discuss the use of NMR in Pharmaceutical analyses
To manipulate NMR instrumentation
To attempt to identify an unknown sample by interpreting NMR spectra
DIRECTED READING
OMED 0104 – LECTURE NOTES
Williams, Dudley H. (Dudley Howard), Spectroscopic methods in organic chemistry,
5th ed. London : McGraw-Hill, (1995. ISBN: 0077091477
Joseph B. Lambert … [et al.]. Organic structural spectroscopy; London: Prentice Hall,
1998 . – ISBN: 0132586908
Ning, Yong-Cheng, Structural identification of organic compounds with spectroscopic
techniques . – Weinheim : Wiley-VCH, 2005 . – ISBN: 3527312404
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INTRODUCTION
NMR spectroscopy is a very powerful technique, with typical applications that
include the following:
1. determination of the structure and conformation of molecules,
2. quality control issues,
3. investigating interactions between molecules,
4. kinetic study of reactions,
5. to obtain information about molecular motion.
One of the most common, standard, NMR experiments that is carried out, is the 1H
single pulse sequence. When analysing the data from this experiments it is important
to consider the following:
Important aspects involved in analysing a 1H spectrum include:
1. chemical shift. A range of chemical shifts (peaks) can be observed in a
NMR spectrum due to the magnetic environment experienced by each
nucleus. These differences are caused by the variation in electron density
in the neighbourhood of each nucleus.
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2. 1H-1H splitting patterns. One of the rules that governs splitting patterns is:
a nucleus, equally coupled to n others over three bonds 3JHH, will give rise
to a signal with n+1 lines. For example, a proton can be observed as a
doublet, triplet or quartet whenever it is 3J coupled equally to either, one,
two, or three protons.
3. integration. The area under each set of peaks is proportional to the number
of protons in resonance at that frequency. Therefore sets of peaks from a
methyl (CH3) group coupled to a methylene (CH2) group would give an
integral ratio of 3:2
4. the coupling constant (J). For a set of split peaks (multiplet), the separation
between the peaks is measured in Hertz and is known as the coupling
constant J. Additionally for a 1H-1H coupling in for example CH3-CH2 ,
both sets of multiplets will have the same coupling constant. J values can
differ widely – from 1Hz to 18Hz.
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EXPERIMENTAL
• Dissolve between 10-20 mg of your sample in approximately 1 ml of
deuterated solvent.
• Sample B is soluble in D2O
• Samples A, C and D are soluble in dimethyl sulphoxide d6 (DMSO d6)
• Using a glass capillary tube add 1 drop of tetramethyl silane (TMS) to your
solution. TMS is a calibration standard in NMR spectroscopy.
• Add your prepared solution into a 5mm diameter glass NMR tube. Put a cap
on the NMR tube and invert the sample a few times to enable thorough
mixing.
• Your sample is now ready for NMR analysis. There are a large number of
different NMR experiments (pulse sequences) which can be applied to any one
sample, but the pulse sequence of interest for this practical is the 1H single
pulse experiment.
• Take your sample to the NMR lab G020 for analysis.
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QUESTIONS
1. Why is it beneficial to have a homogeneous sample for NMR analysis?
2. Why is it important to use deuterated solvents in solution state NMR spectroscopy?
3. From the recorded NMR spectrum, try to assign the NMR peaks and elucidate the
identity of your unknown sample. Please give a full explanation to your reasoning of
your results.