1
EXPERIMENT 4
CHARACTERISATION OF AN UNKNOWN SAMPLE BY MASS
SPECTROMETRY
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 |
| Methanol | Extremely flammable and very toxic. Toxic by inhalation, in contact with skin and if swallowed |
Do not breathe vapour, do not ingest, avoid skin contact, wear gloves. 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:………………………
2
EXPERIMENT 4
CHARACTERISATION OF UNKNOWN SAMPLES. MASS
SPECTROMETRY
___________________________________________________________
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 two
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
3
EXPERIMENT 4
MASS SPECTROMETRY
LEARNING AIMS
To gain familiarity with the use of laboratory MS equipment
To record a MS spectrum of your unknown compound
LEARNING OUTCOMES
To critically discuss the use of MS in Pharmaceutical Analysis
To analyse and interpret a MS spectrum
DIRECTED READING
OMED 0104 Lecture notes
Pharmaceutical Analysis 2nd Ed; Watson (2005) Elsevier, ISBN: 0443074453;
Chapter 9, pp: 187-220 (nice introduction to MS, GC/MS and HPLC/MS)
Mass Spectrometry for Biotechnology, G. Siuzdak, Academic press, 1996
Mass Spectrometry, Principles and Applications. De Hoffman and van Stroobant. 2nd
Ed. Wiley 2001.
_____________________________________________________________________
INTRODUCTION
Mass Spectrometry is a powerful analytical technique that is used to identify unknown
compounds, to quantify known substances and to elucidate the structure of molecules.
The analysis is carried out with minute quantities of material – (nanogram or
picogram amounts) at very low concentrations.
Typical applications include:
• Forensic analysis, such as confirmation and quantification of drugs of abuse.
• Determination of how drugs are used by the body.
• Sequencing of biopolymers such as proteins.
• Identify structures of bio molecules such as carbohydrates, nucleic acids and
steroids.
• Analysis of environmental pollutants.
Mass Spectrometers are usually coupled to Gas/Liquid Chromatographs. This ensures
that components enter the instrument as separated pure compounds. These techniques
are abbreviated to GC/MS and LC/MS.
These hybrid techniques combine the value of chromatographic sample introduction
with the highly sensitive (trace-analysis) mass spectrometer: after chromatographic
separation, the components of a mixture may be identified and quantified by mass
spectrometry.
_____________________________________________________________________
4
EXPERIMENTAL
This experiment is designed to use both Electrospray ionisation (ESI) and Electron
Impact (EI) techniques to examine for the presence of the Mass ion of your unknown
compound.
Sample preparation for GC/MS
For samples A, C and D, dissolve 1mg of your unknown compound in 1ml of
dichloromethane. Dilute this further by taking 100 μL of the unknown solution and
adding it to 1ml of dichloromethane.
For sample B, dissolve 1mg of your unknown compound in 1ml of methanol. Dilute
this further by taking 100 μL of the unknown solution and adding it to 1ml of
methanol.
Sample preparation for LC/MS
Dissolve 1mg of your unknown compound in 1ml of methanol. Dilute this further by
taking 100 μL of the unknown solution and adding it to 1ml of 50% methanol : 50%
water and 0. 1% formic acid.
MS Electrospray Ionisation (ESI)
• The sample is introduced from an HPLC instrument into an ion source at
atmospheric pressure.
• The analyte is ionised by electrospray (an acid-base chemical reaction) which
takes place in the aerosol formed from a steel needle kept at high voltage.
• The HPLC solvent is a mixture of methanol and water (50:50 v/v) with 0. 1%
formic acid.
• The ions formed are passed through a series of very small apertures in order to
maintain a good vacuum in the analyser. The eluent from the HPLC column is
scanned in a repetitive manner and the data interrogated, to obtain an ESI
spectrum.
MS Electron impact (EI)
• Gas chromatography is probably the most common technique for introducing
samples into a mass spectrometer for analysis by EI. Complex mixtures are
routinely separated by gas chromatography and mass spectrometry is used to
identify and quantify the individual components.
• The entire flow from the GC enters the mass spectrometer.
• Consult with the demonstrator regarding instrument settings and parameters.
_____________________________________________________________________
QUESTIONS
1. Explain what you understand by negative ion detection and positive ion detection
used in analysing your samples by ESI MS?
2. From your recorded mass spectra try to elucidate the identity of your unknown
samples. Please give a full explanation to your reasoning of your results.
3. In this experiment you have used the ESI and EI ionisation techniques. Using your
knowledge of MS, explain what you know about the ionisation technique known as
CI?