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EXPERIMENT 4 CHARACTERISATION OF AN UNKNOWN SAMPLE BY MASSSPECTROMETRY
The chemicals listed below will be used in this experiment. The likely hazardsassociated with each of the chemicals are noted and recommended procedures forhandling are given. You must read this page and the experimental descriptioncarefully before starting the experiment and before coming into the laboratory. Noteany potential hazards and adopt precautions as your safe lab practice. When you aresatisfied that you understand any possible difficulties that might arise and therecommended procedures for dealing with them, sign the declaration and have itinitialled by a demonstrator. This must be done prior commencing lab work. At thebeginning of the lab session demonstrators will quiz you about the safety informationand experimental procedure in order to identify your ability to work safely andefficiently. If you fail to prove ability for safe and efficient work you will not beallowed to start lab practical. Please note, that it is your own responsibility tocomplete the lab practical during time that is allocated to you. Be sure to requestinformation or help if you are in doubt on any point.ChemicalHazardPrecautionsUnknown compound AHarmful, skin, eye andrespiratory irritant.Do not ingest, avoid skin/eyecontact, wear glovesUnknown compound BHarmfulDo not ingest, avoid skin/eyecontact, wear glovesUnknown compound CHarmful, skin, eye andrespiratory irritant.Do not ingest, avoid skin/eyecontact, wear glovesUnknown compound DToxic, May cause cancer,Harmful if swallowedDo not ingest, avoid skin/eyecontact, wear glovesMethanolExtremely flammable andvery toxic. Toxic byinhalation, in contact withskin and if swallowedDo not breathe vapour, do notingest, avoid skin contact, weargloves. Keep away from source ofignition, keep container tightlyclosedDeclaration – I have read and understood the contents of the safety information sheetand the script for the experimentSigned (student): ……………………………………………………..Checked (demonstrator): ………………………………………….. Date:………………………2EXPERIMENT 4CHARACTERISATION OF UNKNOWN SAMPLES. MASSSPECTROMETRY___________________________________________________________OVERVIEWA supplier has made an error in the labelling of 4 different chemical compounds. Thechemicals are Phenacetin, Aspirin, Ibuprofen Na salt and trans cinnamic acid. Thesamples have now been labelled as A, B, C and D. Your task will be to indentify twoof the unknown samples.Please work in pairs throughout the following experiments.SUPPLEMENTARY INFORMATIONPhenacetin:Molecular weight = 179.2 g/molλmax in methanol= 248.6 nmMolecular structureAspirin:Molecular weight = 180.2 g/molλmax in methanol= 271.8 and 224.0 nmMolecular structuretrans Cinnamic acid: Ibuprofen Na saltMolecular weight = 148.2 g/mol Molecular weight = 228.3 g/molλmax in methanol= 267.0 and 214.2 nm λmax in methanol= 221.4 nmMolecular structure Molecular structure3EXPERIMENT 4MASS SPECTROMETRYLEARNING AIMS To gain familiarity with the use of laboratory MS equipment To record a MS spectrum of your unknown compoundLEARNING OUTCOMES To critically discuss the use of MS in Pharmaceutical Analysis To analyse and interpret a MS spectrumDIRECTED READINGOMED 0104 Lecture notesPharmaceutical 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, 1996Mass Spectrometry, Principles and Applications. De Hoffman and van Stroobant. 2ndEd. Wiley 2001._____________________________________________________________________INTRODUCTIONMass Spectrometry is a powerful analytical technique that is used to identify unknowncompounds, to quantify known substances and to elucidate the structure of molecules.The analysis is carried out with minute quantities of material – (nanogram orpicogram 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 andsteroids.• Analysis of environmental pollutants.Mass Spectrometers are usually coupled to Gas/Liquid Chromatographs. This ensuresthat components enter the instrument as separated pure compounds. These techniquesare abbreviated to GC/MS and LC/MS.These hybrid techniques combine the value of chromatographic sample introductionwith the highly sensitive (trace-analysis) mass spectrometer: after chromatographicseparation, the components of a mixture may be identified and quantified by massspectrometry._____________________________________________________________________4EXPERIMENTALThis experiment is designed to use both Electrospray ionisation (ESI) and ElectronImpact (EI) techniques to examine for the presence of the Mass ion of your unknowncompound.Sample preparation for GC/MSFor samples A, C and D, dissolve 1mg of your unknown compound in 1ml ofdichloromethane. Dilute this further by taking 100 μL of the unknown solution andadding it to 1ml of dichloromethane.For sample B, dissolve 1mg of your unknown compound in 1ml of methanol. Dilutethis further by taking 100 μL of the unknown solution and adding it to 1ml ofmethanol.Sample preparation for LC/MSDissolve 1mg of your unknown compound in 1ml of methanol. Dilute this further bytaking 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 ESIspectrum.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._____________________________________________________________________QUESTIONS1. 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?
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