PRESENTATION OF BIOPHYSICAL CHEMISTRY

MASS SPECTROSCOPYRAMAN SPECTROSCOPY

Mass Spectroscopy

Mass spectrometry is an analytical tool used for measuring the molecular mass of a sample. For large samples such as biomolecules, molecular masses can be measured to within an accuracy of 0.01% of the total molecular mass of the sample i.e. within a 4 Daltons (Da) or atomic mass units (amu) error for a sample of 40,000 Da. This is sufficient to allow minor mass changes to be detected, e.g. the substitution of one amino acid for another or a post-translational modification.

An outline of what happens in a mass spectrometer

Atoms can be deflected by magnetic fields - provided the atom is first turned into an ion. Electrically charged particles are affected by a magnetic field although electrically neutral ones aren't.

Stage 1: Ionization

The atom is ionized by knocking one or more electrons off to give a positive ion. This is true even for things which you would normally expect to form negative ions (chlorine, for example) or never form ions at all (argon, for example). Mass spectrometers always work with positive ions.

Stage 2: Acceleration

The ions are accelerated so that they all have the same kinetic energy.

Stage 3: Deflection

The ions are then deflected by a magnet field according to their masses. The lighter they are, the more they are deflected.

The amount of deflection also depends on the number of positive charges on the ion - in other words, on how many electrons were knocked off in the first stage. The more the ion is charged, the more it gets deflected

Stage 4: Detection

The beam of ions passing through the machine is detected electrically. The chart recorder further displays further all the details.

The need for a vacuum

It's important that the ions produced in the ionization chamber have a free run through the machine without hitting air molecules.

Ionization

Diagrammatic view

Where are mass spectrometers used?

Mass spectrometers are used in industry and academia for both routine and research purposes. The following list is just a brief summary of the major mass spectrometric applications:

Biotechnology: the analysis of proteins, peptides, oligonucleotides

Pharmaceutical: drug discovery, combinatorial chemistry, pharmacokinetics, drug metabolism

Clinical: neonatal screening, hemoglobin analysis, drug testing

Environmental: water quality, food contamination

Geological: oil composition

How can mass spectrometry help biochemists?

Accurate molecular weight measurements: sample confirmation, to determine the purity of a sample, to verify amino acid substitutions, to detect post-translational modifications, to calculate the number of disulphide bridges

Reaction monitoring: to monitor enzyme reactions, chemical modification, protein digestion

Raman spectroscopy

PRINCIPLE OF RAMAN SPECTROSCOPY

νi ≠ νsRaman: photon energy change

on interaction (‘collision’) with a molecule

E=h νEi >EsEi ≠ Es

Basic theory

The Raman effect occurs when light impinges upon a molecule and interacts with the electron cloud of the bonds of that molecule. The incident photon excites one of the photons into a virtual state. For the spontaneous Raman effect, the molecule will be excited from the ground state to a virtual energy state, and relax into a vibrational excited state, which generates Stokes Raman scattering. If the molecule was already in an elevated vibrational energy state, the Raman scattering is then called anti-Stokes Raman scattering.

Basic theory

A change in the molecular polarization potential — or amount of deformation of the electron cloud —is required for the molecule to exhibit the Raman effect. The amount of the polarizability change will determine the Raman scattering intensity, whereas the Raman shift is equal to the vibrational level that is involved.

we are interested in the energy (wave number) difference between the excitation and the Stokes lines, the excitation source should be monochromatic. This forms the characteristic of a substance.

The difference can be represented as E’-E=h(f-f’) This difference in frequency is called

RAMAN SHIFT. It falls in the range of 100-4000 per cm.

Basic experimental set up

Intense monochromatic radiation from a source consisting of a large spiral discharge tube with mercury electrode is allowed to fall on the cell containing the sample

When the electric discharge passes through the tube mercury emits lines in its spectrum, the most intense of which at 43.58 nm serves as the exciting line.

The scattered light is observed at right angles to the direction of incident radiation

The detector is either a photographic plate or a photo multiplier.

Applications

Raman spectroscopy is commonly used in chemistry, since vibrational information is specific for the chemical bonds in molecules. It therefore provides a fingerprint by which the molecule can be identified. For instance, the vibrational frequencies of SiO, Si2O2, and Si3O3 were identified and assigned on the basis of normal coordinate analyses using infrared and Raman spectra

find the crystallographic orientation of a sample. As with single molecules, a given solid material has characteristic photon modes that can help an experimenter identify it.

Applications

can be used to discover counterfeit drugs without opening their internal packaging, and for non-invasive monitoring of biological tissue

Raman spectroscopy can be used to investigate the chemical composition of historical documents and contribute to knowledge of the social and economic conditions at the time the documents were produced

Raman spectroscopy is being investigated as a means to detect explosives for airport security

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