Mass Spectroscopy 1Dr. Nikhat Siddiqi

Mass spectrometry is a powerful analytical technique that is used to identify unknown compounds, to quantify known compounds, and to elucidate the structure and chemical properties of molecules. Detection of compounds can be accomplished with very minute quantities (as little as g, moles for a compound of mass 1000 Daltons). This means that compounds can be identified at very low concentrations (one part in ) in chemically complex mixtures. 2Dr. Nikhat Siddiqi

Uses of Mass Spectroscopy Detect and identify the use of steroids in athletes. Monitor the breath of patients by anesthesiologists during surgery. Determine whether honey is adulterated with corn syrup. Monitor fermentation processes for the biotechnology industry. Detect dioxins in contaminated fish. Determine gene damage from environmental causes. 3Dr. Nikhat Siddiqi

Identify structures of biomolecules, such as carbohydrates, nucleic acids and steriods. Sequence biopolymers such as proteins and oligosaccharides. Determine how drugs are used by the body. Analyze for environmental pollutants. Identify and quantitate compounds of complex organic mixtures. 4Dr. Nikhat Siddiqi

Mass Spectrometer A mass spectrometer is an instrument that measures the masses of individual molecules that have been converted into ions, i.e., molecules that have been electrically charged. Since molecules are so small, it is not convenient to measure their masses is kilograms, or grams, or pounds. In fact, the mass of a single hydrogen atom is approximately 1.66 X grams. We therefore need a more convenient unit for the mass of individual molecules. This unit of mass is often referred to by chemists and biochemists as the dalton (Da for short), and is defined as follows: 1 Da=(1/12) of the mass of a single atom of the isotope of carbon-12( 12 C). This follows the accepted convention of defining the 12 C isotope as having exactly 12 mass units. 5Dr. Nikhat Siddiqi

A mass spectrometer does not actually measure the molecular mass directly, but rather the mass-to-charge ratio of the ions formed from the molecules. It follows that the charge on an ion is denoted by the integer number z of the fundamental unit of charge, and the mass-to-charge ratio m/z therefore represents daltons per fundamental unit of charge. In many cases, the ions encountered in mass spectrometry have just one charge (z=1) so the m/z value is numerically equal to the molecular (ionic) mass in Da. 6Dr. Nikhat Siddiqi

PRINCIPLES OF MASS SPECTROMETRY Any moving charged species of mass,m, and velocity, v, will be deflected by an applied magnetic field. The magnitude of this deflection will depend on the momentum, μ, of the species which is given by Equation µ = m. v 7Dr. Nikhat Siddiqi

Species with large momentum are deflected less than those with small momentum. Thus, if a stream of atoms and small molecules of identical velocity and charge but different mass in the gas phase is passed through a magnetic field, the deflection experienced by each atom or molecule depends on its mass. This deflection can therefore provide an accurate measure of mass. Larger molecules are uncharged in the gas phase so, in order to deflect these, it is necessary to confer a charge upon them. This may be achieved by irradiating the molecules with a beam of electrons and is called electron impact (EI) ionization. 8Dr. Nikhat Siddiqi

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10Dr. Nikhat Siddiqi

Since the beam of electrons is of quite high energy, this ionization method can cause extensive breakdown of molecular structure, splitting a single molecule into a number of fragment ions of different mass (Figure 4.3) each of which may be deflected differently in the magnetic field. This generates a mass spectrum (MS) which is characteristic of the chemical structure of the molecule. In fact, the complete structure of small biomolecules may be determined by electron impact MS (EI/MS) analysis alone. 11Dr. Nikhat Siddiqi

12Dr. Nikhat Siddiqi

Overview of MS Experiment A mass spectrometer consists of three distinct components viz., a source, analyzer and detector all maintained under a powerful vacuum ( ∼ 1 mPa). The source is an ionization chamber where the stream of ions is generated. The analyzer maintains either a magnetic or electric field which accelerates the stream of ions to a single velocity and then differentially deflects them so that they can be detected. This separates ions based on differences in their m/z ratio. 13Dr. Nikhat Siddiqi

14Dr. Nikhat Siddiqi

Provided all ions carry the same charge, they will deflect in the analyzer according to their m/z ratio that is according to their different mass. A spectrum of differing mass can therefore be generated from a single chemical species. It is possible to generate ions with z > 1, thus allowing analysis of large mass species. 15Dr. Nikhat Siddiqi

The ion beam may be detected by a photomultiplier detector and converted into an electric signal. This signal is amplified by a factor of up to 106 resulting in extremely sensitive detection. 16Dr. Nikhat Siddiqi

17Dr. Nikhat Siddiqi

Formation of gas phase samples ions is an essential prerequisite to the mass sorting and detection processes that occur in a mass spectrometer. The sample, which may be a solid, liquid, or vapor, enters the vacuum chamber through an inlet. Depending on the type of inlet and ionization techniques used, the sample may already exist as ions in solution, or it may be ionized in conjunction with its volatilization or by other methods in the ion source. 18Dr. Nikhat Siddiqi

The gas phase ions are sorted in the mass analyzer according to their mass-to-charge (m/z) ratios and then collected by a detector. In the detector the ion flux is converted to a proportional electrical current. The data system records the magnitude of these electrical signals as a function of m/z and converts this information into a mass spectrum. 19Dr. Nikhat Siddiqi

20Dr. Nikhat Siddiqi