6. Mass Spectrometry Adv. Inst. Techs

How does it work? a very small amount of sample is bombarded by a beam of high energy (usually electron beam) produces a positively charged species M  M + + e known as the molecular ion (same mass as the parent compound) target molecules fly apart into a number of smaller pieces all the positive ions are separated by masses to produce the spectrum peaks corresponding to the break up of the molecule are called the fragmentation pattern

m/e (mass to charge ratio) fragmentation pattern intensity (%) molecular ion base peak

Interpreting mass spectra 1. Molecular weight in most cases, some of the molecular ion remains unfragmented it produces a peak in the spectrum which is the highest mass peak this mass peak is most likely equal to the molecular weight of the compound known as the molecular ion example spectrum most intense peaks will have a peak 1 higher (due to presence of C-13)

each molecule will always fragment in the same way compounds may be positively identified by a comparison of their mass spectra MS of very similar compounds are very similar therefore, not as good a “fingerprint” as IR 2. Molecular fingerprint

MS shows a peak for each isotope of a mixture see the peaks due to both the 37 Cl and 35 Cl and these always occur in the natural 3:1 ratio bromine shows peaks for 79 Br and 81 Br in 1:1 ratio 3. Isotope peaks

a molecule of even number MW must contain either no nitrogen or an even number (or no) of N’s an odd MW signifies an odd number of Ns it doesn’t matter what other elements are in the compound 4. The “nitrogen rule”

some functional groups fragment in a particular and consistent way eg ethyl esters always lose the OCH 2 CH 3 group show a strong peak 45 less than the molecular ion Exercise 6.1 What loss would you expect to see from the molecular ion if the compound was a methyl ester? 31 (CH 3 O) 5. Characteristic fragmentation losses 105 <= <= 144

Exercise 6.2(a) FW 165 loss of 45 => possibly ethyl ester odd FW: odd no. Ns

Exercise 6.3(b) FW 214 & 216 Presence of Br even FW: 0 or even no. Ns

Instrumental components Sample Inlet Fragmentation Chamber Mass Analyser Detector high vacuum

Sample inlet a very small quantity of sample (  g) is required in the mass spectrometer also necessary that the sample is carried into the high vacuum within the instrument without leaks for substances with relatively high volatility, the sample is introduced into a heated injection port at 300  C, very much like one in a GC the sample inlet can be a connection to the “end” of a GC column if the material is not volatile or is unstable when heated, then a direct injection method is used, where the sample is held in a tiny cup, and inserted directly into the fragmentation chamber

Ion source MS relies on the detection of charged fragments of the original molecules the analyte must be ionised first a number of methods for achieving this, including: electron impact – the most common; the only one you need to know chemical ionisation fast atom bombardment

Electron impact ionisation a beam of high energy electrons fired across the path of the analyte molecules an electron is removed by the beam, and energy is added M + e → M +* → F 1 +, F 2 +, F 3, F 4 - only the positive (the most prevalent) fragments are analysed the energy of the beam is controlled by the applied voltage (70 V is normal); lower voltage means less energy means less fragmentation

Caffeine fragmentation

Vacuum is very substantial: around torr (1 atm is 760 torr) requires two levels of vacuum pump: one to remove the bulk of the air, and the ultra high vacuum pump to remove the last traces Exercise 6.3 Why is such a high vacuum required? to remove contaminants (eg O 2, N 2 ) to increase the stability of the fragments by reducing chance of collision

Mass analyser the equivalent of a monochromator separates the fragments on the basis of their different masses before the mass analyser is an ion filter, which blocks any non-positive fragment from passing into the mass analyser employs a series of negatively charged plates, with increasing voltages F+F-FF+F-F

Magnetic sector mass analyser particles moving through a magnetic field have their path bend at a radius proportional to their mass: the heavier the mass, the wider the curve of its path the magnetic field is changed during the scan to allow different masses through magnetic field curved path particle with wrong mass for curve particle with correct mass for curve

Quadrupole mass analyser answers to Exercise 6.4 four metals poles aligned to create electrical fields that allow one mass through at a given setting

Comparison the magnetic sector (MS) is physically larger than the quadrupole (Q) because of the large magnet the MS cannot scan as quickly as the Q the MS costs more than the Q the MS is capable of much better resolution (0.001 amu) than the Q (0.1 amu) Q is ideal as a detector for another instrument eg GC, HPLC, ICP (known as hyphenated instruments) MS is better for freestanding MS instruments

GC-MS the MS needs very small quantities, which equates to the amounts of analytes coming from capillary GC columns the carrier gas is a problem because it would swamp the MS separated and removed by differences in momentum for light He GC column MS inlet vacuum pump

How does the MS produce a chromatogram? at least once a second, the MS does a full mass scan the electronics generate two measures from the detector: the mass spectrum which is stored the total number of fragments (of all masses) in the scan – this is plotted on the chromatogram when only carrier gas is eluting, few fragments are generated when a compound elutes, more fragments are generated, giving a peak the chromatogram then looks the same as if a TC or FI detector had been used

The great advantage of a MSD each point in the chromatogram is linked to a MS this can be used to identify the compound (in conjunction with a spectral library) no need for spiking or RT comparison

Making the MSD selective setting the mass analyser to a fixed mass which is characteristic of the analyte (equivalent to quantitative analysis on UV-VIS) known as single (or selected) ion monitoring no scanning involved (no ID possible, but not necessary as you know what you are looking for) peaks only generated by compounds producing a fragment of the specified mass simplifies chromatogram increases sensitivity (all fragments of that mass are detected – in TIC, most are lost)

Comparison with other GC detectors MS is as sensitive as FI MS is much more expensive than other detectors no other detectors can identify the peak

HPLC-MS the problem of removing the mobile phase is much harder with a liquid must be instantaneous and must not “damage” analytes solution was electrospray uses a high voltage electrical field applied to a nebuliser this vapourises the solvent and ionises the analytes at the same time HPLC-MS is the standard “drugs-in-sport” testing method

ICP-MS same front end: plasma torch not used to produce characteristic radiation, but a stream of ions obviously different to GC- & HPLC-MS which analyse organic molecules this analyses elements no fragmentation occurs (these are atoms already!!) very expensive far more sensitive than any commercially available instrument (ng/L) able to distinguish between isotopes of same element