Mass spectrometry can be instrumental in determining the structure of unknown compounds. When a compound is ionised, particularly using electron impact ionisation, it often fragments into smaller ions. These fragments are characteristic of the structure of the original molecule. By analysing the mass-to-charge ratios of these fragments, a mass spectrometer can provide vital clues about the molecular structure. The pattern of fragmentation essentially serves as a 'fingerprint' for the compound. For example, the presence of specific fragment ions in the spectrum can indicate particular functional groups or linkages within the molecule. Additionally, the intensity of the peaks can suggest the relative stability of these fragments. Combined with other analytical techniques like infrared spectroscopy or NMR, mass spectrometry can be a powerful tool for elucidating the structure of unknown compounds, especially when combined with theoretical models and databases.

Mass spectrometers differentiate between isotopes of the same element based on their mass-to-charge ratio. Isotopes of an element have the same number of protons (and hence the same chemical properties) but differ in the number of neutrons, resulting in different atomic masses. In a mass spectrometer, these differences in mass, albeit often very small, can be detected and measured. When a sample containing isotopes is ionised and analysed, each isotope produces a distinct peak on the mass spectrum corresponding to its unique mass-to-charge ratio. The precision of modern mass spectrometers allows them to distinguish between these slight differences in mass. Therefore, the presence and abundance of different isotopes in a sample can be accurately determined by examining the mass spectrum. This capability is particularly valuable in fields like geochemistry and radiopharmacology, where isotopic composition is crucial.

While Time of Flight (TOF) mass spectrometry is a powerful analytical tool, it has certain limitations. One significant limitation is its sensitivity to the shape and stability of the ionisation pulse, which can affect accuracy and resolution. The quality of the vacuum in the flight tube also plays a crucial role; any interference, like residual gas molecules, can skew results. Additionally, TOF mass spectrometry may have limitations in analysing very high molecular weight compounds due to challenges in ionisation and detection of such large ions. Furthermore, the technique relies heavily on the purity of the sample, as contaminants can interfere with the ionisation process and obscure the results. Finally, while TOF is excellent for determining mass-to-charge ratios, it may not always provide enough structural information for complete structural elucidation of complex molecules without supplementary techniques or prior knowledge of the sample composition.

Different ionisation methods in mass spectrometry are used to accommodate the diverse nature of samples. The choice of ionisation technique can significantly impact the analysis as it determines which ions are formed and how they are formed. Electron impact ionisation, which involves bombarding the sample with high-energy electrons, is suitable for volatile and thermally stable compounds. It typically results in the formation of positively charged ions and is known for causing fragmentation, which can be useful for structural analysis. However, for larger, non-volatile, or thermally sensitive molecules, such as proteins or polymers, electrospray ionisation is more appropriate. This method produces ions by applying a high voltage to a liquid sample, creating a fine spray. The resulting ions are often multiply charged, which is advantageous for analysing high-mass molecules. The choice of ionisation method thus depends on the physical and chemical properties of the sample and the information required from the analysis.

The flight tube in a Time of Flight (TOF) mass spectrometer is crucial for its accuracy and resolution. The tube's primary function is to allow ions to travel from the acceleration point to the detector. The length of the flight tube directly impacts the resolution of the spectrometer. Longer flight tubes offer greater separation between ions of slightly different masses, enhancing resolution. This is because ions have more time and space to spread out according to their mass-to-charge ratios. Additionally, the vacuum within the flight tube is essential to prevent ions from colliding with air molecules, which would impede their flight and affect accuracy. A high vacuum ensures that ions travel unimpeded, allowing for a more accurate measurement of their time of flight. The design and maintenance of the flight tube, therefore, play a critical role in ensuring that the mass spectrometer provides precise and reliable results.