Accurate mass

A Listing of Elemental Compositions vs. Accurate Mass at Nominal Integer Mass of 58 

if a mass spectrometer can separate two masses (100, 101), then AM = 1, M = 100, and R = 100. For conventional accurate mass measurement, R needs to be as large as possible, typically having a value of 20,000. At this sort of resolution, a mass of 100.000 can be separated from a second mass at 100.0050 (AM = M/R = 100/20,000 - 0.005) 

The section on high resolution (HR) already anticipated accurate mass to a certain extent. In fact, HR and accurate mass measurements are closely related and depend on each other, because mass accuracy tends to inprove as peak resolution is improved. Nevertheless, they should not be confused, as performing a measurement at high resolution alone does not equally imply measuring the accurate mass. High resolution separates adjacent signals, accurate mass can deliver molecular formulas [32-34]. 

Until the early 1980s, accurate mass measurements were nearly restricted to electron ionization, and for a while, the technique even seemed to become abandoned. New options available through FT-ICR instrumentation then revived the value of accurate mass measurements. The newly developed orbitrap and a new generation of oaTOF analyzers contributed to an increased demand for accurate mass data. Nowadays, formula elucidation can be performed using any ionization method [35], their widespread application thus demanding a thorough understanding of their potential and limitations [33]. 

Although a spectral feature reveals the mass of an ion, the value is not necessarily accurate enough for rehable identification of the corresponding molecule. In low-resolution mass spectra, the isotopic pattern of an ion may be buried within a single spectral feature. The measurement of m/z may result in an observation closer to the average mass than the exact mass of the ion (as discussed in Section 5.3.2). Even in the case of a well-resolved isotopic pattern, mass measurement may still be affected by the width and shape of the spectral feature. Deconvolution of spectral features contaminated with multiple ions of similar mass is important for accurate molecular identification but beyond the scope of this book. Here we will focus on the determination of accurate masses of single-component samples. 

There are several ways to determine accurate mass, including spectral local maxima, centroids, and least-squares curve-fitting [3,4], We will first introduce the related terminology. 

The centroid mass (/ (. ) of spectral feature is derived fi om the average of its integrated intensity. It can be calculated using the equation  

Least-squares curve-fitting enables determination of accurate mass by finding the maximum of a simulated curve that best describes an observation. The Gaussian function is one of the most frequently used functions to describe features in mass spectra. Fitting peaks is done with software tools which rely on the least-squares method. Other functions can also be used to fit spectral features, such as Lorentzian and polynomial. Notably, 

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Positive-ion electrospray mass spectrum of human hemoglobin (a) as initially obtained with all the measured masses, and (b) after calculation of true mass, as in Figure 8.3. The spectrum transforms into two main peaks representing the main alpha and beta chains of hemoglobin with accurate masses as given. This transformation is fnlly automated. The letters A, B, C refer to the three chains of hemoglobin. Thus, A13 means the alpha chain with 13 protons added. 

Electrospray alone is a reasonably sensitive technique for use with many classes of compounds. Spectacular, unprecedented results have been obtained with accurate mass measurement of high-... 

In general terms, the main function of the magnetic/electric-sector section of the hybrid is to be able to resolve m/z values differing by only a few parts per million. Such accuracy allows highly accurate measurement of m/z values and therefore affords excellent elemental compositions of ions if these are molecular ions, the resulting compositions are in fact molecular formulae, which is the usual MS mode. Apart from accurate mass measurement, full mass spectra can also be obtained. The high-resolution separation of ions also allows ions having only small mass differences to be carefully selected for MS/MS studies. 

Ultimate resolving power (accurate mass determination) + +++... 

High-Resolution, Accurate Mass Measurement Elemental Compositions... 

Calculation of molecular mass from a molecular formula for several simple substances. Accurate masses are shown in parentheses. 

Finally, accurate mass measurement can be used to help unravel fragmentation mechanisms. A very simple example is given in Figure 38.2. If it is supposed that accurate mass measurements were made on the two ions at 203.94381 and 77.03915, then their difference in mass (126.90466) corresponds exactly to the atomic mass of iodine, showing that this atom must have been eliminated in the fragmentation reaction. 

Chemical name Formula Integer Mass Accurate Mass... 

Compositions are read from left to right. Thus, the fifth entry in the table would be C2H4NO, with an accurate mass of 58.053096. 

Therefore, for accurate mass measurement, a standard mass peak (M,) is selected, and the accelerating voltage (V) is changed until the sample ion peak (M ) exactly coincides with the position of Mj. This technique is called peak matching, and the ratio between the original and new voltages (VA ) multiplied by mass (Mj) gives the unknown mass, M . 

By high-resolution mass spectrometry, ions of known mass from a standard substance can be separated from ions of unknown mass derived from a sample substance. By measuring the unknown mass relative to the known ones through interpolation or peak matching, the unknown can be measured. An accurate mass can be used to obtain an elemental composition for an ion. If the latter is the molecular ion, the composition is the molecular formula. 

Accurate mass measurement requires high resolving power. The difference in degrees of difficulty between measuring an m/z of 28 and one of 28.000 is likely to be large. Table 39.3 shows the broad mass ranges achievable with various analyzers. 

The electrospray source can be coupled directly to a liquid chromatographic (LC) column so that, as components of a mixture emerge from the column, they are passed through the source to give accurate mass data. As an example, a mixture of the peptides shown in Figure 40.8(a) was separated by LC and accurately mass-analyzed by ES. 

Once the peaks have been collected and stored, the computer can be asked to work on the data to produce a mass spectrum and print it out, or it can be asked to carry out other operations such as library searching, producing a mass chromatogram, and making an accurate mass measurement on each peak. Many other examples of the use of computers to process mass data are presented in other chapters of this book. 

Assuming that the mass spectrometer has sufficient mass resolution, the computer can prepare accurate ma.ss data on the m/z values from an unknown substance. To prepare that data, the system must acquire the mass spectrum of a known reference substance for which accurate masses for its ions are already known, and the computer must have a stored table of these reference masses. The computer is programmed first to inspect the newly acquired data from the reference compound in comparison with its stored reference spectrum if all is well, the system then acquires data from the unknown substance. By comparison and interpolation techniques using the known reference... 

When multicharged ions are formed, the simple rule of thumb used widely in mass spectrometry that m/z = m because, usually, z = 1 no longer applies for z > 1 then m/z < m, and the apparent mass of an ion is much smaller than its true mass. Accurate mass measurement is much easier at low mass than at high, and the small m/z values, corresponding to high mass with multiple charges, yield accurate values for the high mass. 

In fact, atomic masses are not integers. On the atomic scale, carbon is given a value of 12.0000. On this accurate mass scale, oxygen is 15.9949, nitrogen is 14.0031, hydrogen is 1.0078, and so on. 

Even for large molecules, the ability to measure accurate mass means that elemental compositions can be obtained from the accurately measured molecular mass. 

A simple mass spectrometer of low resolution (many quadrupoles, magnetic sectors, time-of-flight) cannot easily be used for accurate mass measurement and, usually, a double-focusing magnetic/electric-sector or Fourier-transform ion cyclotron resonance instrument is needed. 

Accurate mass measurement on a molecular ion of any substance gives directly the molecular formula for fragment ions, similar measurement gives their elemental compositions. 

Once the mass spectral information has been acquired, various software programs can be employed to print out a complete or partial spectrum, a raw or normalized spectrum, a total ion current (TIC) chromatogram, a mass chromatogram, accurate mass data, and metastable or MS/MS spectra. 

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