Mass spectrum calibration

Principles and Characteristics Mass spectrometry can provide the accurate mass determination in a direct measurement mode. For a properly calibrated mass spectrometer the mass accuracy should be expected to be good to at least 0.1 Da. Accurate mass measurements can be made at any resolution (resolution matters only when separating masses). For polymer/additive deformulation the nominal molecular weight of an analyte, as determined with an accuracy of 0.1 Da from the mass spectrum, is generally insufficient to characterise the sample, in view of the small mass differences in commercial additives. With the thousands of additives, it is obvious that the same nominal mass often corresponds to quite a number of possible additive types, e.g. NPG dibenzoate, Tinuvin 312, Uvistat 247, Flexricin P-1, isobutylpalmitate and fumaric acid for m = 312 Da see also Table 6.7 for m = 268 Da. Accurate mass measurements are most often made in El mode, since the sensitivity is high, and reference mass peaks are readily available (using various fluorinated reference materials). Accurate mass measurements can also be made in Cl... 

Note that the dominant peak in the observed mass spectrum is from the overexpressed protein. The observation of several ribosomal proteins allows for an internal calibration to be performed on the mass spectrum, greatly improving the mass measurement accuracy. 

Mass Calibration (time-of-flight) A means of determining m/z values from their times of detection relative to initiation of acquisition of a mass spectrum. Most commonly this is accomplished using a computer-based data system and a calibration file obtained from a mass spectrum of a compound that produces ions whose m/z values are known. 

Any mass spectrometer requires mass calibration before use. However, the procedures to perform it properly and the number of calibration points needed may largely differ between different types of mass analyzers. Typically, several peaks of well-known m/z values evenly distributed over the mass range of interest are necessary. These are supplied from a well-known mass calibration compound or mass reference compound. Calibration is then performed by recording a mass spectrum of the calibration compound and subsequent correlation of experimental m/z values to the mass reference list. Usually, this conversion of the mass reference list to a calibration is accomplished by the mass spectrometer s data system. Thereby, the mass spectrum is recalibrated by interpolation of the m/z scale between the assigned calibration peaks to obtain the best match. The mass calibration obtained may then be stored in a calibration file and used for future measurements without the presence of a calibration compound. This procedure is termed external mass calibration. 

Fig. 4.37. El mass spectrum of perfluorotributylamine (mass calibrant FC43) to demonstrate unit resolution of a quadmpole analyzer. The expanded views a-c show peaks separated to almost identical degree.

A mass spectrum of a calibration solution is acquired (calibration file) and matched against a table of the expected masses of the peaks in the calibration solution that are stored in a reference file. 

In ICP-MS a multi-element tuning solution is applied for the mass calibration of mass spectra. Figure 6.3 shows the mass spectrum for phosphorus determination at m/z = 31. In this case, the mass calibration was performed with the aid of a solution of a phos-phorus/sulphur mixture. From the known masses of the isotopes of the atomic ions, the masses of the polyatomic ions occurring were determined and identified in accordance with the isobaric polyatomic ions (15N160+ and 14N16OH+). Mass accuracy is the deviation of experimental determined mass of an atomic, polyatomic, cluster, molecular or fragment ion from the exact mass of species expected. Polyatomic, cluster and molecular ions were calculated from the atomic masses (see Appendix I), the mass accuracy is usually presented in ppm. 

Quantitative results were produced for each compound on the basis of internal standard method calculations. A three-point calibration curve was generated for each compound by using peak areas of a quantitation ion extracted from the mass spectrum of the compound. The ion was selected on the basis of it being a uniquely characteristic mass of the compound. The use of extracted ion quantitation produces more accurate results than total ion-current quantitation in cases in which two or more components are not completely resolved chromatographically. This situation is generally the case in complex mixture analysis. The quantitation ions selected for each of the compounds in the mix are listed in the box. 

Masses computed with Equation 22-8 appear in the last column of Table 22-3. A limitation on the accuracy of molecular mass determination is the accuracy of the mlz scale of the mass spectrum. Proteins of known mass can be used to calibrate the instrument. 

There are several other chemometric approaches to calibration transfer that will only be mentioned in passing here. An approach based on finite impulse response (FIR) filters, which does not require the analysis of standardization samples on any of the analyzers, has been shown to provide good results in several different applications.81 Furthermore, the effectiveness of three-way chemometric modeling methods for calibration transfer has been recently discussed.82 Three-way methods refer to those methods that apply to A -data that must be expressed as a third-order data array, rather than a matrix. Such data include excitation/emission fluorescence data (where the three orders are excitation wavelength, emission wavelength, and fluorescence intensity) and GC/MS data (where the three orders are retention time, mass/charge ratio, and mass spectrum intensity). It is important to note, however, that a series of spectral data that are continuously obtained on a process can be constructed as a third-order array, where the three orders are wavelength, intensity, and time. 

It is also possible to improve the accuracy of the mass calibration by making use of a peak of known composition to correct the electric field term for changes in the number of ions [16], In other words, if the identity of some component of the mixture is known by its retention time and/or mass spectrum, a peak of known mass in the mass spectrum may be used to apply a correction factor, x, to the electric field term in the calibration equation, which may be rewritten as ... 

Quantitation. Combined GC-SICM has been used mainly for quantitation. For a particular GA the absolute intensity of a characteristic ion in its mass spectrum is related to the amount of GA present, using standards to calibrate the instrument. Frydman et al. (35) used this "external standard method" to measure the levels of a number of GAs throughout the development of pea seeds. An alternative and preferable approach employs... 

The presence of DCB is proved by the appearance of other set of peaks in the mass spectrum at m/z = 50(10) 51(4) 73(5) 74(8) 75(21) 111(37) 113(12) 146(100) 147(7) 148(65) 149(5) 150(10) with much lower intensities in comparison with these observed for IPA. Using different mixtures of pure IPA and DCB we calibrated the relative intensities of the peaks m/z=45 and m/z = 146 for our experimental conditions. Based on this calibration and experimental mass spectra we calculated the ratio of IPA/DCB (y) for gas mixtures eliminated from the fullerene at different temperatures. The y value increased with the increase of temperature (see Fig. 4) remaining, however less than it was used for precipitation of fullerene from the liquid phase (y=7.37). It means that mainly molecules of DCB but not IPA are incorporated into the substances under study. This is in agreement with the IR data discussed earlier. (Fig. 3). 

Accurate mass measurement of a pseudomolecular ion such as [M+nH] " found in a mass spectrum can be used to determine possible molecular formulae. Accuracy of 0.5 ppm is achievable using FT-ICR-MS, and typically TOF instruments can achieve 2 ppm with appropriate calibration. 

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