Calibration mass

Calibration of peak position for accurate mass determination can be performed internally or externally to minimize systematic errors. Internal calibration can be conducted when compounds with known molecular weight (called calibration compounds or calibrants) are mixed with the sample prior to the introduction into the ion source. This calibration can be performed, for example, by adding the calibrant to the liquid-phase sample while diluting it prior to analysis. The best result is achieved when multiple calibration signals are used to interpolate the m/z of ions within the range of interest. In proteomics, a tryptic digest of albumin from horse heart is typically used as the calibrant because it covers a wide m/z range (e.g., m/z 800-3000) that is ideal for mass calibration of low- to medium-sized peptides. In external calibration, the calibrants are analyzed before the analysis of real samples. The peaks of the calibrants are used to create and set the calibration equation in the data acquisition software. This method provides less mass accuracy because the instrument condition may still vary between the calibration and analyses of real samples. However, external calibrations save time and calibration compounds, and such methods also make analyses of analytes free from interferences caused by calibrants. 

To operate a GC-MS system, calibration of the mass scale is necessary. The cah-bration converts the voltage or time values controlling the analyser into m/z values. For the calibration of the mass scale, a mass spectrum of a known chemical compound is used, where both the fragments (m/z values) and their intensities are known and stored in the data system in the form of a reference table. 

Name Formula M m/z max. Instrument used Magnetic sector Quadrupole/lon trap m/z m/z  

00054857991 Da was taken into account for the calculation of the ionic masses (Audi and Wapstra, 1995 Mohr and Taylor, 1999). 

For GC-HRMS (high mass resolution) systems, an internal mass calibration (scan-to-scan) for accurate mass determinations by control of the data system is employed. At a given resolution (e.g., 10 000), a known reference is used which is continuously leaked into the ion source during analysis. The analyser is positioned on the exact mass of the substance ion to be analysed relative to the measured centroid of the known reference. At the beginning of the next scan, the exact position of the centroid of the reference mass is determined again and is used as a new basis for the next scan (see Chapter 2.3.4.3 Lock-Plus-Cali Mass Technique). 

The usability of the calibration depends on the type of instrument and can last for a period of up to several days or weeks. All tuning parameters, in particular the adjustment of the ion source, affect the calibration as described above. In particular the analyzer scan speed has a strong impact on the mass calibration with many instruments. Special attention should also be paid to a constant temperature of the ion source. Regular mass calibration using analysis conditions is recommended to comply with the lab internal QA/QC (quality assurance/quality control) procedures. 

So far, the concepts of exact mass, mass accuracy and resolution have been introduced without considering the means by which accurate mass measurements can be realized. The key to this problem is mass calibration. Resolution alone can separate ions of different m/z value, but it does not automatically include the information where on the m/z axis the respective signals precisely are located. 

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. 

Note The numerous ionization methods and mass analyzers in use have created a demand for a large number of calibration compounds to suit their specific needs. Therefore, mass calibration compounds will occasionally be addressed later in the chapters on ionization methods. It is also not possible to specify a general level of mass accuracy with external calibration. Depending on the type of mass analyzer and on the frequency of recalibration, mass accuracy can be as high as 1 mmu or as low as 0.5 u. 

If high-resolution measurements are performed in order to assign elemental compositions, internal mass calibration is almost always required. The calibration compound can be introduced from a second inlet system or be mixed with the analyte before the analysis. Mixing calibration compounds with the analyte requires some operational skills in order not to suppress the analyte by the reference or vice versa. Therefore, a separate inlet to introduce the calibration compound is advantageous. This can be achieved by introducing volatile standards such as PFK from a reference inlet system in electron ionization, by use of a dual-target probe in fast atom bombardment, or by use of a second sprayer in electrospray ionization. 

Internal mass calibration typically yields mass accuracies as high as 0.1-0.5 mmu with Fourier transform ion cyclotron resonance, 0.5-5 mmu with magnetic sector, and 2-10 mmu with time-of-flight analyzers. 

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GC/MS. GC/MS is used for separation and quantification of the herbicides. Data acquisition is effected with a data system that provides complete instrument control of the mass spectrometer. The instrument is tuned and mass calibrated in the El mode. Typically, four ions are monitored for each analyte (two ions for each herbicide and two ions for the deuterated analog). If there are interferences with the quantification ion, the confirmation ion may be used for quantification purposes. The typical quantification and confirmation ions for the analytes are shown in Table 4. Alternative ions may be used if they provide better data. 

Tables 6.27 and 6.31 show the main characteristics of ToF-MS. ToF-MS shows an optimum combination of resolution and sensitivity. ToF-MS instruments provide up to 40000 spectra s-1, a mass range exceeding 100000 (in principle unlimited), a resolution of 5000, and peak widths as short as 200 ms. This is better than quadruples and most ion traps can handle. Unlike the quadrupole-type instrument, the detector is detecting every introduced ion (high duty factor). This leads to a 20- to 100-times increase in sensitivity, compared to QMS used in scan mode. The mass range increases quadratically with the time range that is recorded. Only the ion source and detector impose the limits on the mass range. Mass accuracy in ToF-MS is sufficient to gain access to the elemental composition of a molecule. A single point is sufficient for the mass calibration of the instrument. ToF mass spectra are commonly calibrated using two known species, aluminium (27 Da) and coronene (300 Da). ToF is well established in combination with quite different ion sources like in SIMS, MALDI and ESI.

Ledford, E. B., Jr. Rempel, D. L. Gross, M. L. Space charge effects in Fourier transform mass spectrometry Mass calibration. Anal. Chem. 1984,56, 2744-2748. 

One of the specificities of ToF-SIMS is the possibility to switch easily from positive to negative ion mode by reversing the extraction potential. The mass calibration of spectra is internal, using low mass ions (H+, H2+, H3+, C+, CH+, CH2+ and CH3+ ion peaks in... 

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. 

A mass calibration for FTICR analyzers with superconducting magnets is very stable and is valid for many days for normal applications. Mass accuracy < 1 ppm can be obtained over a fairly wide mass range. Unique elemental composition can be determined for masses over 800 Da [262]. Recently, 0.1 ppm mass accuracy, which required a mass resolving power >300,000, has been achieved for several thousand peaks by a 14.5 T instrument [263] and commercial instruments with mass accuracy <0.2 ppm are available. As with the orbitrap (see Section 2.2.5) the frequency is... 

Viscometry is used to measure average molar masses too [1-3]. It is an indirect method, since the measured quantity is the intrinsic viscosity (TV), which is related to the average molar masses calibration by a peculiar formula, called the Mark-Huwink-Sakurada equation [1-3]. 

Example Cesium iodide is frequently used for mass calibration in fast atom bombardment (FAB) mass spectrometry (Chap. 9) because it yields cluster ions of the general formula [Cs(CsI)n] in positive-ion and [I(CsI)J in negative-ion mode. For the [Cs(CsI)io] cluster ion, m/z 2730.9 is calculated instead of the correct value m/z 2731.00405 by using only one decimal place instead of the exact values Mi33Cs = 132.905447 and M1271 = 126.904468. T e error of 0.104 u is acceptable for LR work, but definitely not acceptable if accurate mass measurements have to be performed. 

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.

Fig. 6.34. LR- (a) and HR-EI (b) mass spectra of 2-(l-methylpropyl)-phenol. The elemental compositions as obtained from accurate mass measurement are directly attached to the corresponding peaks. Peaks with small-lettered labels belong to PFK and residual air used for internal mass calibration (Chap. 3.3).

Note PEGS ranging from PEG 300 to PEG 2000 are often used for mass calibration. They are particularly useful as internal reference (Chap. 3.3.5) for accurate mass measurements in positive-ion FAB-MS. 

Fig. 9.13. Positive-ion FAB spectrum of a cationic fluorescent marker dye with PEG 600 admixed for internal mass calibration. By courtesy of K. H. Drexhage, University of Siegen and J. Wolfrum, University of Heidelberg.

Note Scanning of a magnet is affected by hysteresis. This causes the reproducibility of mass calibration to improve after several scan cycles have passed. For best results with dual-target probes, it is therefore recommended to skip the first few scans. 

MALDI is the method of choice for the analysis of synthetic polymers because it usually provides solely intact and singly charged [62] quasimolecular ions over an essentially unlimited mass range. [22,23] While polar polymers such as poly(methylmethacrylate) (PMMA), [83,120] polyethylene glycol (PEG), [120,121] and others [79,122,123] readily form [M+H] or [M+alkali] ions, nonpolar polymers like polystyrene (PS) [99,100,105,106] or non-functionalized polymers like polyethylene (PE) [102,103] can only be cationized by transition metal ions in their l-t oxidation state. [99,100] The formation of evenly spaced oligomer ion series can also be employed to establish an internal mass calibration of a spectrum. [122]... 

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