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Liquid chromatography coupled with mass spectrometry (LC–MS) is one of the most powerful analytical tools for organic compound analysis.
Liquid chromatography coupled with mass spectrometry (LC–MS) is one of the most powerful analytical tools for organic compound analysis. The key advantages of using a mass detector with HPLC methods include:
A mass detector consists of three major components: an ion source that generates ions at atmospheric pressure, a mass analyser that filters ions and a detector that detects ions. As chromatographic peaks elute from the HPLC column and transfer to the ion source, two main processes occur. First, the ion source produces charged molecules or ions and second the mobile phase is removed. Once the ions are created, they are extracted from the ion source and transferred to the mass analyser. Sample ions are then filtered by the quadrupole mass analyser according to their mass-to-charge ratio (m/z) prior to detection.
Atmospheric pressure ionization is regarded as a 'soft' ionization process. Two atmospheric pressure ionization techniques predominate — electrospray ionization (ESI) and atmospheric chemical ionization (APCI). Highly suitable for ionization of polar and ionic small molecules, ESI is also suitable for large biomolecules and synthetic polymers. In contrast, APCI facilitates ionization of less polar molecules.
Both ESI and APCI techniques create mass spectra in which the largest peak, called the base peak, corresponds to the molecular weight of the compound, which is protonated in the positive ion mode (M+H)+ or deprotonated in the negative ion mode (M–H)–.
Source fragmentation can be induced to form diagnostic fragment ions for structural confirmation. However, in some instances, the molecular ion is relatively unstable, which creates fragment ions. With a mass detector, it is possible to make structural predictions from the mass difference of the molecular ion and fragment ions seen in a mass spectrum. The degree of fragmentation can be precisely controlled by adjusting the mass detector's source voltage.
The Thermo Scientific MSQ Plus Mass Detector has intuitive instrument software, with simple interactive method set-up and point-and-click sequences available from standard templates. It features automated mass calibration, system Autotune and instrument set-up that does not require user input. The user can mix and overlap scan events, enabling both confirmation and quantification in a single method.
Regardless of flow-rate and mobile phase composition, the peak shape is consistent between conventional HPLC detection and mass detection.
Mass detection is not constrained by chromatographic resolution because mass selectivity can be used to isolate components of interest from a complex chromatogram. Provided that the compounds differ in molecular weight, specific mass chromatograms can unequivocally locate the compounds of interest (Figure 1).
Figure 1: Baseline resolution is not a prerequisite for chromatographic analysis. Two compounds which are not chromatographically resolved can be isolated and analysed as mass chromatograms, which represent the signal generated by ions with a particular m/z.
The mass spectrum of a compound is reproducible, allowing libraries of spectra to be created and subsequently searched to enable positive identification.
A mass detector is not affected by changes in the mobile phase composition, unlike conventional HPLC detectors that often result in changing baselines whether working with 100% water or 100% solvent.
The Thermo Scientific ESI FastLoc probe for the MSQ Plus Mass Detector can be used with HPLC flow-rates ranging from 10 μL/min to 2 mL/min without splitting. With the Thermo Scientific APCI FastLoc Probe, HPLC flow-rates from 0.2 to 2 mL/min can be used without splitting.
The MSQ Plus Mass Detector has revolutionized the scope of LC–MS applications. Constraints imposed by nonvolatile buffers and sample matrices, which are common with HPLC methods, are no longer applicable. The patented self-cleaning source effectively analyses compounds in phosphate buffers and easily accommodates limited sample preparation protocols such as those for analysis of protein precipitated plasma samples. Unlike other instruments, the MSQ Plus mass detector is designed for robustness, increasing productivity through dependable simplicity. A unique patented cone wash system maintains instrument performance under the most rigorous conditions. A titanium entrance cone provides increased durability and greater chemical resistance. And a tool-free, triple-orthogonal Thermo Scientific M-Path Source guarantees the highest level of robustness (see Figure 2).
Figure 2: A mass detector's sample pathway
The mass detector helps liquid chromatographers run routine HPLC applications more efficiently. Unknown chromatographic peaks can appear during the development and manufacturing of drugs, chemicals, natural products and environmental samples. Mass detection enables a chromatographer to quickly and effectively suggest a number of possibilities for these unknown peaks.
Mass detection adds additional confidence to your quantification enabling you to obtain limits of detection, which can be up to 1000 times greater than traditional HPLC detection (see Figure 3). The MSQ Plus Mass Detector is designed to provide simple mass information about specific analytes at an extremely cost effective outlay without the need to be an expert in the details of LC–MS. Sensitivity and specificity both prevail in high abundance with mass detection, which decreases costs and increases confidence.
Figure 3: Calibration curve of 1 ppb to 10000 ppb.
Mass detection can provide a wealth of information that cannot be matched using conventional HPLC detection methods. Mass detection provides:
– Applicable to a wide range of compounds.
– Applicable to a wide range of HPLC conditions.
– Highly selective and easy to use.
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