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Technical Note: 61757

Introduction to LC/MS for Chromatographers

Diab Elmashni, Thermo Fisher Scientific, San Jose CA, USA

The Advantages of LC/MS Detection

Key Words · MSQ PlusTM · High-throughput · LC/MS Detection · Quantitative HPLC · Sensitivity Liquid chromatography coupled with mass spectrometry detection (LC/MS) is one of the most powerful analytical tools for organic compound analysis. The key advantages of using LC/MS methods over HPLC methods include: · Selectivity ­ Co-eluting peaks can be isolated by mass selectivity and are not constrained by chromatographic resolution · Peak assignment ­ A unique chemical fingerprint for the compound of interest is generated, ensuring correct peak assignment in the presence of complex matrices · Molecular weight information ­ Confirmation and identification of known and unknown compounds · Structural information ­ Controlled fragmentation enables structural elucidation · Rapid method development ­ Provides easy identification of eluted analytes without retention time validation · Sample matrix adaptability ­ Decreases sample preparation time · Quantitation ­ Quantitative and qualitative data can be obtained easily with limited instrument optimization

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 analyzer. Sample ions are then filtered by the quadrupole mass analyzer according to their mass-to-charge ratio (m/z) prior to detection.

The Results of LC/MS Detection

Atmospheric pressure ionization is regarded as a `soft' ionization process. This results in a mass spectrum typically dominated by a single ion that 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)-. For example, a compound with a molecular weight of 200 Da in the positive ion mode will result in a spectrum with a `base peak' at 201 m/z. In negative ion mode (dependent on the chemistry of the compound in question), the spectrum will be dominated by an ion at 199 m/z. Source fragmentation can be induced to form diagnostic fragment ions for structural confirmation. However, in some cases, the molecular ion is relatively unstable, which creates fragment ions. Using an LC/MS 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 MSQ Plus source voltage.

How LC/MS Detection Works

An LC/MS detector consists of three major components: an ion source that generates ions at atmospheric pressure, a mass analyzer which filters ions, and a detector that detects ions. As chromatographic peaks elute from the LC column and transfer to the ion source, two main processes

ESI or APCI Probe

MSQ Plus M-Path

Molecular Weight Information Structural Information

Sample Cone

Cone Wash Exit Cone RF/DC Pre-Filter Lens Main Quadrupole Analyzer

Positive Identification Quantitative Information

Figure 1: LC/MS Sample Pathway

Obtaining Data from the MSQ Plus LC/MS Detector

The MSQ Plus has intuitive instrument software, with simple interactive method set up and point-and-click sequences available from standard templates. It features a built-in auto mass scale calibration, a full system Autotune, and automated instrument set up that does not require user input. It also allows the user to mix and overlap scan events, enabling both confirmation and quantitation in one single method. This software works on Microsoft® Windows® operating environments and utilizes the Microsoft Office software package.

Chromatographic Integrity in LC/MS

Regardless of flow rate and mobile phase composition, the peak shape is consistent between conventional LC detection and LC/MS detection.

The Selectivity of LC/MS Detection

LC/MS detection is not constrained by chromatographic resolution since 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 2).

The Reproducibility of Mass Spectra

The mass spectrum of a compound is reproducible, allowing libraries of spectra to be created and subsequently searched to enable positive identification.

Gradient Elution with LC/MS Detection

In gradient elution, LC/MS is not affected by changes in the mobile phase composition. Unlike conventional UV detection which often results in changing baselines whether it is working with 100% water or 100% solvent.

Figure 2: Chromatographic resolution is no longer a pre-requisite for LC analysis. Two compounds which are not completely resolved can be isolated using `mass chromatograms'

Wider Flow Rate Range for Increased Flexibility

The ESI FastLoc Probe for the MSQ Plus can be used with HPLC flow rates from capillary LC to conventional analytical LC, with flow rates ranging from 10 µL/min to 2 mL/min, without splitting. With the APCI FastLoc Probe, all conventional HPLC columns with flow rates from 0.2 to 2 mL/min can be used without splitting.

TM

Increased Detection with Less Restrictions

The MSQ Plus 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 selfcleaning source has proven to be effective in routinely analyzing compounds in phosphate buffers, and working with limited sample preparation protocols such as protein precipitated plasma samples. Unlike other instruments, the MSQ Plus features a unique patented cone wash facility, which maintains instrument performance under the most rigorous conditions. The new titanium entrance cone provides increased durability and greater chemical resistance. In addition, the new square quadrupole RF/DC pre-filter lens has been designed for high efficiency ion transmission and protection from contamination for the analyzing quadrupole.

LC/MS Applications with the MSQ Plus

The MSQ Plus helps 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. LC/MS detection enables a chromatographer to quickly and effectively suggest a number of possibilities for these unknown peaks. Specially designed for high-throughput applications, the MSQ Plus is ready for combinatorial chemistry, right out of the box.

Figure 3: Calibration curve for 4-nitrophenol determined by electrospray ionization in the negative ion mode

The Benefits of LC/MS Quantitation

LC/MS adds additional confidence to your quantitation enabling you to obtain limits of detection which can be up to 1000 times greater than UV detection. The MSQ Plus 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 multistage MS/MS. Sensitivity and specificity both prevail in high abundance with the MSQ Plus LC/MS detector, which decrease costs and increases confidence.

Summary

LC/MS detection can give a range of information that cannot be matched using conventional LC detection methods. LC/MS detection provides: · Molecular weight information · Structural information · Positive identification from user libraries · Quantitative information from LC/MS detection is: ­ Applicable to a wide range of compounds ­ Applicable to a wide range of LC conditions ­ Highly selective and easy to use

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TN62650_E 01/08S

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TN61758 Intro to LC/MC_.qxd

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