HPLC with Mass Spectrometry

One of the limitations of GC and consequently of GC-MS is that in many cases polymer additives are insufficiently volatile to be separated on GC columns operating at even 

Hewlett Packard, for example, snpply the HP 5988A and HP 5987A mass-selective detectors for use with liquid chromatographs (LC) (see Appendix 1). The particle beam liquid chromatograph-mass spectrometer uses the same switchable EI-CI source and the same software and data systems that are used for a GC-MS system. Adding a GC creates a versatile particle beam LC-GC-MS system that can be switched from LG-MS to GC-MS in an instant. 

El spectra from the system are reproducible and can be searched against standard or custom libraries for positive compound identification. Cl spectra can also be produced. 

The particle beam system is a simple transport device, very similar to a two-stage jet separator. The solvent vapour is pumped away, while the analyte particles are concentrated in a beam and allowed to enter the mass spectrometric source. Here they are vaporised 

The different ways a particle beam LC-MS can be configured reflect the versatility of the system in accommodating both the application and the availability of existing instrumentation. The system consists of the following elements  

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What are the advantages of linking HPLC with mass spectrometry ... 

The combination of HPLC with mass spectrometry therefore allows more definitive identification and the quantitative determination of compounds that are not fully resolved chromatographically. 

In this regard several sophisticated chromatographic methods, with a quantification limit down to about 0.2 ng/g, have been developed and published for the determination of zearalenone. The methods were mainly based on high-performance liquid chromatography (HPLC) with fluorescence detection (Krska 1998 Visconti and Pascale 1998 Schuhmacher et al. 1998 Tanaka et al. 2000), but HPLC with mass spectrometry detection was also used (Shirai et al. 2000 Josephs et al. 2001). 

Perhaps the most mechanically complex solution ever developed for uniting HPLC with mass spectrometry was the moving belt interface [54]. The heart of this system was a mechanically driven continuous belt (analogous to an escalator or moving walkway) to which the HPLC eluent was applied. The majority of the mobile phase was evaporated by a heat source (ideally hot enough to vaporize the solvents but not to... 

More recently, butterfat composition has been analyzed by capillary GC on polarizable liquid phases (122,123), reversed-phase high-performance liquid chromatography (124,125), and GC and HPLC with mass spectrometry (MS) (126), as well as by MS-MS (127). These studies have led to an extensive resolution of butterfat triacylglycerols, but the possible presence of small amounts of positional and reverse isomers as well as of species containing two or three short-chain acids per molecule has not been addressed. 

Direct interfacing of hplc with mass spectrometry is a coupled or hyphenated technique similar to GC-Mass Spectrometry (p. 108) which provides structural information on separated sample components. Its development has been slow because ofdifliculties inherent in removing the liquid mobile phase whilst allowing only the analytes to pass into the mass spectrometer, particularly as reverse phase hplc often employs mobile phases containing aqueous... 

Column separation of proteins and peptides is used for both preparative and analytical purposes. Generally, in the former case low- or medium-pressure systems are preferred, whereas in the latter, high-performance-liquid-chromatography (HPLC) is the method of choice. These systems are coupled with a spectrophotometric detector normally set at wavelengths 280 and (around) 220 nm for proteins and peptides, respectively. Coupling of HPLC with mass spectrometry allows structural identification of compounds after the separation. 

Mass spectrometry is an extremely versatile detection system for gas chromatography. Interfacing an HPLC system to a mass spectrometer is a much more difficult task, however. Describe the major reasons why it is more difficult to combine HPLC with mass spectrometry than it is to combine GC with mass spectrometry. 

AP Bruins. Developments in interfacing microbore HPLC with mass spectrometry (a review). J Chromatogr 323 99—111, 1985. 

In the past the efficient on-line characterization of flavor progenitors by mass spectrometry has been limited by the polar and labile character of these compounds. Combining HPLC with mass spectrometry could have been the method of choice for the analysis of glycoconjugates. But in order to transform molecules from solution to ions in the gas-phase one had to deal with three major incompatibilities (/) ... 

SEC suffers from poor resolution and low sensitivity [5], while GC is limited by the high molecular weight and polar nature of many antioxidants and light stabilisers, which are designed to be reactive and so decompose when exposed to heat [9]. HPLC the most widely used instrumental method also has limitations [10-12]. HPLC lacks a simple sensitive universal detector that is compatible with all liquid mobile phases. UV or fluorescence detectors, which are commonly used, require that additives have a chromophoric moiety, while the universal refractive index detector only functions under isocratic conditions. As a result, Vargo and Olson have coupled HPLC with mass spectrometry (MS) for this type of application by using a moving belt interface [13]. 

The major hurdle standing in the way of directly coupling HPLC with mass spectrometry was that, until recently, ion sources needed a high vacuum. This was difficult to reconcile with the introduction of large volumes of solution. The discovery of electrospray ionization (ESI) provided a means to ionize fragile... 

One of the latest techniques that facilitates the analysis of ultra trace amounts of compounds is the combination of HPLC with mass spectrometry (HPLC-MS). This allows the separation of very small amounts of compounds in a multi-component mixture, and analysis of their quality and quantity (Heudi et al. 2006 Rychlik 2011). 

The introduction of liquid samples into the mass spectrometer has proved especially difficult. However, the advent of electrospray ionisation (ESI) and atmospheric pressure chemical ionisation (APCI) have overcome this problem and revolutionized the interfacing of HPLC with mass spectrometry. This success has pointed the way toward a major change in the mass spectrometric analysis of vitamin D, its analogs, and their metabolites. A disadvantage at the present time is that ionization using ESI and APCI is less efficient than the EI(+) used in GC-MS, but the immediate advantage is that it obviates the need for derivatiza-tion. 

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