LC-MS interface

Coupling of liquid chromatography to mass spectrometry has not only led to a wide variety of interfaces, but also initiated the development of new ionization methods. [8-13,62] In retrospect, the moving belt interface seems rather a curiosity than a LC-interface. The LC effluent is deposited onto a metal wire or belt which is heated thereafter to desolvate the sample. Then, the belt traverses a region of 

Nowadays, moving belt, PBI, API, TSP, and CF-FAB have mostly been replaced by ESI [1,25], APCI, [23,71-73] and APPI [74,75] (Chap. 11). ESI, APCI, and APPI intrinsically represent perfect FC-MS interfacing technologies. Even FC-nanoESI operation is feasible. [18] 

Example UV photodiode array (PDA) and ESI-TOF detection can be combined if the effluent is split or the PDA precedes the ESI interface. The detection methods complement each other in that their different sensitivities towards components of a mixture prevent substances from being overlooked. RICs help to differentiate a targeted compound - an unknown impurity in this case - from others and to identify eventually present isomers. Finally, accurate mass measurement helps in the identification of the unknown (Fig. 12.9). [25] 

In tandem MS, two or more stages of mass analysis are combined in one experiment. [79,80] Each stage provides an added dimension in terms of isolation, selectivity, or structural information to the analysis. Therefore, a tandem MS stage is equivalent to a chromatographic separation, provided the separation of isomers is not required. While chromatography distinguishes substances by their retention time, tandem MS isolates them by mass. [2,3,25] The principles of tandem MS have been discussed and some applications for stmcture elucidation and quantitation have already been shown (Table 12.1). However, the aspect of increased selectivity has not been addressed so far. [81] 

6 tandem MS with magnetic sector instruments example B E = constant linked 

---

Dynamic/continuous-flow FAB allows a continuous stream of liquid into the FAB source hence it constitutes an LC/MS interface for analyses of peptide mixtures. 

Liquid chromatograph/mass spectrometer (LC/MS) interface. An interface between a liquid chromatograph and a mass spectrometer that provides continuous introduction of the effluent from a liquid chromatograph to a mass spectrometer ion source. 

The ion spray liquid chromatography/mass spectrometry (LC-MS) interface coupled via a postsuppressor split with an ion chromatography (IC) has been used in the analysis of alcohol sulfates. The IC-MS readily produces the molecular weight while the tandem mass spectrometric detection IC-MS-MS provides structural information [305]. 

Seven different LC-MS interfaces are described in Chapter 4, with particular emphasis being placed on their advantages and disadvantages and the ways in which the interface overcomes (or fails to overcome) the incompatibilities of the two techniques. The earlier interfaces are included for historical reasons only as, for example, the moving-belt and direct-liquid-introduction interfaces, are not currently in routine use. The final chapter (Chapter 5) is devoted to a number of illustrative examples of the way in which LC-MS has been used to solve various analytical problems. 

He then joined the Central Research Establishment of the Home Office Forensic Science Service (as it then was) at Aldermaston where he developed thermogravimetry-MS, pyrolysis-MS, GC-MS and LC-MS methodologies for the identification of analytes associated with crime investigations. It was here that his interest in LC-MS began with the use of an early moving-belt interface. This interest continued during periods of employment with two manufacturers of LC-MS equipment, namely Kratos and subsequently Interion, the UK arm of the Vestec Corporation of Houston, Texas, the company set up by Marvin Vestal, the primary developer of the thermospray LC-MS interface. 

One of the functions of an LC-MS interface is to remove the mobile phase and this results in buffer molecules being deposited in the interface and/or the source of the mass spectrometer with a consequent reduction in detector performance. Methods involving the use of volatile buffers, such as ammonium acetate, are therefore preferred. 

The effect of the mobile-phase composition on the operation of the different interfaces is an important consideration which will be discussed in the appropriate chapter of this book but mobile-phase parameters which affect the operation of the interface include its boiling point, surface tension and conductivity. The importance of degassing solvents to prevent the formation of bubbles within the LC-MS interface must be stressed. 

With the LC-MS interfaces now available, a wide range of analytes, from low-molecular-weight drugs and metabolites (<1000 Da) to high-molecular-weight biopolymers (>100000 Da), may be studied. 

Figure 4.1 Schematic of a moving-belt LC-MS interface. From applications literature published by Micromass UK Ltd, Manchester, UK, and reproduced with permission.

When optimum experimental conditions have been obtained, all of the mobile phase is removed before the analyte(s) are introduced into the mass spectrometer for ionization. As a consequence, with certain limitations, it is possible to choose the ionization method to be used to provide the analytical information required. This is in contrast to the other LC-MS interfaces which are confined to particular forms of ionization because of the way in which they work. The moving belt can therefore provide both electron and chemical ionization spectra, yielding both structural and molecular weight information. 

The direct-liquid-introduction (DLI) interface was made available commercially just after the moving-belt interface to which, as no company produced both types, it was an alternative. At this time, therefore, the commercial LC-MS interface used within a laboratory was dictated by the manufacturer of the mass spectrometer already in use unless a new instrument was being purchased solely for LC-MS applications. The development of LC-MS in the early 1980s was such that this was very rare and it was therefore unusual that a scientific evaluation was carried out to assess the ability of a type of interface to solve problems within a particular laboratory. 

The advantages and disadvantages of this type of interface, particnlarly in comparison to the moving-belt interface which was available at the same time, are listed below. This was one of the first LC-MS interfaces to be made commercially available and, although used in a number of laboratories, its development was halted premamrely by the introduction of the thermospray interface (as we shall see later). 

The introduction of the dynamic-FAB LC-MS interface allowed the study of more non-volatile and ionic compounds than was possible with the other interfaces available at that time. It was, however, necessary to attach such an interface to a mass spectrometer with sufficient mass range to allow this potential advantage to be realized. This had significant financial implications on any laboratory... 

Arguably the ultimate LC-MS interface would be one that provides El spectra, i.e. a spectrum from which structural information can be extracted by using famihar methodology, and this was one of the great advantages of the moving-belt interface. There is, however, an incompatibility between the types of compound separated by HPLC and the way in which electron ionization is achieved and therefore such an interface has restricted capability, as previously discussed with respect to the moving-belt interface (see Section 4.2 above). 

Although each of the previously described interfaces has advantages for particular types of analyte, there are also clear limitations to their overall performance. Their lack of reliability and the absence of a single interface that conld be used for the majority of analytes did nothing to advance the acceptance of LC-MS as a rontine technique. Their application, even with limitations, did, however, show very clearly the advantages that were to be gained by linking HPLC to MS and the efforts of many to find the ideal LC-MS interface were intensified. 

The introduction of the thermospray interface provided an easy-to-use LC-MS interface and was the first step in the acceptance of LC-MS as a routine analytical technique. It soon became the most widely used LC-MS interface of those available in the mid to late 1980s. 

Electrospray ionization occurs by the same four steps as listed above for thermospray (see Section 4.6). In contrast to thermospray, and most other ionization methods nsed in mass spectrometry, it shonld be noted that electrospray ionization nnnsnally takes place at atmospheric pressure. A similar process carried out under vacuum is known as electrohydrodynamic ionization and gives rise to qnite different analytical results. This technique has not been developed into a commercial LC-MS interface and will not be considered further. 

Figure 4.9 Schematics of electrospray LC-MS interfaces with (a) a heated capillary and (b) a heated block to allow high mobile-phase flow rates. From applications literature published by (a) Thermofinnigan, Kernel Hempstead, UK, and (b) Micromass UK Ltd, Manchester, UK, and reproduced with permission.

The advent of the electrospray interface has allowed the full potential of LC-MS to be achieved. It is now probably the most widely used LC-MS interface as it is applicable to a wide range of polar and thermally labile analytes of both low and high molecular weight and is compatible with a wide range of HPLC conditions. 

In this chapter, seven types of LC-MS interfaces have been described and their performance characteristics compared. Any modifications to the HPLC conditions that are required to allow the interface to operate effectively have been highlighted. 

In this book, a number of different LC-MS interfaces have been described, where some of these have been included primarily from an historical standpoint. Currently, the most widely used interfaces are, undoubtedly, the electrospray and APCI interfaces and it is these that will be concentrated upon (a search of the Science Direct database [1] for 2001 nsing the term thermospray , previously the most widely used interface, yielded only one paper). 

In this study, the effect of mobile-phase flow rate, or more accurately, the rate of flow of liquid into the LC-MS interface, was not considered but as has been pointed out earlier in Sections 4.7 and 4.8, this is of great importance. In particular, it determines whether electrospray ionization functions as a concentration-or mass-flow-sensitive detector and may have a significant effect on the overall sensitivity obtained. Both of these are of great importance when considering the development of a quantitative analytical method. 

Polyimide belt The continuous belt used in the moving-belt LC-MS interface. 

Flgure 9.6 Schematic diagram of a heated pnewaatic nebulizer LC/MS interface combined with an APCI ion source and cross-sectional view of the nebulizer probe. 

The mass spectra of mixtures are often too complex to be interpreted unambiguously, thus favouring the separation of the components of mixtures before examination by mass spectrometry. Nevertheless, direct polymer/additive mixture analysis has been reported [22,23], which is greatly aided by tandem MS. Coupling of mass spectrometry and a flowing liquid stream involves vaporisation and solvent stripping before introduction of the solute into an ion source for gas-phase ionisation (Section 1.33.2). Widespread LC-MS interfaces are thermospray (TSP), continuous-flow fast atom bombardment (CF-FAB), electrospray (ESP), etc. Also, supercritical fluids have been linked to mass spectrometry (SFE-MS, SFC-MS). A mass spectrometer may have more than one inlet (total inlet systems). 

Thermospray was quite popular before the advent of electrospray, but has now given way to the more robust API techniques, although TSP sources continue to operate. Developed as an LC-MS interface, this technique calls for a continuous flow of sample in solution. 

Flow limitations restrict application of the DFI interface for pSFC-MS coupling. pSFC-DFI-MS with electron-capture negative ionisation (ECNI) has been reported [421], The flow-rate of eluent associated with pSFC (either analytical scale - 4.6 mm i.d. - or microbore scale 1-2 mm, i.d.) renders this technique more compatible with other LC-MS interfaces, notably TSP and PB. There are few reports on workable pSFC-TSP-MS couplings that have solved real analytical problems. Two interfaces have been used for pSFC-EI-MS the moving-belt (MB) [422] and particle-beam (PB) interfaces [408]. pSFC-MB-MS suffers from mechanical complexity of the interface decomposition of thermally labile analytes problems with quantitative transfer of nonvolatile analytes and poor sensitivity (low ng range). The PB interface is mechanically simpler but requires complex optimisation and poor mass transfer to the ion source results in a limited sensitivity. Table 7.39 lists the main characteristics of pSFC-PB-MS. Jedrzejewski... 

On-line LC-MS undoubtedly is a more important and versatile identification technique than LC-FTIR. However, there is no single universal LC-MS interface available every interface has its specific limitations with regard to flow-rate and composition of the LC eluent, polarity and molecular mass of the analytes, and/or ionisation technique(s) that can be used. For the non-mass spectroscopist, LC-MS developments have been a rather confusing matter. The developments of 30 years of LC-MS can be summarised as follows ... 

Scheme 7.7 Comparative analyte ranges for the major LC-MS interfaces...

Different options are available for LC-MS instruments. The vacuum system of a mass spectrometer typically will accept liquid flows in the range of 10-20 p,L min-1. For higher flow-rates it is necessary to modify the vacuum system (TSP interface), to remove the solvent before entry into the ion source (MB interface) or to split the effluent of the column (DLI interface). In the latter case only a small fraction (10-20 iLrnin ) of the total effluent is introduced into the ion source, where the mobile phase provides for chemical ionisation of the sample. The currently available commercial LC-MS systems (Table 7.48) differ widely in characteristics mass spectrometer (QMS, QQQ, QITMS, ToF-MS, B, B-QITMS, QToF-MS), mass range m/z 25000), resolution (up to 5000), mass accuracy (at best <5ppm), scan speed (up to 13000Das-1), interface (usually ESP/ISP and APCI, nanospray, PB, CF-FAB). There is no single LC-MS interface and ionisation mode that is readily suitable for all compounds... 

Three general strategies for LC-MS interfacing were developed ... 

Table 7.49 LC-MS interfaces, ionisation modes and mass spectrometers...

---