The Electrospray Interface

High performance liquid chromatography is an effective technique for the separation of compounds of high molecular weight. There are, however, two major problems with the use of mass spectrometry for the study of this type of molecule 

The problem of the successful ionization of thermally labile molecules has been addressed by the introduction of energy-sudden techniques, such as fast-atom bombardment (FAB), which rely on the fact that energy may be provided to the molecule so rapidly that desorption takes place before decomposition may occur. 

The use of the dynamic-FAB probe (see Section 4.4 above) has allowed the successful coupling of HPLC to this ionization technique but there is an upper limit, of around 5000 Da, to the mass of molecules which may be successfully ionized. Problem solving, therefore, often involves the use of chemical methods, such as enzymatic hydrolysis, to produce molecules of a size more appropriate for ionization, before applying techniques such as peptide mapping (see Section 5.3 below). 

The mass range requirement invariably means that FAB is used in conjunction with a magnetic sector instrument. Conventional detectors, such as the electron multiplier, are not efficient for the detection of large ions and the necessary sensitivity is often only obtained when devices such as the post-acceleration detector or array detector are used. Instruments capable of carrying out high-mass investigations on a routine basis are therefore costly and beyond the reach of many laboratories. 

Electrospray is an ionization method that overcomes both of the problems previously described. 

---

The pump must provide stable flow rates from between 10 ttlmin and 2 mlmin with the LC-MS requirement dependent upon the interface being used and the diameter of the HPLC column. For example, the electrospray interface, when used with a microbore HPLC column, operates at the bottom end of this range, while with a conventional 4.6 mm column such an interface usually operates towards the top end of the range, as does the atmospheric-pressure chemical ionization (APCI) interface. The flow rate requirements of the different interfaces are discussed in the appropriate section of Chapter 4. 

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. 

The electrospray interface, described in the previous section, enables mass spectra to be obtained from highly polar and ionic compounds. 

An involatile ion-pairing reagent would be deposited in the electrospray interface and lead to a reduction in performance. Some interfaces have been specifically designed to minimize this by removing the line-of-sight between the spray and the entrance to the mass spectrometer, and are thus more tolerant to involatile buffers. The performance of the interface will be improved by the use of volatile alternatives. 

The MS/MS response for each analyte must first be optimized on the specific instrument to be used. This is usually done by infusing a solution of the analyte into the HPLC mobile phase without a column present. The composition of the mobile phase should match that expected at the time of analyte elution within 25%. The instrument is first operated in the LC/MS mode, and the settings for the electrospray interface are... 

Although the electrospray interface is a powerful tool, especially in the analysis of highly polar... 

Somsen, G. W, Mol, R., and de Jong, G. J. (2003). On-line micellar electrokinetic chromatography-mass spectrometry feasibility of direct introduction of non-volatile buffer and surfactant into the electrospray interface. /. Chromatogr. A 1000, 953—961. 

Ikonomou, M.G. Blades, A.T. Kebarle, P. Investigations of the electrospray interface for liquid chromatography/mass spectrometry. Anal. Chem. 1990, 62, 957-967. 

Confirmation of FLU in catfish muscle by electrospray LC/MS was done (199). Residues of CIPRO, ENRO, SARA, and DIFLX were positively identified at 10-80 /ug/kg. The extraction procedure was based on LLE with acidic ethanol. Extracts were cleaned up on a PRS SPE cartridge. Analytes were eluted with 30% ammonium hydroxide-MeOH (1 4). Chromatographic conditions were optimized to be compatible with the electrospray interface. To obtain maximum sensitivity, separate MS acquisition programs were developed for CIPRO/ENRO and SARA/ DIFLX pairs. This method was used to confirm residues in tissues fortified in the 10-80-ppb range. All relative abundances were within 10% of the value calculated for standard compounds. 

Ionization is accomplished in the electrospray interface by passing the HPLC effluent down a heated metal capillary tube along which an electric charge differential is applied. The evaporating liquid sprays out of the tube end as charge droplets rapidly decreasing in size. A gas nebulizer often... 

The choice of interface is dependent on both the particular analysis and the instrumentation available. Some interfaces require the use of very low flow rates and therefore necessitate the use of either microbore or capillary LC equipment, or a sample splitter if standard-bore equipment is used. Thermospray ionization is the most frequently quoted interface, owing to compatibility with standard-bore instruments. However, the upper molecular weight limit for thermospray ionization is low, and the electrospray interface is becoming popular. The maximum flow rates for different interfaces are listed in Table 3.8.54... 

The evolution of the ESI source has been marked by the use of electrospray devices as interfaces between the separation systems such as HPLC or CE and MS detectors, the earliest instances of which were reported by Yamashita and Fenn [59] and Aleksandrov et al. [60] in the mid-1980s. Because ESI-MS is used in many areas of chemistry, a vast number of articles reporting specific modifications of the electrospray interface has been published so far. Also, instrument manufacturers have provided innovative solutions for more sensitive and reliable mass spectrometers. 

Another popular and efficient inlet system for the LC/MS combination is the atmospheric pressure chemical ionization process. This system has some similarity to the electrospray interface and can also cope with flow rates of up to 2 ml/min. and thus the total column eluent can be utilized without splitting the flow. 

---