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In atmospheric ionization, the ions are formed at atmospheric pressure by nebulization in the source region. Ionization at atmospheric pressure has certain advantages. It avoids the problems that arise when a liquid flows directly into a vacuum, and, if used with some type of separation system, it allows it to operate under ambient conditions. This is advantageous when low flow rates are employed, such as with microbore columns and capillary electrophoresis. A diagram of the atmospheric ionization interface is shown in figure 45.
The atmospheric pressure ionization (API) process is similar in some respects to the electrospray ionization source, and can cope with a range of column flow rates, up to a about 2 ml/min. Consequently, the total mobile phase can be utilized without splitting the flow. However, although all the solute may enter the interface, not all the solute molecules are ionized, and not all the ions that are formed enter the mass spectrometer. There are three forms of the API source: one uses a heated nebulizer with a corona discharge, another employs an atmospheric electrospray and finally one uses an ion spray. The interface that employs a heated nebulizer and a corona discharge ionization process is that depicted in figure 45. The solvent is nebulized by a gas flow, which is then swept by a second stream of gas (the sheath gas or make-up gas) through a quartz tube heater and the solvent is vapourized. The sample then drifts through a chamber containing a corona discharge (set up by a potential of about 2000 volts, applied across a simple electrode arrangement). The charged solvent vapor molecules are used as the ionizing agents (by a Chemical Ionization process). The reactant ions, formed in the corona discharge, collide with the sample molecules and give sample molecule plus a proton (hydrogen positive ions), i.e. [M+H]+. The ions are then drawn by means of an electric field to a plate with an orifice, over which passes another flow of gas called the curtain or barrier gas. The barrier gas helps to prevent uncharged molecules from entering the ion source but the charged ions (due to the effect of the electric field) pass through the orifice into the next chamber. The ions pass through an aperture in the next plate (the skimmer plate) and the space between the sampling plate, and the skimmer plate, is connected to the first vacuum pump. Having passed through the skimmer plate, the ions then pass through an aperture in a third plate into the mass spectrometer analyzer. The space between the skimmer plate and the final plate is connected to a second vacuum pump. By means of this differential pumping, the necessary low pressure can be maintained in the mass spectrometer. As the charged molecules entering the mass spectrometer are virtually parent ions already, the fragmentation pattern is similar to that obtained in MS/MS. In this way, the system differs fundamentally from the electrospray interface. Ionization is soft, sensitive and gives good characteristic spectra appropriate for sample identification and structural elucidation.
A recent modification of the API technique involved a low dead volume interface and was described by Thomson et al. . These workers employed packed microbore columns in conjunction with a low-volume, wall-coated capillary column connections. The total ion current chromatogram of a tryptic digest sample, comprising 1 picomole of human growth hormone, is shown in figure 46. Flow rates of about 80 to 100 μl per minute were used, with about 3 μl passing to the capillary column and entering the interface.
Courtesy of the Perkin Elmer SCIEX Corporation
It is seen that a good separation is obtained and apparently with little resolution lost in the capillary interface. The mass Spectrum of the peak marked T2 in the chromatogram is shown in figure 47.
Courtesy of the Perkin Elmer SCIEX Corporation
Good spectra can be obtained for up to ion masses of at least 900. Such a combination of techniques can be invaluable in biochemical research.
Cai and Henion  used a complex combination of sampling techniques to assay LSD and its analogs in urine. Affinity chromatography was fist employed to extract the substances of interest from the urine. The sample was then displaced from the affinity column and collected in a trap, from which the materials of interest were then displaced onto an LC column for separation.
The mobile phase from the column was then passed through an atmospheric pressure ionization interface to the mass spectrometer. A diagram of their apparatus is shown in figure 48.
Initially, the immuno–affinity column was equilibrated with the PBS. Then 30 μl of PBS-diluted antibody solution (10% antibody–90% PBS) was injected onto the column. Human urine, diluted with PBS (50% urine–50 % PBS) was then pumped through the protein G column and immediately flushed with PBS to remove any weakly bound impurities. During this process, the trapping was equilibrated with the mobile phase. The PBS was then pumped through the affinity column and the trap, which desorbed the materials from the affinity column and re-adsorbed them on the trap. The trap was then back–flushed, and the desorbed materials eluted through the LC column, through an API interface and into the mass spectrometer. An example of the results obtainable by the method is shown in figure 49. The original concentration of LSD in the urine was 0.9 ng/ml, which shows the very high sensitivities that can be obtained by utilizing selective extraction by affinity chromatography. It should also be noted that substance identification is also confirmed by the mass Spectrum, making the procedure also useful for forensic purposes.
Huang et al., in their article on atmospheric pressure ionization, demonstrated the use of the API interface in the LC/MS analysis of some benzodiazepine s. The mass spectrometer scanned the solution between m/z values of 100 and 350 at a scan rate of 3 s/scan.
The separation was developed isocratically and 25 ng of each benzodiazepine was present in the sample mixture. The results obtained are shown in figure 50.
The top chromatogram is the total ion current chromatogram with background subtracted. All the benzodiazepines were well separated and the analysis was complete in less than seven minutes. The positive ion mass spectra obtained for each component are shown below.
Clear and unambiguous (M+H)+ ions are obtained for each benzodiazepine with little fragmentation of the parent ions. The lack of smaller fragments confirms the gentle nature of the API ionizing source and offers great promise for extended use in LC/MS analyses, and may well become more popular, than the electrospray interfaces. Its great advantage is that it operates at ambient pressures and temperature. In addition, the device does not need the extensive pumping support required by the electrospray interface.
About the Author
RAYMOND PETER WILLIAM SCOTT was born on June 20 1924 in Erith, Kent, UK. He studied at the University of London, obtaining his B.Sc. degree in 1946 and his D.Sc. degree in 1960. After spending more than a decade at Benzole Producers, Ltd. Where he became head of the Physical Chemistry Laboratory, he moved to Unilever Research Laboratories as Manager of their Physical Chemistry department. In 1969 he became Director of Physical Chemistry at Hoffmann-La Roche, Nutley, NJ, U.S.A. and subsequently accepted the position of Director of the Applied Research Department at the Perkin-Elmer Corporation, Norwalk, CT, U.S.A.
In 1986 he became an independent consultant and was appointed Visiting Professor at Georgetown
University, Washington, DC, U.S.A. and at Berkbeck College of the University of London; in 1986 he retired but continues to write technical books dealing with various aspects of physical chemistry and physical chemical techniques. Dr. Scott has authored or co-authored over 200 peer reviewed scientific papers and authored, co-authored or edited over thirty books on various aspects of physical and analytical chemistry. Dr. Scott was a founding member of the British chromatography Society and received the American Chemical society Award in chromatography (1977), the M. S. Tswett chromatography Medal (1978), the Tswett chromatography Medal U.S.S.R., (1979), the A. J. P. Martin chromatography Award (1982) and the Royal Society of Chemistry Award in Analysis and Instrumentation (1988).
Dr. Scott’s activities in gas chromatography started at the inception of the technique, inventing the Heat of Combustion Detector (the precursor of the Flame Ionization Detector), pioneered work on high sensitivity detectors, high efficiency columns and presented fundamental treatments of the relationship between the theory and practice of the technique. He established the viability of the moving bed continuous preparative gas chromatography, examined both theoretically and experimentally those factors that controlled dispersion in packed beds and helped establish the gas chromatograph as a process monitoring instrument. Dr. Scott took and active part in the renaissance of liquid chromatography, was involved in the development of high performance liquid chromatography and invented the wire transport detector. He invented the liquid chromatography mass spectrometry transport interface, introduced micro-bore liquid chromatography columns and used them to provide columns of 750,000 theoretical plates and liquid chromatography separations in less than a second. Dr. Scott has always been a “hands-on” scientist with a remarkable record of accomplishments in chromatography ranging from hardware design to the development of fundamental theory. He has never shied away from questioning “conventional wisdom” and his original approach to problems has often produced significant breakthroughs.