Ion Chromatography - The Electrospray Interface 2



Courtesy of the Hewlett Packard Company.


Figure 45. The Total Ion Current Chromatogram of a Sample from the Tryptic Digest of Lysozyme


It follows, that the production of multiple charged ions, increases the mass range of the spectrometer. A chromatogram of a sample from the tryptic digest of lysozyme obtained by total ion monitoring is shown in figure 46.


The column was a Vydac C-18 reversed phase column, 25cm long, 2.1mm I.D., and thermostatted at 50oC. The flow rate was 200 ml per min and the separation was developed by gradient elution. The mobile phase composition was programmed from pure solvent A (0.1% trifluoro-acetic acid (TFA) in water) to 60% solvent B (O.1% TFA in acetonitrile) over a period of 60minutes. The total column eluent was passed into the interface but, as seen from the diagram of the interface and its mode of operation, only a small portion of the solute actually enters the interface and only a fraction of that is carried into the mass analyzer. The mass spectrometer employed was the Hewlett-Packard MS Engine Quadrapole Mass Spectrometer. The mass spectrum of the peak eluted at 30.35 minutes is shown in figure 46.



Courtesy of the Hewlett-Packard Company.


Figure 46. The Mass Spectrum of the Peak Eluted at 30.35 Minutes in the Tryptic Digest Chromatogram of Lysozyme


The spectrum shown in figure 46 exhibits only three peaks at m/z values of 554.1, 876.0 and 1106.8. It is seen from figure 45, that the peak was not completely resolved from its neighbours. Consequently, it was necessary to determine whether all three peaks originated from the same solute, or whether any pair of the peaks resulted from multiple charges on the same molecule. A simple program is included in the Hewlett-Packard data handling software that tests the interrelationship between the individual mass peaks of the mass spectrum. The software determines whether the heights of any pair or group of peaks are linearly related to each other. If the respective peaks increase or decrease proportionally with one another, then they must originate from the same parent molecule, and probably represent multiple charges on the same molecule. The peaks in figure 46 were tested in this way, and the results from the correlation program are shown in figure 47. The data processing demonstrated that the peak at 876.0 m/z was not related to the other two and, furthermore, the peaks at 554.1 and 1106.9 were doubly and singly charged species of the same ion.