Advantages of the IonSpec Fourier Transform mass spectrometers:
Now that we know a little bit about how the ions are moving inside our analyzer cell, and some of the basic concepts about FTMS detection, the question arises of why we would want to use FTMS? Three main reasons are:
These are direct benefits of using Fourier Transform methods to derive the mass spectra. We should note that the high resolution and mass accuracy we can achieve are a direct consequence of the fact that our cyclotron frequencies have no dependence on the velocity of the ions in the analyzer cell or the radius of their orbit. It is also important to remember that resolution and mass accuracy both increase linearly with the strength of the magnetic field used. Our electrospray unit is the most sensitive of any mass spectrometer available, and our ability to reach ppm mass accuracy is critical when trying to identify an unknown.
One of the key features of the Explorer system is the quick switch that can be done between the MALDI/EI and Electrospray vacuum carts. It is simply a matter of rolling one vacuum cart out of the superconducting magnet and rolling the other vacuum cart into the magnet from the other side. The switchover can be done in less than 2 minutes. Both carts take advantage of our patented RF quadrupole ion guide which efficiently transports ions from the source region, through the magnetic field, to the analyzer cell. The fastest commercially available computers are used so that we can calculate large Fast Fourier Transforms (FFT’s), and our windows-based software is very easy to learn and use. Even novice users can learn and use the software in less than a day. Over the last 19 years the electronics have become continuously more modern and compact, with fewer cables, fewer connections, and more integration for increased reliability. Installation is accomplished in less than a week.
Ease of Use:
The above picture is of IonSpec’s main line product, the Explorer FT Mass Spectrometer. This instrument leads the way as the highest performance mass spectrometer available. However, the Explorer FTMS is actually two mass spectrometers in one. Positioned on the right side of the superconducting magnet is the Electrospray vacuum system, and positioned on the left side of the superconducting magnet is the MALDI vacuum system. The two vacuum systems are totally separate and independent from each other. The MALDI vacuum system has its own set of pumps and its own set of electronics, and the same is true for the Electrospray. However, both vacuum systems are controlled by the Omega Data Station. Switching between the MALDI and Electrospray ionization sources is extremely fast and efficient because the two vacuum carts are mounted on wheels, so they are able to roll in and out of the magnet very easily. Having two separate vacuum carts is highly efficient for all types of laboratory environments. In a service laboratory it is impossible to know whether ESI or MALDI samples will be sent in for analysis. With the Explorer FTMS, the instrument operator can be running ESI samples while the MALDI samples are being prepped. When it comes time to switch the operator simply rolls the ESI vacuum system out of the magnet and rolls the MALDI vacuum system in. The rest of the switching is taken care of by the OMEGA Data Station.
EI and MALDI Source:
Now we are going to move on and start talking in more detail about each of the carts. The first cart that we will discuss is the combined electron impact and MALDI (matrix-assisted laser desorption ionization) source cart. EI and MALDI are combined in the same cart because in both cases the ions are generated in high vacuum.
For MALDI, we have a laser that will be beamed in with mirrors and lenses to a sample plate. Ions will be blasted off of the sample plate, into the source region that is pumped down to about 10-6 Torr. Those ions are then electrostatically pulled through a mechanical shutter and into the RF quadrupole ion guide. The shutter used by IonSpec maintains a lower pressure in the analyzer cell which results in high mass resolution. The ions pass down the center of the quadrupole ion guide and are injected into the center of the FTMS cell. The ions are trapped in the analyzer cell by pulsing the voltages on the trapping plate. This method puts the ions at the center of the magnetic field, in the most homogeneous region, which results in higher mass resolution than the "kick sideways" method used by another manufacture. It is the fringing fields of the magnet that require us to use a quadrupole ion guide. Without the ion guide, the fringing fields would repel the ions, and they would be deflected away from the analyzer cell.
Various stages of pumping are present. The two pumps nearest the analyzer cell can be either cryogenic pumps or turbomolecular pumps. In the source region we would use a split-turbomolecular pump. All of the turbomolecular pumps used would be backed by one mechanical pump. This system features revolving powers in excess of 800,000, mass accuracy on the order of 1 ppm and sensitivity on the order of 1 femtomole. We can do MS/MS on the ions that are generated to get structural information out of those ions. We also have data base searching tools available for people who are doing protein and peptide work. A variety of magnets are available: 4.7T, 7.0T or 9.4T, and 12.0T actively-shielded magnets.
Ion Injection:
Ion injection is a key step, so we will discuss it in more detail. A schematic of the RF quadrupole ion guide is shown above. The quadrupole ion guide is able to inject a broad mass range, and it collimates the ion beam tightly in the x,y direction so that the ions are kept at the center of the quadrupole. As a result, the ions are at the center of the cell after injection and ready to be manipulated as desired. It is not necessary to pre-cool the ions down to the center of the cell. The RF ion guide has the highest ion transfer efficiency and lowest injection energy (<10 eV). All the major FTMS research labs and ThermoFinnigan use the same RF ion guide method as IonSpec. Another nice feature of the quadrupole guide is that it can be run with very low voltages, so we are not accelerating the ions in the z direction to an appreciable extent. The ion guide is usually set to anywhere between 5 and 15 volts. This very gentle voltage is able to get the ions to move all the way down to the cell while the quadrupole field keeps the ions trapped in their trajectories and pushes them through the magnetic field without being deflected. The RF quadrupole ion guide is quite easy to use. Other than the single adjustable voltage, it has two operating frequencies that allow us to run at a lower (100-1000 m/z) and a higher (400-2500 m/z) mass range.
Since we hold the patent on this technology, other instrument vendors are not able to use the quadrupole ion guide. As a result, they have to smash the ions through the magnetic field quickly to keep them from being deflected away from their analyzer cell. This requires high voltages of a couple thousand volts. Those high voltages have a couple of bad effects. One thing they will do is impart a lot of energy to the ions and fragment them in an uncontrolled fashion as they go into the analyzer cell. Another issue is the need to slow those ions down from an acceleration of two thousand volts to zero volts once they are in the analyzer cell. Needless to say this is quite difficult and you will likely lose a lot of ions in the process. This will manifest itself as a serious loss of sensitivity.
Mass Calibration (EI/MALDI):
(All experiments were performed at 7.0 tesla)
When we are running in electron impact (EI) or MALDI, it is important to have good mass calibration. For electron impact experiments we will use something like Perfluorotributylamine to do the calibration. Of course, almost any compound that gives a large number of fragment peaks over a wide mass range will be a good choice. In the case of perfluorotributylamine it does give a large number of fragment ions from the EI ionization.
The experiment was run in broadband mode, and it extended from 69 to 502 m/z units. The final data is an average of 5 scans. Our instrument specification is to get an average deviation of 1.5 ppm, and as shown in the table we are much lower than this. As an example, in our FTMS the ion at m/z 313.98336 had a measured m/z of 313.98350. We are only off by 0.00014 m/z units. This corresponds to an error of 0.5 ppm. Our average deviation over all of the observed peaks is 0.4 ppm, which is well within the specification. It is impressive that the errors we observed were in the fourth and fifth decimal places of the measured masses. That allows us to actually do identifications of materials based solely on the mass that we have measured with no other information.
Mass Accuracy (EI/MALDI):
In order to get good mass measurements we need to be able to compare the spectra of interest to a previous calibration spectrum. In such a case, the peaks in the calibration spectrum are the external references that generate the mass calibration curve.
To demonstrate an external calibration a sample of methyl stearate was used. Our external reference will be the previous perfluorotributylamine spectrum. We are again taking a broadband spectrum from 69 to 502 m/z units and acquiring 5 scans. Our mass accuracy specification with external referencing is 2.0 ppm at m/z 298. But our average deviation over several peaks is actually 0.9 ppm. As an example, the ion with an accurate m/z ratio of 199.16950 gave a measured m/z ratio of 199.16926, for an error of 0.00024 m/z units or 1.2 ppm. The peak at m/z 298 was in error by only 1.0 ppm. So again we achieve highly accurate results.
Mass Resolution (EI/MALDI):
A great advantage of FTMS is its high resolving power. In MALDI, we generally use
substance P for our resolution specification.
In this example we are taking a narrowband spectrum to maximize our resolution. In narrowband mode, we limit ourselves to a 20 m/z range centered on the mass of interest. This means that the spacing of the data points we take across the mass range (a maximum of four million) will be much closer than in a broadband experiment, which could cover a range of 2000 m/z. In this example we are only taking one scan to generate our spectrum, and only 1 pmol of substance P has been deposited in the 2,5-dihydroxybenzoic acid (DHB) matrix. The resolution specification in narrowband mode is 600,000, but in the spectrum that is shown we are actually getting a resolution of 869,000. These peaks are extremely narrow, and the spacing between each peak is 1 m/z unit.
Sensitivity (EI/MALDI):
The sensitivity in the MALDI is extremely impressive. The spectra below shows an example of the sensitivity we can achieve. Substance P is again the compound we use for our test, and the specification is that we put a hundred femtomoles on the probe tip, deposited with DHB as the matrix, and get a signal-to-noise (S/N) ratio of 50:1.
In this case we are looking at a broadband from 400 to 2500 m/z units. We acquired 20 scans to generate our spectrum, and we do meet our specification for this parameter. In fact, we can do this 20 scan acquire several times before the signal dies away, which implies that on each laser blast we are only picking up about 1 femtomole of material to take into the analyzer cell.