In my experience, fragmentation predisposition depends on the maldi matrix used (eg CHCA) and the biochemistry of the analyte compound eg Pro-Pro linkages, however the most influential factor appears to be the laser power used in the desorption process rather than the subsequent acceleration of these desorped ions, hence the higher power in PSD. Of course you can talk about the effect of the high kV on and the instability of the ions (metastability) but these have not been a problem for me, at least for my analytes of interest and the optimisation i have performed on my platform for signal detection.

The acceleration voltage alone doesn't cause fragmentation by itself. You need also collisions, and that is what happens in a CID cell or partially in the laser plume in the first stages of its development when the density is still high. Of course assuming that the laser plume and background pressure are the same, a higher acceleration voltage will lead to higher fragmentation because the energy of the collisions will be higher. In the Delayed Extraction method they allow the laser produced plume to expand for hundreds of nanoseconds in voltage free conditions, and the extraction voltage is applied only when the plume density has decreased so much that the fragmentation by collisions is less probable.

I completely agree with Vladimir that high acceleration voltage itself does not cause fragmentation and that you need collisions in the laser plume and a "hot" matrix together with higher laser energy to see fragmentation. The great advantage of high acceleration energy is that the impact of differences in initial energy and position of analyte ions in the plume on mass accuracy is reduced. To obtain higher resolution, the initial energy (and thus the differences in initial energy) should be low compared to the kinetic energy in the drift path

Thanks to all those who have given answers for my query. In this connection, I want you all to go through the details about the functioning of Ultraflex ToF/ToF (Bruker Daltonics), which employs a novel method called "LIFT" for fragmenting molecular ions: ‘A novel MALDI LIFT-TOF/TOF mass spectrometer for proteomics’ D. Suckau, A. Resemann, M. Schuerenberg, P. Hufnagel, J. Franzen & A. Holle, Anal. Bioanal. Chem. 2003; 376, 952-965. Awaiting your comments on this article.

Interesting paper about the Bruker Ultraflex TOF/TOF. I am still reading it but regarding the main question of this post I can say that what was said before continues to be valid: Acceleration (or retarding) voltages do not cause fragmentation. This instrument uses PSD (post source decay) in which the trick is to produce analytes with high internal energies that fragment in the first field free TOF zone (because of their high internal energy). They select one of the families of parent and fragments and analyze it with high resolution and sensitivity in the second TOF stage. Additionally the instrument can use gas collisions (high energy CID) or laser radiation (LID) to induce fragmentation. The LIFT thing is basically delayed extraction applied to the package of selected ions.

Vladimir and Jens are completely correct. The acceleration voltage will not cause fragmentation. First order decay due to internal energy, excitation due to photon absorption, or collision with other molecules are the only mechanisms available. High electric field strengths can cause fragmentation in polar ions but the field drop over the length of the ion must exceed the ion's internal bond energies for that to happen. Even with such high acceleration voltages, the field strength is nowhere near that level.

Acceleration voltages will not increase internal vibrational energy, and only become relevant if you are doing collision studies.

Dear Chris Enke and Roger Laine, Thanks very much for your answers. But, can there not be (some) molecular ions, which can possess some fragile covalent bonds that could be susceptible to fragmentation due to 18 - 20 kV, even before those are subjected to collisions or conditions of post-source decay in MALDI? The reason to ask this question is: I know of some molecules that undergo fragmentation under electrospray ionization (ESI) conditions (perhaps called as in-source fragmentation), i.e., fragmentation taking place during spray or during travel from spray region towards mass analyzer. The typical voltages applied for ESI are in the range 3 - 5 kV. Though, the mechanism of ionization by MALDI and ESI are entirely different, there is a huge difference in the voltage values applied for MALDI and ESI. In the case of ESI, molecular ions do get dissociated due to application of 'cone voltage (in some instruments), 'skimmer voltage (in some instruments)', whose values are again very much lower than the acceleration voltage applied in MALDI. How can this be explained?

In the electrospray region, ions are accelerated toward the inlet. These ions will collide with air and solvent molecules and lose this acceleration. But they could convert some of the collision energy into internal energy in the process. Normally, the ions will remain cooled by repeated frequent collisions. With increased field strength (and again, I stress that it is the field strength, not the voltage that provides the acceleration) it is possible that the collision energy could build up internal energy, stored as vibrational and rotational energy, faster than it is lost. In fact this repeated collision process is used to fragment ions in ion traps. The depth of the potential well in such devices is less than the energy of covalent bonds so single-collision fragmentation is not possible.

So what I think may be going on in your observations is that there is a region where the product of field strength and mean free path between collisions is sufficient to ramp up the internal energy with each collision and that there are enough of them to result in the internal energy reaching covalent bond energy levels. If that is the case, there should be a correlation between fragmentation efficiency and field strength.

Dear Prof. Enke, Thanks a lot for your answer. You have mentioned, "In the electrospray region, ions are accelerated toward the inlet." As far as I know, in electrospray ionization mass spectrometers, there is no acceleration voltage applied to make the ions move from the source region towards analyzer. The electrosprayed droplets/ions (which happen at atmospheric pressure) move into the mass spectrometer due to pressure and potential gradients. In other words, the sprayed droplets/ions are pulled into the mass spectrometer due to differences in the pressure and potentials. Due to this pull, the ions could get accelerated. Am I right? Can you clarify, why you have mentioned that ions are accelerated toward the inlet? Secondly, by 'Field Strength', do you mean V/cm (i.e. Voltage-per-centimetre)?

First, on nomenclature. A potential (voltage) difference between two conductors separated by an insulator (in this case, the gaseous and vacuum regions of the instrument) creates an electric field or potential gradient across this insulator. The strength of this field, as you said, has the units of voltage over distance. However, the field strength is not uniform in this region unless the two conductors are flat parallel plates. Mobile charge carriers (in our case, ions) in the gaseous region will be accelerated in a direction orthogonal to the isopotential lines in the space. The amount of that acceleration is proportional to the field strength and the ion charge. The acceleration will continue until the ion collides with something. At atmospheric pressure, the mean free path of molecules (essentially the distance over which acceleration can take place) and the mean time between collisions is so short that little energy is gained by the ion. Nevertheless, the field serves to direct the general ion motion in the downfield direction.

So ions released by the electrospray into this potential gradient are directed toward the metallic part of the instrument that contains the entrance orifice. Once through the orifice, the electric fields continue with skimmers, ion guides, etc. while the gas pressure decreases and the mean free path increases. In this region, increased field strength along with increased time between collisions can enable sufficient energy gain over enough collision to cause fragmentation. People have used this effect intentionally for some time.

My suggestion was that possibly, at your very high voltages, the field strength somewhere in the region between the esi tip and the inlet, is high enough so that even with the short time between collisions, sufficient energy is gained over repeated collisions to break weak covalent bonds. This hypothesis can be tested by both calculation and experiment. There is also the possibility of corona discharge when such high potentials are applied.

Dear Varatharajan, recurring on your question of fragmentation in the MALDI-ToF instrument: this is not only dependent on acceleration voltage and collisions in the MALDI plume, but also on a) the choice of matrix and b) mode of analysis. Re a), the amount of energy imparted on your analyte during the ionization is moderated differently by different matrices, e.g. there are "soft" matrices (e.g. DHB) and "harsh" matrices (e.g. CHCA) for the same applications. Look for publications from Karas and Glückmann. re b), the actual timescale for fragmentation is sometimes longer than it takes to accelerate the ions out of the MALDI source, therefore it will often happen during the transfer through the field-free drift tube. If you use linear mode detection you will therefore not detect the fragments - once they are in the field-free region they travel along at the same speed as the precursor. Only if you apply the reflectron will you resolve the fragments from the original molecule. This is often used to be able to detect labile molecules e.g. glycopeptides, in spite of the lower resolution and accuracy of the linear mode it can lead to better results.

Please pardon my discussion of electrospray ionization sources in the context of this MALDI question. I lost track of the initial question when forming my response. I agree with the LENZ comment above regarding the most likely source of high internal energies and delayed fragmentation.

Dear Prof. Enke, I started asking about 'Electrospray Ionization (ESI)', though first I started with MALDI. So, why are you saying this? Sabareesh.

I see there was a discussion of ESI after the initial MALDI question so my comments on ESI still stand. There are many similarities in the MALDI and ESI methods in that both involve a high field acceleration of the ions created. But, of course, there are great differences between ionization in vacuum and atmosphere and between an instantaneous and continuous ionization process. I wrote nothing incorrect, but I lost track of the middle part of the discussion where ESI came up.

Dear Prof. Enke, What I meant in my earlier message (posted a few hours ago) was not technical; meaning I did not say that your technical comments were incorrect. "Why are you saying this?" mean "why should you say pardon?" Sabareesh.

I said "pardon" because I thought I had made an error about the nature of the question.

Dear Dr. Enke

I think your comments were right on the subject, once the question about the possibility of ions fragmenting in intense electric fields became a question about ion fragmentation in the first stages of ESI.