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MEETING NEWS Quantitative Synthetic Polymer Mass Spectrometry Workshop at the National Institute of Standards and Technology—Michael J. Felton and Cheryl M. Harris report from Gaithersburg, Md.
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(a)
6000 4000
[C132H34]+
preparation for solvent-based MALDI.” However, the solventless method needs about 10 times as much sample as the solvent-based method to make up for the analyte and matrix sticking to the container
[C132H90]+
2000 0 (b)
(c)
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1.0 0.8 0.6 0.4 0.2 0.0 1550
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1600
[C132H90]+
1650 m/z
1700
1750
(a) Solventless preparation produces greater resolution for a defined mixture of an insoluble polycyclic aromatic hydrocarbon product and its dendrite precursor than (b) solvent-based preparation and more closely resembles (c) simulated spectra.
ing considerably higher laser power to obtain a good analyte signal, she says. With solventless MALDI, a more homogeneous distribution of the analyte and matrix occurs. “You can go straight to that [hot] spot and get beautiful spectra and more reproducible results,” says Trimpin. Finally, solvent-based MALDI is more complex because one has to have the right solvent, the right pH, the right cations—“the right everything,” adds Trimpin. “Sometimes . . . you just have to try and play around, and it’s really timeconsuming to find the optimized sample
A N A LY T I C A L C H E M I S T R Y / F E B R U A R Y 1 , 2 0 0 3
during mixing, says Trimpin. NIST scientists, however, discovered they could use less than half the laser power needed for solvent-based MALDI. The hard part of the solventless method is getting over the fact that grinding together a sample and a matrix and pressing the powder onto the target plate are very “unquantitative”, says Wallace. “It’s kind of ‘black art’, and yet it can work better than the [preparations] that are very quantitative, that you can write down specifically how you’ve prepared the sample. It’s really been quite a thing!”
COURTESY OF SARAH TRIMPIN
Sarah Trimpin of Oregon State University and Klaus Müllen at the Max Planck Institute for Polymer Research in Germany have successfully abandoned using solvents with a popular analytical tool that characterizes macromolecules. And they’ve turned nonbelievers into excited followers in the process. Fifteen years ago, Koichi Tanaka’s group in Japan and Franz Hillenkamp and Michael Karas in Germany introduced MALDI to the analytical community as a method for gentle desorption of large macromolecules. Since then, it’s been ingrained into the minds of many researchers to follow the Hillenkamp and Karas method, in which the analyte and the matrix have to be dissolved for homogenization prior to measurement. But what happens if one wants to analyze insoluble compounds, such as pigments, polycyclic aromatic hydrocarbons, polyfluorene, or poly(dithiathianthrene)? In that case, Trimpin recommends that scientists start grinding away to mix the analyte into matrix powder for a “solventless” MALDI. Among those who have taken a liking to Trimpin’s method are scientists at the National Institute of Standards and Technology (NIST), who about a year ago were skeptical of the technique. Yet, they’ve tried it successfully on polyethylenes that can only be dissolved at temperatures above 120 °C, which is difficult because of the volatility of the solvents at high temperatures, says Bill Wallace of NIST. “We learned from Sarah how to do it, and more and more people are doing it,” he says. Trimpin describes the technique as being between the methods established by Hillenkamp and Karas, in which a lowmolecular-weight organic compound is the matrix, and by Tanaka, in which a cobalt powder serves as the matrix. Trimpin has found that solvent-based MALDI could actually be worse than previously thought
because it creates a very inhomogeneous crystal sample as the solution dries on the target plate. This can make it hard for researchers to find the right “hot spot” on the target plate and might lead to apply-
Arbitrary intensities
MALDI without the solvent? Go for it!
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MALDI, where are the ions from? Although MALDI is popular and was recently celebrated with the Nobel Prize, our collective understanding of it is still somewhat limited. For this reason, Richard Knochenmuss at Novartis Pharmaceutical (Switzerland) probed the MALDI reactions that produce ions for MS detection. Knochenmuss suggests that MALDI is a superposition of primary and secondary ionization, in which primary ionization generates kinetically favorable ions and secondary ionization generates thermodynamically favorable ions from collisions with the primary ions. Primary ionization occurs as the matrix absorbs the laser energy, whereas secondary ionization occurs as the plume is formed above the matrix surface and travels toward the mass spectrometer. The two-stage ion process presents a problem; the reactions in the secondary ionization mask the ionizations of the first, since they may never reach the mass spectrometer. “There are many indications
of the extensive role of secondary reactions. Among the more dramatic are matrix suppression and analyte suppression,” says Knochenmuss. In matrix suppression, a high concentration of analyte can completely suppress matrix ions, and vice versa for analyte suppression. Either suppression can occur regardless of the analyte or matrix ion type (radical, protonated, sodiated, etc.). In many cases, gas-phase charge transfer thermodynamics predicts the resulting mass spectra, providing evidence that thermodynamic secondary ionization reactions are occurring. However, Knochenmuss explains, “A prerequisite for thermodynamic equilibrium is that the plume is sufficiently dense, leading to many collisions. At the lowest laser fluences (pulse energy/area), ion–molecule equilibrium and thermodynamic predictability may not be achieved.” A prevalent assumption for the primary ionization mechanism in UV-MALDI has been two-photon ionization; however, the ionization potentials for the matrix and analyte clusters are too high for this to be practical, he says. A more likely possibility
is excitation pooling, where neighboring excited particles “react” with each other to localize the energy on one of them. In addition, some ions may be preformed in the matrix and are simply separated from the counterions during desorption. Knochenmuss builds quantitative models on the basis of the suggested reactions to determine if the theories might be correct. The model correctly predicts the laser fluence threshold that is commonly observed by MALDI users. Until the fluence reaches a certain value, there is very little signal; however, the signal rapidly increases after the threshold. “The reason for it was unclear. Is it a desorption phenomenon or an ionization phenomenon?” asks Knochenmuss. “My model shows that it is largely a desorption event.” Further results from secondary ionization are being collected, but suppression effects are correctly predicted. Knochenmuss says, “The model gives wide-ranging quantitative or near-quantitative success for a variety of observed [phenomena]. It suggests we now understand some of the basics of UV-MALDI.”
Autumn Meeting of the Swiss Chemical Society—Veronika Meyer reports from Basel, Switzerland. Happy highland cows give healthy milk In many parts of the Alps, the cows spend the summertime on meadows that are located at an altitude of 5000 feet or higher. For centuries, the cowherds and cheese-makers have been convinced that this is not only for the benefit of the animals and for the efficient use of the land, but that the milk and cheese from these high places are of higher quality. This has now been confirmed by a study performed by Misaël Ecoeur under the guidance of Romolo Cicciarelli at the University of Applied Sciences–Sion (Switzerland). The fatty acid composition of 20 milk samples from lowland cows was compared with 20 samples from highland cows. (Samples were taken at four different sites each.) Analysis of the
milk samples was performed by GC after transesterification of the milk fat with potassium hydroxide and methanol. Significant concentration differences were reported for four fatty acids. The content of saturated palmitic acid (C16:0, which has 16 carbon atoms and no double bonds) is lower in the milk of cows that enjoy the grass growing at high places; this acid increases the total cholesterol and low-density lipoprotein cholesterol in humans and, therefore, increases
the risk of cardiovascular disease. On the other hand, the contents of linolenic acid, rumenic acid, and transvaccenic acid are significantly higher in the milk of the highland cows. Linolenic acid (C18:3, cis9, cis12, cis15) helps prevent cardiovascular disease, and both rumenic acid (C18:2, cis9, trans11) and transvaccenic acid (C18:1, trans11) have anticancer potential. No notable differences were found for two other fatty acids, linoleic acid (C18:2) and oleic acid (C18:1). Tourists visiting the Alps should therefore not only take pictures of cowherds in front of glaciated peaks but also drink many glasses of fresh milk. As one can imagine, the locals do only the latter.
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