CHAPTER A2 LASER DESORPTION IONIZATION AND MALDI
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1 Back to Basics Section A: Ionization Processes CHAPTER A2 LASER DESORPTION IONIZATION AND MALDI TABLE OF CONTENTS Quick Guide...27 Summary...29 The Ionization Process...31 Other Considerations on Laser Desorption Ionization...33 Use of a Matrix...35 Types of Laser...35 Secondary Ionization...37 Uses of Lasers Conclusion...39 Micromass UK Limited Page 25
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3 Quick Guide A laser is a device for producing ultraviolet, visible or infrared light of a definite wavelength unlike most other light sources, which give out radiation over a range of wavelengths. The output of a single wavelength of light is described as being coherent. Lasers may be tuneable, viz., although only one wavelength is emitted at any one setting, the actual wavelength can be varied over a small range by changing the setting of the laser. Other notable characteristics of the laser are concerned with the intensity of the light emitted, its pulsed nature and the fine focusing that is possible. For many lasers used in scientific work, the light is emitted in a short pulse, lasting only a few nanoseconds but the pulses can be repeated at very short intervals. Other lasers produce a continuous output of light. The emitted beam of coherent radiation is narrow and can be focused into a very small area. This means that the density of radiation that can be delivered for any one pulse over a small area is very high, much higher than can be delivered by conventional light sources operating with similar power inputs. If the target at which a laser beam is directed can absorb light of the laser wavelength then the target will absorb a large amount of energy in a very small space in a very short time. The absorption of so much energy by a small number of target molecules in such a short time means that their internal energy is greatly increased rapidly and the normal processes of energy dissipation (such as heat transfer) do not have time to occur. Much of this excess of energy is converted into kinetic energy so that the target molecules are vaporized (ablated) and leave the target zone. Some of the target molecules gain so much excess of internal energy in a short space of time that they lose an electron and become ions. These are the molecular cation-radicals found in mass spectrometry by the direct absorption of radiation. However, these initial ions may react with accompanying neutral molecules, as in chemical ionization, to produce protonated molecules. Micromass UK Limited Page 27
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5 The above direct process does not produce a high yield of ions but it does form a lot of molecules in the vapour phase. The yield of ions can be greatly increased by applying a second ionization method (e.g., electron ionization) to the vaporized molecules. Therefore, laser desorption is often used in conjunction with a second ionization step, such as electron ionization, chemical ionization or even a second laser ionization pulse. Laser desorption is particularly good for producing ions from analytically difficult materials. For example, they may be used with bone, ceramics, high molecular mass natural and synthetic polymers and rock or metal specimens. Generally, few fragment ions are formed. Improved ionization may be obtained in many cases by including the sample to be investigated in a matrix formed from sinapic acid, nicotinic acid or other materials. This variant of laser desorption is known as matrix-assisted laser desorption ionization (MALDI). The laser may be used as a finely focused beam, which with each pulse, drills deeper and deeper into the specimen giving depth profiling. Alternatively, the beam can be defocused and moved over an area at lower power so as to explore only surface features of a specimen. Summary Lasers are used to deliver a focused high density of monochromatic radiation to a sample target, which is vaporized and ionized. The ions are detected in the usual way by any suitable mass spectrometer to produce a mass spectrum. The yield of ions is often increased by using a secondary ion source or a matrix. Micromass UK Limited Page 29
6 Laser pulse Neutral molecules and ions begin to desorb (a) (b) Sample surface Absorbed energy starting to be converted into kinetic energy of melted sample Neutral molecules pumped away Ions drawn into mass spectrometer analyser (c) After a few nanoseconds, the absorbed energy has been dissipated Figure 1 A laser pulse strikes the surface of a sample (a), depositing energy which leads to melting and vaporization of neutral molecules and ions from a small confined area (b). A few nanoseconds after the pulse, the vaporized material is either pumped away or, if it is ionic, it is drawn off into the analyser of a mass spectrometer (c). Micromass UK Limited Page 30
7 LASER DESORPTION IONIZATION The Ionization Process A molecule naturally possesses rotational, vibrational and electronic energy. If it is a liquid or a gas, it will also have kinetic energy of motion. Under many everyday circumstances, if a molecule or group of molecules have their internal energy increased (e.g., by heat or radiation) over a relatively long period of time (which may only be a few microseconds), the molecules can equilibrate the energy individually and together so that the excess of energy is dissipated to the surroundings without causing any change in molecular structure. Beyond a certain point of too much energy in too short a time, the energy cannot be dissipated fast enough so that the substance melts and then vaporizes as internal energy of vibration and rotation is turned into translational energy (kinetic energy or energy of motion); simultaneous electronic excitation may be sufficient that electrons may be ejected from molecules to give ions. Thus, putting a lot of energy into a molecular system in a very short space of time can cause melting, vaporization, possible destruction of material and, importantly for mass spectrometry, ionization (Figure 1). A laser is a device that can deliver a large density of energy into a small space. The actual energy given out by a laser is normally relatively small but, as it is focused into a very tiny area of material, the energy delivered per unit area is very large. The analogy may be drawn of sunlight which, although representing a lot of light, will not normally cause an object to heat up so that it burns. However, if the sunlight is focused into a small area by means of a lens, it becomes easy to set an object on fire or to vaporize it. Thus, a low total output of light radiation concentrated into a tiny area actually gives a high density or flux of radiation (we could even say a high light pressure ) - this is typical of a laser. As an example, a Nd-YAG laser operating at 266 nm can deliver a power output of about 10 Watts, somewhat like a sidelight on a motor car. However, this energy is delivered into an area of about 10-7 cm 2 so that the power focused onto the small irradiated area is about 10/10-7 =10 8 Watts/cm 2 =10 5 Kilowatts/cm 2 (the same effect as focusing the heat energy from 100,000 one bar electric fires onto the end of your finger! Micromass UK Limited Page 31
8 Laser energy, E' (a) Laser energy, E' (b) Increasing absorption Absorption peak Increasing absorption Absorption trough Increasing wavelength Increasing wavelength Laser beam (c) Sample desorbed as ions and neutral molecules Laser beam Laser beam reflected (d) Sample surface Figure 2 In (a), a pulse of laser light of a specific wavelength of energy, E, strikes the surface of a specimen which has a light absorption spectrum with an absorption peak near to the laser wavelength. The energy as absorbed, leading to the ablation of neutral molecules and ions (c). In (b), the laser strikes the surface of a specimen that does not have a corresponding absorption peak in its absorption spectrum. The energy is not absorbed but is simply reflected or scattered (d), depending on whether the surface is smooth or rough. Micromass UK Limited Page 32
9 No wonder sample molecules get agitated by the laser, even if it is only a few of them that are affected because of the small area which is irradiated). A molecular system exposed to a laser pulse (or beam) has its internal energy vastly increased in a very short space of time, leading to melting (with increased rotational and vibrational and electronic energy), vaporization (desorption; increased kinetic or translational energy), some ionization (electronic excitation energy leading to ejection of an electron) and possibly some decomposition (increase in total energy sufficient to cause bond breaking). If enough energy is deposited into a sample in a very short space of time, it has no time to dissipate the energy to its surroundings and it is simply blasted away from the target area because of a large gain in kinetic energy (the material is said to be ablated). Laser desorption ionization is the process of beaming laser light, continuously or in pulses, onto a small area of a sample specimen so as to desorb ions, which are examined in the usual way by a mass spectrometer. With continuous lasers (for example an argon ion laser), the energy delivered is usually much less than from pulsed ones and the focusing is not so acute. Thus, the irradiated area of the sample is more like 10-4 cm 2 rather than 10-7 cm 2 and the energy input is much less at about 100 Kilowatts/cm 2 rather than the 100,000 Kilowatts/cm 2 described above. Other Considerations on Laser Desorption Ionization Consider a laser emitting radiation of energy, E. For a substance to absorb that energy, it must have an absorption spectrum (ultraviolet, visible or infrared) that matches that energy. Figure 2 shows two cases, one (a), in which a substance can absorb the energy, E,andone (b), in which it cannot absorb this energy. In the second case, since energy cannot be absorbed, the laser radiation is reflected and none of its energy is absorbed. In the second case, much or all of the available energy can be absorbed and must then be dissipated somehow by the system. This dissipation leads to the effects itemized above. It follows that the capacity of a laser to desorb or ionize a substance will depend on three factors, one the actual wavelength (energy, E ) of the laser light, two the power of the laser and three, the absorption spectrum of the substance being irradiated. Micromass UK Limited Page 33
10 Increasing absorption (a) Increasing wavelength Emission wavelength of laser (energy, E ') Absorption spectrum of matrix Absorption spectrum of sample Laser beam Matrix desorbed as ions and neutral molecules (b) Surface of matrix material plus sample Figure 3 In a MALDI experiment, the sample is mixed or dissolved in a matrix material, which has an absorption spectrum that matches the laser wave length of energy, E. The sample may not have a matching absorption peak (a) but this is not important because the matrix material absorbs the radiation, some of which is passed on to the dissolved sample. Neutral molecules and ions from both sample and matrix material are desorbed (b). Micromass UK Limited Page 34
11 When the first and third factors match most closely and a lot of power is available (large light flux), a lot of the laser energy can be absorbed by the substance being examined; when the first and third factors mismatch, whatever the power, little or none of the laser energy is absorbed. Therefore, for any one laser wavelength, there will be a range of responses for different substances and, for this reason, it is often advantageous to use a tuneable laser so that various wavelengths of irradiation can be selected to suit the substance being examined. Use of a Matrix There is another way of allowing for the above variability of ionization during laser irradiation. Suppose there is a sample substance (a matrix material) having an absorption band that matches closely the energy of the laser radiation. On irradiating this material. it will be rapidly increased in energy and will desorb and ionize quickly, as described above. Now suppose that it is not just the matrix material alone but is a mixture or solution (a matrix) of a substance to be examined with the matrix material. Now, at least some of the energy absorbed by the matrix can be passed on to the sample substance causing it to desorb and ionize (Figure 3a,b). This technique depends on the laser energy matching an absorption band in the matrix and a match with the sample substance is unnecessary so that the method becomes general. It is called, matrix-assisted laser desorption ionization (MALDI). Commonly, sinapic acid (3,5-dimethoxy-4-hydroxycinnamic acid) or nicotinic acid are used as matrix materials for examining organic and other compounds. The ions produced are usually protonated molecules, [M + H] +, with few fragment ions. Types of Laser In theory, any laser can be used to effect desorption and ionization provided it supplies a enough energy of the right wavelength in a short space of time to a sample substance. In practice, for practical reasons, the lasers, which are used tend to be restricted to a few types. The laser radiation can be pulsed or continuous (continuous wave). Typically, laser energies corresponding to the ultraviolet or near visible region of the electromagnetic spectrum (e.g., 266 or 355 nm) or the far infrared (about 20 mm) are used. The lasers are often tuneable over a range of energies. Micromass UK Limited Page 35
12 Ejected (ablated) ions and neutral molecules Laser pulse (a) Three layers A, B, C through the depth of a specimen A B C Yield of ions at each laser pulse A + ions Number of laser pulses B+ions C+ ions (b) Mass spectrometer recording of ion type and yield Figure 4a & b A laser pulse strikes the surface of a specimen (a), removing material from the first layer, A. The mass spectrometer records the formation of A + ions (b). As the laser pulses ablate more material, eventually the layer, B, is reached, at which stage, A + ions begin to decrease in abundance and B + ions appear instead. The process is repeated when the B/C boundary is reached so that B + ions disappear from the spectrum and C + ions appear instead. This method is very useful for depth profiling through a specimen, very little of which is needed. Micromass UK Limited Page 36
13 The so-called peak power delivered by a pulsed laser is often far greater than that for a continuous one. Whereas many substances absorb radiation in the ultraviolet and infrared regions of the electromagnetic spectrum, relatively few substances are coloured. Therefore, a laser which emits only visible light will not be so generally useful as ones emitting in the ultraviolet or infrared ends of the spectrum. Further, with a visible band laser, coloured substances absorb more or less energy depending on the colour. Thus, two identical polymer samples, one dyed red and one blue, would desorb and ionize with very different efficiencies. Secondary Ionization Much of the energy deposited in a sample by a laser pulse or beam desorbs neutral material and not ions. Ordinarily, the neutral substances are simply pumped away and the ions are analysed by the mass spectrometer. To increase the number of ions formed, there is often a second ion source to produce ions from the neutral materials, thereby enhancing the total ion yield. This secondary or additional mode of ionization may be effected by electrons (electron ionization, EI), reagent gases (chemical ionization, CI) or even a second laser pulse. The additional ionization is usually organized as a pulse (electrons, reagent gas or laser), which follows very shortly after the initial laser desorption. Uses of Lasers Laser desorption methods are particularly useful for substances of high mass such as natural and synthetic polymers. Glycosides, proteins, large peptides, enzymes, paints, ceramics, bone and large polymers are all amenable to laser desorption mass spectrometry, with the sample being examined either alone or as part of a prepared matrix. Because of the large masses involved, for pulsed laser desorption, the method is frequently used with time-of-flight or ion trap instruments, which need pulses of ions. For MALDI, sample preparation can be crucial, the number of ions produced varying greatly with both the type of matrix material and with the presence of impurities. Fragment ions are few but the true molecular mass can be misinterpreted because of the formation of adduct ions between the matrix material and the substance under investigation; these adduct ions have greater mass than the true molecular mass. Some impurities, as with common ionic detergents, may act as suppressants to ion formation. Micromass UK Limited Page 37
14 A + and B + ions Different laser pulses B + ions Laser pulse (c) A B Surface of specimen Yield of ions at each laser pulse Number of laser pulses B+ ions A + ions (d) Mass spectrometer recording of ion type and yield Figure 4c & d In (c), less power is used and the laser beam is directed at different spots across a specimen. Where there is no surface contamination only B + ions appear but, where there is surface impurity then ions A + from the impurity also appear in the spectrum (d). Micromass UK Limited Page 38
15 The laser approach without a matrix can be employed in two main ways. Since the intensity and spot size of the laser pulse or beam can be adjusted, the energy deposited into a sample may range from a very large amount confined to a small area of sample to much less spread over a larger area. Thus, in one mode, the laser can be used to penetrate down through a sample, each pulse making the previously ablated depression deeper and deeper. This is depth profiling, which is useful for examining the variation in composition of a sample with depth (Figure 4a). For example, gold plating on ceramic would show only gold ions for the first laser shots until a hole had been drilled right through the gold layer; there would then appear ions such as sodium and silicon that are characteristic of the ceramic material and the gold ions would mostly disappear. By using a laser with less power and the beam spread over a larger area, it is possibly to sample a surface. In this approach, after each laser shot, the laser is directed onto a new area of surface, giving surface profiling (Figure 4c). At the low power used, only the top few nanometers of surface are removed and the method is suited to investigation of surface contamination. The normal surface yields characteristic ions but, where there are impurities on the surface, additional ions appear. Laser desorption is commonly used for pyrolysis/mass spectrometry, in which small samples must be heated very rapidly to high temperatures to vaporize them before they are ionized. In this application of lasers, very small samples are used and the intention is not simply to vaporize intact molecules but also to cause characteristic degradation (the Back-to-Basics guide on pyrolysis/ mass spectrometry should be consulted). Conclusion Lasers may be used in either pulsed or continuous mode to desorb material from a sample, which may be examined as such or may be mixed or dissolved in a matrix. The desorbed (ablated) material contains relatively few or sometimes even no ions and a second ionization step is frequently needed to improve the yield of ions. Molecular or quasimolecular ions are mostly produced with few fragment ions. By adjusting the laser focusing and power, laser desorption can be used for either depth or surface profiling. Micromass UK Limited Page 39
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