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Chapter A2 Laser Desorption Ionization and Maldi Back to Basics Laser Desorption Ionization and MALDI 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.................................................. 37 Conclusion ...........................................................39 Micromass UK Limited Page 25 Back to Basics Laser Desorption Ionization and MALDI This page is intentionally blank. Micromass UK Limited Page 26 Back to Basics Laser Desorption Ionization and MALDI 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 Back to Basics Laser Desorption Ionization and MALDI This page is intentionally blank. Micromass UK Limited Page 28 Back to Basics Laser Desorption Ionization and MALDI • 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 Back to Basics Laser Desorption Ionization and MALDI 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 Ions drawn into mass Neutral molecules spectrometer pumped away 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 Back to Basics Laser Desorption Ionization and MALDI LASER DESORPTION IONIZATION The Ionization A molecule naturally possesses rotational, vibrational and electronic Process 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 side- light on a motor car. However, this energy is delivered into an area of about 10-7 cm2 so that the power focused onto the small irradiated area is about 10/10-7 =108 Watts/cm2 =105 Kilowatts/cm2 (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 Back to Basics Laser Desorption Ionization and MALDI Laser energy, E' Laser energy, E' (a) (b) Absorption peak Absorption trough absorption absorption Increasing Increasing Increasing wavelength Increasing wavelength (c) (d) Laser beam Laser beam Laser beam reflected Sample desorbed as ions and neutral molecules 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 Back to Basics Laser Desorption Ionization and MALDI 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.
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