«Zur Erlangung des akademischen Grades eines Dr.rer.nat vom Fachbereich Bio- und Chemieingenieurwesen der Universität Dortmund genehmigte ...»
1.2 Conceptions On an intact TLC plate during the scanning process, the acquirement of analyte molecules should be efficient (which means a high recovery rate of the analyte), fast, and possess high spatial resolution. One parameter effecting the performance is the scanning speed. Lower speed normally causes higher sample recovery but longer analysis time. There are two methods to acquire molecules from TLC: desorption with energy or extraction with a liquid. The latter is based on the same principle as the ‘scrape-elute’ mode, but with a probe to sample continuously and does not destroy the plate. The solvent containing extracted analytes is transferred to the ion source of the mass spectrometer and subsequently obtain the in situ mass spectra. The choice of the ionization method for such a device is normally electrospray ionization (ESI). However, such a sampling mode involves the choice of solvent, which is critical for the extraction efficiency, and an inevitable diffusion of the analyte molecules on the TLC plate due to the use of the liquid. A desorption with energy has inherent advantages in respect to spatial resolution. In fact, it is more widely used compared to the extraction mode. Several methods to provide the —————————————————————————————————
-6Introduction ————————————————————————————————— desorption energy exist, such as thermal desorption, laser desorption or desorption with fast atoms or ions. Except for thermal desorption, which was only used in a few studies in the early stage of TLC-MS coupling, the other desorption methods can also be used to perform ionization of the desorbed molecules in situ. In the applications adopting these methods on a piece of TLC plate (i.e. a small area cut from an entire TLC plate), ionization can actually also be realized by energy. Then ionisation takes place nearly at the same time as the desorption. However, it is not suitable for a plate scanning system, in which the transfer of desorbed molecules or ions from the surface of the TLC plate to the ion source have to be carried out since the ion source of a mass spectrometer is not big enough to contain a whole TLC plate. Unfortunately, the transfer of ions is more complicated than the transfer of molecules, which require major modifications to the mass spectrometer and decrease the applicability of such a device. Accordingly, an ideal sampling method should only desorb molecules for more convenient transport, and the ionisation should happen in the ion source of mass spectrometry. Obviously, this involves the decoupling of the desorption and ionization step.
The transport part is necessary for scanning an intact TLC plate. In an extraction based system, this part is relatively simply because analytes are contained in liquid. Such a transport part normally means a tube connecting the sampling probe to the ion source of the mass spectrometer.
In an energy desorption based system, if not to modify the mass spectrometer, analytes are transferred with a gas flow. Before the appearance of atmospheric pressure ionization (API) techniques, the desorbed materials were transferred under vacuum or reduced pressure.
This results in the requirement of pumping time for the correct pressure and more manufactural work for a sealing system. The API techniques allow a simpler design and more flexible installation of transport part.
-7Introduction ————————————————————————————————— However, transport under atmospheric pressure also causes more sample losses. Partially, these losses can be decreased by heating the transport lines.
The ionization method critically decides the performance of a TLC/MS interface. As a matter of fact, all popular ionization methods for mass spectrometry have been employed in TLC/MS, which will be discussed in detail in the next chapter. An API method has the advantage to be a ‘soft’ ionization technique with fewer fragments, simpler sample manipulation and, in our case, easier instrumental design and experimental arrangement.
The most popular API techniques include ESI, atmospheric pressure chemical ionization (APCI) and AP-matrix-assisted laser desorption /ionization. These techniques are chosen for a certain application in dependence on the analytes or the purpose of the investigation. The combination of laser desorption and APCI was recently developed, in which the desorption by laser and ionization by APCI were well decoupled. This combination can be easily incorporated into TLC/MS system. Such a system benefits from the high spatial resolution of the laser, simple transfer of analyte molecules, compatibility with modern mass spectrometric systems and less fragmentation under atmospheric pressure. One drawback of such a system is that the cost for a traditional pulsed laser system is relatively high, which somehow counteracts the advantage of TLC in the low costs. The size of the laser system is also not ideal for a miniaturization of the whole analytical system.
Accordingly, another choice of the laser system was done here: the application of diode lasers. Diode lasers are reliable, compact, costs effective, easy to use and have a high efficiency. It can be anticipated that they play a more important role in the future. For the purpose of desorption, a continuous wave (cw) diode laser can continuously desorb molecules with the correct power and therefore keep desorption process —————————————————————————————————
-8Introduction ————————————————————————————————— uninterrupted. However, diode lasers have never been used for laser desorption in the past. To introduce diode lasers into this scope, a graphite substrate was applied here to improve the absorption efficiency of laser irradiance. In doing so, the necessary power density for laser desorption decreased by two orders of magnitude. As a consequence, graphiteassisted diode laser induced desorption/atmospheric pressure chemical ionization can successfully be used for a TLC/MS interface system with plate scanning.
The experimental work of this thesis starts with studies on graphite plate targets for the testing of the feasibility of the method and basic optimization. Then, the studies on TLC pieces cut from the entire plates with graphite suspension covered are carried out to investigate the real situation with the appearance of TLC material. Subsequently, a plate scanning system is built up based on the results of above studies. Finally, a quantification method is developed for this system.
-9Theoretical Background —————————————————————————————————
2. Theoretical Background
2.1 Basic techniques involved in this work This work is focused on the establishment of a combination between a chromatography technique and mass spectrometry, and cw diode lasers are employed for laser desorption for the first time. To give a general impression on the basic techniques involved, they are introduced as follows.
2.1.1 Diode laser Since 1962, the first diode laser was reported by Nathan et al,1 many types of diode lasers emitting in the near-infrared or far-red region have been developed. Recently, green and blue diode lasers become commercially available. Diode lasers are widely used in bar-code readers, compact disks and laser printers.
- 10 Theoretical Background ————————————————————————————————— In a diode laser the lasing medium is a semiconductor p-n junction similar to that found in a light emitting diode (LED). As shown in Fig. 2-1, when a diode is forward biased, holes from the p-region are injected into the n-region, and electrons from the n-region are injected into the p-region.
If electrons and holes are present in the same region, they may radiatively recombine—that is, the electron "falls into" the hole and emits a photon with the energy of the band gap. This is called spontaneous emission.
Under suitable conditions, the electron and the hole may coexist in the same area for quite some time (on the order of microseconds) before they recombine. If a photon of exactly the right frequency appears within this time period, recombination may be stimulated by the photon. This causes another photon of the same frequency to be emitted, with exactly the same direction, polarization and phase as the first photon. In a laser diode, the semiconductor crystal is shaped somewhat like a sheet of paper—very thin in one direction and rectangular in the other two. The top of the crystal is n-doped, and the bottom is p-doped, resulting in a large, flat p-n junction.
The two ends of the crystal are cleaved so as to form perfectly smooth, parallel edges; two reflective parallel edges are called a Fabry-Perot cavity. Photons emitted in precisely the right direction will be reflected several times from each end face before they are emitted. Each time they pass through the cavity, the light is amplified by stimulated emission.
Hence, if there is more amplification than loss, the diode begins to "lase".2,3 Fig. 2-2 shows a picture of a diode laser. It was taken by a camera mounted onto a microscope. The laser diode (circled part) was mounted on a copper stage, which is helpful for heat elimination. The size of the laser diode is about 0.8 x 0.8 mm2. As introduced above, the laser beams exit the diode in two dimensions, in this picture, upward and downward. The upward laser beam is the laser output, and the downward laser beam, —————————————————————————————————
- 11 Theoretical Background ————————————————————————————————— called ‘reference beam’ is monitored by a photo diode, so that the power output of a diode laser is accurately tuneable.
Due to reliability, compactness, tunability, long lifetime, and easy operation and maintenance, diode lasers have been used successfully in analytical spectroscopy, such as molecular absorption spectroscopy, fluorescence spectroscopy, atomic absorption spectroscopy and optogalvanic spectroscopy.4-8 However, diode lasers have never been applied in the laser desorption technique until now. Generally, diode lasers can be distinguished into two categories: single-mode and multimode.4 Single mode diode lasers are mostly employed in atomic absorption spectroscopy. Compared with hollow cathode lamps, which are the classic light sources in atomic absorption spectroscopy, single-mode diode lasers have the advantages of higher light intensity, narrower linewidth, more —————————————————————————————————
- 12 Theoretical Background ————————————————————————————————— stable output power, and smaller dimension. However, they only have output powers of up to 150 mW, which are not suitable for our application.
Accordingly, all diode lasers used in this work are multimode diode lasers.
Today, there are multimode diode lasers with powers up to 50 W commercially available. This power is sufficient for laser desorption.