«Zur Erlangung des akademischen Grades eines Dr.rer.nat vom Fachbereich Bio- und Chemieingenieurwesen der Universität Dortmund genehmigte ...»
- 55 A new interface to couple mass spectrometry with thin-layer chromatography for full-plate scanning —————————————————————————————————
Figure 5-5. Fragmentation of 18:0-18:2 phosphoserine.
5.3.2 Scanning of a mixture after separation The measurements discussed before by using a single compound after separation demonstrated that the system can well desorb, transport and ionize analyte molecules during a scanning process. The subsequent experiment was carried out by using a complex mixture of sphingomyelin and lecithin. For this purpose, 1µl SPM solution (100 ng), and 1 µl PC solution (500 ng) and a mixture of 1 µl SPM/PC (100 ng sphingomyelin and 500 ng lecithin) was developed on a TLC plate.
The first scan was carried out along the direction of development on the lane of SPM/PC mixture, as shown in Fig. 5-6a. A TIC-signal with two peaks from sphingomyelin (SPM) and lecithin (PC) measured during the scan as well as the related mass spectra are shown in Fig. 5-6b-d. As mentioned before, sphingomyelin and lecithin are mixtures of a group of glycerophospholipids. The different species of sphingomyelin as well as lecithin cannot be separated by conventional TLC techniques because all species in the group have nearly the same migrating distance so that they are located in the same sample spot. The mass spectra depicted in Fig. 5c, d show that sphingomyelin and lecithin can easily be identified. As shown in the spectra, signals of different molecular species of sphingomyelin and lecithin were obtained at the same time although they —————————————————————————————————
- 56 A new interface to couple mass spectrometry with thin-layer chromatography for full-plate scanning —————————————————————————————————
- 57 A new interface to couple mass spectrometry with thin-layer chromatography for full-plate scanning —————————————————————————————————
5.4 Scanning on the TLC plate with graphite assistance 5.4.1 Graphite covered plates A high power density of laser beam is not always desired because it may cause fragmentation and destroy the TLC layer. Therefore, similar experiments were carried out on surfaces covered with five different graphite particle densities. The graphite suspension was sprayed on the TLC plates and the graphite densities were determined by measuring the gray scale of photos taken with a camera mounted on a microscope, as shown in Fig. 5-7.
- 58 A new interface to couple mass spectrometry with thin-layer chromatography for full-plate scanning ————————————————————————————————— Peak Area TLC plates with 5 different grey scales were scanned with different laser power densities and a scan velocity of 0.6 mm/s. The signal area measured during these experiments is shown in Fig. 5-8 as a function of the laser power density. The SPM signal was first obtained when the laser power was higher than 500 mW focused on a spot with a diameter of 50 µm (power density: 2.5x104 W/cm2) and when the TLC plate with the highest graphite density was used. Therefore, the necessary power density is about 30 times lower than that measured when a TLC plate was used without graphite coating. A power of 4 W (power density: 2x105 W/cm2) was enough to measure an intensive signal when the plate with the grey scale value 1 was used. Increasing graphite particle density results in a reduced demand on laser power density for desorption. For the relatively ‘lightcoloured’ plates with the grey scale values 1, 2 and 3 the peak area —————————————————————————————————
- 59 A new interface to couple mass spectrometry with thin-layer chromatography for full-plate scanning ————————————————————————————————— increased with higher laser power. However, for the darker TLC plates 4 and 5, the peak area decreased with higher laser powers. Ignited tracks were observed on these two plates at the sites where the laser beam was scanned. Obviously, these conditions caused fragmentation of the analytes and reduced signal intensity.
5.4.2 Graphite embedded plates The requirement for desorption energy can be well reduced on a graphite covered TLC plate, and the method can be applied to all kinds of commercial TLC plates, but it results in more laboratorial work for the preparation of graphite layer at the same time. Because visualization methods, such as staining techniques, are mostly used to locate sample spots on TLC plate, commercially available TLC plates are white, and graphite particles have to be applied on the surface of a plate. However, a ‘grey’ plate with graphite particles embedded in TLC materials probably supplies the similar function as a graphite covered plate.
A quick protocol was adopted to investigate the feasibility of the detection on such a plate with simple tools. Graphite particles were added into silica gel 60 particles with a quantitative ratio of 2:100. Afterwards, 4 ml water was added into 1 g of such a particle mixture, and then it was mixed by a glass rod to form slurry. The slurry was deposited on a piece of glass (5 cm x 10 cm) and then pressed over with a glass rod horizontally.
Two ends of the glass rod were covered with teflon tubes to form a space between the glass tube and the table surface. The space ensured the slurry distributed homogeneously on the plate after the glass rod was pushed over from one edge to another of the plate, as shown in Fig. 5-9. The plate was subsequently dried in an oven for 15 min under 150 °C. Finally, the plate had a grey and a relatively smooth surface.
- 60 A new interface to couple mass spectrometry with thin-layer chromatography for full-plate scanning —————————————————————————————————
- 61 A new interface to couple mass spectrometry with thin-layer chromatography for full-plate scanning —————————————————————————————————
In this work, the plate was built up without further technical tool for TLC plate making and not based on an optimized protocol, but the experimental results had no unacceptable drawback and proved that the approach was feasible. It is reasonable to anticipate that it works better with a more sophisticated instrumentation for plate preparation. However, only after commercial graphite embedded TLC plates had become available, laser —————————————————————————————————
- 62 A new interface to couple mass spectrometry with thin-layer chromatography for full-plate scanning ————————————————————————————————— desorption on such TLC plates can benefit from both reduced requirement for laser power and a quicker experimental procedure.
5.5 Influence of scanning speed Figure 5-12. Peak area of the SPM signal at different scanning speeds The scanning speed is an important factor for the system. Higher speed causes faster analysis, but possibly results in an incomplete desorption of analyte molecules. A low scanning speed can theoretically give benefits on sensitivity and spatial resolution, but needs more time for a scan. A compromise is necessary between speed and sensitivity. To probe the influence of scanning speed on the signal, 5 samples with identical amount of sphingomyelin (100 ng) on a TLC plate were developed and then scanned with different velocity: 0.3 mm/s, 0.6 mm/s, 1.8 mm/s, 3.0 mm/s,
3.6 mm/s. As shown in Fig. 5-12, except for the data spot at 0.3 mm/s, the peak area decreases with higher scanning speed. This result is interpretable to be a result of an insufficient evaporation of analyte molecules at higher —————————————————————————————————
- 63 A new interface to couple mass spectrometry with thin-layer chromatography for full-plate scanning ————————————————————————————————— scanning speed. In this case, the sample spot was irradiated by the laser beam for a shorter period of time, and not enough energy was conducted to the analyte molecules. However, the wall of the transport system keeps capturing analyte molecules for a longer time at lower scanning speeds.
This can cause more sample loss during transfer. If a more sufficient desorption cannot compensate the larger sample losses at slower scan rates, the signal peak area will decrease, such as in the case of 0.3 mm/s.
Therefore, scanning speed of 0.6 mm/s was chosen for the following studies. This speed is much higher than the speed selected for the plate scanner based on ESI, which has to be operated at very low scanning speeds (several tens µm/s),52 since the extraction of the analyte from TLC material is a much more time-consuming step than desorption with energy.
5.6 Rapid screening One of the important applications of the TLC technique is rapid screening for specific substances in complex samples. This can be conveniently realized with the device described here. To demonstrate the feasibility of the technique, the screening of SPM in a SPM/PC mixture was performed.
One reference sample containing only SPM and four synthetic samples containing SPM/PC mixtures were developed simultaneously on a TLC plate. The SPM concentration was identical in all 5 samples. First, the migrating distance of SPM can be identified by scanning the reference sample along the migrating direction. Then, the scan was continued from the center of the SPM spot in the reference sample in orthogonal direction of the development, as shown in Fig. 5-13a. SPM signals were obtained from all synthetic samples at the same migrating distance of the plate. The signal areas (as shown in Fig. 5-13b) of the 4 mixed samples were observed to be close to that of the reference sample. The average relative —————————————————————————————————
- 64 A new interface to couple mass spectrometry with thin-layer chromatography for full-plate scanning —————————————————————————————————
Figure 5-13. (a) Schematic diagram of rapid screening; (b) Total ion chromatogram of a SPM screening experiment. Ref. and #1, #2, #3, #4 means reference sample and four synthetic samples; RT is the retention time; AA means integral peak area.
In this chapter, a novel interface to realize the coupling of TLC and MS was demonstrated. A diode laser, which is compact, easy to use and cost effective, was employed as the laser desorption source. The TLC plate was placed on a moving x-y stage to realize full-plate scanning. Two scan modes were used: a scan along the direction of the analyte development, which is appropriate for detecting all the —————————————————————————————————