«Zur Erlangung des akademischen Grades eines Dr.rer.nat vom Fachbereich Bio- und Chemieingenieurwesen der Universität Dortmund genehmigte ...»
- 65 A new interface to couple mass spectrometry with thin-layer chromatography for full-plate scanning ————————————————————————————————— components in one sample and a scan across several sample spots orthogonally to the direction of development, which is suitable for high throughput analysis of the same compound or compounds with the same Rf from several samples. The mass spectrometer was coupled to provide an identifying ability to TLC technique, which is important for the analysis of unknown substances or overlapping spots as well as to improve the reliability of the analysis. A gas jet pump connected with heated glass tubes enabled transportation of the desorbed analyte molecules to the ionization region.
The measurement was carried out on both non-pretreated and graphite-coated TLC plates. Using the graphite-coated surface, the necessary laser power for desorption decreased down to 104 W/cm2, about 30 times lower than that needed for the desorption from white surfaces.
- 66 Quantification with the plate scanning device —————————————————————————————————
6. Quantification with the plate scanning device
6.1 Quantification in mass spectrometry The goal of quantitative analysis in mass spectrometry is the correlation the intensity of the signals with the quantity of the compound present in the sample. Generally, an external or an internal standard is applied for this purpose.
In an external standard method, a synthetic sample containing a known quantity of the molecule to be measured (Mste) is introduced into mass spectrometer, and then the signal intensity (Iste) is recorded. Under the same analytical conditions, an equal volume of the solution containing the analyte molecules to be quantified (Mx) is introduced into the mass spectrometer and its response signal intensity (Ix) is measured. Because the volumes that are introduced are equal, there is a proportionality between the response intensities and the quantities as long as the response signal intensity remains linear with respect to the concentration and as long as the
signal intensity is zero at zero concentration:
Mx = Ix • Mste / Iste
Because this linearity is not always good over a wide concentration range and for all ionization methods, verification using a calibration curve is necessary. In order to do this, equal volumes of a series of synthetic samples containing an increasing quantity of the molecules to be measured are introduced into the mass spectrometer and the intensities of their response signals are recorded. In fact, the visual quantification method in TLC is also an external method.
- 67 Quantification with the plate scanning device ————————————————————————————————— An internal standard method allows the elimination of various error sources compared with an external standard method. In this method, an internal standard with chemical and physical properties as close as possible to the properties of the molecules to be measured is added into the sample. A calibration curve is constructed by measuring synthetic samples containing the same known quantity of the internal standard and increasing quantities of the compound to be measured. This allows a mathematical relationship to be obtained between the intensities of the signals corresponding to the compound to be analyzed and the internal standard (Ix/Isti) and the quantity of compound present in sample (Mx). The measurements are then carried out with the unknown samples after a constant quantity of internal standard had been added to them. Internal standards can be classified into three categories: structural analogs that are labeled with stable isotopes, structural homologs and compounds from the same chemical family.
6.2 Quantification in TLC-MS In TLC-MS, often the quantification is done off-line and represents a complicated and time-consuming procedure. The sample spot has to be located by a visualization method first. Then, the stationary phase containing the analyte has to be removed from the plate followed by extracting the analyte with a solvent. Finally, the usual quantification procedure has to be carried out for the analyte solution.30 Another approach is the extraction of the analyte directly from the plate with a liquid51 and the direct transport of the extraction liquid to a mass spectrometer. However, the measurement is still performed intermittently from one sample spot to another, which is not really suitable for high throughput analysis. In contrast, the experimental setup presented here is —————————————————————————————————
- 68 Quantification with the plate scanning device ————————————————————————————————— able to provide a quick and continuous on-line quantitative measurement technique. Nevertheless, reliable quantification is a critical aspect using desorption techniques. Obviously, the surface condition is an important factor for laser desorption. Especially on TLC plates, the inhomogeneity of the microscopic surface condition at different locations results in differences in the desorption efficiency of analyte molecules.
Consequently, the use of an internal standard is highly beneficial. The ideal internal standard would be the application of isotopically labelled analyte species (e.g. C-labelled) because they are located in the same position as the analyte after separation. However, just very few isotopically labelled biomolecules are commercially available. Another possibility is the use of structural homologs, e.g. other phospholipids, as internal standards. However, even slightly different structures can lead to a separation from the analytes during the TLC run. To overcome the problem, a suitable reference compound was added into the mobile phase.
After the development of the sample this compound was distributed homogeneously over the whole area below the solvent front on the plate.
The ‘background’ signal of this compound was then used as an internal standard. In this way, the chromatographic property of the internal standard is no longer important and a wide range of compounds can be chosen for this purpose. Here, reserpine (RSP) was used as the internal standard (0.6 g/l in the mobile phase), a compound that is often used for calibration of LC-MS systems.
6.3 Quantification for the TLC plate scanning system In a normal quantification procedure, the ratio between the signal area of analyte (Ax) and the signal area of the internal standard is used to build a calibration curve. In this work, the internal standard yields a steady signal.
- 69 Quantification with the plate scanning device ————————————————————————————————— As shown in Fig. 6-1, one possibility is to use the integral area of the internal standard (Ast) during the appearance of the analyte signal.
However, this area is subject of change due to the chromatographic reproducibility, i.e. it will change with the width of the analyte signal although its signal area might be the same. To reduce this kind of deviations, the average intensity of the internal standard (Ist) was used, which is defined here as the integral area during the appearance of the analyte signal divided by the number of scans (n) that is recorded by mass
Ist = Ast / n A calibration curve is constructed based on the ratio between the signal area of analyte and the average intensity of the internal standard (Ax/Ist).
Figure 6-1. Schematic diagram about the quantification strategy —————————————————————————————————
- 70 Quantification with the plate scanning device ————————————————————————————————— For this study, sphingomyelin was chosen as the analyte. Its strongest signal (m/z 502.6) was employed as the basis to calculate the signal area.
For each point in the calibration curve five synthetic samples with the same SPM concentration were measured. The results are listed in Table 6-1.
At first, the calibration curve was constructed only based on the signal area of SPM (Ax) without involving the signal of reserpine. It is actually an external method. As shown in Fig. 6-2, it shows a poor linearity (R2 = 0.9520). However, if the calibration curve was constructed based on the ratio of the signal area and the average intensity of RSP (Ax/Ist), a good —————————————————————————————————
- 71 Quantification with the plate scanning device ————————————————————————————————— linear correlation (R2 = 0.9991) could be obtained (see Fig. 6-3). Besides the benefit of a better linear correlation, the use of Ax/Ist also improves the reproducibility of the method compared with the use of only Ax, which is especially important for a laser desorption technique.
The internal standard used here shows no structural conformity with the analyte. In principle, also isotopically labelled compounds or structural homologs could be used as internal standards in the same way as described here, which possibly would even result in a better accuracy. However, since a relatively large amount of internal standard is necessary for providing enough signal intensity, this kind of internal standard application would not be suited for high-cost internal standard materials.
Although the device is mainly designed for qualitative work, also analyte quantification was carried out. The internal standard was —————————————————————————————————
- 72 Quantification with the plate scanning device ————————————————————————————————— added into the mobile phase to yield a ‘background’ signal, which was used as a reference signal for the quantification. This method has advantages in linear correlation and reproducibility compared with an external method.
- 73 Conclusions—————————————————————————————————
A novel interface for TLC/MS, in which a full-plate scanning can be carried out on a TLC plate to recover the chromatographic information and real-time acquire mass spectra for separated analytes, was developed. In this system, a compact, easy to use and cost effective diode laser was employed to desorb molecules into the gas phase. The desorbed molecules were transferred by gas flow to the area in front of the heated capillary of the mass spectrometer and subsequently ionized by a corona discharge at atmospheric pressure. This system enables fast detection, simple sample preparation, high throughput analysis, the capacity for identifying unknown compounds and compatibility for most modern LC/MS systems.
An optional plate pretreating method with graphite assistance was employed to decrease the requirement for desorption power density and consequently increased the possible choices for the laser source.
The first part of the work was carried out on a graphite target to investigate the feasibility of the application of a diode laser induced desorption. At the same time, basic optimizations of the mass spectrometer and the experimental arrangement were performed. A simple ion source with tunable geometric parameters was development for this purpose. The results showed a diode laser can be well applied for laser desorption with the assistance of graphite, and a power density in the order of 104 W/cm2 was sufficient for desorbing biochemical analytes with moderate molecular weight. This power density is two orders of magnitude lower than the necessary power density for a white surface.