In planar chromatography light is used for detecting separated sample spots by illuminating the TLC plate from the top with light of known intensity (I0). If the illuminating light shows higher intensity than the reflected light (J), a fraction of light must be absorbed by the sample (the analyte) or the TLC layer. If a sample spot absorbs light, the reflected light intensity of this spot (J) is smaller than the illuminating light. The difference between these light intensities is absorbed by the sample. The definition of the total absorption coefficient a is
Iabs¼ I0 J ¼ aI0: (8:1)
Increasing sample amounts will induce a decreasing light reflection. Therefore a transformation algorithm is needed which turns decreasing light intensities into increasing signal values. Ideally there should be a linear relationship between the transformed measurement data (TMD) and the analyte amount [4–7].
Theoretical considerations lead to the following equation for transformation purposes that show linearity between the TMD and the absorption coefficient [8]:
TMD(l) ¼ k I0
J J I0
þ J
I0
1
¼ a
1 a, (8:2)
where
k is the backscattering factor (k 0 and k 1)
I0represents the illuminating light intensity at different wavelengths J is the intensity of reflected light at different wavelengths
a represents the absorption coefficient
Equation 8.2 is split into two parts, the absorption share and afluorescence share.
The first term in Equation 8.2 describes the light absorption and is dependent on the backscattering factor, whereas the second term describes the fluorescence of the analyte [8,9]. The value of the backscattering factor k is in the range between 0 and 1. The backscattering factor depends on the scattering quality of the stationary phase.
8.2.1.1 The Reversal Reflectance Formula
For k¼ 1, Expression 8.2 describes a situation where all the light is reflected from the plate surface. No inner parts of the TLC layer are illuminated and light absorption occurs only at layer-top. With k¼ 1, Expression 8.3 can be derived from Equation 8.2.
TMD(l) ¼ I0
J 1
¼ a
1 a: (8:3)
Expression 8.3 transforms the light losses caused by absorptions into positive values [8,9]. The measurement result of a single TLC track is best visualized as a contour plot.
A contour plot comprises the measurement data of a single track at different wavelengths. To measure a contour plot, a track of a TLC plate is scanned by use of a diode-array detector. Usually the plate is moved below an interface, which illumin-ates the plate at different wavelengths and detects the reflected light. For each wavelength the reflected light intensity (J) and the light intensity (I0) of the illumin-ating lamp are measured. A contour plot comprises the TMD data at different wavelengths and different track locations. In other words, it summarizes the meas-ured spectra in the y-axis at different separation distances. Mostly ten spectra are measured within 1 mm separation distance.
Figure 8.1 shows a photo of a ginkgo biloba extract separation and the contour plot of the track. In y-axis the different compounds of the ginkgo extract are visualized as peaks at different wavelengths. In the x-axis the positive values of Equation 8.4 are represented as spectra at different separation distances. The wavelength range is 200–600 nm.
8.2.1.2 The Kubelka=Munk Theory
The Kubelka=Munk theory was first published in the year 1931 and is based on the assumption that half of the scattered flux is directed forwards and half
Absorption 600
0 30 60 90
mm
200 400 nm
FIGURE 8.1 Contour plot of a ginkgo biloba extract separation calculated according to Expression 8.3. The track was stained by using the Anisaldehyde–sulfuric acid reagent after separation on silica gel with ethyl acetate, acetic acid, formic acid, and water 100þ 11 þ 11 þ 26 (v=v).
Identification of Compounds Online 177
backwards [4–6]. Both fluxes show the same intensity. According to Kubelka and Munk [5] scattering in each layer will illuminate the next layer above and below with half of its nonabsorbed light intensity. With the abbreviation R0¼ J=I0 and with k¼ 1=2 the following expression results:
TMD(l, k ¼ 1=2) ¼(1 R0)2 2R0 ¼ a
1 a: (8:4)
The Kubelka=Munk equation comprises both the absorption and the fluorescence signals of an analyte [8]. This expression is therefore recommended to get a quick overview of which kind of substances are being separated.
8.2.2 DIRECTDETECTION INFLUORESCENCE
For k¼ 0 no incident light is reflected to the plate top [8]. Light leaving the TLC plate at the top must therefore befluorescence light.
TMD(l, k ¼ 0) ¼ J I0 1
: (8:5)
In general, thefluorescent light is shifted to higher wavelengths (lF) in comparison with the absorbed light (lA). That means that thefluorescence usually shows lower energy than the absorbed light. A contour plot evaluated by thefluorescence formula instantly reveals compounds at the track showingfluorescence.
Figure 8.2 shows left the fluorescence contour plot of a ginkgo biloba extract evaluated with Equation 8.5 in the wavelength range from 400 to 600 nm. In y-axis the different compounds of the ginkgo extract are visualized as peaks at different wavelengths. In the x-axis the positive values of Equation 8.5 are represented as spectra at different separation distances. The fluorescence densitogram (right) is evaluated according to thefluorescence formula (8.5).
It is well known thatfluorescence from an RP-18 phase looks much brighter than from a silica-gel plate, because the coating of RP-18 material blocks a nonradiative deactivation of the activated sample molecules. By spraying a TLC plate with a viscous liquid, e.g., paraffin oil dissolved in hexane (20%–67%), the fluorescence of a sample can be tremendously enhanced. The mechanism behind fluorescence enhancement is to keep molecules at a distance either from the stationary layer or from other sample molecules [10]. Therefore not only paraffin oil but also a number of different molecules show this enhancement effect.
8.2.3 FLUORESCENCE QUENCHING
For the detection of UV absorbing substances simply by eye, TLC plates are very often prepared with a fluorescence indicator. Commonly an inorganic dye (manganese-activated zinc-silicate) is used. This dye absorbs light at 254 nm show-ing a green fluorescence at ~520 nm. Sample molecules in the layer cover the fluorescent dye and inhibit its light absorption. In comparison with an uncovered
area, sample spots or sample bands show lower light intensity in the vis region because the coveredfluorescent dye cannot transform absorbed light into a fluores-cence emission. Dark zones on a bright fluorescent background will indicate the position of the components. The term‘‘fluorescence quenching’’ is often used for this decrease of reflected light intensity. However, the sensitivity of this detection method is lower than the sensitivity of reflection measurements.
8.2.4 MASSSPECTROMETRYONLINE
The combination of TLC and mass spectroscopy has often been described [11–14].
For desorption from the plate and for ionization several techniques are published such as matrix-assisted laser desorption=ionization mass spectrometry (MALDI techniques using an UV=IR-Laser) [15–19], by atmospheric pressure chemical ion-ization (APCI) [20–22], single ion beam (SIMS) [23], or desorption electro spray ionization (DESI) [24–26]. All these methods are able to measure the molecule mass and characteristic fragments produced during the measurement process. This is very important information to identify—in combination with UV–Vis Spectra—the nature of TLC zones. Nevertheless, quantification is possible only by using an internal standard, because desorption processes are difficult to reproduce in all methods.
Recent publications of three new methods offer a quantitative TLC–MS approach without using an internal standard. Firstly this is the use of a ‘‘ChromeX-traktor’’ device [27], the coupling of TLC- or HPTLC plates with a DART-device (Direct Analysis in Real Time) [28] and the APGD-method (Atmospheric Pressure Glow Discharge) [29]. The TLC-Extractor is a 43 2 mm stamp, which is set on the
Fluorescence 500 nm 600
400 0 30 60 90
mm
FIGURE 8.2 Fluorescence contour plot of a of a ginkgo biloba extract evaluated with Equation 8.5 (left), the stained track by use of NEU-reagent (middle) and thefluorescence densitogram according to thefluorescence formula (8.5).
Identification of Compounds Online 179
sample zone. An HPLC-pump pump mobile phase through the part of the TLC plate which is covered by the stamp and extract the analyte which is transported to an MS-device. The spatial resolution of this simple and robust working method is 2 mm. The detection limit is in the pg range [27]. The DART system works with an excited helium gas stream, forming protonated water clusters from the surrounding air. These clusters transfer their energy to the analyt, forming molecule cations.
The spatial resolution of this device is better than 7 mm and the detection limit is in the lower ng range.
In the APGD method a simple ion source using plasma under atmospheric pressure desorbs and ionizes the analyt. The spatial resolution of this technique is better than 2.5 mm. All TLC–MS systems offer structural information about the analyt and extend the scope of the TLC technique [32].