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Evaluation of quantitative thin layer chromatography using staining reagents
Journal of Chromatography A, 1164 (2007) 298–305
Evaluation of quantitative thin layer chromatography using staining reagents Richard Johnsson a , Gustav Tr¨aff b , Martin Sund´en a , Ulf Ellervik b,∗ a
Organic Chemistry, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden b Sweday, S¨ olvegatan 8c, SE-223 62 Lund, Sweden
Received 14 May 2007; received in revised form 10 July 2007; accepted 17 July 2007 Available online 21 July 2007
Abstract Thin layer chromatography (TLC) using staining reagents is a superior method for analyzing organic compounds without chromophores. It is fast, versatile and sometimes the only viable method. We have investigated quantitative TLC using staining reagents, in combination with modern image analysis software. Our results show that it is possible to get reliable measurements, suitable for high-throughput screening or physical organic investigations. The range of detection and the errors for the different parts of the process are illustrated. We show that the errors are largely due to the staining process and can be diminished by measuring ratios of compounds. © 2007 Elsevier B.V. All rights reserved. Keywords: TLC; Image analysis software; Quantification; Staining reagents
1. Introduction Thin layer chromatography (TLC) is one of the most widespread analytical methods used in the organic chemistry laboratory. The rationale is several fold. It is an inexpensive method that only consumes small amounts of sample as well as solvents. There are almost no restrictions in the use of solvent and a variety of stationary phases is commercially available, although SiO2 is by far the most used. Besides scanning in the UV-region, there is also an abundance of developing methods including absorption of iodine vapor and dipping or spraying for example sulfuric acid, permanganate or anisaldehyde solutions. Since there are no requirements for UV-activity (as for HPLC), paramagnetic properties (as for NMR) or volatility (as for GC) this makes TLC one of the most general analytical methods. For example, due to the absence of chromophores and the high boiling points, underivatized carbohydrates are difficult to analyze by other methods than TLC. TLC only uses the chromatographic surface once and the whole analyte is detected, which means that there is no risk of accumulation of the analyte on the chromatographic surface. Finally, TLC is an ideal method for analysis
∗
Corresponding author. Tel.: +46 46 2228220; fax: +46 46 2228209. E-mail address: [email protected] (U. Ellervik).
0021-9673/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2007.07.029
of combinatorial reactions since it is easy to analyze several samples, each containing several compounds, simultaneously. Quantitative analysis is a powerful tool and the information gathered from quantitative studies can be used in a multitude of chemical disciplines, in particular analytical, physical organic and industrial chemistry. However, due to inadequate sample application, limited detectability and sensitivity and also reproducibility TLC was for long not regarded a quantitative method. However, with the development of high-performance TLC and the use of slit scanning densitometers, the accuracy has dramatically increased [1]. Unfortunately, with slit scanners each lane has to be scanned individually and the width of the scanning area has to be set to the same value as the diameter of the largest spot, which results in high signal-to-noise ratio [2,3]. Today the slit scanners have been replaced by charge-coupled device (CCD) detection. Scanning the plate using a CCD detector has been shown to be much faster and efficient. The CCD scans the plate at once and has shown to achieve fast and reliable results [1,4,5]. The CCD has a low signal-to-noise ratio and can operate in regions from UV to near IR [1]. It is also possible to use an ordinary office scanner as a densitometer. By using colored samples or by staining the plate with a suitable developing solution it is possible to scan the plate and use image analysis software to process the digital image. This way of quantification is fast, inexpensive and uncomplicated.
R. Johnsson et al. / J. Chromatogr. A 1164 (2007) 298–305
Image analyzers are thought to be a powerful tool in use with TLC [6]. However, research and software development in this area is scarce and at the moment there are only a handful of published articles available on the subject [7–12]. We decided to develop new image analysis software tailored for quantitative TLC image analysis, JustTLC [13].
299
2.5. Error in sample application Rose Bengal (4,5,6,7-tetrachloro-2 ,4 ,5 ,7 -tetraiodofluorescein disodium salt) (11 mg, 0.011 mmol) was dissolved in methanol (15 mL). The solution was applied to a TLC plate with 1 L, 2 L and 5 L microcaps 10 times each and analyzed using JustTLC.
2. Experimental section 2.6. Method of staining 2.1. General experimental details All samples were applied using Drummond “microcaps” 1 L, 2 L and 5 L (Drummond Scientific Company) TLC plates were alumina plates coated with silica gel 60 F254 (Merck). The samples were visualized using anisaldehyde (15 mL anisaldehyde and 2.5 mL H2 SO4 in 250 mL EtOH), vanillin (6 g vanillin and 2.5 mL H2 SO4 in 250 mL EtOH) and sulfuric acid (8 mL H2 SO4 in 100 mL EtOH). All plates were scanned using an Epson Perfection 2400 photo scanner. All files were scanned as tiff-images at 1200 dpi unless otherwise stated. 2.2. Useful range for detection 1,2,3,4,6-Penta-O-acetyl--d-glucopyranose (181 mg, 0.46 mmol) was dissolved in CH2 Cl2 (0.928 mL) to give a stock solution of 0.5 M. The stock solution was diluted with CH2 Cl2 to give 0.05, 0.005, 0.0005 and 0.00005 M solutions. 1,2,3,4,6Penta-O-acetyl--d-glucopyranose (81 mg, 0.21 mmol) was dissolved in CH2 Cl2 (1.034 mL) to give a stock solution of 0.2 M. The stock solution was diluted with CH2 Cl2 to give 0.02, 0.002, 0.0002 and 0.00002 M solutions. 1,2,3,4,6-PentaO-acetyl--d-glucopyranose (41 mg, 0.11 mmol) was dissolved in CH2 Cl2 (1.058 mL) to give a stock solution of 0.1 M. The stock solution was diluted with CH2 Cl2 to give 0.01, 0.001, 0.0001 and 0.00001 M solutions. One microliter of the different concentrations were spotted and eluted (heptane/EtOAc 1:1) three times each and stained with anisaldehyde, vanillin and sulfuric acid. The TLC plates were analyzed using JustTLC. 2.3. Error in scanning process Three samples (1 L) of a solution of 1,2,3,4,6-penta-Oacetyl--d-glucopyranose (0.05 M in CH2 Cl2 ) were added to a TLC plate, eluted (heptane/EtOAc 1:1) and developed with anisaldehyde. The TLC plate was scanned five times and analyzed using JustTLC. The TLC plate was also scanned at 72, 150, 300 and 600 dpi once each and then analyzed using JustTLC. 2.4. Precision of algorithm A solution of 1,2,3,4,6-penta-O-acetyl--d-glucopyranose in CH2 Cl2 , 0.05 M was used. Three spots of 1 L were added on a TLC plate and eluted (heptane/EtOAc 1:1) and developed with anisaldehyde. The TLC plate was then scanned and analyzed using JustTLC five times.
Five samples (1 L) of a solution of 1,2,3,4,6-penta-O-acetyl-d-glucopyranose (0.05 M in CH3 CN) were added to 20 TLC plates which were eluted (heptane/EtOAc 1:1). Ten TLC plates were sprayed with anisaldehyde while the remaining 10 plates were dipped in an anisaldehyde solution. The plates were then scanned and analyzed using JustTLC. 2.7. Staining reagent and heating method Five samples (1 L) of a solution of 1,2,3,4,6-penta-O-acetyl-d-glucopyranose (0.05 M in CH3 CN) were added to 20 TLC plates which were eluted (heptane/EtOAc 1:1). The TLC plates were dipped in anisaldehyde. Ten TLC plates were heated in oven at 150 ◦ C while the remaining plates were heated with a hot air gun at 5–600 ◦ C. The TLC plates were then scanned and analyzed using JustTLC. The procedure was repeated using vanillin or sulfuric acid. 2.8. Internal standard 1-O-Isopropyl-2,3,4,6-tetra-O-acetyl-␣-d-glucopyranoside and 1-O-isopropyl-2,3,4,6-tetra-O-acetyl--d-glucopyranoside (53 mg, 0.136 mmol and 6 mg, 0.015 mmol in CH3 CN 2.532 mL) to give α:β 90:10 (25 mg, 0.064 mmol and 8 mg, 0.020 mmol in CH3 CN 1.422 mL) to give α:β 75:25 (34 mg, 0.087 mmol and 34 mg, 0.087 mmol in CH3 CN 2.860 mL) to give α:β 50:5 (18 mg, 0.046 mmol and 6 mg, 0.015 mmol in CH3 CN 1.033 mL) to give α:β 25:75 and (9 mg, 0.023 mmol and 79 mg, 0.202 mmol in CH3 CN 3.740 mL) to give α:β 1:9. All solutions had a total concentration of 0.06 M. Five samples (1 L) of each of the five mixtures were added to a TLC plate and eluted (heptane/EtOAc 4:3). The procedure was repeated using 10 TLC plates and stained using anisaldehyde. The TLC plates were then scanned and analyzed using JustTLC. 2.9. Anomerization 1-O-Isopropyl-2,3,4,6-tetra-O-acetyl--d-glucopyranoside (17 mg, 0.044 mmol) was dissolved in CH2 Cl2 (0.450 mL) and BF3 • OEt2 (0.017 mL, 0.134 mmol) was added. Samples (0.050 mL) were collected and partitioned between ether (0.200 mL) and NaHCO3 (sat aq, 0.500 mL). The phases were separated and TLC samples were taken from the organic phase. The organic phase was then concentrated and analyzed using NMR.
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3. Result and discussion
An example of an actual TLC plate, scanned by an ordinary office scanner and analyzed by JustTLC is shown in Fig. 1.
3.1. Image analysis software 3.2. Useful detection range The software, JustTLC, is commercially available from Sweday [13] and treats the image as a three-dimensional topography, where each spot can be seen as a mountain in a landscape. The program works with digital images (tif, jpeg and bmp) and is not specific for flatbed scanned images. Spots are automatically or manually detected. The quantification starts by reducing noise and finding the statistically best separation between each spot and the background. Then, the background is modeled and subtracted from the image. Finally, the interior of each spot is integrated so that each corresponding spot volume is found. The major challenges in this procedure are noise, background variation and spot overlapping. The noise is reduced by applying a diffusion filtering technique, which preserves the spot shape while removing the random Gaussian noise in a robust manner. Varying background is more difficult, but since its properties significantly differ compared to the spots, it is possible to model and subtract it from the image. This makes each spot within an image to have the same background level. Spots located too close to each other in the image are split at the intersected area so that a true spot volume is obtained.
Fig. 1. TLC plate, scanned by an ordinary office scanner and analyzed by JustTLC. The dots mark each spot and the contours mark the separation between the spots and the background.
At first the useful detection range for sample analysis using different staining reagents was investigated. The study was limited to carbohydrates, a compound class which due to high polarity and few chromophores is difficult to detect by
Fig. 2. 1,2,3,4,6-Penta-O-acetyl--d-glucopyranose stained with (a) anisaldehyde, (b) vanillin and (c) sulfuric acid. Filled squares, circles and diamonds indicate three different series in each experiment.
R. Johnsson et al. / J. Chromatogr. A 1164 (2007) 298–305
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Fig. 3. Illustration of (a) saturated, (b) normal and (c) faint spots.
other methods. Three standard staining reagents were chosen; anisaldehyde, vanillin and sulfuric acid. A series of solutions of 1,2,3,4,6-penta-O-acetyl--dglucopyranose with concentrations from 0.0001 to 0.5 M were applied to TLC plates (1 L samples), thus resulting in amounts from 1 × 10−10 to 5 × 10−7 mol. The plates were eluted and stained using anisaldehyde, vanillin and sulfuric acid. Each experiment was performed in triplicate and analyzed using JustTLC (Fig. 2). As can be seen from Fig. 2, the errors are higher at large and small amounts of material. The spots could be detected down to 2 × 10−10 mol but not practically be quantified below 2 × 10−9 mol. The errors for low concentrations are due to faint
spots close or equal to the background level. The errors at high concentration are due to saturated spots which become underrated, hence the slightly bent curves in Fig. 2. Examples of spots with varying intensities are shown in Fig. 3. 3.3. Estimation of errors To evaluate quantitative TLC using staining reagent the different errors in the process were estimated. The error in the scanning process was estimated by applying three spots of 5 × 10−8 mol 1,2,3,4,6-penta-O-acetyl--d-glucopyranose to one TLC plate, developed by staining with anisaldehyde solution. The plate was then scanned five times and analyzed using
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Fig. 4. Differences between dipping TLC plates in staining reagent or spraying the reagent onto the plate. (a) Results from 10 plates with five spots each (dipped in anisaldehyde). (b) Example of TLC plate dipped in anisaldehyde. (c) Three-dimensional picture of TLC plate dipped in anisaldehyde. (d) Results from ten plates with five spots each (sprayed with anisaldehyde). (e) Example of TLC plate sprayed with anisaldehyde. (f) Three-dimensional picture of TLC plate sprayed with anisaldehyde.
JustTLC. The error was calculated to 0.3%. Different scanner resolutions, from 72 up to 1200 dpi, were also tested. It was seen that the relative intensities of the spots on a plate were independent of the scanner resolution. A fixed resolution of 1200 dpi was later used for all experiments. To confirm the precision of the algorithm used in the image analysis software, the same scanned TLC plate was evaluated five times with no variation in the results. The errors in the sample application were estimated by applying 30 spots of Rose Bengal (4,5,6,7-tetrachloro2 ,4 ,5 ,7 -tetraiodofluorescein disodium salt) to a TLC plate using “microcaps” with the volumes 1, 2 and 5 L (10 spots each). The plate was analyzed and the errors calculated to 1.6% for 1 L, 1.8% for 2 L and 5.0% for 5 L spots. As expected, the errors increase by increased volume of the capillar. There are several methods to apply staining reagent to a TLC plate. The differences between dipping the TLC plate in the staining reagent compared to spraying the reagent onto the plate were investigated. Ten plates with five spots each were subjected to the two different methods and developed by heating with a
hot air gun (Fig. 4). Fig. 4 shows that dipping of TLC plates results in less error. The average spot volume for dipped plates was 5.3 ± 0.4 × 107 , an error of about 7%. For sprayed plates the average spot volume was 6.8 ± 1.5 × 107 , an error of about 22%. From the actual plates it can be seen that spraying gives increased background variation as well as splitting of the peaks (Fig. 4e and f), which explains the larger error. All further experiments were based on dipping TLC plates to minimize errors. Fig. 5 illustrates that there are substantial variation in the errors for different staining reagents as well as heating method. The errors are summarized in Table 1. Finally, the errors in the developing process were evaluated. Different staining reagents were tested in combination with two heating methods; an oven at 150 ◦ C and a hot air gun with an estimated temperature of 500–600 ◦ C. In the oven, the plates were heated for a fixed time (15 min) and with the hot air gun they were heated until the plate did not grow visually darker. For each method, five spots of 5 × 10−8 mol 1,2,3,4,6-penta-Oacetyl--d-glucopyranose were added to ten TLC plates, which were eluted and stained. The results are displayed in Fig. 5.
R. Johnsson et al. / J. Chromatogr. A 1164 (2007) 298–305
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Fig. 5. Errors using different staining reagents in combination with hot air gun or oven. (a) Anisaldehyde, hot air gun, (b) anisaldehyde, oven, (c) vanillin, hot air gun, (d) vanillin, oven, (e) sulfuric acid, hot air and (f) sulfuric acid, oven. Each column represents the five spots of one TLC plate.
3.4. Measurement of ratios for minimization of errors in quantitative TLC To summarize, there are substantial errors connected with the staining process. Especially the variation between different plates adds to the errors. To minimize this problem, measurement of ratios between two different compounds in the same sample were investigated. The anomeric mixture of 1-O-isopropyl-2,3,4,6-tetra-O-acetyl-d-glucopyranoside
which is easy to separate by TLC was used. Five spots of a 1:1 mixture of the ␣- and -glucosides were applied to 10 TLC plates which were eluted and stained using the anisaldehyde/hot air gun combination. The percentage of the ␣-glucoside was calculated for each spot and the result is shown in Fig. 6. Similar experiments for 10, 25, 75 and 90% of the ␣-glucoside were also performed and the measured ratios from TLC were plotted versus the actual amounts (Fig. 7).
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Table 1 Errors using different developing techniques Entrya
Staining reagent
Heating method
Average spot volume (×10−7 )
a b c d e f
Anisaldehyde Anisaldehyde Vanillin Vanillin Sulfuric acid Sulfuric acid
Hot air gun Oven Hot air gun Oven Hot air gun Oven
5.3 2.6 6.0 1.6 5.2 3.4
a
± ± ± ± ± ±
0.4 0.7 0.8 0.3 1.0 0.2
Error (%) 7 27 13 17 19 6
The different entries connects to Fig. 5.
Fig. 6. Percentage of ␣-glucoside in a mixture of ␣- and -glucosides (1:1). In this figure it is shown that the errors are dramatically smaller both in each TLC plate but, and more importantly, between different plates. The average error is 2%.
To finally evaluate quantitative TLC using staining reagents, the anomerization of 1-O-isopropyl-2,3,4,6-tetra-O-acetyl--dglucopyranoside to the corresponding ␣-glucopyranoside was investigated. The reaction was performed in CH2 Cl2 with
Fig. 8. Anomerization of 1-O-isopropyl-2,3,4,6-tetra-O-acetyl--dglucopyranoside followed by TLC (filled circles) and NMR (filled squares).
BF3 • OEt2 as promoter. Samples were collected at 0, 5, 10, 15, 30, 60, 90 and 120 min, quenched and analyzed with TLC and NMR in triplicate. The result is shown in Fig. 8. The outcome of the two methods gives similar result. The reaction was assumed to follow pseudo-first order kinetics and the k -values were calculated. From TLC k = 1.0 × 10-4 , from NMR k = 1.2 × 10−4 . 4. Conclusion We have shown that it is possible to perform quantitative TLC using staining reagents and image analysis software. In order to get reliable results the combination of staining reagent and heating method must be considered. The best reliability is obtained by measuring ratios between different compounds, since errors can be quite high for singular data points. It is thus possible to get results useful for analytical and physical organic investigations. Acknowledgments
Fig. 7. Plot of ␣-glucoside (%) measured from TLC vs. the actual amounts. As can be seen from this figure, the result from TLC analysis correlates well with the actual ratios with excellent linearity (R > 0.99).
This work was supported by the Swedish Research Council and the Crafoord Foundation. We thank Dr. Clas Wes´en for help with initial studies.
R. Johnsson et al. / J. Chromatogr. A 1164 (2007) 298–305
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.chroma.2007.07.029. References [1] R.E. Simon, L.K. Walton, Y. Liang, B. Denton, Analyst 126 (2001) 446. [2] I. Vovk, M. Prosek, J. Chromatogr. A 779 (1997) 329. [3] H. Agrawal, N. Kaul, A.R. Paradkar, K.R. Mahadik, J. Pharma. Biomed. Anal. 36 (2004) 33.
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[4] Y. Liang, M.E. Baker, B.T. Yeager, M.B. Denton, Anal. Chem. 68 (1996) 3885. [5] M. Petrovic, M. Kastelan-Macan, K. Lazaric, S. Babic, J. AOAC Int. 82 (1999) 25. [6] C.F. Poole, J. Chromatogr. A 856 (1999) 399. [7] A.L. Pires Valente, F. Augusto, A.C. Lemes, K. Lukjanenko, Anal. Commun. 34 (1997) 193. [8] A.V. Gerasimov, J. Anal. Chem. 59 (2004) 348. [9] F.-T. Chau, T.-P. Chan, J. Wang, Bioinformatics 14 (1998) 540. [10] M.E. Johnson, J. Chem. Ed. 77 (2000) 368. [11] S. Garbacia, R. Touzani, O. Lavastre, J. Comb. Chem. 6 (2004) 297. [12] L. Zhang, X. Lin, J. Chromatogr. A 1109 (2006) 273. [13] JustTLC 1.1, Sweday, Lund, Sweden.
Evaluation of quantitative thin layer chromatography using staining reagents Richard Johnsson a , Gustav Tr¨aff b , Martin Sund´en a , Ulf Ellervik b,∗ a
Organic Chemistry, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden b Sweday, S¨ olvegatan 8c, SE-223 62 Lund, Sweden
Received 14 May 2007; received in revised form 10 July 2007; accepted 17 July 2007 Available online 21 July 2007
Abstract Thin layer chromatography (TLC) using staining reagents is a superior method for analyzing organic compounds without chromophores. It is fast, versatile and sometimes the only viable method. We have investigated quantitative TLC using staining reagents, in combination with modern image analysis software. Our results show that it is possible to get reliable measurements, suitable for high-throughput screening or physical organic investigations. The range of detection and the errors for the different parts of the process are illustrated. We show that the errors are largely due to the staining process and can be diminished by measuring ratios of compounds. © 2007 Elsevier B.V. All rights reserved. Keywords: TLC; Image analysis software; Quantification; Staining reagents
1. Introduction Thin layer chromatography (TLC) is one of the most widespread analytical methods used in the organic chemistry laboratory. The rationale is several fold. It is an inexpensive method that only consumes small amounts of sample as well as solvents. There are almost no restrictions in the use of solvent and a variety of stationary phases is commercially available, although SiO2 is by far the most used. Besides scanning in the UV-region, there is also an abundance of developing methods including absorption of iodine vapor and dipping or spraying for example sulfuric acid, permanganate or anisaldehyde solutions. Since there are no requirements for UV-activity (as for HPLC), paramagnetic properties (as for NMR) or volatility (as for GC) this makes TLC one of the most general analytical methods. For example, due to the absence of chromophores and the high boiling points, underivatized carbohydrates are difficult to analyze by other methods than TLC. TLC only uses the chromatographic surface once and the whole analyte is detected, which means that there is no risk of accumulation of the analyte on the chromatographic surface. Finally, TLC is an ideal method for analysis
∗
Corresponding author. Tel.: +46 46 2228220; fax: +46 46 2228209. E-mail address: [email protected] (U. Ellervik).
0021-9673/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2007.07.029
of combinatorial reactions since it is easy to analyze several samples, each containing several compounds, simultaneously. Quantitative analysis is a powerful tool and the information gathered from quantitative studies can be used in a multitude of chemical disciplines, in particular analytical, physical organic and industrial chemistry. However, due to inadequate sample application, limited detectability and sensitivity and also reproducibility TLC was for long not regarded a quantitative method. However, with the development of high-performance TLC and the use of slit scanning densitometers, the accuracy has dramatically increased [1]. Unfortunately, with slit scanners each lane has to be scanned individually and the width of the scanning area has to be set to the same value as the diameter of the largest spot, which results in high signal-to-noise ratio [2,3]. Today the slit scanners have been replaced by charge-coupled device (CCD) detection. Scanning the plate using a CCD detector has been shown to be much faster and efficient. The CCD scans the plate at once and has shown to achieve fast and reliable results [1,4,5]. The CCD has a low signal-to-noise ratio and can operate in regions from UV to near IR [1]. It is also possible to use an ordinary office scanner as a densitometer. By using colored samples or by staining the plate with a suitable developing solution it is possible to scan the plate and use image analysis software to process the digital image. This way of quantification is fast, inexpensive and uncomplicated.
R. Johnsson et al. / J. Chromatogr. A 1164 (2007) 298–305
Image analyzers are thought to be a powerful tool in use with TLC [6]. However, research and software development in this area is scarce and at the moment there are only a handful of published articles available on the subject [7–12]. We decided to develop new image analysis software tailored for quantitative TLC image analysis, JustTLC [13].
299
2.5. Error in sample application Rose Bengal (4,5,6,7-tetrachloro-2 ,4 ,5 ,7 -tetraiodofluorescein disodium salt) (11 mg, 0.011 mmol) was dissolved in methanol (15 mL). The solution was applied to a TLC plate with 1 L, 2 L and 5 L microcaps 10 times each and analyzed using JustTLC.
2. Experimental section 2.6. Method of staining 2.1. General experimental details All samples were applied using Drummond “microcaps” 1 L, 2 L and 5 L (Drummond Scientific Company) TLC plates were alumina plates coated with silica gel 60 F254 (Merck). The samples were visualized using anisaldehyde (15 mL anisaldehyde and 2.5 mL H2 SO4 in 250 mL EtOH), vanillin (6 g vanillin and 2.5 mL H2 SO4 in 250 mL EtOH) and sulfuric acid (8 mL H2 SO4 in 100 mL EtOH). All plates were scanned using an Epson Perfection 2400 photo scanner. All files were scanned as tiff-images at 1200 dpi unless otherwise stated. 2.2. Useful range for detection 1,2,3,4,6-Penta-O-acetyl--d-glucopyranose (181 mg, 0.46 mmol) was dissolved in CH2 Cl2 (0.928 mL) to give a stock solution of 0.5 M. The stock solution was diluted with CH2 Cl2 to give 0.05, 0.005, 0.0005 and 0.00005 M solutions. 1,2,3,4,6Penta-O-acetyl--d-glucopyranose (81 mg, 0.21 mmol) was dissolved in CH2 Cl2 (1.034 mL) to give a stock solution of 0.2 M. The stock solution was diluted with CH2 Cl2 to give 0.02, 0.002, 0.0002 and 0.00002 M solutions. 1,2,3,4,6-PentaO-acetyl--d-glucopyranose (41 mg, 0.11 mmol) was dissolved in CH2 Cl2 (1.058 mL) to give a stock solution of 0.1 M. The stock solution was diluted with CH2 Cl2 to give 0.01, 0.001, 0.0001 and 0.00001 M solutions. One microliter of the different concentrations were spotted and eluted (heptane/EtOAc 1:1) three times each and stained with anisaldehyde, vanillin and sulfuric acid. The TLC plates were analyzed using JustTLC. 2.3. Error in scanning process Three samples (1 L) of a solution of 1,2,3,4,6-penta-Oacetyl--d-glucopyranose (0.05 M in CH2 Cl2 ) were added to a TLC plate, eluted (heptane/EtOAc 1:1) and developed with anisaldehyde. The TLC plate was scanned five times and analyzed using JustTLC. The TLC plate was also scanned at 72, 150, 300 and 600 dpi once each and then analyzed using JustTLC. 2.4. Precision of algorithm A solution of 1,2,3,4,6-penta-O-acetyl--d-glucopyranose in CH2 Cl2 , 0.05 M was used. Three spots of 1 L were added on a TLC plate and eluted (heptane/EtOAc 1:1) and developed with anisaldehyde. The TLC plate was then scanned and analyzed using JustTLC five times.
Five samples (1 L) of a solution of 1,2,3,4,6-penta-O-acetyl-d-glucopyranose (0.05 M in CH3 CN) were added to 20 TLC plates which were eluted (heptane/EtOAc 1:1). Ten TLC plates were sprayed with anisaldehyde while the remaining 10 plates were dipped in an anisaldehyde solution. The plates were then scanned and analyzed using JustTLC. 2.7. Staining reagent and heating method Five samples (1 L) of a solution of 1,2,3,4,6-penta-O-acetyl-d-glucopyranose (0.05 M in CH3 CN) were added to 20 TLC plates which were eluted (heptane/EtOAc 1:1). The TLC plates were dipped in anisaldehyde. Ten TLC plates were heated in oven at 150 ◦ C while the remaining plates were heated with a hot air gun at 5–600 ◦ C. The TLC plates were then scanned and analyzed using JustTLC. The procedure was repeated using vanillin or sulfuric acid. 2.8. Internal standard 1-O-Isopropyl-2,3,4,6-tetra-O-acetyl-␣-d-glucopyranoside and 1-O-isopropyl-2,3,4,6-tetra-O-acetyl--d-glucopyranoside (53 mg, 0.136 mmol and 6 mg, 0.015 mmol in CH3 CN 2.532 mL) to give α:β 90:10 (25 mg, 0.064 mmol and 8 mg, 0.020 mmol in CH3 CN 1.422 mL) to give α:β 75:25 (34 mg, 0.087 mmol and 34 mg, 0.087 mmol in CH3 CN 2.860 mL) to give α:β 50:5 (18 mg, 0.046 mmol and 6 mg, 0.015 mmol in CH3 CN 1.033 mL) to give α:β 25:75 and (9 mg, 0.023 mmol and 79 mg, 0.202 mmol in CH3 CN 3.740 mL) to give α:β 1:9. All solutions had a total concentration of 0.06 M. Five samples (1 L) of each of the five mixtures were added to a TLC plate and eluted (heptane/EtOAc 4:3). The procedure was repeated using 10 TLC plates and stained using anisaldehyde. The TLC plates were then scanned and analyzed using JustTLC. 2.9. Anomerization 1-O-Isopropyl-2,3,4,6-tetra-O-acetyl--d-glucopyranoside (17 mg, 0.044 mmol) was dissolved in CH2 Cl2 (0.450 mL) and BF3 • OEt2 (0.017 mL, 0.134 mmol) was added. Samples (0.050 mL) were collected and partitioned between ether (0.200 mL) and NaHCO3 (sat aq, 0.500 mL). The phases were separated and TLC samples were taken from the organic phase. The organic phase was then concentrated and analyzed using NMR.
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3. Result and discussion
An example of an actual TLC plate, scanned by an ordinary office scanner and analyzed by JustTLC is shown in Fig. 1.
3.1. Image analysis software 3.2. Useful detection range The software, JustTLC, is commercially available from Sweday [13] and treats the image as a three-dimensional topography, where each spot can be seen as a mountain in a landscape. The program works with digital images (tif, jpeg and bmp) and is not specific for flatbed scanned images. Spots are automatically or manually detected. The quantification starts by reducing noise and finding the statistically best separation between each spot and the background. Then, the background is modeled and subtracted from the image. Finally, the interior of each spot is integrated so that each corresponding spot volume is found. The major challenges in this procedure are noise, background variation and spot overlapping. The noise is reduced by applying a diffusion filtering technique, which preserves the spot shape while removing the random Gaussian noise in a robust manner. Varying background is more difficult, but since its properties significantly differ compared to the spots, it is possible to model and subtract it from the image. This makes each spot within an image to have the same background level. Spots located too close to each other in the image are split at the intersected area so that a true spot volume is obtained.
Fig. 1. TLC plate, scanned by an ordinary office scanner and analyzed by JustTLC. The dots mark each spot and the contours mark the separation between the spots and the background.
At first the useful detection range for sample analysis using different staining reagents was investigated. The study was limited to carbohydrates, a compound class which due to high polarity and few chromophores is difficult to detect by
Fig. 2. 1,2,3,4,6-Penta-O-acetyl--d-glucopyranose stained with (a) anisaldehyde, (b) vanillin and (c) sulfuric acid. Filled squares, circles and diamonds indicate three different series in each experiment.
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Fig. 3. Illustration of (a) saturated, (b) normal and (c) faint spots.
other methods. Three standard staining reagents were chosen; anisaldehyde, vanillin and sulfuric acid. A series of solutions of 1,2,3,4,6-penta-O-acetyl--dglucopyranose with concentrations from 0.0001 to 0.5 M were applied to TLC plates (1 L samples), thus resulting in amounts from 1 × 10−10 to 5 × 10−7 mol. The plates were eluted and stained using anisaldehyde, vanillin and sulfuric acid. Each experiment was performed in triplicate and analyzed using JustTLC (Fig. 2). As can be seen from Fig. 2, the errors are higher at large and small amounts of material. The spots could be detected down to 2 × 10−10 mol but not practically be quantified below 2 × 10−9 mol. The errors for low concentrations are due to faint
spots close or equal to the background level. The errors at high concentration are due to saturated spots which become underrated, hence the slightly bent curves in Fig. 2. Examples of spots with varying intensities are shown in Fig. 3. 3.3. Estimation of errors To evaluate quantitative TLC using staining reagent the different errors in the process were estimated. The error in the scanning process was estimated by applying three spots of 5 × 10−8 mol 1,2,3,4,6-penta-O-acetyl--d-glucopyranose to one TLC plate, developed by staining with anisaldehyde solution. The plate was then scanned five times and analyzed using
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Fig. 4. Differences between dipping TLC plates in staining reagent or spraying the reagent onto the plate. (a) Results from 10 plates with five spots each (dipped in anisaldehyde). (b) Example of TLC plate dipped in anisaldehyde. (c) Three-dimensional picture of TLC plate dipped in anisaldehyde. (d) Results from ten plates with five spots each (sprayed with anisaldehyde). (e) Example of TLC plate sprayed with anisaldehyde. (f) Three-dimensional picture of TLC plate sprayed with anisaldehyde.
JustTLC. The error was calculated to 0.3%. Different scanner resolutions, from 72 up to 1200 dpi, were also tested. It was seen that the relative intensities of the spots on a plate were independent of the scanner resolution. A fixed resolution of 1200 dpi was later used for all experiments. To confirm the precision of the algorithm used in the image analysis software, the same scanned TLC plate was evaluated five times with no variation in the results. The errors in the sample application were estimated by applying 30 spots of Rose Bengal (4,5,6,7-tetrachloro2 ,4 ,5 ,7 -tetraiodofluorescein disodium salt) to a TLC plate using “microcaps” with the volumes 1, 2 and 5 L (10 spots each). The plate was analyzed and the errors calculated to 1.6% for 1 L, 1.8% for 2 L and 5.0% for 5 L spots. As expected, the errors increase by increased volume of the capillar. There are several methods to apply staining reagent to a TLC plate. The differences between dipping the TLC plate in the staining reagent compared to spraying the reagent onto the plate were investigated. Ten plates with five spots each were subjected to the two different methods and developed by heating with a
hot air gun (Fig. 4). Fig. 4 shows that dipping of TLC plates results in less error. The average spot volume for dipped plates was 5.3 ± 0.4 × 107 , an error of about 7%. For sprayed plates the average spot volume was 6.8 ± 1.5 × 107 , an error of about 22%. From the actual plates it can be seen that spraying gives increased background variation as well as splitting of the peaks (Fig. 4e and f), which explains the larger error. All further experiments were based on dipping TLC plates to minimize errors. Fig. 5 illustrates that there are substantial variation in the errors for different staining reagents as well as heating method. The errors are summarized in Table 1. Finally, the errors in the developing process were evaluated. Different staining reagents were tested in combination with two heating methods; an oven at 150 ◦ C and a hot air gun with an estimated temperature of 500–600 ◦ C. In the oven, the plates were heated for a fixed time (15 min) and with the hot air gun they were heated until the plate did not grow visually darker. For each method, five spots of 5 × 10−8 mol 1,2,3,4,6-penta-Oacetyl--d-glucopyranose were added to ten TLC plates, which were eluted and stained. The results are displayed in Fig. 5.
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Fig. 5. Errors using different staining reagents in combination with hot air gun or oven. (a) Anisaldehyde, hot air gun, (b) anisaldehyde, oven, (c) vanillin, hot air gun, (d) vanillin, oven, (e) sulfuric acid, hot air and (f) sulfuric acid, oven. Each column represents the five spots of one TLC plate.
3.4. Measurement of ratios for minimization of errors in quantitative TLC To summarize, there are substantial errors connected with the staining process. Especially the variation between different plates adds to the errors. To minimize this problem, measurement of ratios between two different compounds in the same sample were investigated. The anomeric mixture of 1-O-isopropyl-2,3,4,6-tetra-O-acetyl-d-glucopyranoside
which is easy to separate by TLC was used. Five spots of a 1:1 mixture of the ␣- and -glucosides were applied to 10 TLC plates which were eluted and stained using the anisaldehyde/hot air gun combination. The percentage of the ␣-glucoside was calculated for each spot and the result is shown in Fig. 6. Similar experiments for 10, 25, 75 and 90% of the ␣-glucoside were also performed and the measured ratios from TLC were plotted versus the actual amounts (Fig. 7).
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Table 1 Errors using different developing techniques Entrya
Staining reagent
Heating method
Average spot volume (×10−7 )
a b c d e f
Anisaldehyde Anisaldehyde Vanillin Vanillin Sulfuric acid Sulfuric acid
Hot air gun Oven Hot air gun Oven Hot air gun Oven
5.3 2.6 6.0 1.6 5.2 3.4
a
± ± ± ± ± ±
0.4 0.7 0.8 0.3 1.0 0.2
Error (%) 7 27 13 17 19 6
The different entries connects to Fig. 5.
Fig. 6. Percentage of ␣-glucoside in a mixture of ␣- and -glucosides (1:1). In this figure it is shown that the errors are dramatically smaller both in each TLC plate but, and more importantly, between different plates. The average error is 2%.
To finally evaluate quantitative TLC using staining reagents, the anomerization of 1-O-isopropyl-2,3,4,6-tetra-O-acetyl--dglucopyranoside to the corresponding ␣-glucopyranoside was investigated. The reaction was performed in CH2 Cl2 with
Fig. 8. Anomerization of 1-O-isopropyl-2,3,4,6-tetra-O-acetyl--dglucopyranoside followed by TLC (filled circles) and NMR (filled squares).
BF3 • OEt2 as promoter. Samples were collected at 0, 5, 10, 15, 30, 60, 90 and 120 min, quenched and analyzed with TLC and NMR in triplicate. The result is shown in Fig. 8. The outcome of the two methods gives similar result. The reaction was assumed to follow pseudo-first order kinetics and the k -values were calculated. From TLC k = 1.0 × 10-4 , from NMR k = 1.2 × 10−4 . 4. Conclusion We have shown that it is possible to perform quantitative TLC using staining reagents and image analysis software. In order to get reliable results the combination of staining reagent and heating method must be considered. The best reliability is obtained by measuring ratios between different compounds, since errors can be quite high for singular data points. It is thus possible to get results useful for analytical and physical organic investigations. Acknowledgments
Fig. 7. Plot of ␣-glucoside (%) measured from TLC vs. the actual amounts. As can be seen from this figure, the result from TLC analysis correlates well with the actual ratios with excellent linearity (R > 0.99).
This work was supported by the Swedish Research Council and the Crafoord Foundation. We thank Dr. Clas Wes´en for help with initial studies.
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