Summary
The present protocol developed a method to estimate the yield of compounds on the TLC plate using the blue-LED illumination technique. The advantages of this approach are that it is safe, effective, inexpensive, and allows the researcher to measure multiple samples simultaneously.
Abstract
Thin-layer chromatography (TLC) is an accessible analytical technique that has been extensively used in organic chemistry research to quantify the yield of unknown samples. The present study developed an effective, cheap, and safe method to estimate the yield of samples on a TLC plate using the blue-LED illuminator. Lovastatin extracted from Aspergillus terreus was the example compound used in the present study. Regression models based on the lovastatin standard were used to evaluate the yield of lovastatin. Three methods were compared: bioassay, UV detection, and blue-LED illumination. The result showed that the blue-LED illumination method is significantly more time-effective than UV detection and bioassay methods. Additionally, the blue-LED illumination was a relatively safe option because of the concern of biological hazards in the bioassay method (e.g., microbial infection) and ultraviolet exposure in the UV detection method. Compared to the expensive methods requiring specialized instruments and long-term training before working independently, such as GC, HPLC, and HPTLC, using the blue-LED illuminator was an economical option to estimate the yield of samples from a TLC plate.
Introduction
Thin-layer chromatography (TLC) is widely used as a qualitative and quantitative technique in the field of organic chemistry1,2,3. The main advantages of TLC are that it provides fast detection, flexible sample requirements, and does not require specialized equipment4. To date, even though many advanced approaches have been established, TLC is still the main method for identifying unknown samples in a mixture. However, the challenge of this approach is the lack of safe and inexpensive equipment for quantifying the sample yield, especially for developing laboratories with limited budgets. The present study, therefore, aimed to develop an efficient, safe, and inexpensive method combining with TLC to estimate the yield of the samples.
Unlike high-performance TLC (HPTLC), high-performance liquid chromatography (HPLC), and gas chromatography (GC) with strict sample requirements, time-consuming, and involvement of multistep for sample preparation1,5, TLC showed several advantages. First, for sample preparation, the HPLC and GC cannot detect the crude extract because the crude extract may plug the column of HPLC and GC. Second, when the samples are not UV-suitable (important for HPLC analysis) or with low volatility (important for GC analysis), TLC can be applied to these samples, and the use of visualization reagent makes the isolated samples visible on thin layers6,7,8. Third, for general users, HPLC and GC generally require a relatively long time pre-training before working independently, compared to TLC. In addition, quantitative TLC analysis, known as high-performance TLC (HPTLC), can digitize the information on a TLC plate with a highly sensitive scanner. However, the cost of the HPTLC system is relatively expensive. As such, developing a cost-effective and fast approach to quantify samples on the TLC plate is an important topic.
Similar methods have been developed for TLC yield quantification; for example, Johnson9 reported a technique that allows the quantification of the samples on a TLC plate by using a flatbed scanner attached to a computer. In 2001, El-Gindy et al.10 developed the TLC- densitometric method, which was used to detect the compound with optical density, and the technique was also applied by Elkady et al.11. In 2007, Hess2 presented the digitally enhanced-TLC (DE-TLC) method applied to detect the yield of a compound on a TLC plate using a digital camera combined with UV light. Hess also compared the cost differences between HPTLC and DE-TLC method and concluded that the DE-TLC method could be used in high school and college labs because of its affordable cost2. However, the cost of the TLC-densitometric method was still expensive, and the operation of ultraviolet light requires adequate pre-training in case the users might get exposed to ultraviolet radiation. Therefore, compatible with TLC, developing an efficient, safe, and inexpensive method to quantify the sample yield is desirable.
The present study described a protocol for detecting the sample on a TLC plate using the blue-LED illuminator, and developed a regression model with high reliability (high R-square value) to measure the dimensions of the bands, and then determine the compound yield. Finally, it was found that the blue-LED illumination method is a relatively safe (vs. UV-detection method), cheap (vs. GC, HPLC, and HPTLC), and effective (vs. bioassay method) approach for yield quantification.
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Protocol
The present protocol is described using lovastatin as an example. Lovastatin was extracted from one-week-old Aspergillus terreus.
1. Compound extraction
NOTE: For details on compound extraction, please see Figure 1.
- Culture Aspergillus terreus on the potato dextrose agar (PDA, see Table of Materials) medium at 30 °C.
- Dry the culture at 40 °C for 24 h. Transfer the dried culture into a 50 mL tube using sterilized tweezers and add 15 mL ethyl acetate.
- Shake the mixture vigorously by vortexing for 1 min and incubate for 1 h at 40 °C with shaking at 200 rpm.
- Sonicate the mixture using a 40 kHz ultrasonic bath (see Table of Materials) at 40 °C for 1 h.
- Centrifuge the mixture at 5,000 x g for 1 min at room temperature and filter through an 11 µm filter paper.
- Extract the filtrate with an equal volume of sterile water in a separatory funnel.
- After phase separation, collect the organic layer, and then evaporate in a rotary evaporator (see Table of Materials). Dissolve the residue in 2 mL of ethyl acetate.
2. Separation of the crude extract by normal phase (NP) adsorption column
- Pack the column with NP silica gel as the stationary phase, and use n-hexane:ethyl acetate:trifluoroacetic acid (H:E:T; 80:20:0.1, v/v/v) as the mobile phase.
- Load 2 mL of the extract (step 1) onto the column and add the mobile phase solvent at a flow rate of 1 mL/min to elute the extract.
NOTE: The flow rate was manually controlled using a stopcock. - Verify the effluent by TLC to confirm the presence of lovastatin, and then evaporate in a rotary evaporator at 45 °C until the solvent gets removed. This step takes approximately 20-25 min.
- Dissolve the residue in 1 mL of ethyl acetate, and then mix with an equal volume of 1% trifluoroacetic acid.
- Centrifuge the mixture at 5,000 x g for 1 min at room temperature and collect the organic layer in a new glass tube.
3. Preparation and loading of thin-layer chromatogram (TLC) plates
- Spot 5 µL of samples and lovastatin standards (see Table of Materials) onto the baseline of the TLC plate using a capillary pipette, leaving a border of 1 cm on the sides of the TLC plate.
- Dry the TLC plate in a fume hood for 5 min at room temperature.
- Place the plate gently by forceps in a saturated glass chamber containing the mobile phase solvent. Cover the chamber with a glass lid and allow the plate to develop fully.
- Remove the plate from the chamber when the solvent line reaches 1 cm from the top of the plate.
4. Analysis by the blue-LED illuminator
- Mark the solvent line with a pencil. Dry the plate in the fume hood for 10 min at room temperature.
- After drying, immediately soak the plate in 10% H2SO4 solvent, and then dry in the fume hood for 10 min at room temperature.
- Place the plate on the heating panel until the brown spots appear. Ensure that the plate is not overheated, as this might make visualization of lovastatin difficult.
- Transfer the plate to the blue-LED illuminator and scan using a compatible freeware (MiBio Fluo) (see Table of Materials).
5. Yield estimation by the regression model
- Measure the dimension of bands using the ImageJ software (see Table of Materials).
- Establish a regression model using data analysis and graphing software (see Table of Materials) based on the descending concentrations of lovastatin standards, including 1 mg/mL, 0.75 mg/mL, 0.5 mg/mL, and 0.25 mg/mL.
- Apply the regression model to estimate the yield of the samples.
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Representative Results
This study presented the blue-LED illumination method to estimate the yield of compounds, and this method was validated and compared to bioassay and UV-detected methods (Table 1). The regression models were developed based on the dimensions of bands and concentration of standards for three methods, respectively, to predict the yield of samples. First, in the results of the bioassay method, the R-square between the dimensions of the inhibition zone and lovastatin standards was 0.99, and the sample yield was 0.56 mg predicted by the regression model (Figure 2). Second, in the UV-detection method, the R-square between the lovastatin standards and the dimension of bands on the TLC plate was 0.97, and the yield of the sample predicted by the regression model was 0.53 mg (Figure 3). Notably, the band's edges were blurry, and relatively low signal intensity bands were observed (Figure 3A). Third, in the blue-LED illumination method, the R-square between lovastatin standards and the bands' dimension on the TLC plate was 0.98, and the sample yield was 0.54 mg predicted by the regression model (Figure 4). The predicted yield using the blue-LED illuminator was closer to the bioassay method (set as control). The dimension of the band was proportional to the amount of lovastatin, and the clear bands were obtained by the blue-LED illumination method. In addition, the working hours of the bioassay, UV-detected, and blue-LED illumination methods were approximately 24 h, 2 h, and 1 h, respectively; (Note: working hour means the total time spent on the yield examination of lovastatin).
Figure 1: The working flow of the protocol. Please click here to view a larger version of this figure.
Figure 2: Bioassay method. (A) Bioassay of lovastatin against Neurospora crassa (incubated for 24 h at 30 °C). At the end of the trial, the 90 mm agar plates were photographed under visible light. (B) The concentrations of six lovastatin standards were: No. 1 (1 mg/mL), No. 2 (0.75 mg/mL), No. 3 (0.5 mg/mL), and No. 4 (0.25 mg/mL). Sample No. 5 was diluted to 0.25× (1:4). Sample No. 6 was diluted to 0.5× (1:2). The inhibition zone dimension (mm2) was measured by the imaging software. (C) A regression model was developed using data analysis and graphing software based on the inhibition zone dimension of standards. Please click here to view a larger version of this figure.
Figure 3: Thin-layer chromatogram (TLC) plate exposed to UV light. (A) The n-hexane:ethyl acetate (2:3 v/v) was used as the mobile phase in the TLC analysis, and the TLC plate was exposed to UV light (365 nm) after soaking in the developer (10% H2SO4). (B) The concentrations of six lovastatin standards were: No. 1 (1 mg/mL), No. 2 (0.75 mg/mL), No. 3 (0.5 mg/mL), and No. 4 (0.25 mg/mL). Sample No. 5 was diluted to 0.25× (1:4). Sample No. 6 was diluted to 0.5× (1:2). The inhibition zone dimension (mm2) was measured by the imaging software. (C) A regression model was developed using data analysis and graphing software based on the dimension of the lovastatin standard bands on the TLC plate. Please click here to view a larger version of this figure.
Figure 4: Thin-layer chromatogram (TLC) plate scanned by the blue-LED illuminator. (A) The n-hexane:ethyl acetate (2:3 v/v) was used as the mobile phase in the TLC analysis, and the TLC plate was scanned by the blue-LED illuminator. (B) The concentrations of six lovastatin standards were: No. 1 (1 mg/mL), No. 2 (0.75 mg/mL), No. 3 (0.5 mg/mL), and No. 4 (0.25 mg/mL). Sample No. 5 was diluted to 0.25× (1:4). Sample No. 6 was diluted to 0.5× (1:2). The inhibition zone dimension (mm2) was measured by the imaging software. (C) A regression model was developed using data analysis and graphing software based on the dimension of the lovastatin standard bands on the TLC plate. Please click here to view a larger version of this figure.
| Bioassay | Blue-LED Illuminator | UV-detected | |
| Results observation | Eyes | Blue-LED illuminator and eyes | UV light and eyes |
| Image resolution | Medium | High | Low (blur and faint image) |
| Approximate time cost | 24 h | 1 h | 2 h |
| Analysis skill required | Medium | Low | Medium |
| Safety | Microbial infection | Very safe | UV light exposure |
| Regression equation | y = 0.0019x + 0.0304 | y = 0.0399x - 0.1271 | y = 0.0657x - 0.6405 |
| R-square | 0.99 | 0.98 | 0.97 |
| Slope | 0.0019 | 0.0399 | 0.0657 |
| Intercept | 0.0304 | -0.1271 | -0.6405 |
| Standard error of slope | 8.94E-05 | 3.54E-03 | 6.28E-03 |
| Standard error of intercept | 0.03032 | 0.07115 | 0.12375 |
Table 1: Comparison of the three detection methods used in this study.
| TLC-densitometric method | TLC-image analysis | |||||||
| El-Gindy et al.10 |
Elkady et al.11 |
Musharraf et al.12 |
Johnson9 | Hess2 | Blue-LED Illuminator method (This study) |
|||
| Sample | Acebutolol HCL | Ciprofloxacin HCL | Metronidazole | Danazol | Cholesterol | Vanillin | Nicotinamide | Lovastatin |
| Results | UV detector |
TLC scanner |
TLC scanner |
Flatbed scanner | Digital camera with UV lamp |
Blue-LED illuminator | ||
| Wavelength | 230 nm | 280 nm | 280 nm | 291 nm | NA | 254 nm | NA | |
| Correlation coefficient | 0.996a | 0.9991a | 0.9994a | 0.996a | 0.998a | 0.971b | 0.987b | 0.99a 0.98b |
| Regression equation | NA | y = 5.7853x +19.9383 |
y = 1.1104x + 6.9755 |
y = 7.949x + 2460 | y = 0.96x | NA | NA | y = 0.0399x -0.1271 |
| a: Pearson correlation coefficient | ||||||||
| b: R-square | ||||||||
Table 2: Comparison of previous methods and the current study.
Supplementary Figure 1: The thin-layer chromatogram (TLC) plate with ampicillin was scanned by the blue-LED illumination method. (A) Ethyl acetate:methanol (9:13 v/v) was used as the mobile phase in the TLC analysis, and the TLC plate was scanned by the blue-LED illuminator. (B) The concentration of four ampicillin standards were: No. 1 (100 mg/mL), No. 2 (75 mg/mL), No. 3 (50 mg/mL), and No. 4 (25 mg/mL). The bands' dimension was measured by the imaging software. (C) A regression model was developed using data analysis and graphing software based on the dimension of the ampicillin standard bands on the TLC plate. Please click here to download this File.
Supplementary Figure 2: Thin-layer chromatogram (TLC) plate with apramycin scanned by the blue-LED illumination method. (A) Methanol:water (6:5 v/v) was used as the mobile phase in the TLC analysis and the TLC plate was scanned by the blue-LED illuminator. (B) The concentration of four apramycin standards were: No. 1 (50 mg/mL), No. 2 (40 mg/mL), No. 3 (30 mg/mL), and No. 4 (20 mg/mL). The bands' dimension was measured by the imaging software. (C) A regression model was developed using data analysis and graphing software based on the dimension of the apramycin standard bands on the TLC plate. Please click here to download this File.
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Discussion
The present study described a new approach, the blue-LED illuminator, to quantify compounds without using expensive and specialized equipment, such as HPTLC, HPLC, and GC method, and the method was compared with the bioassay and UV-detected methods to evaluate quantification performance. As a result, it was concluded that the blue-LED illumination method is a relatively safe and effective protocol used to quantify the yield of targeted compounds on the TLC plate.
Previous studies have reported several quantitative methods without using specialized quantitative equipment, and all the studies showed that the accuracy of the quantitation was close to that of the use of specialized equipment2,9,10,11,12 (Table 2). For instance, El-Gindy et al.10 compared the accuracy of the estimated yield based on the HPLC and TLC-densitometric methods, and the results showed that there were no significant differences between the two methods (p-value < 0.05). Compared with the blue-LED illumination method in this study, the TLC-densitometric method developed by El-Gindy et al.10 required a special wavelength detector, while the blue-LED illumination method required a simple and inexpensive blue-LED scanner. Meanwhile, the blue-LED scanner could also be used for other purposes, such as gel electrophoresis scanning, but the specialized TLC scanner developed by Elkady et al.11 was used only for the TLC-densitometric method.
In addition to the TLC-densitometric method10,11, the flatbed scanner and the digital camera method were developed to detect the yield of samples. For example, Johnson9 established a rapid TLC detection method using the flatbed scanner, and then measured the absorption using commercial image software. However, the image software used in the blue-LED illumination method was free and easy to use. Hess2 developed the "TLC analyzer" software to estimate the dimension of bands on TLC plate images taken by a digital camera, which was similar to the UV-detected method in this study. However, both the methods may interact potentially with UV light hazards.
The regression models based on the dimension of standards' bands were developed for bioassay, blue-LED illuminator, and UV-detected method, and the R-square value was 0.99, 0.98, and 0.97, respectively (Table 1). As high linearity (R-square) was achieved, it is suggested that the regression model could measure the yield of samples. The X-value in the regression model was substituted by the dimension of standards' bands, and the estimated yield (predicted Y-value in the regression model) was approximately 5.6, 5.4, and 5.3, determined by bioassay, blue-LED, and UV-detection methods, respectively. The results indicated that the R-square and estimated yield using the blue-LED illuminator was closer to the bioassay method (set as control) than the UV-detected method (Table 1).
To understand the limitations of this approach, the approach was also applied to detect two other compounds, including ampicillin and apramycin; the results are shown in Supplementary Figure 1 and Supplementary Figure 2. Two important steps must be noted in this approach: (1) after drying, the plate must be visualized immediately; delayed visualization may affect the spot detection; (2) overexposure of the plate is not recommended as the scanned image from the plate comes with high background and makes difficult to measure the areas of the spots.
To conclude, the blue-LED illumination method is a relatively safe, time-saving, inexpensive, and less laborious approach to speed the quantifying of the yield of samples compared to other quantitative chromatographic methods; therefore, this approach is an ideal protocol for use in developing labs with limited budgets. Meanwhile, the TLC plate images retrieved from the blue-LED illumination method were much clearer than those from the UV detection method (Figure 3A vs. Figure 4A), which also helped to precisely determine the dimension of the bands on the TLC plate, and also to estimate the yield of the samples.
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Disclosures
All the authors declare that they have no conflicts of interest.
Acknowledgments
This study was supported by the Ministry of Science and Technology, Taiwan (MOST 108-2320-B-110-007-MY3).
Materials
| Name | Company | Catalog Number | Comments |
| American bacteriological Agar | Condalab | 1802.00 | |
| Aspergillus terreus | ATCC 20542 | ||
| Blue-LED illuminator | MICROTEK | Bio-1000F | |
| Centrifuge | Thermo Scientific | HERAEUS Megafuge 8 | |
| Compact UV lamp | UVP | UVGL-25 | |
| Ethyl Acetate | MACRON | MA-H078-10 | |
| Filter Paper 125mm | ADVANTEC | 60311102 | |
| ImageJ | NIH | Freeware | https://imagej.nih.gov/ij/download.html |
| Lovastatin standard | ACROS | A0404262 | |
| MiBio Fluo | MICROTEK | V1.04 | |
| n-Hexane | C-ECHO | HH3102-000000-72EC | |
| OriginPro | OriginLab | 9.1 | https://www.originlab.com/origin |
| Potato dextrose broth H | STBIO MEDIA | 110533 | |
| Rotary evaporator | EYELA | SB-1000 | |
| Sulfuric acid | Fluka | 30743-2.5L-GL | |
| TLC silica gel 60 F254 | MERCK | 1.05554.0001 | |
| Trifluoroacetic acid | Alfa Aesar | 10229873 | |
| Ultrasonic vibration machine | DELTA | DC600 |
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