This study was supported by the Ministry of Science and Technology, Taiwan (MOST 108-2320-B-110-007-MY3).

The present protocol is described using lovastatin as an example.&#160;Lovastatin was extracted from one-week-old Aspergillus terreus.
1. Compound extraction
NOTE: For details on compound extraction, please see Figure 1.
<ol>
	<li>Culture Aspergillus terreus on the potato dextrose agar (PDA, see Table of Materials) medium at 30 &#176;C.</li>
	<li>Dry the culture at 40 &#176;C for 24 ...

<ol>
	<li>Pyka, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+progress+of+selected+drugs+in+TLC.">Detection progress of selected drugs in TLC.</a> BioMed Research International. 2014, 732078 (2014).</li><li>Hess, A. V. 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=First+derivative+spectrophotometric,+TLC-densitometric,+and+HPLC+determination+of+acebutolol+HCL+in+presence+of+its+acid-induced+degradation+product.">First derivative spectrophotometric, TLC-densitometric, and HPLC determination of acebutolol HCL in presence of its acid-induced degradation product.</a> Journal of Pharmaceutical and Biomedical Analysis. 24 (4), 527-534 (2001).</li><li>Elkady, E. F., Mahrouse, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversed-phase+ion-pair+HPLC+and+TLC-densitometric+methods+for+the+simultaneous+determination+of+ciprofloxacin+hydrochloride+and+metronidazole+in+tablets.">Reversed-phase ion-pair HPLC and TLC-densitometric methods for the simultaneous determination of ciprofloxacin hydrochloride and metronidazole in tablets.</a> Chromatographia. 73 (3-4), 297-305 (2011).</li><li>Musharraf, S. 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All the authors declare that they have no conflicts of interest.

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...

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>American bacteriological Agar</td>
			<td>Condalab</td>
			<td>1802.00</td>
			<td></td>
		</tr>
		<tr>
			<td>Aspergillus terreus&#160;</td>
			<td></td>
			<td>ATCC 20542</td>
			<td></td>
		</tr>
		<tr>
			<td>Blue-LED illuminator</td>
			<td>MICROTEK</td>
			<td>Bio-1000F</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Thermo Scientific&#160;</td>
			<td>HERAEUS Megafuge 8</td>
			<td></td>
		</tr>
		<tr>
			<td>Compact UV lamp</td>
			<td>UVP</td>
			<td>UVGL-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl Acetate</td>
			<td>MACRON</td>
			<td>MA-H078-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter Paper 125mm</td>
			<td>ADVANTEC</td>
			<td>60311102</td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>NIH</td>
			<td>Freeware</td>
			<td>https://imagej.nih.gov/ij/download.html</td>
		</tr>
		<tr>
			<td>Lovastatin standard</td>
			<td>ACROS</td>
			<td>A0404262</td>
			<td></td>
		</tr>
		<tr>
			<td>MiBio Fluo&#160;</td>
			<td>MICROTEK</td>
			<td>V1.04</td>
			<td></td>
		</tr>
		<tr>
			<td>n-Hexane</td>
			<td>C-ECHO</td>
			<td>HH3102-000000-72EC</td>
			<td></td>
		</tr>
		<tr>
			<td>OriginPro</td>
			<td>OriginLab</td>
			<td>9.1</td>
			<td>https://www.originlab.com/origin</td>
		</tr>
		<tr>
			<td>Potato dextrose broth H</td>
			<td>STBIO MEDIA</td>
			<td>110533</td>
			<td></td>
		</tr>
		<tr>
			<td>Rotary evaporator</td>
			<td>EYELA</td>
			<td>SB-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Sulfuric acid</td>
			<td>Fluka</td>
			<td>30743-2.5L-GL</td>
			<td></td>
		</tr>
		<tr>
			<td>TLC silica gel 60 F254</td>
			<td>MERCK</td>
			<td>1.05554.0001</td>
			<td></td>
		</tr>
		<tr>
			<td>Trifluoroacetic acid</td>
			<td>Alfa Aesar</td>
			<td>10229873</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasonic vibration machine</td>
			<td>DELTA</td>
			<td>DC600</td>
			<td></td>
		</tr>
	</tbody>
</table>

estimating the yield of compounds on the tlc plate via the blue-led illumination technique

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.

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 w...

Watch this Scientific Journal Video about Estimating the Yield of Compounds on the TLC Plate via the Blue-LED Illumination Technique at JoVE.com

Estimating the Yield of Compounds on the TLC Plate via the Blue-LED Illumination Technique

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.

Estimating the Yield of Compounds on the TLC Plate via the Blue-LED Illumination Technique

Thin-layer chromatography (TLC) is an accessible analytical technique that has been extensively used in organic chemistry research to quantify the yield ...

This protocol developed a method to estimate the yield of compounds on the TLC plate using the blue-LED illumination technique. The advantages of this method are that the blue-LED illumination technique is a safe, effective and cheap method, and allows the researcher to measure multiple samples at the same time. This method also can be applied to biochemistry and natural products chemistry.To begin, dry the culture at 40 degrees Celsius for 24 hours. Transfer the dried culture into a 50 milliliter tube using sterilized tweezers and add 15 milliliters of ethyl acetate. Shake the mixture vigorously by vortexing for one minute and incubate the tube for one hour at 40 degrees Celsius with shaking at 200 RPM.Then sonicate the mixture using a 40 kilohertz ultrasonic bath at 40 degrees Celsius for one hour. Next, centrifuge the mixture at 5, 000 G for one minute at room temperature and filter through an 11 micrometer filter paper. Extract the filtrate with an equal volume of sterile water in a separatory funnel.After phase separation, collect the organic layer, evaporate it in a rotary evaporator, and dissolve the residue in two milliliters of ethyl acetate. Pack the column with normal phase silica gel as the stationary phase, and use n-hexane ethyl acetate and trifluoroacetic acid as the mobile phase. Load two milliliters of the extract onto the column and add the mobile phase solvent at a flow rate of one milliliter per minute to elute the extract.After verifying the effluent by TLC to confirm the presence of lovastatin, evaporate in a rotary evaporator at 45 degrees Celsius until the solvent gets removed. Dissolve the residue in one milliliter of ethyl acetate and then mix with an equal volume of 1%trifluoroacetic acid. Centrifuge the mixture at 5, 000 G for one minute at room temperature, and collect the organic layer in a new glass tube.Using a capillary pipette spot five microliters of samples and lovastatin standards onto the baseline of the TLC plate, leaving a border of one centimeter on the sides of the TLC plate. Then dry the TLC plate in a fume hood for five minutes 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 solvents line reaches one centimeter from the top of the plate. Mark the solvent line with a pencil and dry the plate in the fume hood for 10 minutes at room temperature.After drying, immediately soak the plate in 10%sulfuric acid solvent, and then dry in the fume hood for 10 minutes at room temperature. Next, place the plate on the heating panel until the brown spots appear. Transfer the plate to the blue-LED illuminator and scan using compatible freeware.The results obtained from the bioassay method demonstrated that the R square between the dimensions of the inhibition zone and lovastatin standards was 0.99, and the sample yield was 0.56 milligrams as predicted by the regression model. The UV detection method shows that the R square between the lovastatin standards and the dimension of bands on the TLC plate was 0.97, and the sample yield predicted by the regression model was 0.53 milligrams. However, the band's edges were blurry and relatively low signal intensity bands were observed.For the blue-LED illumination method, the R square between lovastatin standards and the band's dimension on the TLC plate was 0.98, and the sample yield was 0.54 milligrams as predicted by the regression model. The predicted yield using the blue-LED illuminator was closer to the bioassay method and clear bands were obtained. If the plate is overheated, the visualization of lovastatin may be difficult to observe.

estimating-yield-compounds-on-tlc-plate-via-blue-led-illumination

Research

JoVE Journal

Biology

1.5K visualizações.  National Sun Yat-sen University. Este protocolo desenvolveu um método para estimar o rendimento de compostos na placa TLC usando a técnica de iluminação azul-LED. As vantagens deste método são que a técnica de iluminação azul-LED é um método seguro, eficaz e barato, e permite ao pesquisador medir várias amostras ao mesmo tempo. Este método também pode ser aplicado à bioquímica e química de produtos naturais.Para começar, seque a cultura a 40 graus Celsius por 24 horas. Transfira a cultura seca para u...