Name: estimating the yield of compounds on the tlc plate via the blue-led illumination technique
Uploaded: 2022-10-06T13:10:00.000+00:00
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Description: Watch this Scientific Journal Video about Estimating the Yield of Compounds on the TLC Plate via the Blue-LED Illumination Technique at JoVE.com

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 h. Transfer the dried culture into a 50 mL tube using sterilized tweezers and add 15 mL ethyl acetate.</li>
	<li>Shake the mixture vigorously by vortexing for 1 min and incubate for 1 h at 40 &#176;C with shaking at 200 rpm.</li>
	<li>Sonicate the mixture using a 40 kHz ultrasonic bath (see Table of Materials) at 40 &#176;C for 1 h.</li>
	<li>Centrifuge the mixture at 5,000 x g for 1 min at room temperature and filter through an 11 &#181;m filter paper.</li>
	<li>Extract the filtrate with an equal volume of sterile water in a separatory funnel.</li>
	<li>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.</li>
</ol>
2. Separation of the crude extract by normal phase (NP) adsorption column
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>Verify the effluent by TLC to confirm the presence of lovastatin, and then evaporate in a rotary evaporator at 45 &#176;C until the solvent gets removed. This step takes approximately 20-25 min.</li>
	<li>Dissolve the residue in 1 mL of ethyl acetate, and then mix with an equal volume of 1% trifluoroacetic acid.</li>
	<li>Centrifuge the mixture at 5,000 x g for 1 min at room temperature and collect the organic layer in a new glass tube.</li>
</ol>
3. Preparation and loading of thin-layer chromatogram (TLC) plates
<ol>
	<li>Spot 5 &#181;L 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.</li>
	<li>Dry the TLC plate in a fume hood for 5 min at room temperature.</li>
	<li>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.</li>
	<li>Remove the plate from the chamber when the solvent line reaches 1 cm from the top of the plate.</li>
</ol>
4. Analysis by the blue-LED illuminator
<ol>
	<li>Mark the solvent line with a pencil. Dry the plate in the fume hood for 10 min at room temperature.</li>
	<li>After drying, immediately soak the plate in 10% H2SO4 solvent, and then dry in the fume hood for 10 min at room temperature.</li>
	<li>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.</li>
	<li>Transfer the plate to the blue-LED illuminator and scan using a compatible freeware (MiBio Fluo) (see Table of Materials).</li>
</ol>
5. Yield estimation by the regression model
<ol>
	<li>Measure the dimension of bands using the ImageJ software (see Table of Materials).</li>
	<li>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.</li>
	<li>Apply the regression model to estimate the yield of the samples.</li>
</ol>

<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. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Digitally+enhanced+thin-layer+chromatography:+An+inexpensive,+new+technique+for+qualitative+and+quantitative+analysis.">Digitally enhanced thin-layer chromatography: An inexpensive, new technique for qualitative and quantitative analysis.</a> Journal of Chemical Education. 84 (5), 842-847 (2007).</li><li>Ullah, Q., Mohammad, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vitamins+determination+by+TLC/HPTLC-a+mini-review.">Vitamins determination by TLC/HPTLC-a mini-review.</a> Journal of Planar Chromatography - Modern TLC. 33 (5), 429-437 (2020).</li><li>Chen, Z., Tao, H., Liao, L., Zhang, Z., Wang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quick+identification+of+xanthine+oxidase+inhibitor+and+antioxidant+from+Erycibe+obtusifolia+by+a+drug+discovery+platform+composed+of+multiple+mass+spectrometric+platforms+and+thin-layer+chromatography+bioautography.">Quick identification of xanthine oxidase inhibitor and antioxidant from Erycibe obtusifolia by a drug discovery platform composed of multiple mass spectrometric platforms and thin-layer chromatography bioautography.</a> Journal of Separation Science. 37 (16), 2253-2259 (2014).</li><li>Duncan, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chiral+separations:+A+comparison+of+HPLC+and+TLC.">Chiral separations: A comparison of HPLC and TLC.</a> Journal of Liquid Chromatography. 13 (14), 2737-2755 (1990).</li><li>Sherma, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thin-layer+chromatography+in+food+and+agricultural+analysis.">Thin-layer chromatography in food and agricultural analysis.</a> Journal of Chromatography A. 880 (1-2), 129-147 (2000).</li><li>Boche&#324;ska, P., Pyka, A., Boche&#324;ska, P., Boche&#324;ska, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+acetylsalicylic+acid+in+pharmaceutical+drugs+by+TLC+with+densitometric+detection+in+UV.">Determination of acetylsalicylic acid in pharmaceutical drugs by TLC with densitometric detection in UV.</a> Journal of Liquid Chromatography. 35 (10), 1346-1363 (2012).</li><li>Poole, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Planar+chromatography+at+the+turn+of+the+century.">Planar chromatography at the turn of the century.</a> Journal of Chromatography A. 856 (1-2), 399-427 (1999).</li><li>Rapid Johnson, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=simple+quantitation+in+thin-layer+chromatography+using+a+flatbed+scanner.">simple quantitation in thin-layer chromatography using a flatbed scanner.</a> Journal of Chemical Education. 77 (3), 368-372 (2000).</li><li>El-Gindy, A., Ashour, A., Abdel-Fattah, L., Shabana, M. 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. G., Ul Arfeen, ., Shoaib, Q., M, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+validation+of+TLC-densitometric+method+for+the+quantification+of+a+steroidal+drug,+danazol+in+its+pharmaceutical+formulations.">Development and validation of TLC-densitometric method for the quantification of a steroidal drug, danazol in its pharmaceutical formulations.</a> Journal of Planar Chromatography - Modern TLC. 25 (4), 331-337 (2012).</li></ol>

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><tbody><tr><td>American bacteriological Agar</td><td>Condalab</td><td>1802.00</td><td></td></tr><tr><td>&lt;em&gt;Aspergillus terreus&amp;nbsp;&lt;/em&gt;</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&amp;nbsp;</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&amp;nbsp;</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 ...

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1.7K Views.  National Sun Yat-sen University. 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.

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

Thin-Layer Chromatography

Chromatography
Chromatography is a technique used in organic chemistry to separate compounds in a mixture based on their differences in solubility between two different phases. The concept is similar to liquid-liquid extraction, except in chromatography, the two phases consist of the stationary phase and the mobile phase. The stationary phase is a solid &#8212; typically a microscale hydrogel bead &#8212; while the mobile phase is a carrier solvent.
In traditional chromatography, the stationary phase is packed into a vertical column, and the solution mixture introduced at the top of the column. As the mixture flows through the stationary phase, the compounds partition between the stationary phase and the mobile phase based on their structures and polarities, forming discrete bands. Solutes that interact weakly with the stationary phase move more quickly through the column and exit &#8212; or elute &#8212; first. Solutes that interact more strongly with the stationary phase move slowly through the column and elute later. The bands can be collected individually in order to isolate and purify compounds in a mixture.
There are several types of chromatography, each harnessing a different chemical property to achieve separation. For example, in ion-exchange chromatography, the stationary phase beads can be positively or negatively charged, attracting molecules with opposite charge only. In size exclusion chromatography, the porous stationary phase is composed of beads such as agarose or dextran polymers. Smaller molecules can enter the pores more readily, while the larger molecules flow past the pores and elute faster.
<h4>Thin-Layer Chromatography</h4>
Thin-layer chromatography (TLC) is a type of chromatography technique that separates compounds based on their polarity. Like traditional chromatography, there are three components of a TLC system: the stationary phase, the mobile phase, and the solute. However, unlike traditional chromatography, the stationary phase is arranged in a thin layer on a plate rather than packed into a column. TLC most often uses polar silica gel, a form of silicon dioxide, as the stationary phase. The stationary phase forms hydrogen bonds due to the OH groups on its surface.
First, a starting line is drawn on the bottom of the TLC plate using a pencil. The compounds or mixture being analyzed are spotted on the starting line using a thin capillary. Then, the bottom of the plate is immersed in the mobile phase, which is usually an organic solvent that is less polar than the stationary phase. The solvent travels up the plate by capillary action, passing the solute spots and carrying some of each component with it.
As the solvent travels up the plate, the components partition to either the mobile phase or the stationary phase. If the component is polar, it interacts more with the polar stationary phase. It travels slowly and only moves a short distance on the TLC plate. If the component of the sample is less polar &#8212; and more soluble in the mobile phase than it is in the stationary phase &#8212; it interacts more with the mobile phase and travels farther on the TLC plate. The extent of the polarity of the component and the mobile phase are essential to understanding and predicting the separation.
TLC plates typically contain a UV-reactive fluorescent dye that will glow under a UV source of 254 nanometers. Therefore, TLC plates can be analyzed by observing them under UV light. Compounds within the TLC plate, such as the solutes of interest, will show up as dark spots compared to a green background. By circling the spots with a graphite pencil, the distance the compounds traveled relative to the solvent front can be measured. The spot of the organic compound, if not fluorescent itself, masks the fluorescence of the plate and shows up as a dark spot. Some organic compounds are UV-active and emit light when exposed to UV light. These are typically conjugated compounds, meaning those with alternating double and single bonds, and can be identified by the wavelength emitted.
<h4>Retardation Factor</h4>
By analyzing the retardation factor (Rf) of a component with a specific solvent, an unknown solute can be determined using TLC. The retardation factor is the ratio of the distance traveled by a component to the distance traveled by the mobile phase.
<img src="https://www.jove.com/CDNSource/lm/labs/62/62_Concepts_1.jpg" />
The distance traveled by the solute is measured from the starting line to the center point of the spot, and the distance traveled by the mobile phase is measured from the same starting line to the solvent front. The retardation factor of a compound is dependent on the mobile phase used. The retardation factor is large for compounds that are highly non-polar with a non-polar mobile phase. Low retardation factor values are seen for polar components with a non-polar mobile phase.
In the case of a highly non-polar mobile phase, some polar components may not move at all. This results in an extremely low retardation factor and an insufficient separation. A highly polar mobile phase causes the compound to move with the solvent and yields an extremely high retardation factor. This results in very little separation between the components.
For separation to be effective, the retardation factors of the components should be about 0.3 - 0.7 apart. To find the efficient mobile phase, trial and error is employed. Often, a mixture of two solvents proves most effective.
<h4>References</h4>
<ol>
	<li style="font-size:17px">Harris, D.C. (2015). Quantitative Chemical Analysis. New York, NY: W.H. Freeman and Company.</li>
</ol>

Thin-Layer Chromatography: Experimental Setup and Separation - Concept

Chromatography
Chromatography is a technique used in organic chemistry to separate compounds in a mixture based on their differences in solubility between ...

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Thin-layer Chromatographic (TLC) Separations and Bioassays of Plant Extracts to Identify Antimicrobial Compounds

A common screen for plant antimicrobial compounds consists of separating plant extracts by paper or thin-layer chromatography (PC or TLC), exposing the chromatograms to microbial suspensions (e.g. fungi or bacteria in broth or agar), allowing time for the microbes to grow in a humid environment, and visualizing zones with no microbial growth.&#160;The effectiveness of this screening method, known as bioautography, depends on both the quality of the chromatographic separation and the care taken with microbial culture conditions.&#160;This paper describes standard protocols for TLC and contact bioautography&#160;with a novel application to amino acid-fermenting bacteria. The extract is separated on flexible (aluminum-backed) silica TLC plates, and bands are visualized under ultraviolet (UV) light.&#160;Zones are cut out and incubated face down onto agar inoculated with the test microorganism.&#160;Inhibitory bands are visualized by staining the agar plates with tetrazolium red.&#160;The method is applied to the separation of red clover (Trifolium pratense cv. Kenland) phenolic compounds and their screening for activity against Clostridium sticklandii, a hyper ammonia-producing bacterium (HAB) that is native to the bovine rumen.&#160;The TLC methods apply to many types of plant extracts&#160;and other bacterial species (aerobic or anaerobic), as well as fungi, can be used as test organisms if culture conditions are modified to fit the growth requirements of the species.

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Current automated radiosynthesizers are designed to produce large clinical batches of radiopharmaceuticals. They are not well suited for reaction ...

Analysis of the Lipid Composition of Mycobacteria by Thin Layer Chromatography

Mycobacteria species can differ from one another in the rate of growth, presence of pigmentation, the colony morphology displayed on solid media, as well as other phenotypic characteristics. However, they all have in common the most relevant character of mycobacteria: its unique and highly hydrophobic cell wall. Mycobacteria species contain a membrane-covalent linked complex that includes arabinogalactan, peptidoglycan, and long-chains of mycolic acids with types that differ between mycobacteria species. Additionally, mycobacteria can also produce lipids that are located, non-covalently linked, on their cell surfaces, such as phthiocerol dimycocerosates (PDIM), phenolic glycolipids (PGL), glycopeptidolipids (GPL), acyltrehaloses (AT), or phosphatidil-inositol mannosides (PIM), among others. Some of them are considered virulence factors in pathogenic mycobacteria, or critical antigenic lipids in host-mycobacteria interaction. For these reasons, there is a significant interest in the study of mycobacterial lipids due to their application in several fields, from understanding their role in the pathogenicity of mycobacteria infections, to a possible implication as immunomodulatory agents for the treatment of infectious diseases and other pathologies such as cancer. Here, a simple approach to extract and analyze the total lipid content and the mycolic acid composition of mycobacteria cells grown in a solid medium using mixtures of organic solvents is presented. Once the lipid extracts are obtained, thin-layer chromatography (TLC) is performed to monitor the extracted compounds. The example experiment is performed with four different mycobacteria: the environmental fast-growing Mycolicibacterium brumae and Mycolicibacterium fortuitum, the attenuated slow-growing Mycobacterium bovis bacillus Calmette-Gu&#233;rin (BCG), and the opportunistic pathogen fast-growing Mycobacterium abscessus, demonstrating that methods shown in the present protocol can be used to a wide range of mycobacteria.

A protocol is presented to extract the total lipid content of the cell wall of a wide range of mycobacteria. Moreover, extraction and analytical protocols of the different types of mycolic acids are shown. A thin-layer chromatographic protocol to monitor these mycobacterial compounds is also provided.

Mycobacteria species can differ from one another in the rate of growth, presence of pigmentation, the colony morphology displayed on solid media, as well ...

Fast Colony Forming Unit Counting in 96-Well Plate Format Applied to the Drosophila Microbiome

The intestines of animals are colonized by commensal microbes, which impact host development, health, and behavior. Precise quantification of colonization is essential for studying the complex interactions between host and microbe both to validate the microbial composition and study its effects. Drosophila melanogaster,&#160;which has a low native microbial diversity and is economical to rear with defined microbial composition, has emerged as a model organism for studying the gut microbiome. Analyzing the microbiome of an individual organism requires identification of which microbial species are present and quantification of their absolute abundance. This article presents a method for the analysis of a large number of individual fly microbiomes. The flies are prepared in 96-well plates, enabling the handling of a large number of samples at once. Microbial abundance is quantified by plating up to 96 whole fly homogenates on a single agar plate in an array of spots and then counting the colony forming units (CFUs) that grow in each spot. This plating system is paired with an automated CFU quantification platform, which incorporates photography of the plates, differentiation of fluorescent colonies, and automated counting of the colonies using an ImageJ plugin. Advantages are that (i) this method is sensitive enough to detect differences between treatments, (ii) the spot plating method is as accurate as traditional plating methods, and (iii) the automated counting process is accurate and faster than manual counting. The workflow presented here enables high-throughput quantification of CFUs in a large number of replicates and can be applied to other microbiology study systems including in vitro and other small animal models.

Fast Colony Forming Unit Counting in 96-Well Plate Format Applied to the Drosophila Microbiome

This method quantifies microbial abundance using a 96-well plate format to plate colony forming units (CFUs) and is applied to the Drosophila microbiome in whole fly homogenate samples. CFUs are counted with an automated image analysis software provided here.

The intestines of animals are colonized by commensal microbes, which impact host development, health, and behavior. Precise quantification of colonization ...

Performing 1D Thin Layer Chromatography

Source: Laboratory of Dr. Yuri Bolshan &#8212; University of Ontario Institute of Technology
Thin layer chromatography (TLC) is a chromatographic method used to separate mixtures of non-volatile compounds. A TLC plate consists of a thin layer of adsorbent material (the stationary phase) fixed to an appropriate solid support such as plastic, aluminum, or glass1. The sample(s) and reference compound(s) are dissolved in an appropriate solvent and applied near the bottom edge of the TLC plate in small spots. The TLC plate is developed by immersing the bottom edge in the developing solvent consisting of an appropriate mobile phase. Capillary action allows the mobile phase to move up the adsorbent layer.&#160;As the solvent moves up the TLC plate, it carries with it the components of each spot and separates them based on their physical interactions with the mobile and stationary phases.

Thin Layer Chromatography: Principle, Procedure, Applications

Source: Laboratory of Dr. Yuri Bolshan &#8212; University of Ontario Institute of Technology
Thin layer chromatography (TLC) is a chromatographic method ...

JoVE Science Education

Organic Chemistry

Thin Layer Chromatography-Based Separation of Mycolic Acid Variants: A Method to Separate Mycobacterial Cell Wall Lipids Based on Polarity

Source: Guallar-Garrido, S., et al.&#160; <a href="https://www.jove.com/v/62368">Analysis of the Lipid Composition of Mycobacteria by Thin Layer Chromatography</a>. J. Vis. Exp. (2021).
This video demonstrates the thin-layer chromatography-based separation of mycolic acids. This technique separates the different mycolic acids&#8212;long-chain lipids present in the cell wall of mycobacteria&#8212;based on their polarity.

Source: Guallar-Garrido, S., et al.&#160; Analysis of the Lipid Composition of Mycobacteria by Thin Layer Chromatography. J. Vis. Exp. (2021).
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JoVE Encyclopedia of Experiments

Biological Techniques

Chromatography Techniques

Thin Layer Chromatography Based Enzyme Activity Assay: A Technique to Determine In Vitro Pyrophosphokinase Activity Using Thin Layer Chromatography

Source: Pokhrel, A., et al. A <a href="https://www.jove.com/v/58547">Purification and In Vitro Activity Assay for a (p)ppGpp Synthetase from Clostridium difficile</a>. J. Vis. Exp. (2018).
This video demonstrates a thin-layer chromatography-based enzyme assay to determine pyrophosphokinase activity using radiolabeled nucleotides. The method provides insights into nucleotide synthesis and phosphotransfer reactions.

Source: Pokhrel, A., et al. A Purification and In Vitro Activity Assay for a (p)ppGpp Synthetase from Clostridium difficile. J. Vis. Exp. (2018).
This ...

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

Summary

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Optimization of Radiochemical Reactions using Droplet Arrays

Analysis of the Lipid Composition of Mycobacteria by Thin Layer Chromatography

Fast Colony Forming Unit Counting in 96-Well Plate Format Applied to the Drosophila Microbiome

Performing 1D Thin Layer Chromatography

Thin Layer Chromatography-Based Separation of Mycolic Acid Variants: A Method to Separate Mycobacterial Cell Wall Lipids Based on Polarity

Thin Layer Chromatography Based Enzyme Activity Assay: A Technique to Determine In Vitro Pyrophosphokinase Activity Using Thin Layer Chromatography