We thank the late Dr. Norm Taylor, Dept. Plant and Soil Science at the University of Kentucky, for allowing us to use samples from his red clover plots for this study. This project was funded by the United States Department of Agriculture.

1. Preparation of Plant Extract
<ol>
	<li>See Kagan and Flythe10 for extraction of phenolic compounds from Trifolium pratense cv. Kenland.&#160;</li>
	<li>To extract other compounds in other plants, check the phytochemical analysis literature for plant- or metabolite-specific extraction methods (many are described), or look for protocols such as those of Khurram et al.7,8 which &#160;isolate many compounds with a wide range of polarities.</li>
</ol>
2. Preparation of Thin-layer Plates
<ol>
	<li>Clean TLC plates by developing in one or more polar, neutral solvents, in order to move adsorbed contaminants away from the zone of development.
	<ol>
		<li>In a fume hood, prepare enough cleaning solvent (e.g. 15-100 ml of ethyl acetate-methanol 2:1, v/v) to cover the bottom of the TLC developing chamber, as well as the lower edge of a TLC plate when set inside the chamber.</li>
		<li>Use commercially available glass TLC developing chambers (different sizes available, with lids) or foil-covered Pyrex beakers or preserving jars.</li>
		<li>Use scissors to cut aluminum- or plastic-backed (flexible) silica gel plates, which come in various sizes (20 cm x&#160;20 cm and smaller), to fit the available developing chamber. (Caution:&#160;silica can cause lung damage if inhaled. Work in a fume hood, and handle TLC plates with gloves to avoid getting skin oils onto the silica.)</li>
		<li>Insert the plates into the chamber, with the tops leaning against the chamber walls.&#160;Plates should not touch each other.&#160;Cover the chamber and let the solvent move up the plate by capillary action.</li>
		<li>When solvent has reached the top of the plates, remove plates from chamber and arrange in a standing position within the fume hood until solvent has evaporated.</li>
		<li>Check to see if impurities have migrated near the top of the TLC plate by looking for a yellow band under visible light, or a fluorescent band under ultraviolet (UV) light (see the &#8220;impurity front&#8221; or IF in Figure 2B).&#160;If the majority of the plate still has a yellowish tinge, repeat the cleaning process.</li>
		<li>After removing TLC plates from the chamber, discard the solvent. Allow residual solvent to evaporate completely before using the chamber for Protocol 2.</li>
		<li>To remove residual moisture that can affect migration of compounds on silica16, prop the plates upright in a drying oven at 100 &#176;C&#160; (10-15 min for a 20 cm x&#160;20 cm plate, and 5 min for 7 cm x&#160;10 cm plates).</li>
		<li>If a 100 &#176;C drying oven is not available, heat plates for a longer period of time at lower temperatures (i.e. 40 min at 60 &#176;C).</li>
		<li>After the plates are dry, let them cool to ambient temperature before loading.</li>
	</ol>
	</li>
</ol>
3. Preparation of Developing Chambers for Extract Separation
<ol>
	<li>Use scissors to cut a piece of filter paper slightly below chamber height, and about half the chamber perimeter in width.&#160;This paper acts as a wick to draw solvent up the chamber wall and saturate the chamber with solvent vapors, thus improving reproducibility of separations1.</li>
	<li>In a fume hood, mix solvents (ethyl acetate-methanol 4:1, v/v, for this study). Pour solvent mixture into the chamber&#160;and cover. Wait until the entire wick is wet with solvent, indicating chamber saturation, to put plates into chamber.</li>
</ol>
4. Loading and Development of TLC Plates
<ol>
	<li>Lightly mark the origin with pencil.&#160;If the TLC plate adsorbent is soft and easily damaged, make marks at edges. Compounds should be above the surface of the developing solvent when plates are inserted into the TLC chamber.</li>
	<li>Dissolve extracts in enough organic solvent (in this case, methanol) to have a concentrated solution instead of a turbid suspension.</li>
	<li>Load samples and standards as narrow bands with a microliter syringe or capillary micropipettes, leaving a 1&#160;cm border on the sides of the plate. Allow the bands to dry (fanning the plate or loading it in a fume hood helps).</li>
	<li>If a greater concentration of sample is needed on a plate, &#34;overspot&#34; by loading samples again on the dried bands.</li>
	<li>With forceps or tongs, set plate(s) inside the saturated TLC chamber.&#160;Plates should not touch the wick because it may provide solvent to the plates at points of contact, thus altering the path of compound migration.&#160;Cover chamber and let plates develop.</li>
</ol>
5. Preparation of Plates for Bioassay
<ol>
	<li>Remove plate(s) from TLC tank before the solvent front reaches the top of the plate, and mark the height of the solvent front with a pencil.&#160;Let plate dry in fume hood.
	<ol>
		<li>Develop any remaining TLC plates in the same TLC chamber, which is generally usable for an entire day if kept closed.&#160;Remake solvent mixture for TLC chamber if the amount of solvent in the chamber decreases notably.</li>
	</ol>
	</li>
	<li>After plates are dry, visualize bands under visible or UV light, and delineate bands with a pencil.&#160;A viewing chamber with a portable UV lamp is convenient, especially if the lamp can detect compounds at both 254 nm (short-wave UV) and 365 nm (long-wave UV). 
	Note: At this point or after the next step, plates can be wrapped in plastic wrap, covered with foil, and stored at -20 &#176;C. Maximum storage time depends on compound stability. Because silica is not neutral, some compounds may degrade while on the TLC plate.</li>
	<li>Photograph or draw plates.
	<ol>
		<li>Use a gel photodocumentation system if one equipped with overhead UV and/or visible lights is available.</li>
		<li>If no commercial photodocumentation system is available, photograph plates inside of a box lined with dark paper or cloth, with a camera set on a tripod and a portable UV light clamped to a ring stand17. When photographing plates that have no fluorescent indicator, use a blue light filter over the camera lens to improve band appearance17.</li>
	</ol>
	</li>
	<li>To aid in characterizing bands, calculate retention factor (Rf) values by measuring distances traveled by compound and solvent front, and dividing the former by the latter (Figure 2A).</li>
</ol>
6. Bacterial Culture and Assay
<ol>
	<li>Prepare and inoculate media under anaerobic conditions.&#160;The culture used in this case was Clostridium sticklandii strain SR, which was obtained from the culture collection of James B. Russell, Cornell University, Ithaca, NY.
	<ol>
		<li>See Flythe and Kagan13 and the materials list for a description of HAB media.</li>
	</ol>
	</li>
	<li>Grow the culture to exponential or stationary phase. Use sterile anaerobic techniques when working with anaerobic microorganisms.</li>
	<li>Inoculate the culture (1% v/v) into molten agar after the temperature of the agar (0.75% w/v) has decreased to less than 60 &#176;C.&#160;Mix gently and immediately bring into an anaerobic chamber (95% CO2-5% H2 atmosphere for HAB media) to pour.</li>
	<li>Pour into 15 mm x&#160;100 mm plastic Petri dishes, and allow to solidify.</li>
	<li>With scissors, cut the TLC plate into zones containing band(s) of interest.&#160;Lay bands face down onto agar plates, marking bands on the plate backing to keep track of original orientation on TLC plate.&#160;Lay an unused TLC strip onto an agar plate as a control.</li>
	<li>Incubate agar plates, agar side down, in the anaerobic chamber (24 hr, 39 &#176;C).</li>
</ol>
7. Visualization of Bioassayed Agar Plates
<ol>
	<li>With forceps, remove bands from agar plates while they are in the anaerobic chamber.</li>
	<li>Add 1% (w/v) tetrazolium red, prepared in water, dropwise onto the surfaces of the agar plates.&#160;Allow the color to develop for at least 20 min, or until the control plate turns completely red, before removing from the anaerobic chamber. The anaerobic bacteria will begin to lose viability immediately after removal from the anaerobic chamber, but the color is stable for more than 24 hr.</li>
	<li>Photograph under visible light.</li>
</ol>

<ol>
	<li>Stahl, E., Ashworth, M. R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Thin-layer chromatography. , (1969).</li><li>Marston, A. <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+with+biological+detection+in+phytochemistry.">Thin-layer chromatography with biological detection in phytochemistry.</a> J. Chromatogr. A. 1218 (19), 2676-2683 .</li><li>Homans, A. L., Fuchs, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+bioautography+on+thin-layer+chromatograms+as+a+method+for+detecting+fungitoxic+substances.">Direct bioautography on thin-layer chromatograms as a method for detecting fungitoxic substances.</a> J. Chromatogr. 51, 327-329 .</li><li>Lund, B. M., Lyon, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+inhibitors+of+Erwinia+carotovora+and+E.+herbicola+on+thin-layer+chromatograms.">Detection of inhibitors of Erwinia carotovora and E. herbicola on thin-layer chromatograms.</a> J. Chromatogr. 110, 193-196 (1975).</li><li>Betina, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioautography+in+paper+and+thin-layer+chromatography+and+its+scope+in+the+antibiotic+field.">Bioautography in paper and thin-layer chromatography and its scope in the antibiotic field.</a> J. Chromatogr. A. 78, 41-51 (1973).</li><li>Weltzien, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ein+biologischer+Test+f&#252;r+fungizide+Substanzen+auf+dem+Papierchromatogramm.">Ein biologischer Test f&#252;r fungizide Substanzen auf dem Papierchromatogramm.</a> Naturwissenschaften. 45, 288-289 (1958).</li><li>Khurram, M., Khan, A. M., Hameed, A., Abbas, N., Quayum, A., Inayat, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibacterial+activities+of+Dodonaea+viscosa+using+contact+bioautography+technique.">Antibacterial activities of Dodonaea viscosa using contact bioautography technique.</a> Molecules. 14 (3), 1332-1341 (2009).</li><li>Khurram, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+anticandidal+potential+of+Quercus+baloot+Griff.+using+contact+bioautography+technique.">Evaluation of anticandidal potential of Quercus baloot Griff. using contact bioautography technique.</a> Afr. J. Pharm. Pharmacol. 5 (12), 1538-1542 (2012).</li><li>Robinson, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Organic Constituents of Higher Plants. , (1963).</li><li>Kagan, I. A., Flythe, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factors+affecting+the+separation+and+bioactivity+of+red+clover+(Trifolium+pratense)+extracts+assayed+against+Clostridium+sticklandii,+a+ruminal+hyper+ammonia-producing+bacterium.">Factors affecting the separation and bioactivity of red clover (Trifolium pratense) extracts assayed against Clostridium sticklandii, a ruminal hyper ammonia-producing bacterium.</a> Nat. Prod. Commun. 7 (12), 1605-1608 (2012).</li><li>Mattila, P., Kumpulainen, J. <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+free+and+total+phenolic+acids+in+plant-derived+foods+by+HPLC+with+diode-array+detection.">Determination of free and total phenolic acids in plant-derived foods by HPLC with diode-array detection.</a> J. Agric. Food Chem. 50 (13), 3660-3667 (2002).</li><li>Hamburger, M. O., Cordell, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+direct+bioautographic+TLC+assay+for+compounds+possessing+antibacterial+activity.">A direct bioautographic TLC assay for compounds possessing antibacterial activity.</a> J. Nat. Prod. 50 (1), 19-22 (1987).</li><li>Flythe, M., Kagan, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antimicrobial+effect+of+red+clover+(Trifolium+pratense)+phenolic+extract+on+the+ruminal+hyper+ammonia-producing+bacterium,+Clostridium+sticklandii.">Antimicrobial effect of red clover (Trifolium pratense) phenolic extract on the ruminal hyper ammonia-producing bacterium, Clostridium sticklandii.</a> Curr. Microbiol. 61, 125-131 .</li><li>Rahalison, L., Hamburger, M., Hostettmann, K., Monod, M., Frenk, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+bioautographic+agar+overlay+method+for+the+detection+of+antifungal+compounds+from+higher+plants.">A bioautographic agar overlay method for the detection of antifungal compounds from higher plants.</a> Phytochem. Anal. 2 (5), 199-203 (1991).</li><li>Chomnawang, M. T., Trinapakul, C., Gritsanapan, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+antigonococcal+activity+of+Coscinium+fenestratum+stem+extract.">In vitro antigonococcal activity of Coscinium fenestratum stem extract.</a> J. Ethnopharmacol. 122, 445-449 (2009).</li><li>Stahl, E., Kaldewey, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spurenanalyse+physiologisch+aktiver,+einfacher+Indolderivate.">Spurenanalyse physiologisch aktiver, einfacher Indolderivate.</a> Hoppe-Seyler&#8217;s Z. Physiol. Chem. 323, 182-191 .</li><li>Kagan, I. A., Hammerschmidt, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arabidopsis+ecotype+variability+in+camalexin+production+and+reaction+to+infection+by+Alternaria+brassicicola.">Arabidopsis ecotype variability in camalexin production and reaction to infection by Alternaria brassicicola.</a> J. Chem. Ecol. 28 (11), 2121-2140 (2002).</li><li>Kline, R. M., Golab, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+technique+in+developing+thin-layer+bioautographs.">A simple technique in developing thin-layer bioautographs.</a> J. Chromatogr. 18, 409-411 (1965).</li><li>Wedge, D. E., Nagle, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+2D-TLC+bioautography+method+for+the+discovery+of+novel+antifungal+agents+to+control+plant+pathogens.">A new 2D-TLC bioautography method for the discovery of novel antifungal agents to control plant pathogens.</a> J. Nat. Prod. 63 (8), 1050-1054 (2000).</li><li>Beck, A. B., Knox, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+acylated+isoflavone+glycosides+from+subterranean+clover+and+red+clover.">The acylated isoflavone glycosides from subterranean clover and red clover.</a> Aust. J. Chem. 24 (7), 1509-1518 (1971).</li><li>Kahn, R. A., Bak, S., Svendsen, I., Halkier, B. A., M&#248;ller, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+reconstitution+of+cytochrome+P450ox+and+in+vitro+reconstitution+of+the+entire+biosynthetic+pathway+of+the+cyanogenic+glucoside+dhurrin+from+sorghum.">Isolation and reconstitution of cytochrome P450ox and in vitro reconstitution of the entire biosynthetic pathway of the cyanogenic glucoside dhurrin from sorghum.</a> Plant Physiol. 115 (4), (1997).</li><li>Peterson, C. A., Edgington, L. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+estimation+of+the+fungicide+benomyl+using+a+bioautograph+technique.">Quantitative estimation of the fungicide benomyl using a bioautograph technique.</a> J. Agr. Food Chem. 17 (4), 898-899 (1969).</li></ol>

Mention of trade names or commercial products in the article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the USDA. The authors declare no competing financial interest.

This protocol describes a simple method for separating an extract into subsets of compounds and assaying those subsets by contact bioautography.&#160;The method is quite similar to one used by Chomnawang et al.15 to screen for plant metabolites inhibitory to gonorrhea-causing bacteria. The type of bioautography employed to screen for antimicrobial plant compounds depends on many factors, including the test microorganism, the laboratory setup, and the preferences of the person(s) performing the bioassa...

Assaying for antimicrobial compounds in plants requires separating the components of a plant extract, exposing a test microorganism to those components, and determining whether the microorganism&#8217;s growth is inhibited by any of the compounds. Separations by paper or thin-layer chromatography (PC or TLC) are convenient because many compounds can be separated on a planar surface.&#160;Separation is based on polarity, with some compounds binding tightly to the adsorbent (cellulose in the case of PC, and a variety of adsorbents in the case of TLC) and migrating less than others1. Figure 1 provides an example of the relative positions of polar and nonpolar phenolic compounds after separation on a silica TLC plate.
<img alt="Figure 1" fo:content-width="5in" fo:src="/files/ftp_upload/51411/51411fig1highres.jpg" src="/files/ftp_upload/51411/51411fig1.jpg" /> 
Figure 1. Diagram illustrating distributions of compounds of different polarities after separation on a silica thin-layer chromatographic (TLC) plate.&#160;Phenolic compounds of red clover (Trifolium pratense L.) are used as an example.&#160;Polar compounds, such as clovamide, have a strong affinity for a polar adsorbent like silica and remain near the origin (OR), while less polar compounds, such as the three isoflavones near the solvent front (SF), partition more readily into the solvents (which are less polar than silica unless water, acids, or bases are included) and migrate farther up the plate.
After separation of an extract on a TLC plate, test microorganisms can be exposed to all compounds on the plate, thus speeding the identification of the active components of an extract2.&#160;If a fungal or bacterial culture is exposed to the chromatogram, microbial growth will occur everywhere except over areas with growth-inhibitory compounds. Zones of inhibition then can be visualized by observing the contrast between mycelial growth and the growth-free areas if fungi have been applied3&#160;or by spraying with compounds that change color when reduced or hydrolyzed by living cells4.&#160;Although the use of paper or thin-layer chromatograms for antimicrobial assays was first applied to antibiotics5 and fungicides3,6, plant extracts are now frequently screened for antimicrobial compounds with this method, often referred to as bioautography.&#160;The protocols described herein apply to bioautography of thin-layer chromatograms.&#160;TLC is widely used because it is relatively rapid and can be performed on different adsorbents (e.g. silica, starch, alumina), as well as providing good resolution and sensitivity1.
Plant extracts can be prepared for TLC in many ways.&#160;Common methods include extracting plant material in alcohol-water mixtures such as 80% ethanol7,8, possibly with the addition of acid or base9. Following an extraction in such solvents, which contain some water and are possibly acidic or basic, extracts must be concentrated so that they can be applied to TLC plates in a minimal volume.&#160;The concentration of alcohol-water extracts can be achieved by partitioning with water-immiscible organic solvents8&#160;or with a mixture of such solvents, such as ethyl acetate-ethyl ether (1:1, v/v)10,11. Different plant metabolites are extracted into different organic solvents, depending on their polarities.&#160;To ensure that plant organic acids or bases are extracted into organic solvents at this stage, the pH of an alcohol-water extract can be raised or lowered with a water-soluble acid or base to convert dissociated analytes into their nondissociated forms, which are then soluble in neutral organic solvents9. The organic phase can then be evaporated under reduced pressure or under nitrogen&#160;and adjusted to the desired volume for TLC.&#160;The pH of the extract is unlikely to be lethal to bioassay microorganisms due to the partitioning of analytes into neutral solvents, small final volume, and evaporation of the extract on the TLC plate prior to separation.
Both fungi and bacteria are employed as test microorganisms in bioautography of plant extracts2.&#160;Spores of some fungi, such as Cladosporium cucumerinum, germinate on TLC plates (apart from areas with inhibitory compounds) if sprayed onto plates in a nutrient solution and incubated in a moist environment for several days3.&#160;The dark mycelium of C. cucumerinum on noninhibitory zones provides a sharp contrast to zones free of mycelial growth.&#160;Although bacteria have been applied to thin-layer chromatography (TLC) plates in the same manner4,12, bacteria are also poured over TLC plate surfaces in agar overlays13,14.&#160;Yeast, such as Candida albicans, can be applied in agar overlays as well14.&#160;Alternatively, TLC plates can be placed face down onto agar inoculated with bacteria10,15 or yeast8, a method known as contact bioautography2.
We describe a method for contact bioautography to screen for antimicrobial phenolic compounds from red clover (Trifolium pratense cv. Kenland).&#160;The test microorganism is Clostridium sticklandii, a ruminal hyper ammonia-producing bacterium (HAB) and obligate anaerobe.&#160;Although the separations used do not resolve all components of the extract, they facilitate identification of zones of antimicrobial activity, thus narrowing the pool of possible antimicrobial compounds. The protocol utilizes standard procedures for TLC1. The protocol also describes some of the techniques required for culturing obligate anaerobes for such an assay, a usage of contact bioautography15&#160;and a visualization method with a tetrazolium salt, which stains living cells2,4.

<table border="0" cellpadding="0" cellspacing="0" width="739">
	<tbody>
		<tr>
			<td>Silica F254 TLC plates, aluminum-backed, 0.2 mm thickness, 20 &#215; 20 cm</td>
			<td>EMD Chemicals&#160;</td>
			<td>5554/7</td>
			<td>These plates are coated with silica that contains an indicator fluorescing at 254 nm.&#160; Compounds absorbing at that wavelength appear dark on a fluorescent green background.&#160; Alternative sources include Analtech, Selecto Scientific, Fluka.&#160; Adsorbents other than silica may be needed.&#160; Plastic-backed plates may be suitable, depending on the solvents to be used.&#160;&#160;</td>
		</tr>
		<tr>
			<td>Sharp, heavy-duty scissors&#160;</td>
			<td>any sewing supply company</td>
			<td>similar to Fiskars&#160; 175800-1002</td>
			<td>For cutting TLC plates.&#160; A paper cutter with a sharp blade can be used as well.&#160; Do not inhale silica dust.</td>
		</tr>
		<tr>
			<td>Drying oven at 100 &#176;C (mechanical convection)</td>
			<td>Thermo Scientific</td>
			<td>PR305225M</td>
			<td>Quincy Lab, Inc, Chicago, IL (www.quincylab.com); Cascade Technical Sciences, Hillsboro, OR (www.cascadetek.com)</td>
		</tr>
		<tr>
			<td>TLC chamber</td>
			<td>Kimble Chase&#160;</td>
			<td>416180-0000</td>
			<td>Alternative sources:&#160; Aldrich. Pyrex beakers or preserving jars can be used for small plates (i.e. 5 &#215; 10 cm).&#160; Cover with aluminum foil (jar lids may contain material extractable by solvent vapors).</td>
		</tr>
		<tr>
			<td>50-&#181;L syringe with flat needle tip</td>
			<td>Hamilton</td>
			<td>80965</td>
			<td>For loading amounts of standard or sample exceeding 5-10 &#181;L.&#160; Alternative sources are equivalent.</td>
		</tr>
		<tr>
			<td>micropipets</td>
			<td>Drummond</td>
			<td>2-000-001</td>
			<td>For loading small amounts of standards or samples.&#160; Alternative sources:&#160; VWR.&#160; Also, Pasteur pipets can be stretched to a thinner diameter with a butane torch.&#160;&#160;</td>
		</tr>
		<tr>
			<td>Filter paper (#1 grade)</td>
			<td>Whatman</td>
			<td>1001 917</td>
			<td>Serves as a chamber wick.&#160; Other grades of filter paper are OK.&#160; This size can be trimmed for the chambers holding 20 &#215; 20 cm plates.&#160;&#160;&#160;&#160;</td>
		</tr>
		<tr>
			<td>Beaker tongs</td>
			<td>Fisher Scientific</td>
			<td>15-186</td>
			<td>For putting plates in and out of a large TLC chamber.&#160; Alternate sources: VWR&#160;</td>
		</tr>
		<tr>
			<td>Flat-edge forceps&#160;</td>
			<td>Fisher Scientific</td>
			<td>10-275</td>
			<td>For putting plates in and out of a small chamber.&#160;&#160; Alternate sources: VWR&#160;</td>
		</tr>
		<tr>
			<td>Small portable UV lamp with 4-Watt or 6-Watt bulbs for short- and long-wave UV light illumination (254 and 365 nm, respectively)</td>
			<td>Ultraviolet Products&#160;</td>
			<td>95-0271-01</td>
			<td>Alternate sources: Spectronics Corporation (www.spectroline.net)</td>
		</tr>
		<tr>
			<td>Viewing cabinet for use with hand-held UV lamp</td>
			<td>Ultraviolet Products&#160;</td>
			<td>Chromato-Vue C-10E</td>
			<td>UV-active bands are more easily circled if plates can be set in here.&#160; Alternate sources: Spectronics Corporation.&#160;</td>
		</tr>
		<tr>
			<td>Photodocumentation system with overhead UV lamp and visible lamp</td>
			<td>Kodak&#160;</td>
			<td>Gel Logic 200</td>
			<td>&#160;Alternate sources: Ultraviolet Products (www.uvp.com).&#160; See protocol for homemade alternative.</td>
		</tr>
		<tr>
			<td>Anaerobic Chamber, Type A, Vinyl</td>
			<td>Coy&#160;</td>
			<td>7150000</td>
			<td>This chamber is appropriate for anaerobic bacteria, like Clostridium sticklandii, as described. &#160;However, growth conditions must be tailored to organism used in the assay.&#160; A biosafety cabinet and other precautions should be taken if pathogenic organisms are used. Alternate sources: Anaerobe Systems, BioRad, Plas Labs, others&#160;</td>
		</tr>
		<tr>
			<td>Tetrazolium red</td>
			<td>Sigma-Aldrich</td>
			<td>T8877</td>
			<td>Alternate sources: MP Biomedicals, Santa Cruz Biotechnology, Alfa Aesar</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Ingredients for HAB media</td>
		</tr>
		<tr>
			<td>Pyridoxamine &#183; 2 HCl</td>
			<td>Sigma-Aldrich</td>
			<td>P9380</td>
			<td>For this and for all the other reagents in this table, alternative sources are equivalent.</td>
		</tr>
		<tr>
			<td>Riboflavin</td>
			<td>Sigma-Aldrich</td>
			<td>R4500</td>
			<td></td>
		</tr>
		<tr>
			<td>Thiamine HCl</td>
			<td>Sigma-Aldrich</td>
			<td>T3902</td>
			<td></td>
		</tr>
		<tr>
			<td>Nicotinamide</td>
			<td>Sigma-Aldrich</td>
			<td>N3376</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium D-Pantothenate</td>
			<td>Sigma-Aldrich</td>
			<td>C8731</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipoic Acid&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>T5625</td>
			<td></td>
		</tr>
		<tr>
			<td>p-Aminobenzoic acid&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>A9878</td>
			<td></td>
		</tr>
		<tr>
			<td>Folic acid</td>
			<td>Sigma-Aldrich</td>
			<td>F8798</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotin</td>
			<td>Sigma-Aldrich</td>
			<td>B4639</td>
			<td></td>
		</tr>
		<tr>
			<td>Cobalamine&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>C3607</td>
			<td></td>
		</tr>
		<tr>
			<td>Pyridoxal HCl</td>
			<td>Sigma-Aldrich</td>
			<td>P9130</td>
			<td></td>
		</tr>
		<tr>
			<td>Pyridoxine</td>
			<td>Sigma-Aldrich</td>
			<td>P5669</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>&#160;E6758</td>
			<td></td>
		</tr>
		<tr>
			<td>Iron sulfate &#183; 7 H2O</td>
			<td>Sigma-Aldrich</td>
			<td>&#160;F8263</td>
			<td></td>
		</tr>
		<tr>
			<td>Zinc sulfate &#183; 7 H2O</td>
			<td>Sigma-Aldrich</td>
			<td>Z0251</td>
			<td></td>
		</tr>
		<tr>
			<td>Manganese chloride &#183; 4 H2O</td>
			<td>Sigma-Aldrich</td>
			<td>M8054</td>
			<td></td>
		</tr>
		<tr>
			<td>Boric acid</td>
			<td>Sigma-Aldrich</td>
			<td>B6768</td>
			<td></td>
		</tr>
		<tr>
			<td>Cobalt chloride &#183; 6 H2O</td>
			<td>Sigma-Aldrich</td>
			<td>&#160;C8661</td>
			<td></td>
		</tr>
		<tr>
			<td>Copper chloride &#183; 2 H2O</td>
			<td>Sigma-Aldrich</td>
			<td>459097</td>
			<td></td>
		</tr>
		<tr>
			<td>Nickel chloride &#183; 6 H2O</td>
			<td>Sigma-Aldrich</td>
			<td>203866</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium molybdate &#183; 2 H2O</td>
			<td>Sigma-Aldrich</td>
			<td>331058</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Trypticase (Pancreatic digest of casein)</td>
			<td>Thermo Fisher</td>
			<td>B11921</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic anhydrous</td>
			<td>Thermo Fisher</td>
			<td>P284</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium carbonate &#183; H2O&#160;</td>
			<td>Thermo Fisher</td>
			<td>S636</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Thermo Fisher</td>
			<td>50841063</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium sulfate &#183; 6 H2O</td>
			<td>Thermo Fisher</td>
			<td>7791-18-6</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride &#183; 2 H2O</td>
			<td>Thermo Fisher</td>
			<td>BP510</td>
			<td></td>
		</tr>
		<tr>
			<td>Cysteine HCl</td>
			<td>Thermo Fisher</td>
			<td>19464780</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium phosphate dibasic anhydrous</td>
			<td>Thermo Fisher</td>
			<td>P290</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Thermo Fisher</td>
			<td>BP358</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Representative silica TLC separations of red clover (Trifolium pratense cv. Kenland) extracts, containing phenolic compounds, are shown in Figure 2. Separation of red clover extract in ethyl acetate-hexane (9:1, v/v), over 8.5 cm, resulted in five bands, one incompletely resolved from the origin (Figure 2A). However, Figure 2B demonstrates that about twice as many bands were revealed when a different sample of red clover extract (from the same cultivar, but grow...

Watch this Scientific Journal Video about Thin-layer Chromatographic TLC Separations and Bioassays of Plant Extracts to Identify Antimicrobial Compounds at JoVE.com

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

Methods are described for thin-layer chromatographic (TLC) separation of plant extracts and contact bioautography to identify antibacterial metabolites. The methods are applied to the screening of red clover phenolic compounds inhibiting hyper&#160;ammonia-producing bacteria (HAB) native to the bovine rumen.

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 ...

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67.5K Views.  United States Department of Agriculture. Methods are described for thin-layer chromatographic (TLC) separation of plant extracts and contact bioautography to identify antibacterial metabolites. The methods are applied to the screening of red clover phenolic compounds inhibiting hyper ammonia-producing bacteria (HAB) native to the bovine rumen.

Video: Thin-layer Chromatographic TLC Separations and Bioassays of Plant Extracts to Identify Antimicrobial Compounds

Profiling the Triacylglyceride Contents in Bat Integumentary Lipids by Preparative Thin Layer Chromatography and MALDI-TOF Mass Spectrometry

The mammalian integument includes sebaceous glands that secrete an oily material onto the skin surface. Sebum production is part of the innate immune system that is protective against pathogenic microbes. Abnormal sebum production and chemical composition are also a clinical symptom of specific skin diseases. Sebum contains a complex mixture of lipids, including triacylglycerides, which is species-specific. The broad chemical properties exhibited by diverse lipid classes hinder the specific determination of sebum composition. Analytical techniques for lipids typically require chemical derivatizations that are labor-intensive and increase sample preparation costs. This paper describes how to extract lipids from mammalian integument, separate broad lipid classes by thin-layer chromatography, and profile the triacylglyceride contents using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. This robust method enables a direct determination of the triacylglyceride profiles among species and individuals, and it can be readily applied to any taxonomic group of mammals.

Mammalian integument contains solvent-extractable lipids that can provide chemical compositions characteristic of individual species. This paper presents a routine method for separating broad lipid classes isolated from integumentary tissues using thin layer chromatography and determining the triacylglyceride profile by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

The mammalian integument includes sebaceous glands that secrete an oily material onto the skin surface. Sebum production is part of the innate immune ...

Methods to Identify the NMR Resonances of the 13C-Dimethyl N-terminal Amine on Reductively Methylated Proteins

Nuclear magnetic resonance (NMR) spectroscopy is a proven technique for protein structure and dynamic studies. To study proteins with NMR, stable magnetic isotopes are typically incorporated metabolically to improve the sensitivity and allow for sequential resonance assignment. Reductive 13C-methylation is an alternative labeling method for proteins that are not amenable to bacterial host over-expression, the most common method of isotope incorporation. Reductive 13C-methylation is a chemical reaction performed under mild conditions that modifies a protein&#39;s primary amino groups (lysine &#949;-amino groups and the N-terminal &#945;-amino group) to 13C-dimethylamino groups. The structure and function of most proteins are not altered by the modification, making it a viable alternative to metabolic labeling. Because reductive 13C-methylation adds sparse, isotopic labels, traditional methods of assigning the NMR signals are not applicable. An alternative assignment method using mass spectrometry (MS) to aid in the assignment of protein 13C-dimethylamine NMR signals has been developed. The method relies on partial and different amounts of 13C-labeling at each primary amino group. One limitation of the method arises when the protein&#39;s N-terminal residue is a lysine because the &#945;- and &#949;-dimethylamino groups of Lys1 cannot be individually measured with MS. To circumvent this limitation, two methods are described to identify the NMR resonance of the 13C-dimethylamines associated with both the N-terminal &#945;-amine and the side chain &#949;-amine. The NMR signals of the N-terminal &#945;-dimethylamine and the side chain &#949;-dimethylamine of hen egg white lysozyme, Lys1, are identified in 1H-13C heteronuclear single-quantum coherence spectra.

Methods to Identify the NMR Resonances of the 13C-Dimethyl N-terminal Amine on Reductively Methylated Proteins

Two methods for assigning the &#945;- and &#949;-dimethylamine nuclear magnetic resonance signals of a reductively 13C-methylated N-terminal lysine are described. One method utilizes the pH-induced selectivity of the reductive methylation reaction, and the other uses aminopeptidase to selectively remove the N-terminal lysine.

Nuclear magnetic resonance (NMR) spectroscopy is a proven technique for protein structure and dynamic studies. To study proteins with NMR, stable magnetic ...

Analysis of Volatile and Oxidation Sensitive Compounds Using a Cold Inlet System and Electron Impact Mass Spectrometry

This video presents a protocol for the mass spectrometrical analysis of volatile and oxidation sensitive compounds using electron impact ionization. The analysis of volatile and oxidation sensitive compounds by mass spectrometry is not easily achieved, as all state-of-the-art mass spectrometric methods require at least one sample preparation step, e.g., dissolution and dilution of the analyte (electrospray ionization), co-crystallization of the analyte with a matrix compound (matrix-assisted laser desorption/ionization), or transfer of the prepared samples into the ionization source of the mass spectrometer, to be conducted under atmospheric conditions. Here, the use of a sample inlet system is described which enables the analysis of volatile metal organyls, silanes, and phosphanes using a sector field mass spectrometer equipped with an electron impact ionization source. All sample preparation steps and the sample introduction into the ion source of the mass spectrometer take place either under air-free conditions or under vacuum, enabling the analysis of compounds highly susceptible to oxidation. The presented technique is especially of interest for inorganic chemists, working with metal organyls, silanes, or phosphanes, which have to be handled using inert conditions, such as the Schlenk technique. The principle of operation is presented in this video.

This video presents a protocol for the mass spectrometrical analysis of volatile and oxidation sensitive compounds using electron impact ionization. The presented technique is especially of interest for inorganic chemists, working with metal organyls, silanes, or phosphanes which have to be handled using inert conditions, such as the Schlenk technique.

This video presents a protocol for the mass spectrometrical analysis of volatile and oxidation sensitive compounds using electron impact ionization. The ...

On-line Analysis of Nitrogen Containing Compounds in Complex Hydrocarbon Matrixes

The shift to heavy crude oils and the use of alternative fossil resources such as shale oil are a challenge for the petrochemical industry. The composition of heavy crude oils and shale oils varies substantially depending on the origin of the mixture. In particular they contain an increased amount of nitrogen containing compounds compared to the conventionally used sweet crude oils. As nitrogen compounds have an influence on the operation of thermal processes occurring in coker units and steam crackers, and as some species are considered as environmentally hazardous, a detailed analysis of the reactions involving nitrogen containing compounds under pyrolysis conditions provides valuable information. Therefore a novel method has been developed and validated with a feedstock containing a high nitrogen content, i.e., a shale oil. First, the feed was characterized offline by comprehensive two-dimensional gas chromatography (GC &#215; GC) coupled with a nitrogen chemiluminescence detector (NCD). In a second step the on-line analysis method was developed and tested on a steam cracking pilot plant by feeding pyridine dissolved in heptane. The former being a representative compound for one of the most abundant classes of compounds present in shale oil. The composition of the reactor effluent was determined via an in-house developed automated sampling system followed by immediate injection of the sample on a GC &#215; GC coupled with a time-of-flight mass spectrometer (TOF-MS), flame ionization detector (FID) and NCD. A novel method for quantitative analysis of nitrogen containing compounds using NCD and 2-chloropyridine as an internal standard has been developed and demonstrated.

A method combining comprehensive two-dimensional gas chromatography with nitrogen chemiluminescence detection has been developed and applied to on-line analysis of nitrogen containing compounds in a complex hydrocarbon matrix.

The shift to heavy crude oils and the use of alternative fossil resources such as shale oil are a challenge for the petrochemical industry. The ...

Using Capillary Electrophoresis to Quantify Organic Acids from Plant Tissue: A Test Case Examining Coffea arabica Seeds

Carboxylic acids are organic acids containing one or more terminal carboxyl (COOH) functional groups. Short chain carboxylic acids (SCCAs; carboxylic acids containing three to six carbons), such as malate and citrate, are critical to the proper functioning of many biological systems, where they function in cellular respiration and can serve as indicators of cell health. In foods, organic acid content can have significant impact on taste, with increased SCCA levels resulting in a sour or &#34;acid&#34; taste. Because of this, methods for the rapid analysis of organic acid levels are of particular interest to the food and beverage industries. Unfortunately, however, most methods used for SCCA quantification are dependent on time-consuming protocols requiring the derivatization of samples with hazardous reagents, followed by costly chromatographic and/or mass spectrometric analyses. This method details an alternate method for the detection and quantification of organic acids from plant material and food samples using free zonal capillary electrophoresis (CZE), sometimes simply referred to as capillary electrophoresis (CE). CZE provides a cost-effective method for measuring SCCAs with a low limit of detection (0.005 mg/ml). This article details the extraction and quantification of SCCAs from plant samples. While the method provided focuses on measurement of SCCAs from coffee beans, the method provided can be applied to multiple plant-based food materials.

Using Capillary Electrophoresis to Quantify Organic Acids from Plant Tissue: A Test Case Examining Coffea arabica Seeds

This article presents a method for the detection and quantification of organic acids from plant material using free zonal capillary electrophoresis. An example of the potential application of this method, determining the effects of a secondary fermentation on organic acid levels in coffee seeds, is provided.

Carboxylic acids are organic acids containing one or more terminal carboxyl (COOH) functional groups. Short chain carboxylic acids (SCCAs; carboxylic ...

Raman and IR Spectroelectrochemical Methods as Tools to Analyze Conjugated Organic Compounds

In the presented work, two spectroelectrochemical techniques are discussed as tools for the analysis of the structural changes occurring in the molecule on the vibrational level of energy. Raman and IR spectroelectrochemistry can be used for advanced characterization of the structural changes in the organic electroactive compounds. Here, the step-by-step analysis by means of Raman and IR spectroelectrochemistry is shown. Raman and IR spectroelectrochemical techniques provide complementary information about structural changes occurring during an electrochemical process, i.e. allows for the investigation of redox processes and their products. The examples of IR and Raman spectroelectrochemical analysis are presented, in which the products of the redox reactions, both in solution and solid state, are identified.

A protocol of step-by-step Raman and IR spectroelectrochemical analysis is presented.

In the presented work, two spectroelectrochemical techniques are discussed as tools for the analysis of the structural changes occurring in the molecule ...

Using Cyclic Voltammetry, UV-Vis-NIR, and EPR Spectroelectrochemistry to Analyze Organic Compounds

Cyclic voltammetry (CV) is a technique used in the analysis of organic compounds. When this technique is combined with electron paramagnetic resonance (EPR) or ultraviolet-visible and near-infrared (UV-Vis-NIR) spectroscopies, we obtain useful information such as electron affinity, ionization potential, band-gap energies, the type of charge carriers, and degradation information that can be used to synthesize stable organic electronic devices. In this study, we present electrochemical and spectroelectrochemical methods to analyze the processes occurring in active layers of an organic device as well as the generated charge carriers.

In this article, we describe electrochemical, electron paramagnetic resonance, and ultraviolet-visible and near-infrared spectroelectrochemical methods to analyze organic compounds for application in organic electronics.

Cyclic voltammetry (CV) is a technique used in the analysis of organic compounds. When this technique is combined with electron paramagnetic resonance ...

Electrophoretic Crystallization of Ultrathin High-performance Metal-organic Framework Membranes

We report the synthesis of thin, highly intergrown, polycrystalline metal-organic framework (MOF) membranes on a wide range of unmodified porous and non-porous supports (polymer, ceramic, metal, carbon, and graphene). We developed a novel crystallization technique, which is termed the ENACT approach: the electrophoretic nuclei assembly for the crystallization of highly intergrown thin films (ENACT). This approach allows for a high density of heterogeneous nucleation of MOFs on a chosen substrate via the electrophoretic deposition (EPD) directly from the precursor sol. The growth of well-packed MOF nuclei leads to a highly intergrown polycrystalline MOF film. We show that this simple approach can be used for the synthesis of thin, intergrown zeolite imidazole framework (ZIF)-7 and ZIF-8 films. The resulting 500 nm-thick ZIF-8 membranes show a considerably high H2 permeance (8.3 x 10-6 mol m-2 s-1 Pa-1) and ideal gas selectivities (7.3 for H2/CO2, 15.5 for H2/N2, 16.2 for H2/CH4, and 2655 for H2/C3H8). An attractive performance for C3H6/C3H8 separation is also achieved (a C3H6 permeance of 9.9 x 10-8 mol m-2 s-1 Pa-1 and a C3H6/C3H8 ideal selectivity of 31.6 at 25 &#176;C). Overall, the ENACT process, owing to its simplicity, can be extended to synthesize intergrown thin films of a wide range of nanoporous crystalline materials.

A simple, reproducible, and versatile approach for the synthesis of intergrown, polycrystalline metal-organic framework membranes on a wide range of unmodified porous and non-porous supports is presented.

We report the synthesis of thin, highly intergrown, polycrystalline metal-organic framework (MOF) membranes on a wide range of unmodified porous and ...

Design, Synthesis, and Photochemical Properties of Clickable Caged Compounds

Caged compounds enable the photo-mediated manipulation of the cell physiology with high spatiotemporal resolution. However, the limited structural diversity of currently available caging groups and the difficulties in synthetic modification without sacrificing their photolysis efficiencies are obstacles to expanding the repertoire of caged compounds for live cell applications. As the chemical modification of coumarin-type photo-caging groups is a promising approach for the preparation of caged compounds with diverse physical and chemical properties, we report a method for the synthesis of clickable caged compounds that can be modified easily with various functional units via the copper(I)-catalyzed Huisgen cyclization. The modular platform molecule contains a (6-bromo-7-hydroxycoumarin-4-yl)methyl (Bhc) group as a photo-caging group, which exhibits a high photolysis efficiency compared to those of the conventional 2-nitrobenzyls. General procedures for the preparation of clickable caged compounds containing amines, alcohols, and carboxylates are presented. Additional properties such as the water solubility and cell targeting ability can be readily incorporated into clickable caged compounds. Furthermore, the physical and photochemical properties, including the photolysis quantum yield, were measured and were found to be superior to those of the corresponding Bhc caged compounds. The described protocol could therefore be considered a potential solution for the lack of structural diversity in the available caged compounds.

A protocol for the synthesis and measurement of the photochemical properties of modular caged compounds with clickable moieties is presented.

Caged compounds enable the photo-mediated manipulation of the cell physiology with high spatiotemporal resolution. However, the limited structural ...

Chromatographic Fingerprinting by Template Matching for Data Collected by Comprehensive Two-Dimensional Gas Chromatography

Data processing and evaluation are critical steps of comprehensive two-dimensional gas chromatography (GCxGC), particularly when coupled to mass spectrometry. The rich information encrypted in the data may be highly valuable but difficult to access efficiently. Data density and complexity can lead to long elaboration times and require laborious, analyst-dependent procedures. Effective yet accessible data processing tools, therefore, are key to enabling the spread and acceptance of this advanced multidimensional technique in laboratories for daily use. The data analysis protocol presented in this work uses chromatographic fingerprinting and template matching to achieve the goal of highly automated deconstruction of complex two-dimensional chromatograms into individual chemical features for advanced recognition of informative patterns within individual chromatograms and across sets of chromatograms. The protocol delivers high consistency and reliability with little intervention. At the same time, analyst supervision is possible in a variety of settings and constraint functions that can be customized to provide flexibility and capacity to adapt to different needs and goals. Template matching is shown here to be a powerful approach to explore extra-virgin olive oil volatilome. Cross-alignment of peaks is performed not only for known targets, but also for untargeted compounds, which significantly increases the characterization power for a wide range of applications. Examples are presented to evidence the performance for the classification and comparison of chromatographic patterns from sample sets analyzed under similar conditions.

This protocol presents an approach to fingerprint and explore multi-dimensional data collected by comprehensive two-dimensional gas chromatography coupled to mass spectrometry. Dedicated pattern recognition algorithms (template matching) are applied to explore the chemical information encrypted in the extra-virgin olive oil volatile fraction (i.e., volatilome).