Name: using capillary electrophoresis to quantify organic acids from plant tissue: a test case examining coffea arabica seeds
Uploaded: 2016-11-12T14:00:00
Description: Watch this Scientific Journal Video about Using Capillary Electrophoresis to Quantify Organic Acids from Plant Tissue: A Test Case Examining Coffea arabica Seeds at JoVE.com

The authors would like to acknowledge the financial support of this project by The J.M. Smucker company.

1. Prøvepreparering <ol><li> Monter prøver for kort kjetting karboksylsyre (SCCA) ekstraksjon. Fremstille 1,0 g av kaffe frø ved en tid for å sikre at tilstrekkelig prøve vil være igjen etter behandlingen. <ol><li> Hvis prøvene ble frosset før maleprosessen, holde vevet frosset gjennom hele behandlingen for å hindre at fryse / tine-skade og prøven oksidasjon. Fjern prøven fra fryseren eller sub-null lagring bare etter behov for sliping. </li><li> Flash fryse ferske prøver i flytende nitrogen umiddelbart f?...

<ol>
	<li>Ara&#250;jo, W. L., Nunes-Nesi, A., Nikoloski, Z., Sweetlove, L. J., Fernie, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+Control+and+Regulation+of+the+Tricarboxylic+Acid+Cycle+in+Photosynthetic+and+Heterotrophic+Plant+Tissues:+TCA+Control+and+Regulation+in+Plant+Tissues.">Metabolic Control and Regulation of the Tricarboxylic Acid Cycle in Photosynthetic and Heterotrophic Plant Tissues: TCA Control and Regulation in Plant Tissues.</a> Plant Cell Environ. 35 (1), 1-21 (2012).</li><li>Finkemeier, I., Konig, A. C., 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=Transcriptomic+Analysis+of+the+Role+of+Carboxylic+Acids+in+Metabolite+Signaling+in+Arabidopsis+Leaves.">Transcriptomic Analysis of the Role of Carboxylic Acids in Metabolite Signaling in Arabidopsis Leaves.</a> Plant Physiol. 162 (1), 239-253 (2013).</li><li>Doyle, M. P., Buchanan, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Food Microbiology: Fundamentals and Frontiers. , (2013).</li><li>T&#367;ma, P., Samcov&#225;, E., &#352;tul&#236;k, K. <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+the+Spectrum+of+Low+Molecular+Mass+Organic+Acids+in+Urine+by+Capillary+Electrophoresis+with+Contactless+Conductivity+and+Ultraviolet+Photometric+Detection-An+Efficient+Tool+for+Monitoring+of+Inborn+Metabolic+Disorders.">Determination of the Spectrum of Low Molecular Mass Organic Acids in Urine by Capillary Electrophoresis with Contactless Conductivity and Ultraviolet Photometric Detection-An Efficient Tool for Monitoring of Inborn Metabolic Disorders.</a> Anal Chim Acta. 685 (1), 84-90 (2011).</li><li>L&#243;pez-Bucio, J., Nieto-Jacobo, M. F., Ram&#305;&#769;rez-Rodr&#305;&#769;guez, V., Herrera-Estrella, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organic+Acid+Metabolism+in+Plants:+From+Adaptive+Physiology+to+Transgenic+Varieties+for+Cultivation+in+Extreme+Soils.">Organic Acid Metabolism in Plants: From Adaptive Physiology to Transgenic Varieties for Cultivation in Extreme Soils.</a> Plant Sci. 160 (1), 1-13 (2000).</li><li>Cebolla-Cornejo, J., Valc&#225;rcel, M., Herrero-Mart&#236;nez, J. M., Rosell&#242;, S., Nuez, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+Efficiency+Joint+CZE+Determination+of+Sugars+and+Acids+in+Vegetables+and+Fruits:+CE+and+CEC.">High Efficiency Joint CZE Determination of Sugars and Acids in Vegetables and Fruits: CE and CEC.</a> Electrophoresis. 33 (15), 2416-2423 (2012).</li><li>Rosello, S., Galiana-Balaguer, L., Herrero-Martinez, J. M., Maquieira, A., Nuez, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+Quantification+of+the+Main+Organic+Acids+and+Carbohydrates+Involved+in+Tomato+Flavour+Using+Capillary+Zone+Electrophoresis.">Simultaneous Quantification of the Main Organic Acids and Carbohydrates Involved in Tomato Flavour Using Capillary Zone Electrophoresis.</a> J Sci Food Agr. 82 (10), 1101-1106 (2002).</li><li>Wasielewska, M., Banel, A., Zygmunt, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capillary+Electrophoresis+in+Determination+of+Low+Molecular+Mass+Organic+Acids.">Capillary Electrophoresis in Determination of Low Molecular Mass Organic Acids.</a> Int J Environ Sci Dev. 5 (4), 417-425 (2014).</li><li>Galli, V., Garc&#236;a, A., Saavedra, L., Barbas, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capillary+Electrophoresis+for+Short-Chain+Organic+Acids+and+Inorganic+Anions+in+Different+Samples.">Capillary Electrophoresis for Short-Chain Organic Acids and Inorganic Anions in Different Samples.</a> Electrophoresis. 24 (1213), 1951-1981 (2003).</li><li>Klampfl, C. W. <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+Organic+Acids+by+CE+and+CEC+Methods.">Determination of Organic Acids by CE and CEC Methods.</a> Electrophoresis. 28 (19), 3362-3378 (2007).</li><li>Kenney, B. F. <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+Organic+Acids+in+Food+Samples+by+Capillary+Electrophoresis.">Determination of Organic Acids in Food Samples by Capillary Electrophoresis.</a> J Chromatogr A. 546, 423-430 (1991).</li><li>Galli, V., Barbas, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capillary+Electrophoresis+for+the+Analysis+of+Short-Chain+Organic+Acids+in+Coffee.">Capillary Electrophoresis for the Analysis of Short-Chain Organic Acids in Coffee.</a> J Chromatogr A. 1032 (1-2), 299-304 (2004).</li><li>Schmitt-Kopplin, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capillary+Electrophoresis:+Methods+and+Protocols.">Capillary Electrophoresis: Methods and Protocols.</a> Methods in Molecular Biology. , 384 (2008).</li><li>Nollet, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Chromatographic analysis of the environment 3rd ed. , (2006).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> ElixerOA Organic Acids/Anions Operating and Instruction Manual, MicroSolv Technology Corperation. , (2001).</li><li>Dahlen, J., Hagberg, J., Karlsson, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+low+molecular+weight+organic+acids+in+water+with+capillary+zone+electrophoresis+employing+indirect+photometric+detection.">Analysis of low molecular weight organic acids in water with capillary zone electrophoresis employing indirect photometric detection.</a> Fresenius J. Anal. Chem. 366 (5), 488-493 (2000).</li><li>Ibanez, A. B., Bauer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analytical+method+for+the+determination+of+organic+acids+in+dilute+acid+pretreated+biomass+hydrolysate+by+liquid+chromatography-time-of-flight+mass+spectroscopy.">Analytical method for the determination of organic acids in dilute acid pretreated biomass hydrolysate by liquid chromatography-time-of-flight mass spectroscopy.</a> Biotech. For Biofuels. 7 (145), (2014).</li></ol>

Som med alle analytisk teknikk, er det flere viktige faktorer som i vesentlig grad kan påvirke kvaliteten og påliteligheten av data generert. For det første er det viktig å behandle prøvene effektiv måte, med et minimum av fryse / tine-sykluser. Gjentatt frysing og tining kan kompromittere den kjemiske sammensetning av prøven før behandling eller analyse. For det andre er det viktig å bruke trinnene i denne protokollen til alle prøver konsekvent og jevnt. Tekniske feil som skyldes inkonsekvent prøveopparbeide...

Carboxylic acids are organic compounds containing one or more terminal carboxyl functional groups, each attached to an R-group containing one or more carbons (R-C[O]OH). Short chain, low molecular weight carboxylic acids (short chain carboxylic acids, SCCAs) containing between one and six carbons, are essential components of cellular respiration, and function in several biochemical pathways necessary for cell growth and development. SCCAs play critical roles in cellular metabolism1, cell signaling2, and organismal responses to the environment (such as antibiosis3). Because of this, SCCAs can serve as useful indicators of disruptions to cellular metabolism, plant stress responses4,5, and fruit quality6,7. To date, SCCAs have been quantified primarily through chromatographic techniques such as high performance liquid chromatography (HPLC) or gas chromatography-mass spectroscopy (GC-MS). While these methods, are capable of achieving very low limits of detection, they can be expensive, require the derivatization of target SCCAs using caustic and/or toxic reagents, and include lengthy separation runs on the GC or HPLC. Because of this, interest in the use of free zonal capillary electrophoresis (CZE), which does not require sample derivatization, to quantify organic acids has steadily increased8.
Free zonal capillary electrophoresis (CZE) is a chromatographic separation methodology that, due to its high number of theoretical plates, speed, and relative ease-of-use, is increasingly replacing both GC-MS and high-pressure liquid chromatography as an analytical method for the quantification (particularly for quality control purposes) of anions, cations, amino acids, carbohydrates, and short chain carboxylic acids (SCCAs)8,9,10. CZE-based separation of small molecules, including SCCAs, is based two primary principles: the electrophoretic movement of charged ions in an electrical field established across the buffer filling the capillary; and the electro-osmotic movement of the entire buffer system from one end of the capillary to the other, generally towards the negative electrode. In this system, small molecules move towards the negative electrode at varying speeds, with the speed of each molecule determined by the ratio of the net charge of the molecule to the molecular mass. As the movement of each individual molecule in this system is dependent on the charge state of the molecule and the overall rate of electro-osmotic flow (which is itself based on the ion content of the buffer used to fill the capillary), the buffer pH and ionic composition heavily impact the degree to which molecules can be efficiently separated using CZE. Because of this, SCCAs, with their relatively high charge-to-mass ratios, are ideal targets for CZE-based separation. Metabolites separated using CZE can be detected using a variety of methods, including UV absorbance, spectral absorbance (which is generally performed using a photo-diode array [PDA]), and/or mass spectroscopy (CE-MS or CE-MS/MS)8. The diversity of separation and detection methods provided by CZE makes it an extremely flexible and adaptable technique. Because of this, CZE has been increasingly applied as a standard method of analysis in the areas of food safety and quality11,12, pharmaceutical research13, and environmental monitoring13,14. 
Capillary electrophoresis has been used to detect and quantify short chain carboxylic acids for nearly two decades13. The resolving power (particularly for small, charged molecules), short run time, and low per sample cost of CZE analyses make CZE an ideal technique for the separation and quantification of SCCAs13. This method presents a protocol to utilize CZE to measure the concentration of organic acids from plant tissues. Example data was generated through the successful implementation of this protocol to measure the change in organic acid levels in coffee seeds following a secondary fermentation treatment. The protocol details the critical steps and common errors of CZE-based separation of SCCAs, and discusses the means by which this protocol can be successfully applied to quantify SCCAs in additional plant tissues.

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	<tbody>
		<tr height="19">
			<td height="19" style="height:19px;width:296px;">Ceramic Moarter and Pestle</td>
			<td style="width:123px;">Coorstek</td>
			<td style="width:119px;">60310</td>
			<td style="width:247px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:296px;">Beckman Coulter P/ACE MDQ CE system</td>
			<td style="width:123px;">Beckman Coulter</td>
			<td style="width:119px;">Various</td>
			<td style="width:247px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:296px;">Glass sample vials</td>
			<td style="width:123px;">Fisher Inc.</td>
			<td style="width:119px;">033917D</td>
			<td style="width:247px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:296px;">1.5 ml microcentrifuge tubes&#160;</td>
			<td style="width:123px;">Fisher Inc.</td>
			<td style="width:119px;">02-681-5</td>
			<td style="width:247px;"></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:296px;">LC/MS grade water</td>
			<td style="width:123px;">Fisher Inc.</td>
			<td style="width:119px;">W6-1</td>
			<td style="width:247px;">Milli-Q water (18.2 M&#937;.cm) is also acceptable</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:296px;">15 ml glass tube/ Teflon lined cap&#160;</td>
			<td style="width:123px;">Fisher Inc.</td>
			<td style="width:119px;">14-93331A</td>
			<td style="width:247px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:296px;">Parafilm M</td>
			<td style="width:123px;">Fisher Inc.</td>
			<td style="width:119px;">13-374-12</td>
			<td style="width:247px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:296px;">CElixirOA detection Kit pH 5.4&#160;</td>
			<td style="width:123px;">MicroSolv</td>
			<td style="width:119px;">06100-5.4</td>
			<td style="width:247px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:296px;">BD Safety-Lok syringes</td>
			<td style="width:123px;">Fisher Inc.</td>
			<td style="width:119px;">14-829-32</td>
			<td style="width:247px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:296px;">17 mm Target Syringe filter, PTFE</td>
			<td style="width:123px;">Fisher Inc.</td>
			<td style="width:119px;">3377154</td>
			<td style="width:247px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:296px;">32 Karat, V. 8.0 control software</td>
			<td style="width:123px;">Beckman Coulter</td>
			<td style="width:119px;">285512</td>
			<td style="width:247px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:296px;">capillary electrophoresis (CE) sample vials&#160;</td>
			<td style="width:123px;">Beckman Coulter</td>
			<td style="width:119px;">144980</td>
			<td style="width:247px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:296px;">caps for CE vials&#160;</td>
			<td style="width:123px;">Beckman Coulter</td>
			<td style="width:119px;">144648</td>
			<td style="width:247px;"></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:296px;">Liquid Nitrogen</td>
			<td style="width:123px;">N/A</td>
			<td style="width:119px;">N/A</td>
			<td style="width:247px;">Liquid nitrogen is available from most facilities services</td>
		</tr>
	</tbody>
</table>

using capillary electrophoresis to quantify organic acids from plant tissue: a test case examining coffea arabica seeds

Karboksylsyrer er organiske syrer inneholdende én eller flere terminal karboksylgruppe (COOH) funksjonelle grupper. Kortkjedede karboksylsyrer (SCCAs; karboksylsyrer inneholdende tre til seks karbonatomer), slik som malat og citrat, er kritisk for korrekt funksjon av mange biologiske systemer, hvor de funksjonerer i cellulær respirasjon og kan tjene som en indikator på celle helse. I mat, kan innhold av organisk syre ha betydelig innvirkning på smak, med økte SCCA nivåer som resulterer i en sur eller &quot;syre&quot; smak. På grunn av dette, fremgangsmåter for rask analyse av organiske syrenivåer er av spesiell interesse for næringsmiddelindustrien. Dessverre har imidlertid de fleste fremgangsmåter som benyttes for scca kvantifisering er avhengig tidkrevende protokollene krever derivatisering av prøver med farlige reagenser, fulgt av kromatografisk kostbare og / eller massespektrometrisk analyse. Denne metoden detaljer en alternativ metode for påvisning og kvantifisering av orgANIC syrer fra plantemateriale og matvarer prøver ved hjelp fri sone kapillær elektroforese (CZE), noen ganger bare referert til som kapillær elektroforese (CE). CZE gir en kostnadseffektiv metode for å måle SCCAs med lav deteksjonsgrense (0,005 mg / ml). Denne artikkelen detaljer utvinning og kvantifisering av SCCAs fra planteprøver. Selv om fremgangsmåten gitt fokuserer på måling av SCCAs fra kaffebønner, kan fremgangsmåten gitt anvendes på flere plante-baserte næringsmidler.

Denne protokollen har blitt brukt til å måle effekten av frø behandlinger på SCCA innholdet av grønne kaffe frø. I dette eksperimentet, de seks behandlinger var: en mettet mikrobiell suspensjon av Leuconostoc pseudomesenteroides belastning GCP674 i sitt vekstmedium (1), en vandig suspensjon av GCP674 mikrober i vann (2), en vandig oppløsning av eddiksyre og melkesyre (0,15 og 0,4 mg / ml) (3), en brukt M1 vekstmedium behandling (4), dH 2 O vann (5), og en ubeha...

Watch this Scientific Journal Video about Using Capillary Electrophoresis to Quantify Organic Acids from Plant Tissue: A Test Case Examining Coffea arabica Seeds at JoVE.com

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

Denne artikkelen presenterer en metode for påvisning og kvantifisering av organiske syrer fra plantemateriale ved hjelp av gratis sone kapillær elektroforese. Et eksempel på den potensielle anvendelsen av denne fremgangsmåten, å bestemme virkningene av en sekundær gjæring på organiske syrenivåer i kaffe frø, er gitt.

Bruke Kapillær elektroforese å kvantifisere organiske syrer fra Plant Tissue: En Test Case Undersøke<em&gt; Coffea arabica</em&gt; Seeds

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

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Chemistry

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

Qualitative Identification of Carboxylic Acids, Boronic Acids, and Amines Using Cruciform Fluorophores

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Molecular cruciforms are X-shaped systems in which two conjugation axes intersect at a central core. If one axis of these molecules is substituted with ...

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Gas chromatography (GC) is a highly sensitive method used to identify and quantify the fatty acid content of lipids from tissues, cells, and plasma/serum, ...

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

Synthesis and Characterization of Functionalized Metal-organic Frameworks

Syntese og karakterisering av funksjon Metal-organisk Rammeverk

Metal-organic frameworks have attracted extraordinary amounts of research attention, as they are attractive candidates for numerous industrial and ...

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Free-solution capillary electrophoresis (CE) separates analytes, generally charged compounds in solution through the application of an electric field. ...

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We demonstrate a straightforward protocol to graft pristine multiwalled carbon nanotubes (MWCNTs) with polystyrene (PS) chains at the sidewalls through a ...

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Bruke sykliske Voltammetry, UV-Vis-NIR og EPR Spectroelectrochemistry å analysere organiske forbindelser

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

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