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. नमूना तैयार <ol><li> लघु श्रृंखला कार्बोक्जिलिक एसिड (SCCA) निकासी के लिए नमूने इकट्ठे। वह पर्याप्त नमूना प्रसंस्करण के बाद ही रहेगा सुनिश्चित करने के लिए एक समय में कॉफी के बीज की 1.0 ग्राम तैयार करें। <ol><li> नम?...

<ol>
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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. 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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>

किसी भी विश्लेषणात्मक तकनीक के साथ के रूप में, वहाँ कई महत्वपूर्ण कारक है कि काफी गुणवत्ता और उत्पन्न आंकड़ों की विश्वसनीयता को प्रभावित कर सकते हैं। सबसे पहले, यह / फ्रीज पिघलना चक्र की एक न्यूनतम के स?...

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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	</colgroup>
	<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

कार्बोक्जिलिक एसिड एक या एक से अधिक टर्मिनल carboxyl (COOH) कार्य समूहों से युक्त कार्बनिक अम्ल होते हैं। छोटी श्रृंखला कार्बोक्जिलिक एसिड (SCCAs; कार्बोक्जिलिक एसिड तीन से छह कार्बन युक्त) ऐसे Malate और साइट्रेट के रूप में, कई जैविक प्रणालियों, जहां वे सेलुलर श्वसन में काम करते हैं और सेल स्वास्थ्य के संकेतक के रूप में सेवा कर सकते हैं के समुचित कार्य के लिए महत्वपूर्ण हैं। खाद्य पदार्थों में, कार्बनिक अम्ल सामग्री स्वाद पर महत्वपूर्ण प्रभाव पड़ता है, एक खट्टा या &quot;एसिड&quot; स्वाद में जिसके परिणामस्वरूप वृद्धि हुई SCCA के स्तर के साथ हो सकता है। इस वजह से, कार्बनिक अम्ल के स्तर के तेजी से विश्लेषण के लिए तरीके खाद्य और पेय उद्योगों के लिए विशेष रुचि के हैं। दुर्भाग्य से, हालांकि, ज्यादातर SCCA मात्रा का ठहराव के लिए इस्तेमाल किया तरीकों समय लेने वाली खतरनाक अभिकर्मकों के साथ नमूनों की आवश्यकता होती है derivatization प्रोटोकॉल पर निर्भर कर रहे हैं, महंगा chromatographic और / या बड़े पैमाने पर spectrometric विश्लेषण द्वारा पीछा किया। इस विधि का विवरण पता लगाने और ओआरजी की मात्रा का ठहराव के लिए एक वैकल्पिक विधिसंयंत्र सामग्री और मुक्त जोनल केशिका वैद्युतकणसंचलन (CZE) का उपयोग भोजन के नमूने से anic एसिड होता है, कभी कभी बस केशिका वैद्युतकणसंचलन (सीई) के रूप में भेजा। CZE का पता लगाने के एक कम सीमा (0.005 मिलीग्राम / एमएल) के साथ SCCAs को मापने के लिए एक लागत प्रभावी तरीका प्रदान करता है। यह लेख संयंत्र के नमूने से निकासी और SCCAs की मात्रा का ठहराव का विवरण। प्रदान की विधि कॉफी बीन्स से SCCAs की माप पर केंद्रित है, प्रदान की विधि कई संयंत्र आधारित खाद्य सामग्री के लिए लागू किया जा सकता है।

इस प्रोटोकॉल सफलतापूर्वक हरी कॉफी के बीज की SCCA सामग्री पर बीज उपचार के प्रभाव को मापने के लिए उपयोग किया गया है। इस प्रयोग में, छह उपचार थे: Leuconostoc की एक संतृप्त माइक्रोबियल निलंबन अपनी मध्?...

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

इस लेख का पता लगाने और मुफ्त जोनल केशिका वैद्युतकणसंचलन का उपयोग संयंत्र सामग्री से कार्बनिक अम्ल की मात्रा का ठहराव के लिए एक विधि प्रस्तुत करता है। इस विधि के संभावित आवेदन, कॉफी के बीज में कार्बनिक अम्ल के स्तर पर एक माध्यमिक किण्वन के प्रभाव का निर्धारण करने का एक उदाहरण है, प्रदान की जाती है।

प्लांट टिश्यू से कार्बनिक अम्ल यों केशिका वैद्युतकणसंचलन का उपयोग: एक परीक्षण के मामले की जांच<em&gt; Coffea अरेबिका</em&gt; बीज

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

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

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The Use of Gas Chromatography to Analyze Compositional Changes of Fatty Acids in Rat Liver Tissue during Pregnancy

गर्भावस्था के दौरान चूहा जिगर ऊतक में फैटी एसिड की compositional परिवर्तन का विश्लेषण करने के लिए गैस क्रोमैटोग्राफी का प्रयोग करें

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, ...

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

एक शीत इनलेट सिस्टम और इलेक्ट्रॉन प्रभाव मास स्पेक्ट्रोमेट्री का उपयोग अस्थिर और ऑक्सीकरण संवेदनशील यौगिकों का विश्लेषण

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

Functionalized के संश्लेषण और विशेषता धातु कार्बनिक चौखटे

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

Capillary Electrophoresis to Monitor Peptide Grafting onto Chitosan Films in Real Time

केशिका वैद्युतकणसंचलन रीयल टाइम में chitosan फिल्में पर पेप्टाइड ग्राफ्टिंग नजर रखने के लिए

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

Grafting Multiwalled Carbon Nanotubes with Polystyrene to Enable Self-Assembly and Anisotropic Patchiness

Polystyrene के साथ Multiwalled कार्बन नैनोट्यूब को सक्षम करने के लिए स्व-विधानसभा और अनिसोट्रोपिक Patchiness

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

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

कार्बनिक यौगिकों का विश्लेषण करने के लिए चक्रीय Voltammetry, यूवी विज़-NIR, और EPR Spectroelectrochemistry का उपयोग करना

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

Microprobe Capillary Electrophoresis Mass Spectrometry for Single-cell Metabolomics in Live Frog (Xenopus laevis) Embryos

Microprobe Capillary Electrophoresis Mass Spectrometry for Single-cell Metabolomics in Live Frog Xenopus laevis Embryos

लाइव मेंढक (Xenopus laevis) भ्रूण में एकल कोशिका Metabolomics के लिए Microprobe केशिका ट्रो मास स्पेक्ट्रोमेट्री

The quantification of small molecules in single cells raises new potentials for better understanding the basic processes that underlie embryonic ...

Microfluidic Preparation of Liquid Crystalline Elastomer Actuators

तरल क्रिस्टलीय Elastomer के Microfluidic तैयारी

This paper focuses on the microfluidic process (and its parameters) to prepare actuating particles from liquid crystalline elastomers. The preparation ...