Usando Eletroforese Capilar para quantificar ácidos orgânicos a partir de tecido da planta: um caso de teste de exame<em> Coffea arabica</em> Sementes
Summary
Este artigo apresenta um método para a detecção e quantificação de ácidos orgânicos a partir de material vegetal, utilizando electroforese capilar de zona livre. Um exemplo do potencial aplicação do presente método, a determinação dos efeitos de uma fermentação secundária em níveis de ácidos orgânicos em sementes de café, é fornecida.
Abstract
Os ácidos carboxílicos são ácidos orgânicos que contêm um ou mais carboxilo terminal (COOH) grupos funcionais. ácidos carboxílicos de cadeia curta (SCCAs; ácidos carboxílicos contendo de três a seis átomos de carbono), tais como malato e citrato, são críticas para o funcionamento de muitos sistemas biológicos, onde eles funcionam na respiração celular e podem ser utilizados como indicadores de saúde da célula. Nos alimentos, teor de ácido orgânico pode ter um impacto significativo no sabor, com o aumento dos níveis de SCCA, resultando em um sabor "ácido" azedo ou. Devido a isso, a métodos para a análise rápida dos níveis de ácidos orgânicos são de interesse particular para as indústrias alimentares e de bebidas. Infelizmente, no entanto, a maioria dos métodos utilizados para a quantificação SCCA são dependentes de protocolos demorado que requer a derivação de amostras com reagentes perigosos, seguido de cromatograf ica dispendiosa e / ou de espectrometria de massa. Este método detalha um método alternativo para a detecção e quantificação de Orgácidos Anic de material vegetal e amostras de alimentos que utilizam eletroforese capilar zonal livre (CZE), às vezes chamado simplesmente de eletroforese capilar (CE). CZE fornece um método de custo-eficácia para a medição SCCAs com um baixo limite de detecção (0,005 mg / ml). Este artigo detalha a extração e quantificação de SCCAs a partir de amostras de plantas. Enquanto o método fornecido centra-se na medição de SCCAs de grãos de café, o método fornecido pode ser aplicado a várias substâncias alimentares à base de plantas.
Introduction
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.
Protocol
Representative Results
Discussion
Como acontece com qualquer técnica analítica, existem vários fatores críticos que podem afetar significativamente a qualidade e confiabilidade dos dados gerados. Em primeiro lugar, é importante para processar amostras de forma eficiente, com um mínimo de ciclos de congelamento / descongelamento. ciclos repetidos de congelamento e descongelamento pode comprometer a composição química da amostra antes do processamento ou análise. Em segundo lugar, é crítico para aplicar os passos deste protocolo para todas as …
Disclosures
The authors have nothing to disclose.
Acknowledgements
The authors would like to acknowledge the financial support of this project by The J.M. Smucker company.
Materials
| Ceramic Moarter and Pestle | Coorstek | 60310 | |
| Beckman Coulter P/ACE MDQ CE system | Beckman Coulter | Various | |
| Glass sample vials | Fisher Inc. | 033917D | |
| 1.5 ml microcentrifuge tubes | Fisher Inc. | 02-681-5 | |
| LC/MS grade water | Fisher Inc. | W6-1 | Milli-Q water (18.2 MΩ.cm) is also acceptable |
| 15 ml glass tube/ Teflon lined cap | Fisher Inc. | 14-93331A | |
| Parafilm M | Fisher Inc. | 13-374-12 | |
| CElixirOA detection Kit pH 5.4 | MicroSolv | 06100-5.4 | |
| BD Safety-Lok syringes | Fisher Inc. | 14-829-32 | |
| 17 mm Target Syringe filter, PTFE | Fisher Inc. | 3377154 | |
| 32 Karat, V. 8.0 control software | Beckman Coulter | 285512 | |
| capillary electrophoresis (CE) sample vials | Beckman Coulter | 144980 | |
| caps for CE vials | Beckman Coulter | 144648 | |
| Liquid Nitrogen | N/A | N/A | Liquid nitrogen is available from most facilities services |
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