Usando elettroforesi capillare per quantificare Acidi organici da tessuti vegetali: un banco di prova d'esame<em> Coffea arabica</em> Semi
Summary
Questo articolo presenta un metodo per l'individuazione e la quantificazione di acidi organici provenienti da materiale vegetale che utilizzano gratuitamente zonale elettroforesi capillare. Un esempio del potenziale applicazione di questo metodo, determinare gli effetti di una fermentazione secondaria sui livelli di acidi organici in semi di caffè, è fornito.
Abstract
Acidi carbossilici sono acidi organici contenenti uno o più terminale carbossilico (COOH) gruppi funzionali. A catena corta acidi carbossilici (SCCAs; acidi carbossilici contenenti da tre a sei atomi di carbonio), come malato e citrato, sono fondamentali per il corretto funzionamento di molti sistemi biologici, in cui funzionano nella respirazione cellulare e può servire come indicatori di salute delle cellule. Negli alimenti, contenuto in acidi organici può avere un impatto significativo sul gusto, con un aumento dei livelli di SCCA risultante in un gusto "acide" acida o. Per questo motivo, i metodi per l'analisi rapida dei livelli di acidi organici sono di particolare interesse per le industrie alimentari e bevande. Sfortunatamente, tuttavia, la maggior parte dei metodi impiegati per SCCA quantificazione dipendono protocolli in termini di tempo che richiedono la derivatizzazione dei campioni con reagenti pericolosi, una costosa cromatografica e / o analisi di spettrometria di massa. Questo metodo dettagli un metodo alternativo per l'individuazione e la quantificazione dei orgGli acidi Anic da materiale vegetale e campioni di prodotti alimentari che utilizzano gratuitamente elettroforesi capillare zonale (CZE), a volte semplicemente indicato come elettroforesi capillare (CE). CZE fornisce un metodo conveniente per la misurazione SCCAs con un limite inferiore di rilevazione (0,005 mg / ml). Questo articolo descrive l'estrazione e la quantificazione dei SCCAs da campioni vegetali. Mentre il metodo fornito concentra sulla misura della SCCAs da chicchi di caffè, il metodo fornito può essere applicato a diversi materiali alimentari di origine vegetale.
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
Come con qualsiasi tecnica analitica, ci sono diversi fattori critici che possono influenzare significativamente la qualità e l'affidabilità dei dati generati. In primo luogo, è importante trattare i campioni in modo efficiente, con un minimo di cicli di congelamento / scongelamento. Ripetuti di congelamento e scongelamento possono compromettere la composizione chimica del campione prima della trasformazione o analisi. In secondo luogo, è fondamentale applicare i passi di questo protocollo per tutti i campioni i…
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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