The overall goal of this procedure is to quantify the levels of organic acids present in plant tissues. This method can be used to answer questions in plant biochemistry and physiology, such as the impact of environmental stimuli on root growth, crop development, and food or beverage quality. The main advantages of this method are that sample preparation is minimal, the method is high throughput, and capillary electrophoresis is more cost effective than mass spectrometry.
Though this method is used to elucidate plant physiological responses, it can also be applied to food science and food quality, where organic acids critically impact flavor, safety, and stability. To begin, assemble samples for short chain carboxylic acid, or SCCA, extraction. Prepare one gram of coffee seed at a time to ensure that enough sample will remain after processing.
To attain a uniform particle size for maximal SCCA extraction efficiency, first pre-chill a mortar and pestle with liquid nitrogen. Add the sample to a small volume of liquid nitrogen in the mortar. Using a ladle, add enough liquid nitrogen to the mortar to completely submerge the sample.
Add hard-to-grind samples, such as raw coffee seeds, to the liquid nitrogen. Allow them to freeze for 10 to 30 seconds before grinding. Break the tissue into smaller fragments using a vertical crushing motion.
Then, complete tissue grinding using a circular grinding motion. Repeat the freezing and grinding steps twice more so that samples are ground the total of three times. Typically, three successive rounds of grinding will reduce samples to a powder with a flour-like consistency.
Transfer the powder to glass vials or 1.5 ml micro-centrifuge tubes. Initiate downstream processing immediately after grinding or store samples at 80 degrees Celsius until samples are ready for extraction. Assemble authentic standards for the SCCAs of interest to create external and internal standard solutions for use in determining SCCA concentrations.
For coffee samples, include citric, malic, acetic, and lactic acid as acids of interest, and adipic acid as the internal standard. Create a 10 milligram per milliliter stock solution by adding 100 milligrams of standard to a 10 milliliter volumetric flask. Fill the volumetric flask to the 10 milliliter line with ultra pure water to dissolve the acid.
Transfer each stock solution to a clean 15 milliliter glass tube, with a polytetrafluoroethylene lined cap, and seal with plastic paraffin film. Stock solutions can be stored in sealed tubes at four degrees Celsius for one week. Prepare 50 milliliters of the SCCA extraction solution by diluting the internal standard stock solution to 0.05 milligrams per milliliter in ultra pure water.
Do this by adding 250 microliters of the internal standard stock solution to 49.75 milliliters of water. Next, prepare standard curve samples for SCCAs of interest. Use a minimum of at least five points with concentrations in the linear response range that span the expected SCCA concentrations in the samples.
Dilute each SCCA to the determined concentration in new micro-centrifuge tubes using ultra pure water to a volume of one milliliter. Ensure that each concentration point contains all four acid standards and the internal standard at the correct concentration. Remove samples to be extracted from storage, and place them on ice.
Weigh out 100 milligrams of sample for the SCCA extraction. Measure an amount of sample as close to the target mass as possible, to reduce variability. After weighing each sample, transfer the tissue to a clean 1.5 milliliter micro-centrifuge tube.
Keep track of the mass of each sample measured, as the amounts of SCCAs detected will be normalized using the sample mass. After weighing all samples, add one milliliter of exaction solution to each of the sample tubes. Keep the ratio of extraction solution to tissue mass the same across all extractions.
Mix well by vortexing for 10 seconds. Allow the samples to sit at room temperature for one hour. During this hour, mix each tube every 15 minutes by vortexing for 10 seconds.
After one hour of extraction, mix the samples one final time and transfer the sample tubes to a micro-centrifuge. Centrifuge the samples at four degrees Celsius, 10, 000 times G for 10 minutes, to precipitate the solid material. To filter the samples, prepare syringe-mounted disc filters, ensuring that each filter is correctly attached to the syringe.
Prepare one syringe equipped with a disc filter for each sample to be analyzed, including the standard curve samples. Filter the supernatants directly into a clean micro-centrifuge tube. After filtering, close the tube containing the filtrate, and discard the syringe filter apparatus.
Transfer one milliliter of sample to each capillary electrophoresis, or CE vial, taking care to avoid splashing the sample into the neck of the vial. After transferring the sample, place a CE cap on each vial. Set up the SCCA detection run as described in the text protocol.
Load the vials containing the standard curve points and the vials containing the samples to assayed into the inlet sample tray, as described in the manufacturer's instructions. Note the position of each vial for auto-sampler programming. After conditioning as described in the text protocol, initiate sample separation by opening the Control menu in the software, and selecting Run Sequence.
Monitor the photodiode array, or PDA trays, to ensure proper separation. Observe the first trace and ensure that it has a flat baseline, and cleanly resolve individual acid peaks. To integrate sample peaks, open the first file and set the automatic integration steps to exclude the first three minutes of the run.
In the same menu, set peak selection criteria to a minimum peak width of 50 units, and a peak area of 100 units, to provide a moderately high level of selectivity, separating acid peaks from background noise in the trace. Perform data analysis as described in the text protocol. Shown here, is a comparison of PDA traces highlighting an overloaded sample.
As analyte concentration increases, individual peak geometry may begin to become asymmetric. At 0.05 milligrams per milliliter, acetic acid presents a well defined bilaterally symmetrical peak. As the concentration of acetic acid increases to 0.07 or 0.10 milligrams per milliliter, the peak becomes asymmetrical, and a peak tail forms.
This peak tailing is a good indication that the sample is overloaded. Displayed here are example PDA traces showing organic acids in green coffee samples. Green coffee samples were dilution 1:10 before loading into CE vials.
Acids of interest include acetic acid, citric acid, lactic acid, malic acid, and the internal standard, adipic acid. While attempting this procedure, it's important to remember to grind samples to uniform consistency, and pipette samples carefully, to maximize reproducibility and minimize experimental error. After watching this video, you should have a good understanding of how to quantify organic acids from plant samples using capillary electrophoresis, or CE.You should be able to grind plant samples, extract organic acids, and prepare plant extracts for downstream CE analysis.
