The overall goal of this procedure is to perform a rapid analysis of volatile compounds in fruits. This is accomplished by first cutting and homogenizing the fruit tissue. The vapor phase above the liquid sample is then analyzed with the electronic nose.
Following electronic nose analysis, the data is exported and transformed. The final step of the procedure is to identify covas index windows and consolidate the peaks under a single covas index label using the graphical interface. Ultimately, the results show that differences in abundance of fruit follicles as measured with an electronic nose can be related to experimental treatments such as fruit variety, maturity, and storage.
The main advantage of this technique over existing methods, such as traditional gas chromatography, is that it allows a more rapid analysis of volatile compounds in fruit. When performing this analysis, slight changes in analyte retention times may occur. This could lead to misinterpretation of the data without careful consideration of the peak alignment process.
After harvesting fruits at the desired maturity stage, rinse with tap water. In order to remove dirt and dust, select fruits for analysis. Based on the absence of external and internal defects and size, homogeneity cut fruits longitudinally into wedges to be used for volatile sampling.
If applicable, remove skin seeds, seed cavity tissue, or pit fruit. Tissue selection must be consistent throughout the experiment, and variability within a single fruit should be taken into account. For example, samples should be obtained equally from the equatorial blossom and stem end parts.
Combine the selected fruit tissue, mix it in order to randomize and then weigh out 200 grams into a commercial blender. Add 200 milliliters of saturated calcium chloride solution. The calcium chloride is intended to act as an inhibitor of enzymatic activity, which may occur after cutting and homogenizing the fruit flesh.
Then add 50 microliters of a 100 millimolar solution of two methyl butyl iso ate in methanol. This solution is added as an internal standard to monitor for any possible losses of volatile compounds during the homogenization process. Next, homogenize the mixture in a laboratory blender for 30 seconds at 18, 000 RPM.
Then immediately pour into a glass bottle and seal with a Teflon lid. Keep the homogenate in the bottle until all samples are prepared. Pipette five milliliter aliquots of juice without foam into 20 milliliter glass amber vials preparing at least three vials per sample.
To serve as technical replicates. Seal the vials with steel screw caps fitted with Teflon silicone septa. At this point, samples may be analyzed immediately or flash frozen in liquid nitrogen and stored at ultra low temperature For later analysis.
Load the appropriate analysis method onto the Xenos. Enter the parameters as found in the written protocol. Connect a stainless steel needle with non coring tip to the xenos inlet.
Purge the system several times with ambient air until the baseline is stable and no peaks larger than 200 counts are detected. Prepare to tune the instrument by inserting a needle into the septum of a vial containing a solution of straight chain acas to relieve the pressure. Then insert the needle connected to the instrument inlet into the septum.
Perform the tune by initiating headspace sampling. The tune result is used by the instrument software to convert the retention time of the alluded peaks from time units into covas index or KI units. Consequently, after the system is tuned, the retention times are reported in KI units.
Begin sample analysis following equilibration of the sample for 30 minutes by inserting needles into the septum of the sample vial. As done for the solution of straight chain acas. Initiate headspace sampling manually by clicking on the play button, causing the pump to activate and withdraw the vapors present above the sample.
At the end of the analysis, a chromatogram appears on the screen and the sensor is automatically heated to 150 degrees Celsius for 10 seconds to clean it. When the system status box turns green, again, the instrument is ready to analyze another sample. To ensure a stable baseline and proper system cleaning, run at least one air blank between each sample.
Analyze at least three technical replicates per sample as well as vial blanks. Export the data into a Microsoft Excel file after acquisition using the log stored data function in Mense software. Once the data are exported, add columns containing labels for variables and replicates.
The data format exported from instrument software can be transformed for easier manipulation using the Python 0.6 script generated by this lab. The name of the source file and sheet name for the input data, as well as the desired file name for output are edited directly in the script. This script facilitates data manipulation and analysis through identification of unique kis across all of the samples.
The data are reordered with sample information in rows and unique KIS in columns with each cell representing the corresponding peak area. If a peak is not detected for a KI value in a sample, the corresponding cell remains empty. Next, using a second script generated by this lab import, the data from the file edited in the previous step.
Analysis is based on viewing and analyzing the number of times each KI value was detected. Thus, the program displays a bar graph of KI hits. For each KI value, evaluate the K hits of specific subsets of samples, analyzing each group of technical replicates together.
To do so, analyze every treatment or variable separately by checking or unchecking the corresponding boxes. After identifying the width of each KI window using the graphical interface, randomly select some of the corresponding chromatograms. In mense software evaluate overlapping peaks among the technical replicates.
Once the KI window is individuated, the merge feature in the graphical interface is used to merge the KIS that fall in the window into the most populated ki First. Click on the merge button to activate the feature and select the most populated ki at the center of the window by left clicking the corresponding bar. Once the bar has been selected, it changes color and turns green to merge the KIS that fall within the window into the selected ki right click on the corresponding bars.
This causes the bars to turn red while a blue bar of the corresponding length is added on top of the central ki. Once all the selected kis have been merged into the appropriate central ki, click on the merge button again to accept the changes. This causes the merge button to turn yellow in case of mistakes.
The unmerge button is also available to unmerge. Click the unmerge button in the graphical interface. Then right click on the red bar to be un merg.
From red, the bar turns blue. Click the un merge button again to accept the changes. If one attempts to incorrectly merge two peaks in a single sample into a single KI value, an error message is printed.
Once all of the merging operations have been save the file before proceeding with statistical analysis, the chromatograms of the air and vial blanks are analyzed to monitor for possible contaminations. Once the ki of the peaks and the blanks have been identified, subtract the area of the peak detected in the air and or vial blank from the area of the peak present. In the sample, the electronic nose was able to detect differences in volatile profiles among melon fruit harvested at different maturity stages.
Shown here are examples of chromatograms from early mature fruit and fully ripe fruit, revealing the differences in peak area For numerous peaks, 20 K windows were identified across all samples. An analysis of variants showed that of these different kis, the abundance of 14 peaks detected by the electronic nose varied significantly between two maturity stages. Here, the peak abundance of the early mature fruit for each of these kis is plotted in green, and that of the fully ripe fruit is plotted in orange.
After completing this procedure, other methods like gas chromatography coupled with mass spectrometry can be performed in order to identify the components corresponding to individual peaks on the electronic nose After its development. This technique will provide a rapid analysis tool and allow researchers in the field of plant pathology, plant physiology, post harvest biology, and food science to explore changes in volatile composition in fruit as function of maturity, variety, or storage. After watching this video, you should have a good understanding of how rapidly analyze volatile compounds with an electronic nose and perform peak alignment using our graphical interface.
