Can Orange Essential Oil Testing Be Faster and Use Fewer Resources?

Analysing composition is essential to checking the quality of essential oils. Repeated testing, however, takes time, electricity and gas. Could we obtain the information we need more quickly and with fewer resources?

A study published by Gaia Bechis and colleagues in 2023 improved a quality-control method using sweet and bitter orange essential oils as examples. Alongside the relative amounts of ingredients, the researchers examined the proportions of molecules with mirror-image forms and worked to speed up that analysis.

The same ingredient name can include mirror-image forms

Sweet and bitter orange peel oils both contain abundant limonene and have very similar sets of constituents. A conventional list of ingredients and their percentages may not always distinguish them easily.

This is where enantiomers can help. Some molecules occur as pairs that correspond like a left and right hand: mirror images that cannot be superimposed exactly. This three-dimensional property is called chirality, and the two forms are distinguished using labels such as R and S.

Earlier compositional studies, for example, have found a tendency for R-linalool to predominate in bitter orange oil and S-linalool in sweet orange oil. Measuring the proportions of R and S within the linalool, as well as the percentage of linalool overall, adds another clue.

Mirror-image differences can sometimes affect odour quality or intensity. This study, however, did not ask people to smell the oils and rate their preferences or the quality of the scents. Its aim was to improve analytical quality control.

Using a narrow tube that distinguishes molecular shapes

The researchers used gas chromatography–mass spectrometry capable of separating enantiomers. A sample is vaporized and passes through a narrow tube, or column, coated inside with a special material. Different constituents travel through at different speeds, allowing them to be separated and detected in sequence.

Mirror-image pairs that tend to appear together in conventional analysis can also be separated because they interact differently with this material. The researchers used known standards and information about transit times through the instrument to identify the forms.

They first used one authentic reference oil each of sweet and bitter orange, taking conventional conditions with a 25 m column as their benchmark. This analysis took 87 minutes per run.

Speed is not the only consideration

To accelerate the analysis, they adjusted the temperature programme and made the column shorter and narrower. Raising the temperature too quickly, however, can cause constituents that need to be separated to overlap. Under one set of conditions, the linalyl acetate enantiomers could not be separated; under another, linalool overlapped with other constituents.

The researchers adjusted conditions while retaining separation of important constituents. They reduced the run time to 31 minutes with the same 25 m column, 22 minutes with a 15 m column, and 16 minutes with an even narrower 10 m column. The different methods gave similar enantiomeric proportions for the reference samples.

For the commercial samples, they ultimately chose the 15 m method rather than the fastest, 10 m method. The narrow 10 m column required high pressure and limited the amount of sample that could be introduced. Routine testing needed a practical balance that avoided overloading with abundant constituents such as limonene without losing trace constituents.

The 22-minute method also used less electricity and gas

Electricity consumption measured with a power meter fell from 1.4 kWh per analysis for the conventional method to 0.3 kWh for the selected 15 m method. Helium used to carry the sample also fell from approximately 2 L to approximately 0.4 L per analysis.

The researchers also used AGREE, an indicator for evaluating the environmental aspects of an analytical method. It scores methods from 0 to 1 across 12 considerations, including sample handling, analytical energy requirements, substances used and sample throughput; values closer to 1 are better.

The score improved from 0.57 for the conventional method to 0.70 for the 15 m method. This is neither a score of 70 for the oil’s quality nor a direct measure of a particular percentage reduction in environmental impact. It is an indicator for comparing analytical methods across multiple considerations.

Some changes were not fully captured by the indicator. Although helium consumption decreased, the waste-related score did not improve. That criterion assigns the same low rating to quantities above 200 mL, making it difficult to reflect the large difference in gas use.

The authors noted that this threshold may be reasonable for liquid-based sample preparation but has limitations for comparing gas consumption in gas chromatography. Alongside using indicators for comparison, we need to examine what they measure and which changes their scores do not show.

Distinguishing storage-related changes from enantiomeric proportions

For a quality-control marker, it also matters whether it changes substantially during storage. The researchers re-examined in 2023 two oils that had been prepared and analysed in 2006 and stored at room temperature.

Although limonene showed pronounced degradation, the R-to-S proportions of linalool were almost unchanged from the earlier measurements. The proportions were also retained in linalool isolated from bergamot oil in 2016.

These findings suggest that linalool’s enantiomeric proportions may help track a sample’s characteristics after storage. They do not mean that the oil as a whole did not deteriorate. The samples were also limited, so the results do not guarantee unchanged proportions in every essential oil or under every storage condition.

Differences and overlap among 48 commercial samples

The improved method was also applied to 29 commercial bitter orange samples and 19 sweet orange samples. These formed a separate set from the reference oils and long-stored samples.

The researchers examined relative composition and enantiomeric proportions for constituents including β-pinene, sabinene, limonene and linalool. They compared these numerous values using principal component analysis, which summarizes data so that similarities between samples become visible.

Many samples resembled their respective reference oils. There was also a tendency toward higher R-linalool and certain other constituents on the bitter orange side, and higher S-linalool and others on the sweet orange side. The two groups nevertheless overlapped in part.

This was therefore not a classification-accuracy test demonstrating correct identification of every unknown oil. Rather, enantiomeric proportions supplement differences that may be difficult to see from composition alone, providing information for closer investigation of samples that differ from typical oils.

The work was supported by University of Turin research funding, and reference oils and other materials were supplied by Italian producers. The authors declared no conflicts of interest.

Summary

In orange-oil analysis, looking at mirror-image proportions alongside constituent names and percentages helped characterize samples. By adjusting conditions while retaining that information, the chosen method shortened analysis from 87 to 22 minutes and reduced electricity and helium consumption.

The value of this study lies in designing analytical capability together with the speed and resource use that make ongoing testing practical. It did not assess the environmental footprint of essential-oil products themselves, but offers a concrete example of improving everyday quality-control analyses. It also shows why environmental indicators should be used alongside actual analytical performance and ease of operation.

References

Bechis, G., Minteguiaga, M. A., Sgorbini, B., Marengo, A., Rubiolo, P., & Cagliero, C. (2023). Make the Quality Control of Essential Oils Greener: Fast Enantioselective GC-MS Analysis of Sweet and Bitter Orange as a Case Study. Molecules, 28(17), 6231. DOI: 10.3390/molecules28176231. Full original article.

Original article: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. Published under CC BY 4.0.