PRESSURIZED LIQUID EXTRACTION

What is pressurized liquid extraction?

Czernicka et al. (2020) write of “pressurized liquid extraction or accelerated solvent extraction”, abbreviated ASE.

How is a PLE system built?

According to the review by Barp et al. (2023), the instrumentation basically consists of a solvent tank, a pump, an oven containing the extraction cells, several valves and restrictors, and collection vials. The basic instrumentation can vary depending on whether the process is static, with a fixed volume of extractant, or dynamic, with the extractant flowing continually through the sample. Extraction pressure is controlled by special valves, and there “may” be an inert gas circuit, commonly nitrogen, that helps to remove solvent from the lines after static extraction.

How is the sample prepared?

According to the review by Barp et al. (2023), samples with a high water content, which can prevent non-polar solvents from reaching the analytes within the matrix, are usually oven-dried or freeze-dried before extraction, or previously purified drying agents (sodium sulfate, Na2SO4) are added.

How does PLE compare with other extraction methods, according to one review?

In the comparison, by the review of Barp et al. (2023), of the most commonly used extraction methods for solid samples, PLE is listed with low solvent consumption, a short process time and a sample post-treatment marked “Yes/no (in cell clean-up)”. Its listed advantages are the possibility to perform more extraction cycles and a high sample throughput. Soxhlet (SoxE) is listed in the same comparison with medium solvent consumption, a very long process time and a sample post-treatment marked “Yes”.

What did one study on ginger rhizomes measure?

In the study by Czernicka et al. (2020), fresh ginger rhizomes (500 g) were purchased from a local market in Lublin, Poland.

The plant material was finely cut, thoroughly mixed and directly exposed to the tests; only the SFE extraction was performed from the dried plant material.

How were the extractions run?

For ASE, finely cut rhizomes (1 g) were placed in a stainless steel extraction cell and extracted with n-hexane at 40, 60 and 80 °C, each extraction being performed three times. The other working conditions of the ASE 100 extractor (Dionex) were a static time of 5 min, a flush volume of 60 %, a purge time of 20 s and 3 extraction cycles, the pressure being maintained at about 100 bar throughout the extraction process. The extracts were first evaporated on a rotary vacuum evaporator at 35 °C, then dissolved in n-hexane before the GC-MS analysis. For hydrodistillation, 50 g of finely cut ginger rhizomes were placed in a round-bottomed flask with 130 mL of distilled water, and the process was performed for 3 h in a Deryng apparatus. The oil obtained was mixed with n-hexane, dried from water over silica gel and stored in a sealed vial at 4 °C before the GC-MS analysis. In the authors’ list of the extracts obtained, Deryng apparatus hydrodistillation (HD) is given at atmospheric pressure, a temperature of “>100” °C and 180 min, with water as extractant, and yielded a “Liquid oil extract, light yellow”. The extracts ASE 40, ASE 60 and ASE 80 are given at 100 bar, at 40, 60 and 80 °C respectively, for 3 × 5 min with n-hexane as extractant, and are described as “Liquid extract, dark yellow”.

What did the analyses show?

The authors write that only HD and SFE were found to recover monoterpene hydrocarbons from the plant matrix, the remaining techniques providing extracts rich in more complex constituents, like sesquiterpenes. They also write that classical hydrodistillation and SFE extraction were the only techniques from the tested ones that extracted simple terpene hydrocarbons, such as α- and β-pinene, myrcene, eucalyptol, camphene or others, the other extraction protocols not being efficient enough to recover this group of metabolites from the plant cells. According to the authors, ASE “in all tested temperature settings (40, 60, and 80 °C) was found to be inefficient”, “probably” due to the decomposition of volatiles occurring during the extraction process. The ASE extracts produced at 40 °C did not contain any terpenes, while at higher temperatures their content was “still very low”. In the authors’ table of the major constituents of volatile extracts, in µg/g of extract, ar-curcumene is given at 120.95 ± 3.6 for HD, 0.00 for ASE 40, 45.85 ± 4.2 for ASE 60 and 35.84 ± 7.3 for ASE 80. In the same table, α-zingiberene is given at 320.82 ± 25.4 for HD, 0.00 for ASE 40 and ASE 60, and 110.90 ± 6.9 for ASE 80. α-Sesquiphellandrene is given at 143.07 ± 35.1 for HD, 0.00 for ASE 40 and ASE 60, and 98.86 ± 15.2 for ASE 80. In another table, giving the percentage content of volatile extracts from ginger rhizomes, the authors’ results give α-zingiberene at 27.24 for HD and 18.11 for ASE 80 (accelerated solvent extraction at 80 °C, 3 times 5 min). For ar-curcumene, the same table gives 2.77 for HD and 2.75 for ASE 80.

What did one study on rosemary leaves measure?

According to Hirondart et al. (2020), conventional Soxhlet extraction (CSE) has several disadvantages, such as a long operation time requiring a minimum of hours or days, large solvent volumes and inadequacy for thermolabile analytes. In their study, rosemary leaves, Salvia rosmarinus Spenn. (Rosmarinus officinalis L. in the study), were provided by the company Naturex (Avignon, France) and were collected in Morocco in 2015. Their initial moisture was 8.2 ± 0.2 %; they were ground before extraction with a grinder fitted with a 0.5 mm sieve, and the granulometry of the rosemary powder was 610 ± 22 µm. The extraction solvent was made from food grade ethanol 96° v/v and demineralized water. According to the authors, hydro-alcoholic solutions offer many advantages as a solvent, since they can solubilize both hydrophilic and lipophilic “active compounds”.

How was PLE run in this study?

PLE was carried out on an ASE200 accelerated solvent extractor (Dionex, Thermo Fisher Scientific), which allows extraction of plant material at high pressure (up to 130 bar) and high temperature (up to 200 °C). The optimal loading was determined to be 3.1 g of ground rosemary leaves and 7.3 g of Fontainebleau sand, homogenized in an 11 mL stainless-steel cell fitted with stainless steel frits on both sides and a cellulose filter at the bottom to obtain a filtered extract. In each extraction cycle, the cell was filled with extraction solvent via an HPLC pump, pressurized and placed into the preheated oven; depending on the set extraction temperature, cell preheating lasted between 5 and 9 min, followed by a static period of extraction. The cell was then flushed with fresh solvent (60 % of the extraction cell volume) and purged with a flow of nitrogen for 1 min. According to the authors, several cycles of extraction can be performed to drain “active compounds” from the plant matrix; the extracts were collected into a glass vial and analysed without a concentration step.

Which parameters did the study test?

Preliminary trials evaluated the impact of the solvent, the percentage of dispersant and the flushing volume, the other extraction parameters being fixed according to the literature at 100 °C, 80 bar, a static extraction time of 5 min and 3 extraction cycles. PLE was performed with 0, 20, 40, 60, 80 and 100 % (g/g) ethanol in water. At 0 and 20 % ethanol, RA was extracted but no CA, while from 40 % ethanol CA was extracted as well. The solvent maximizing both RA and CA extraction was 80 % ethanol, with 10.13 ± 0.02 mg RA/g rosemary and 20.6 ± 0.4 mg CA/g rosemary extracted. Fontainebleau sand was used as a dispersant “in order to favor a uniform distribution of sample and maximize the extraction yield”. In trials with 30, 50, 70 and 90 % g/g of dispersant, the proportion of dispersant in the cell had a small impact on extraction mass yield, which varied between 31 ± 2 % and 37 ± 2 %. In extractions with flushing volumes of 40, 60, 80 and 100 % mL/mL, a flushing volume of 60 % mL/mL maximized the extraction mass yield (36.9 ± 2.0 %), while higher flushing volumes decreased it.

What did the experimental design show?

In a multivariate study with 20 experiments, temperature ranged from 40 to 190 °C, pressure from 40 to 130 bar and static extraction time from 3 (3 × 1 min) to 33 min (3 × 11 min). Mass extraction yield ranged from 26.3 to 49.6 %, RA content from 9.93 to 12.24 mg/g rosemary and CA content from 19.90 to 22.12 mg/g rosemary.

At a 95 % confidence level, the linear effect of temperature (A) and all quadratic effects (A2, B2 and C2) were significant, with p-values below 0.05, and the most influential effect was the linear term of temperature. In the summary of the ANOVA for the central composite design, the pressure (B) line gives p-values of 0.6225 for mass yield, 0.7389 for RA content and 0.1756 for CA content. Experimentally, the formation of a Maillard reaction product occurred at 150 °C or above, as evidenced by the brown color of the extract and the burnt smell. The authors state that in this study extraction was investigated for analytical purposes and that the sensory characteristics of the extracts were not considered. The authors write that the degradation of the targeted compounds did not occur, because the RA and CA contents of the extracts were not lower at 190 °C than at 115 °C. There was no significant difference in CO content, which according to the authors indicates that there was no degradation of CA into CO due to oxidation. The settings which simultaneously maximized mass yield, RA content and CA content were a temperature of 183 °C, a pressure of 130 bar and an extraction time of 3 × 1 min. Elsewhere in the paper, the authors write that the experimental design identified an optimized PLE set of a temperature of 183 °C, a pressure of 130 bar and an extraction duration of 3 min.

How did PLE compare with Soxhlet extraction in this study?

In the authors’ table on the reproducibility of extraction and the statistical comparison between PLE and CSE, which lists eight values per procedure and their mean, the mean extraction yield is 27.91 % for CSE and 46.20 % for PLE. In the same table, the mean RA content is 9.86 mg/g for CSE and 10.01 mg/g for PLE, and the mean CA content 17.68 mg/g for CSE and 21.13 mg/g for PLE. According to Hirondart et al. (2020), the mean RA contents are 10 ± 1 mg/g rosemary by PLE and 9.9 ± 0.5 mg/g rosemary by CSE, and the mean CA contents 21 ± 1 mg/g rosemary by PLE and 17.7 ± 0.9 mg/g rosemary by CSE.

References

• Barp L., Višnjevec A.M., Moret S., « Pressurized Liquid Extraction: A Powerful Tool to Implement Extraction and Purification of Food Contaminants », Foods 2023;12(10):2017, doi:10.3390/foods12102017 — https://www.ebi.ac.uk/europepmc/webservices/rest/PMC10217656/fullTextXML
• Czernicka L., Ludwiczuk A., Rój E., Marzec Z., Jarzab A. et al., « Acetylcholinesterase Inhibitors among Zingiber officinale Terpenes—Extraction Conditions and Thin Layer Chromatography-Based Bioautography Studies », Molecules 2020;25(7):1643, doi:10.3390/molecules25071643 — https://www.ebi.ac.uk/europepmc/webservices/rest/PMC7181192/fullTextXML
• Hirondart M., Rombaut N., Fabiano-Tixier A.S., Bily A., Chemat F., « Comparison between Pressurized Liquid Extraction and Conventional Soxhlet Extraction for Rosemary Antioxidants, Yield, Composition, and Environmental Footprint », Foods 2020;9(5):584, doi:10.3390/foods9050584 — https://www.ebi.ac.uk/europepmc/webservices/rest/PMC7278715/fullTextXML
• Govaerts R. (ed.), World Checklist of Vascular Plants (WCVP), Royal Botanic Gardens, Kew — https://sftp.kew.org/pub/data-repositories/WCVP/wcvp.zip