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Supercritical Extraction of Steviol Glycosides from Stevia Leaves


Supercritical fluid extraction (SFE) has emerged as a highly promising and efficient method for the extraction of steviol glycosides from the Stevia rebaudiana plant. The supercritical extraction process utilizes carbon dioxide (CO2) at temperatures and pressures above its critical point, creating a unique supercritical fluid with excellent solvent properties. In the context of steviol glycoside extraction, this method offers numerous advantages, including high selectivity, minimal solvent residue, and reduced degradation of thermally sensitive compounds. Supercritical extraction enables the precise control of extraction parameters, allowing for the customization of conditions to target specific steviol glycosides, thereby optimizing product quality. Furthermore, the environmentally friendly nature of CO2 as a solvent aligns with the growing demand for sustainable and green extraction processes. The potentiality of supercritical extraction in steviol glycoside extraction lies in its ability to deliver a pure and high-quality product while minimizing environmental impact, making it a compelling choice for the food and beverage industry as it seeks healthier and natural sweetening alternatives.

Supercritical fluid extraction (SFE) is a modern and efficient method of extracting compounds from various materials using supercritical fluids as solvents. Supercritical fluids, typically carbon dioxide (CO2) in this context, exhibit properties of both liquids and gases under specific temperature and pressure conditions. The critical point is the set of conditions where the fluid is neither distinctly liquid nor gas but possesses characteristics of both.

In supercritical fluid extraction, CO2 is pressurized and heated beyond its critical point, creating a supercritical state. This supercritical CO2 is then used as a solvent to extract target compounds from solid or liquid materials. The process offers several advantages over traditional extraction methods:

  1. Selectivity: Supercritical fluids can be tuned to selectively extract specific compounds by adjusting temperature and pressure. This allows for the extraction of target compounds without co-extracting unwanted substances.

  2. Mild Processing Conditions: Supercritical fluid extraction is typically performed at lower temperatures compared to other extraction methods, minimizing the degradation of heat-sensitive compounds.

  3. Environmental Friendliness: CO2 is commonly used as the supercritical fluid in extraction due to its low toxicity, non-flammability, and minimal environmental impact. Additionally, it can be easily removed from the extracted product.

  4. Efficiency: Supercritical fluid extraction is generally faster than conventional extraction methods, leading to higher productivity.

  5. Solvent-Free End Product: As the supercritical fluid can be easily separated from the extracted compounds, the final product is often free from residual solvents, ensuring a cleaner and safer end product.


Applications of supercritical fluid extraction are diverse and include the extraction of essential oils, flavors, fragrances, pharmaceuticals, and bioactive compounds from natural sources. The method is widely employed in industries such as food, pharmaceuticals, and natural product extraction due to its efficiency and ability to produce high-quality extracts. Despite its advantages, the equipment required for supercritical fluid extraction can be complex and expensive, limiting its widespread use in certain applications.

What is supercritical fluid extraction ?

Different phases of Carbon Dioxide at different temperature and pressure

Phase diagram.jpg

Properties of supercritical fluids


Liquid like densities

(100 – 1000 times more than gasses)

Diffusivities higher than liquids

(10⁻³ – 10⁻⁴ cm²/S)

Good solvating power

Almost no surface tension

Low viscosity (10 – 100 times less than liquid)

Gas like compressibility


CO2 Phase change

Phase change of carbon dioxide from liquid to supercritical fluid

Major components of a supercritical extraction system

Here is a general overview of the main components found in a typical supercritical extraction system:


Extractant Gas (typically CO2) Supply System:

High-pressure extractant gas is stored in a gas cylinder and regulated to achieve the desired pressure for supercritical fluid extraction. The gas must be of high purity to ensure the final extracted product is free from contaminants.


A high-pressure pump is used to pressurize the gas to levels above its critical point. This pump is a crucial component in maintaining the necessary conditions for the fluid to exist in a supercritical state.


The preheater raises the temperature of the pressurized gas to the desired level, typically above the critical temperature. Heating is a critical step in achieving the supercritical state and enhancing the solubility of target compounds.

Extractor Vessel:

The extractor vessel is a high-pressure chamber where the supercritical fluid comes into contact with the sample material. The sample material (e.g., plant material, biomass, or other substances) is placed in the extractor vessel for extraction.


After extraction, the supercritical fluid carrying the extracted compounds is depressurized in a separator. The change in pressure causes the supercritical fluid to revert to its gaseous state, leaving behind the extracted compounds.

Collection System:

The separated extracted compounds are collected in this system, which may include a collection vessel or other arrangements depending on the specific application.

Temperature and Pressure Controls:

The system is equipped with precise temperature and pressure controls to allow for the fine-tuning of extraction conditions. These controls are essential for adjusting the selectivity and efficiency of the extraction process.

Safety Features:

Given the high pressures and temperatures involved, safety features such as pressure relief valves and emergency shutdown mechanisms are incorporated to ensure the system operates safely.

The addition of a co-solvent in supercritical fluid extraction (SFE) can play a significant role in enhancing the efficiency and selectivity of the extraction process. While supercritical fluids like carbon dioxide (CO2) are excellent solvents under supercritical conditions, they may not be ideal for extracting certain types of compounds. Co-solvents are often introduced to the system to modify the solvating properties of the supercritical fluid, expanding its applicability to a broader range of target compounds. Here are some key roles of co-solvents in supercritical extraction:

  1. Increased Solubility:

    • Co-solvents can enhance the solubility of specific compounds in the supercritical fluid. This is particularly important when the target compounds have limited solubility or are poorly extracted using the supercritical fluid alone.

  2. Improved Selectivity:

    • Co-solvents can improve the selectivity of the extraction process. By altering the solvating power of the supercritical fluid, co-solvents help in selectively extracting certain compounds while minimizing the co-extraction of unwanted substances.

  3. Temperature Adjustment:

    • Co-solvents can influence the critical temperature of the overall solvent system. This allows for extraction at lower temperatures, reducing the potential for thermal degradation of heat-sensitive compounds.

  4. Tuning Extraction Conditions:

    • Co-solvents provide an additional parameter for adjusting the extraction conditions. By varying the composition of the supercritical fluid with the co-solvent, researchers can fine-tune the extraction parameters to optimize the process for specific applications.

  5. Enhanced Mass Transfer:

    • Co-solvents can improve mass transfer rates, facilitating the movement of solutes from the sample matrix to the supercritical fluid. This can lead to faster and more efficient extractions.

  6. Expansion of Applicability:

    • The use of co-solvents widens the range of compounds that can be effectively extracted using supercritical fluids. This makes SFE applicable to a broader spectrum of natural products, including polar and non-polar compounds.

  7. Reduced Extraction Pressures:

    • In some cases, the addition of co-solvents may allow for extraction at lower pressures compared to using supercritical fluids alone. This can have practical benefits in terms of equipment costs and safety considerations.


Common co-solvents used in conjunction with supercritical fluids include ethanol, methanol, water, and others, depending on the nature of the target compounds and the characteristics of the sample matrix. The choice of co-solvent and its concentration is a crucial aspect of optimizing the supercritical extraction process for specific applications.

Role of Co-solvents in Super-critical Extraction

Schematic diagram of a super-critical extraction system

SCFE Scheme.jpg

Extract Out

Pilot SCFE System
Small SCFE equipment.jpg
Industrial SCFE System
Industrial Super-critical Extraction System.jpg

Brief Review of R&D Work Done on Super-critical Fluid Extraction of Steviol glycosides:

Tan et al. (1988) hold a Japanese patent for the extraction of steviol glycosides by supercritical fluid extraction (SCFE) with CO2 and a co-solvent. Methanol, ethanol, and acetone were used as co-solvents. The purification step is accomplished by adsorption

Yoda, Marques, Petenate, and Meireles (2001) identified six classes of chemical substances in CO2-extracts of stevia leaves: alcohols, aliphatic hydrocarbons, labdanic diterpenes, sesquiterpenes and small amounts of steroids and triterpenes.


Glycosides are insoluble in carbon dioxide and soluble in mixtures of carbon dioxide and a polar solvent such as water, ethanol, methanol, etc. SCFE process can be advantageously used by designing a two-step process. The first step consists of a partial removal of the bitter tasting components by drastically reducing the CO2-soluble substances, such as alcohols, aliphatic hydrocarbons, sesquiterpenes, and triterpenes from stevia leaves, followed by SCFE using a polar co-solvent (Pasquel, Meireles, Marques, &Petenate, 2000). Their method involved i) pretreatment of stevia leaves by super critical extraction with CO2 at 200 bar and 30°C followed by ii) extraction of the stevia glycosides by super critical extraction using the following mixtures: CO2 + water, CO2 + ethanol, and CO2 + water + ethanol. Pretreatment was carried out at an average solvent flow rate of 4.82.10⁻⁵kg/s for a period of 12 hours.The second extraction was done at 120 and 200 bar and 16°, 30° and 45°C. Samples of the extract were collected every 30 minutes and the total extraction time was 12 hours. The original glycoside content of the leaves was 5.0 ± 0.1%. The yields were in general very low, except for the assay using water as cosolvent carried out at 120 bar and 16°C. They noticed that CO2 + water mixture is capable of extracting larger amounts of rebaudioside A than the conventional process using water and organic solvents.


The mean total yield for SCFE pretreatment of stevia leaves at 200 bar and 30°C was 3.0% (m/m). Conversely, yields for SCFE with co-solvent of stevia glycosides were below 0.50%, except at 120 bars, 16°C, and 9.5% (molar) water. For this condition, the total yield was 3.4± 0.3%. The yields for the conventional process were approximately equal regardless of whether untreated or pretreated leaves were used. The quality of the glycosidic fraction with respect to its capacity as a sweetener was better for the SCFE in terms of the relative amount of stevioside and rebaudioside A.


Yoda et al (2002) also studied the supercritical fluid extraction of steviol glycosides from stevia leaves using a two-step process: (i) CO2 extraction at 200 bar and 30°C, and (ii) CO+ water extraction. Approximately 72% of the CO2-soluble compounds were recovered and the major compound was austroinulin. The process removed approximately 50% of the original stevioside and about 72% of the rebaudioside A.


Choi et al (2002) evaluated the effect of temperature, pressure, and percentage of co-solvent on the extraction yield. Although sufficient extractability was not obtained by pure CO2 under any conditions of temperature and pressure, the addition of a co-solvent dramatically improved the extraction yield of steviol glycosides, making it comparable to organic solvent extraction. Among the co-solvents evaluated, the mixture of methanol and water showed greater extraction efficiency than the others. The extraction yield by CO2 + methanol + water (80:16:4) was found to be 150% of conventional organic extraction. In addition to improving the extraction yield, SCFE obviously provided a higher purity of steviol glycosides in the final extract.


The work of Erkucuk et al (2009) was aimed for optimization of the glycoside extraction of Stevia rebaudiana leaves using supercritical fluid extraction. They used different values of pressure (150–350 bar), temperature (40–80 °C) and concentration of ethanol-water mixture (70:30) as co-solvent (0–20%) with a CO2 flow rate of 15 g min⁻¹ for 60 min. The observed the optimum extraction conditions as 211 bar pressure, 80°C extraction temperature and 17.4% co-solvent concentration - which yielded 36.66 mg/g stevioside and 17.79 mg/g rebaudioside A. Total glycosides composition were close to those obtained using conventional water extraction (64.49 mg/g) and a little higher than ethanol extraction (48.60 mg/g). The postulated that industrial scale application of super critical extraction of steviol glycosides is technically challenging.


A supercritical fluid extraction process for steviol glycosides was optimized by Ameer et al (2017) by employing a 5-level-3-factor central composite design to achieve maximum target response values for total extract yield, ST yield, Reb-A yield and total phenolic content. The optimized SCFE parameters included a modifier concentration of 40%, an extraction temperature of 45°C, and a pressure of 225 bar. Super critical extraction yielded higher target response values than conventional maceration extraction (24h) and was a faster, lower energy, and greener extraction method with reduced CO2 emissions and lower solvent consumption.

References : 

Tan, S.; Shibuta, Y.; Tanaka, O. (1988)Isolation of sweetener from Stevia rebaudiana .Jpn. Kokai  63, 177, 764


Pasquel, A.; Meireles, MAA; Marques, M.O.M. (1999)Stevia (Stevia rebaudiana Bertoni) leaves pre-treatment with pressurized CO2: an evaluation of the extract composition, Proceedings of the 6thMeeting on Supercritical Fluids: Chemistry and Materials, 501


Pasquel, A; Meireles, MAA; Marques MOM & Petenate, AJ (2000). Extraction of stevia glycosides with CO2+ water, CO2+ ethanol, and CO2+ water+ ethanol. Brazilian Journal of Chemical Engineering. 17. 10.1590


Yoda, SK; Marques MOM; Petenate, AJ; Meireles MAA (2001). Kinetics of SCFE extraction of glycosides from Stevia rebaudiana Bertoni using CO2+ H2O. : Proceedings of the 4th Brazilian Meeting on Supercritical Fluids, Salvador, BA, Brazil.


Choi, YH; Kim, I; Yoon, KD;  Lee, SJ; Kim, CY;  Yoo, KP; Choi, YH & Kim, J (2002): Supercritical fluid extraction and liquid chromatographic-electrospray mass spectrometric analysis of stevioside from Stevia rebaudiana leaves; Chromatographia 55, 617–620


Yoda, SK;Marques MOM; Petenate, AJ; Meireles MAA (2003). Supercritical fluid extraction from Stevia  rebaudiana Bertoni using CO2 and CO2 + water: Extraction kinetics and identification of extracted components. Journal of Food Engineering - J FOOD ENG. 57.10.1016/S0260-8774(02)00281-9.


Erkucuk ,A; Akgun, IH;Yesil-Celiktas, O; (2009) Supercritical CO2 extraction of glycosides from Stevia rebaudiana leaves: Identification and optimization: The Journal of Supercritical Fluids, Volume 51, Issue 1, November 2009, Pages 29-35


Ameer, K; Byung-Soo Chun; Joong-Ho Kwon (2017):Optimization of supercritical fluid extraction of steviol glycosides and total phenolic content from Stevia rebaudiana (Bertoni) leaves using response surface methodology and artificial neural network modeling:Industrial Crops and Products, , Volume 109 Pages 672 - 685

Extraction conditions.jpg

Supercritical Extraction Plant at IIT Mumbai

Challenges for adoption of supercritical extraction processes in industrial scale 

High Equipment Costs: The initial investment for supercritical fluid extraction equipment can be relatively high. This may pose a barrier, especially for smaller producers or companies with limited resources.

Process Optimization: Achieving optimal extraction conditions, such as pressure and temperature, can be challenging. The efficiency of supercritical extraction is highly dependent on finding the right parameters for each specific compound, and the optimization process can be time-consuming.

Selectivity: The selectivity of supercritical CO2 along with the co-solvents identified so far, is not    very high, which may lead to the co-extraction of unwanted components. So, additional downstream steps, such as post-extraction purification, are necessary to obtain high-purity steviol glycosides.

Scale-Up Challenges: Transitioning from laboratory-scale to large-scale production can be complex. Parameters that work well in a small-scale setting might not be directly scalable, and adjustments may be necessary to maintain efficiency and yield.

Energy Consumption: While supercritical fluid extraction is considered a greener alternative to some traditional methods, it still requires energy to pressurize and heat the supercritical fluid. Energy consumption can be a concern, especially if the process is not optimized.

High production cost: Higher investment servicing cost, higher energy cost, low throughput higher maintenance and depreciation cost may add up to a higher and non-competitive production cost.

Characteristics of compressors used in CO2 based suparcritical extraction systems

Supercritical CO2 extraction requires compressors capable of handling high pressures and delivering a fluid in a supercritical state. The compressors used in supercritical CO2 extraction systems are typically of the positive displacement or dynamic (centrifugal) type. Here are the two primary types:

Reciprocating Compressors (Positive Displacement):

  • Piston Compressors: These compressors use one or more pistons to compress the CO2 gas. Piston compressors are known for their high efficiency and are commonly used in smaller-scale supercritical CO2 extraction systems.

  • Diaphragm Compressors: Diaphragm compressors use a flexible diaphragm to displace the gas, providing a pulsation-free and oil-free compression process. These compressors are suitable for applications where product purity is crucial.


Centrifugal Compressors (Dynamic):

  • Centrifugal Compressors: These compressors use a rotating impeller to accelerate the CO2 gas to high velocities, converting kinetic energy into potential energy (pressure). Centrifugal compressors are often used in larger-scale supercritical CO2 extraction systems. They offer high capacity and are suitable for continuous processing.

The selection of a compressor depends on factors such as the scale of the extraction system, desired pressure levels, and the specific requirements of the extraction process. Both reciprocating and centrifugal compressors have their advantages and limitations:

Reciprocating compressors:

  • Advantages: Well-suited for lower to medium-scale applications, high efficiency, and good pressure control.

  • Limitations: Pulsating flow, potential for wear and maintenance issues over time.


Centrifugal compressors:

  • Advantages: Suitable for large-scale applications, continuous operation, lower maintenance requirements, and smooth, pulsation-free flow.

  • Limitations: Lower efficiency at lower pressures compared to reciprocating compressors.


It's essential to choose a compressor that meets the specific requirements of the supercritical CO2 extraction process, considering factors such as pressure range, flow rate, reliability, and maintenance considerations. Additionally, safety measures must be in place to handle the high pressures associated with supercritical CO2 extraction systems.

Unit processes of a supercritical extraction system 

A supercritical extraction system is operated through a series of steps to harness the unique properties of supercritical fluids. The process involves controlling temperature and pressure to achieve and maintain supercritical conditions. Here is a general overview of how a supercritical extraction system is operated:

Loading Raw Material:

The material containing the target compounds is loaded into the extraction vessel. This could be plant material, biomass, or any substance from which specific compounds need to be extracted.

Adjusting Parameters:

The operator sets the parameters for temperature and pressure inside the extraction vessel. These parameters are critical for achieving the supercritical state of the solvent. The critical point of CO2 is at approximately 31.1°C (88°F) and 73.8 atm (1070 psi).


The extraction vessel is pressurized to the desired level. As pressure increases, CO2 transitions from a gas to a supercritical fluid. The high pressure is necessary for maintaining the supercritical state.


The system is heated to the specified temperature. This is typically done to reach the critical temperature of CO2, ensuring it is in the supercritical state.


The supercritical CO2 is introduced into the extraction vessel, where it behaves both like a gas and a liquid. This dual-phase nature allows it to penetrate the raw material and selectively extract the target compounds. The supercritical fluid's unique properties, such as its high diffusivity and low viscosity, enhance the extraction efficiency.


The extracted compounds, along with the supercritical CO2, are then passed to a separation vessel. By adjusting temperature and pressure, the supercritical CO2 is depressurized, reverting to a gas phase and leaving behind the extracted compounds. This separation step is crucial for obtaining the purified extract.


The extracted compounds are collected, and any remaining CO2 is often recycled for further use in the system. The collected extract can undergo additional processing steps, such as evaporation or solvent removal, to obtain the final product.

System Venting and Depressurization:

After extraction and collection, the system is vented and depressurized to return to normal atmospheric conditions. This step allows for safe handling and maintenance of the equipment.

It's important to note that the specific parameters and configurations of a supercritical extraction system may vary based on the target compounds, the nature of the raw material, and the desired product characteristics. Operators must carefully optimize conditions to achieve efficient extraction while minimizing degradation of sensitive compounds. Additionally, safety measures are crucial, given the high pressures involved in the process.

Scale-up challenges

Scaling up supercritical extraction processes from laboratory or pilot scale to commercial production poses several challenges that must be carefully addressed to ensure efficiency, consistency, and safety. Some key scale-up challenges for supercritical extraction processes include:

Equipment Design and Cost:

Designing larger extraction vessels and systems that can handle increased volumes while maintaining the necessary pressure and temperature conditions becomes complex. Moreover, the cost of scaling up equipment can be substantial, and considerations for cost-effectiveness must be factored into the design.

Pressure Management:

As the scale increases, managing and maintaining high pressures become more challenging. Larger vessels may require stronger materials, and pressure differentials across the system need careful consideration to prevent leaks or system failures.


Heat Transfer:

Achieving and maintaining precise temperature control becomes more challenging in larger systems. Efficient heat transfer throughout the system, especially in the extraction vessel, is crucial to ensure uniform extraction and prevent temperature fluctuations that could affect product quality.


Mass Transfer:

Mass transfer limitations may become more prominent at larger scales. Ensuring that supercritical fluid adequately permeates the raw material for efficient extraction becomes more challenging as scale increases.


Scale-Dependent Fluid Properties:

The properties of supercritical fluids, such as density and viscosity, can change at different scales. These variations may impact extraction efficiency and require adjustments to operating parameters to maintain optimal performance.

Safety Concerns:

Safety considerations become more critical as the scale of the extraction process increases. Dealing with larger volumes of supercritical fluids and managing the potential risks associated with high pressures requires robust safety measures and monitoring systems.


Raw Material Variability:

Scaling up may expose the extraction process to greater variability in the properties of raw materials. Ensuring consistent product quality across different batches, especially when dealing with natural materials, becomes a significant challenge.


Environmental Impact:

Larger-scale supercritical extraction processes may have a higher environmental impact, particularly in terms of energy consumption and CO2 emissions. Developing sustainable and energy-efficient practices becomes crucial as the scale increases.

Regulatory Compliance:

Meeting regulatory requirements for larger-scale production adds complexity. Compliance with safety, environmental, and quality standards becomes more demanding, necessitating careful documentation and adherence to guidelines.


Process Optimization:

Optimizing extraction conditions for larger-scale operations requires a thorough understanding of the interplay between parameters. Fine-tuning the process to achieve both efficiency and product quality becomes an ongoing challenge.

Addressing these scale-up challenges often involves a combination of engineering solutions, process optimization, and continuous monitoring. Collaboration between engineers, chemists, and process experts is essential to successfully navigate the complexities associated with scaling up supercritical extraction processes for commercial production.

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