a quick overview of extraction as a bioseparation methodology, along with two scientific articles as examples.

1. EXTRACTION
Course: Bioseparation Engineering.
Code: (BIO606)
Presented by:
Titilayomi Aboluwoye
Yousra Mohamed

2. OUTLINES
 Introduction to Extraction.
 Classes of Extraction.
 Liquid-Liquid Extraction.
 Solid-Liquid Extraction.
o Supercritical Fluid Extraction
 Study Cases.
 References.

3. DEFINITION
 In extraction, a solvent is used to solubilize and
separate a solute from other materials with lower
solubility in the said solvent.
Or is the process by which a solute is transferred from one
phase to a new phase.

4. CLASSES OF EXTRACTION
 Generally, there are two classes of Extraction
processes:
 Liquid-liquid extraction, is a separation process
consisting of the transfer of a solute from one
solvent to another, the two solvents being
immiscible or partially miscible with each other. it is
also known as partitioning.
Examples: Extraction of oxygenated terpenoids from
citrus essential oils using ethanol as a solvent.

5. CLASSES OF EXTRACTION CONT.
2. Solid-liquid extraction, whereby a solute is
extracted from a solid phase with the help of a
solvent. Also termed “leaching” and “elution”
(when applied to the removal of adsorbed solute
from an adsorbent)
Examples: extraction of salt from rock using water as
solvent.

6. SOLID-LIQUID EXTRACTION
The mechanism of solid-liquid extraction involves:
wetting of the solid surface with solvent, penetration
of the solvent into the solid, dissolution of the
extractables, transport of the solutes from
the interior of the solid particles to their surface,
and dispersion of the solutes within the bulk of the
solvent surrounding the solid particles by
agitation.

7. PROCESSING CONDITION OF EXTRACTION
PROCESS IS A CRITICAL FACTOR:
 Temperature [1]:
 Pressure [2].
 Particle size [3].
 Agitation [4].
 Application of pulsed electric field (PEF) to
extraction [5].

8. SUPERCRITICAL FLUID EXTRACTION
 A supercritical fluid (SCF) is a substance at a
temperature and pressure above those of the
critical point.
 Supercritical fluid extraction (SCFE or SFE) is an
extraction process carried out using a supercritical
fluid as a solvent [6] while extraction is usually
performed from a solid, it can sometimes be from
liquid.
 It can effuse through solids like a gas,
and dissolve materials like a liquid

9. SUPERCRITICAL FLUID EXTRACTION CONT.
Figure 1:Phase diagram showing supercritical region
[7].

10. SUPERCRITICAL FLUID EXTRACTION CONT.
 Hence, the critical point C represents the end of the
gas-liquid equilibrium curve on the temperature-
pressure plane.
 The density of supercritical fluids is close to that of
the liquid while their viscosity is low and
comparable to that of a gas.
 The relatively high density imparts to SCFs good
solubilization power while the low viscosity results
in particularly rapid permeation of the solvent into
the solid matrix.

11. STUDY CASES: CASE 1

12. AIM
The aim of this work was to evaluate supercritical
fluid extraction (SFE) for carotenoid recovery from
carrot peels on various carotenoid-rich fruit and
vegetable wastes.
15 matrices, including flesh and peels of sweet
potato, tomato, apricot, pumpkin and peach, as well
as flesh and wastes of green, yellow and red
peppers, were submitted to SFE under optimized
conditions (59 C, 350 bar, 15 g/min CO2, 15.5%
(v/v) ethanol as co-solvent and 30 min of extraction
time)

13. IMPORTANCE OF CAROTENOIDS:
 Carotenoids are molecules especially ubiquitous in red-
and orange-coloured fruits and vegetables.
 They are a central component of human nutrition due to
the important biological functions which they are
involved in and responsible for. These molecules are
also used as food colorants', have potent antioxidant
activities, and can be employed as precursors of aroma
or flavor compounds.
 Due to these reasons, there is a clear interest by the
food, chemical, pharmaceutical and cosmetics sectors in
utilizing Carotenoids for various applications, as
functional and/or bioactive compounds.

14. MATERIALS AND METHODS
1. SAMPLE PREPARATION
 Fifteen matrices of carotenoid-rich fruits and
vegetables were tested, all purchased from a local
supermarket. These included the flesh and peels of
sweet potato, red tomato, apricot, pumpkin, peach,
green, yellow and red bell peppers and their waste
residues (seeds and stems).
 All vegetables were washed and peeled manually.
The samples were then frozen at 20 C for 36–48 h,
freeze dried for 72 h, milled with a home grinder for
2 min and sieved to cut off particles greater than
750 m in diameter.

15. 2. SUPERCRITICAL FLUID EXTRACTION
 For each run, 5.0 g of freeze-dried samples were placed in a
supercritical fluid extractor.
 A total of 95.0 g of inert glass beads were added to fill the
vessel volume in order to avoid dispersion effects and the
samples submitted to a CO2 flow rate of 15 g/min and the
dynamic extraction time was fixed at 30 min.
 These operating conditions were previously optimized for
carrot peels via a Central Composite Design of Experiments
[8] and included: temperature of 59.0 C, pressure of 350 bar
and 15.5% (v/v) of ethanol as co-solvent.
 Runs were performed in duplicates and the results are
presented as the average value for all measurements.
 The extracts were collected, dissolved in ethanol and stored
at 18 C in dark glass containers until further analysis.

16. 3. MOISTURE CONTENT
 The moisture content in the samples was measured
by a halogen moisture analyser.

17. 4. CAROTENOID EXTRACTION AND ANALYSIS:
Briefly, 1.0–2.0 g of initial freeze-dried samples, both of flesh and
peel, were weighed and added to 6 mL of methanol.
 After vigorous mixing, samples were centrifuged for 5 min at
2500 g and the supernatant was separated; a new extraction
was performed twice with 8 mL of a mixture of hexane and
acetone (1:1).
 Subsequently, the organic solvent fractions were combined,
25 mL of saturated NaCl were added, and the mixture was
shaken in a separator funnel.
 After phase separation, the lower, water-phase was re-
extracted with 8 mL of hexane and the resulting supernatant
was combined with the first.
 The combined fractions were evaporated under nitrogen
stream and re-dissolved in methanol prior to High Pressure
Liquid Chromatography (HPLC) analysis.

18. CONT.
 The SFE extracts, in turn, obtained dissolved in the
ethanol used as co-solvent, were directly filtered
and submitted to the HPLC analysis.
 A silica-based reversed-phase column was used in
the separation of carotenoids .
 The injection volume was 100 L and the flow rate
was kept constant at 1.0 mL/min.
 For carotenoid identification and quantification,
previously-built calibration curves of external
commercial standards (-carotene, -carotene, lutein
and lycopene) were used. All detected peaks were
analysed at 450 nm.

19. CONT. STUDY CASES: CASE 2.

20. AIM
1. To develop SFE process to obtain THC extracts
from the cannabis plant. In addition, a solid phase
extraction (SPE) using CO2/EtOH as solvents
was explored as an isolation-purification technique
to obtain a high purity THC standard.
2. To study the effects of the extraction parameters
(temperature, pressure and EtOH concentration)
on the extraction yields and the THC content in
the extracts.
3. To isolate and purify THC from enriched extracts
using a single SPE step.

21. 1. SAMPLE PREPARATION:
 Samples of fully ripe cannabis were selected and
harvested (figure 3)
 A representative amount of vegetal material was
sampled and 300g selected for further use.
 The selected material was dried up at room
temperature, then the dried sample was milled and
sieved to a size of less than 0.5 mm.
 Finally, the vegetal material was stored at 4 °C in
the absence of light until SFE.

22. Figure 3: Showing the general structure of cannabis Sativa

23. 2. SUPERCRITICAL FLUID EXTRACTION
 The extractions were performed under minimal pressure to
avoid the thermal degradation of the target compound.
 SFE was performed using a laboratory scale SFE apparatus
(Fig. 4).
 Temperature in the extraction unit was controlled using an
electrical jacket and regulator.
 The extraction pressure and flow were maintained constant
using a regulator.
 Ethanol as a co-solvent was supplied by a liquid pump and
mixed with the main CO2 stream at a constant rate before at
the extraction cell.
 All extractions were kept constant:
1. the extraction time 4 h.
2. the cannabis sample amount 8 g.
3. the supercritical solvent flow 0.55 kg/h.
(as given by a previous study [9]).

24. Figure 4:

25.  The extractions were carried out in a six-hour
period, using a solvent polarity gradient - first
hexane, then ethyl acetate, and finally ethanol.
 Briefly, raw material was extracted with hexane,
obtaining the non-polarity fraction. Subsequently,
the residual cake was extracted with ethyl acetate,
producing the middle-polarity fraction.
 Finally, the next residual cake was extracted with
ethanol, thus getting the polar fraction.

27. CONT.
 The extraction parameters were varied:
1. Pressure (between 15–33 Mpa).
2. Temperature (40–80°C).
3. Co-solvent concentrations (0–5%) EtOH.
 After each extraction, co-solvent was removed
under vacuum and the extracts were weighed using
an analytical balance to estimate the extraction
yields.
 All extracts were analyzed by gas chromatography
with flame ionization detection (GC-FID) to quantify
their THC content.

28. 3. THC ISOLATION AND PURIFICATION BY SPE
 The isolation and purification of THC were developed
using a solid phase extraction column packed with silica
gel [10].
Solvent A(trifluoroaceticacid 0.05% in water)
solvent B(trifluoroacetic acid 0.05% in acetonitrile)
 The extract was dissolved in solvent A and injected into
the SPE column, then the compounds were eluted
using solvent B from at a constant flow rate.
 Then the solvent was removed under vacuum.

29. CONT.
 The SFE and GC-FID results were used to identify
and select an extract with good extraction yield, the
highest THC content and lowest contamination.
 The resulting free-solvent fraction was lyophilized,
and the yield of the SFE-SPE process was
determined.
 The final fraction was analyzed by RP-HPLC and
NMR to assess the purity of THC obtained.

30. Table 1: Shows that the highest extraction yield was 26.36%, corresponding to
the extract number 6 obtained at 33 MPa, 80 °C and 5% EtOH.

31. CONCLUSION
 At these conditions the supercritical solvent has a
good solvent power because of the high extraction
pressure.
 Also, the presence of co-solvent improves the CO2
solvation power [12,13].

32. REFERENCES:
[1] (Pereira et al., 2016)
[2] (Cacace and Mazza 2007)
[3] Vishwanathan et al., 2011
[4] (Cogan et al., 1967).
[5] (Loginova et al., 2010, 2011a,b; Yan et al., 2012)
[6] (King, 2000).
[7] Berk, Z. (2018). Food process engineering and technology. Academic press.
[8] De Andrade Lima M., Charalampopoulos D., Chatzifragkou A. Optimisation and modelling of supercritical
CO2 extraction process of carotenoids from carrot peels. J. Supercrit. Fluids. 2018;133:94–102. doi:
10.1016/j.supflu.2017.09.028.
[9] H. Perrotin-Brunel, Sustainable Production of Cannabinoids with Supercritical
Carbon Dioxide Tecnologies, TUDelft, 2018 (accessed 14 September 2018), https://
repository.tudelft.nl/islandora/object/uuid%3Ac1b4471f-ea42-47cb-a230-
5555d268fb4c.
[10] W. Kamysz, M. Okroj, E. Lempicka, T. Ossowski, J. Lukasiak, Fast and efficient
purification of synthetic peptides by solid-phase extraction, Acta Chromatogr. 14
(2004) 180–186.
[11] L. Rovetto, N. Aieta, Supercritical carbon dioxide extraction of cannabinoids from
Cannabis sativa L. Plant material, J. Supercrit. Fluids 129 (2017) 16–27, https://doi.
org/10.1016/j.supflu.2017.03.014.
[12] M. Rahoma, S. Marleny, M. Paulo, Extraction of caffeine, theobromine, and cocoa
butter from brazilian cocoa beans using supercritical CO2 and ethane, Ind. Eng.
Chem. Res. 14 (2002) 6751–6758, https://doi.org/10.1021/ie0203936.