Supercritical Fluid Extraction (SFE) has emerged as a promising technique of extraction in past few years in food domain. The presentation reviews the theoretical aspects, instrumentation, applications and some case studies.Supercritical Fluid Extraction (SFE) has emerged as a promising technique of extraction in past few years in food domain. The presentation reviews the theoretical aspects, instrumentation, applications and some case studies.

1. SUPERCRITICAL FLUID EXTRACTION
IN FOOD ANALYSIS
By
Ms. Shruthy Sesharinathan
Ms. Srutee Rout
Mr. Varad Bende
Ms. Zumismita Kalita
M.Tech Food Biotechnology
Institute of Chemical Technology, Mumbai

2. 2
 INTRODUCTION TO FOOD ANALYSIS
 SUPERCRITICAL FLUID
 SUPERCRITICAL FLUID EXTRACTION
 APPLICATIONS OF SFE
 CASE STUDIES
 ADVANTAGES AND DISADVANTAGES
 INDUSTRY OUTLOOK
 CONCLUSIONS AND FUTURE SCOPE
 REFERENCES
CONTENTS

3.  Food analysis is a very important branch of analytical chemistry, able to provide information
about chemical composition, processing, quality control (QC) and contamination of foodstuffs,
ensuring compliance with national and international food standards
 Every technique provides specific information based on physical-chemical interaction of the
molecules present in the food compounds
HAVE YOU EVER WONDERED WHAT ARE THE CONSTITUENTS OF FOOD WE
EAT EVERYDAY?
Techniques used in Food Analysis
Ultraviolet, Visible and Fluorescence Spectroscopy
Infrared Spectroscopy (IR)
High-performance liquid chromatography (HPLC)
Gas chromatography (GC)
Mass spectrometry (MS)
Supercritical fluid chromatography (SFC) and supercritical fluid extraction (SFE)
Nuclear magnetic resonance (NMR)
FOOD ANALYSIS 1

4. Check quality of raw ingredients
Check composition during processing
Evaluating new processes for making food products
Identifying source of problem for unacceptable products
Develop/check nutritional label (plus other government regulations)
 STEPS IN ANALYSIS
FOOD ANALYSIS
Selection and Preparing
the Sample
Performing the assay
Calculation and
Interpreting the result
 WHY FOOD ANALYSIS ?
RAW MATERIALS PROCESSED FOODS FINISHED PRODUCTS
 WHERE FOOD ANALYSIS ?
2

5. • A substance above its critical point
• Liquid and gas phase are indistinguishable
• Possesses solvative power like liquid
• Diffuses like gas
• Carbon dioxide is the most commonly known
• Used as solvent in different separation, purification, analysis processes
• Miscible with each other: Tc (mix) = (Mole fraction A) x TcA + (Mole fraction B) x TcB
5
Density (kg/m2 ) Viscosity (μPaS) Diffusivity (mm2/s)
Gases 1 10 1-10
Supercritical fluids 100-1000 50-100 0.01-0.1
Liquids 1000 500-1000 0.001
Temperature
Pressure
Solid
Liquid
Supercritical Fluid
Critical point
Triple point
For CO2 , TC =31°C, PC =1100 psi
Gas
Fig: Pressure vs. Temperature plot of SCF
SUPERCRITICAL FLUID
 Comparison Between Properties of Fluids
3

6.  Extraction : Process of selectively dissolving mixture components in appropriate solvent
 .Whereas in Supercritical fluid extraction, Supercritical fluid is used as solvent/ mobile
phase; resembles to soxhlet, where bioactive components are extracted
 PRINCIPLE
• Chilled solvent is pressurized followed by heating causes critical condition
• Use of solvative and diffusive property of SF
• Passes through solid like a gas and dissolves active compounds like a liquid
• Fine tuning by small change in pressure and temperature near critical point
• Co- modifier is used for increasing solubility
6
Temperature-> Pressure->
Fig: Phase change of CO2
SUPERCRITICAL FLUID EXTRACTION 4

7. 7
 HISTORY OF SCF AND SFE
• Supercritical fluid (SCF) was discovered by Cagniard de la Tour in 1822. He called this
state ‘the particular state’
• Solvent properties of SCF were first reported well over 100 years ago
• Ability of SCF to dissolve low vapour pressure solid materials was first reported by Hannay
and Hogarth in 1879
• Since 1980s – 1990s SCF has been used in several industries and research as it resembles
Soxlet extraction except solvent used is a supercritical fluid
SUPERCRITICAL FLUID EXTRACTION 5

8.  MODE OF OPERATION
 FACTORS AFFECTING
8
SUPERCRITICAL FLUID EXTRACTION
 SUPERCRITICAL FLUID EXTRACTION
Static Extraction
• Discontinuous recovery of extracted
material
Dynamic Extraction
• Continuous recovery of extracted
material
Extraction Process
Particle size and shape
Moisture content
Surface area and
porosity
Sample size
Vapour pressure
Interaction with SCF
Choice of Solvent
Polarity of analyte
Solubility of analyte
Requirements of the
adjuvants
Critical pressure
Critical temperature
Interaction with
component
6

9. 9
SUPERCRITICAL FLUID EXTRACTION
Solvent Critical Temperature °C Critical Pressure MPa
Water 374.0 22.1
Methanol 34.4 8.0
Carbon dioxide 31.2 7.3
Ethane 32.4 4.8
Nitrous oxide 36.7 7.1
Propane 96.6 42
SOLVENTS IN SUPERCRITICAL FLUID EXTRACTION
CO2 – SOLVENT FOR SUPERCRITICAL FLUID EXTRACTION
•Chemically inert
•Cheap
•Colourless, Odourless
•Non toxic
•Non flammable
•Low critical temperature and pressure
•Recyclable
• Supercritical CO2 behaves like a lipophilic solvent
but, compared to liquid solvents, it has the
advantage that its selectivity or solvent power is
adjustable and can be set to values ranging from
gas-like to liquid-like
• Different solvents can be used along with CO2
depending upon the polarity of analyte
7

10. Fig: Schematic diagram for instrumentation of SFE
SUPERCRITICAL FLUID EXTRACTION - INSTRUMENTATION
 INSTRUMENTATION
 Fluid reservoir
•Gas cylinder for liquid CO2
 Pump
•Reciprocating Pump
•Syringe pump (Pulsefree flow at large range of flow rates)
 Restrictor
•Maintains pressure change in the extraction vessel
 Heater
•For increasing the temperature of SF
 Extraction cell
•Usually stainless steel chamber
•Capable of withstanding high P (300-600 atm)
Separator
•Separates SF and extract
Fig. Typical 1 l SF Extractor
10
8

11.  TECHNICAL SPECIFICATIONS
Fig: Flow diagram of 1 L extractor
Fig: 1 L SCFE system 11
Extraction vessels
•Operating pressure: Upto 400 bar
•Design Temperature: 80°C
•Volume of extraction: 0.1 L-300L
•Material of construction: SS 316
•Product separator
•Operating pressure: 250 bar
•Design temperature: 250°C
•Volume of separation vessel: 0.1-20 L
•Material of construction: SS316
 Liquid CO2 pump
•Operating pressure: 500 bar
•Discharge capacity: Variable
•Material of construction: SS/PTFE
 Liquid CO2 storage tank
• Material of construction: Mild steel
• Temperature: 20°C
• Pressure: 57 bar
• Full length eductor or ‘dip tube’
• No helium filled headspace
• Grade ‘bone dry’, less than 50 ppm H2O
SUPERCRITICAL FLUID EXTRACTION - INSTRUMENTATION 9

12. 12
SUPERCRITICAL FLUID EXTRACTION
 SUPERCRITICAL FLUID EXTRACTION – OPERATIONAL STAGES
From CO2 gas Feed Vessel,
CO2 gas in liquid form will
be fed to Refrigeration unit
and gets cooled to (-) 15°C
High Pressure Liquid CO2
Pump sucks the Liquid CO2,
compresses it up to 300 bar
pressure, stores it in Liquid
CO2 Receiver vessel
High pressure Liquid CO2
heated by Heater unit up to
30 -80°C, Supercritical fluid
CO2 gets stored in CO2 SCF
Vessel at 300 bar pressure
Supercritical fluid CO2
enters in the already charged
Extractor (Temperature: 30 -
80 °C and pressure: 300 bar)
SCF+ extract enters
separation vessel- Decrease
in pressure causes separation
Left out CO2 gas gets re-
circulated in the system via
refrigeration Unit
Fig: Schematic diagram for instrumentation of SFE
Separator
Control
valve
Chiller
CO2
reservoir
PumpHeater
Extractor
10

13.  RECYCLING SFE (TRAPPING SYSTEM)
There can be three ways-
1. Reduction of pressure
2. Reduction of temperature
3. Pumping SCF to expansion tank
13
SUPERCRITICAL FLUID EXTRACTION 10

14. • Used to extract active ingredients or Analytes from various plants and microbial samples and
useful in extraction of unknown natural products
• By manipulating temperature and pressure of the supercritical fluid, we can solubilize and
selectively extract the compound of interest
 COMPARATIVE STUDY
Sr No Solvent Extraction Supercritical Fluid Extraction
1. Impure due to the presence of different
types of solvents
Is totally free of solvents and hence very pure
2. High content of heavy metals may come in
the extract
Extract highly pure, eliminates heavy metals
when not required
3. Contents of inorganic salts is larger
As polar substances get dissolved due to
poor selectivity of solvents
Totally free of inorganic salts as polar
substances do not get dissolved because of
high selectivity of CO2
4. Both polar as well as non polar compounds
are extracted
Only non polar compounds get extracted.
Need modifiers for polar compounds.
14
SFE IN FOOD ANALYSIS 11

15.  Determination of fats and oil samples in meat, eggs, meals, chocolate, dairy products and
food snacks
 Extraction of natural products, flavors and fragrances from seeds, vegetables and plants
using SC-CO2 account for about one-half of all SFE applications in food analysis
 Determination of heavy metals from the food samples
 SFE is also extensively used for isolating analytes such as pesticides, drugs or toxins from
samples
APPLICATIONS OF SFE 12

16. 16
APPLICATIONS OF SFE
40%
19%
10%
6%
25%
Sales
Fat
Determination
Pesticides
Drug residues
Toxin
Flavours and
Fragrances
13

17.  Total Fat
 Major component of food
 Possesses functional, nutritional properties
 Overconsumption may result in obesity
 SCF extraction
1. From meat and meat products [1]
• Temperature: 80° C, Pressure: 345 bar, Flow rate: 10 and 200 ml/min
• Solvent: CO2
• Recovery: 96 to 100%
2. From olives [2]
• Temperature: 50° C, Pressure: 352 bar
• Solvent: CO2
• Recovery: 67-99%
CASE STUDIES 14

18. CASE STUDIES
 STEROIDS
 Structural and signaling molecule
 Medicinal properties
 SCF Extraction
1. Cholesterol from fish muscle [3]
• Temperature: 40-50° C, Pressure: 137-345 bar, flow rate: 7.7 ml/min
• Solvent: CO2
• Recovery: 97-99%
2. Cholesterol from cod liver oil [4]
• Temperature: 60° C, Pressure: 115 bar
• Solvent: CO2
• Recovery: >99%
15

19.  Solvent extraction and solvent distillation are common process operations in the production of
natural flavours and fragrances.
 In order to minimize the competitive edge of synthetic materials, alternative extraction
methodologies that are cost efficient and comply with both consumer preference and regulatory
control must be used like Extraction with super-critical fluids.
CASE STUDIES 16

20.  AROMA COMPOUNDS IN SPICES FRUITS, VEGETABLES AND CEREALS
Sr
No
Spice Compone
nt
Extracted
Technique
used With
SFE
P bar / Temp
C
Comments Ref.
1 Ginger 6-Gingerol SC-CO2 280bar/40C 25.97% of total
extract
[5]
2 Thyme Thymol Dynamic SFE
with SC-CO2
coupled to
GC.
200 bar/ 40°C 95% Recovery [6]
3 Turmeric Turmerone
s
Using
modified CO2
(addition of
methanol)
200-400
bar/313-333
K .
60% Recovery [7]
4 Citrus fruits limonene SC-CO2 125 bar / 308
K
75% yield was
extracted using
ratio 6 kg of CO2
per kg of orange
peel
[8]
20
CASE STUDIES 17

21. 1. AFLATOXIN B1
CASE STUDIES
Sr
No
Food Sample SCF Pressure
bar /
Temp C
Analyte
determinat
ion
Comments Ref.
1 Corn CO2-15% ACN/
MeOH (2 : 1)
345/80 HPLC
Fluroscence
90% Recovery [9]
2 Animal feed CO2+10%
MeOH
397/80 HPLC
Fluroscence
86% Recovery [10]
3 Peanut meal CO2 -MeOH 650/40 HPLC
Fluroscence
80% Recovery [11]
18

22. 2. FUMONISIN B1[12]
• Highly toxic compounds extracted from corn and corn dust
• Fungi : Fusarium moniliforme
• Can cause neurotoxic disease and cancer in humans
• When CO2 is used as solvent , the ability to extract Fumonisin compounds is weak. Hence mixed with
modifiers like methanol, acetonitrile and acetic acid
• Temperature - 40° C and Pressure – 18Mpa
CASE STUDIES
22
19
Extraction method Mean recovered concn (ppm)
Liquid-liquid extraction 2.12
SFE before optimization 24.53
SFE after optimization 78.16
LIQUID-LIQUID EXTRACTION VS. SFE

23. 23
 PESTICIDES
Sr
No
Food
Material
SF P bar /
Temp C
Analyte
Determinatio
n
Comments Ref.
1 Tea CO2 +
Acetone
380/60 GC-MS 70-90% recovery [13]
2 Fish CO2 135-179 /50 GC-MS 46-92% recoveries [14]
3 Spinach CO2 103/40 SFC-MS Upto 100 ppb pesticide
residues extracted
[15]
CASE STUDIES 20

24. 24
 HEAVY METALS
Sr.
No
Food
Material
Analyte
(Heavy
Metal)
SF P bar /
Temp
C
Analyte
Determ
ination
Comments Ref.
1 Digfish Arsenic CO2
-20%
MeOH
380/60 Micellar
LC-
ICP-MS
57% recovery [16]
2 Bovine liver Zinc CO2 203/50 AAS In situ complexation with
tetrabutylammonium
dibutyl-dithiocarbamate.
86% recovery
[17]
3 Seafood Methylmer
cury
CO2 201/50 AAS 89& Recovery [17]
CASE STUDIES
• The reagents used for the supercritical fluid extraction of toxic heavy
metals are Kelex 100, Cyanex 272, 301 and 302, and D2EHTPA
21

25. Selectivity
Efficiency
Reproducibility
Safety
• Chemically inert solvent
• Non flammable solvent
• Good solvent characteristics for non polar and slightly polar solutes
Chemical aspects
• Recyclable
• Hazardous solvent wastes are eliminated to a greater extent
• Non toxic wastes even if generated
• Low energy requirements
Green Technology
• Easily recoverable
• Dissolving power and selectivity is a function of temperature, pressure and modifiers
• Mild operating temperature ensures product stability and quality
Physical aspects
25
ADVANTAGES AND CHALLENGES 22

26. High capital cost
Cannot analyze extremely polar analytes
High pressures required
Use of organic solvents as modifiers makes it less
green
26
ADVANTAGES AND CHALLENGES 23

27. Market growth rate – over 25 % from 2013 to 2018
PROMINENT MANUFACTURERS
• Agilent
• Fischer Scientific
• Helwett Packard
• Chemtron Science Laboratories
• Consafe Science India Private Limited
• Nova Swiss
• NATECO2
Cost of instrument ranges from Rs 10 lac to few crores
MARKET OUTLOOK 24

28. 28
CONLCUSIONS 25
• Supercritical technology is a promising alternative technology for extracting unusual compounds
due to its selectivity, efficiency and speed of operation
• Fine tuning of solvent can be done in supercritical extraction near critical point as it causes huge
changes in density
• Problems with extraction efficiency can occur when the matrix contains large amounts of water
or lipids, which may co-elute with the analyte and impose problems in the subsequent analysis
• SFE is environmentally benign method as it doesn’t require toxic solvents for extraction
• As the time required in experiment is 10-20 mins, it is a rapid method of analysis

29. • Supercritical technology is a promising alternative technology with a bright future
• Current interests indicate that we will see more products on the shelves of our markets processed
with supercritical technology
• High pressures is another concern, modifying more sophisticated instruments which can work
optimally under high pressure
• Operational cost can be decreased by process optimisation
• Recent work has shown that SFE limitations can be overcome by using a water adsorbent such as
Hydromatrix® and/or a fat retainer as basic alumina in the extractor, and by adding a polar
modifier, when nonpolar carbon dioxide is the main extracting fluid
• Ongoing researches can also help us to better understand the full potential of supercritical
technology for developing novel processes and equipment, and that will help to widen the
application areas and to reduce the processing costs
29
FUTURE SCOPE 26

30. 1. Eike Anklam , Hans Berg , Lennart Mathiasson , Matthew Sharman & Franz Ulberth (1998)
“Supercritical fluid extraction (SFE) in food analysis: A review”, Food Additives &
Contaminants, 15:6, 729-750
2. Bruna Aparecida Souza Machado, Camila Gambini Pereira, Silmar Baptista Nunes, Francine
Ferreira Padilha & Marcelo Andres Umsza Guez (2013) “Supercritical Fluid Extraction Using
CO2: Main Applications and Future Perspectives”, Separation Science and
Technology, 48:18, 2741-2760
3. Kooi-Yeong Khaw, Marie-Odile Parat, Paul Nicholas Shaw and James Robert Falconer,
“Solvent Supercritical Fluid Technologies to Extract Bioactive Compounds from Natural
Sources: A Review ”,Molecules, 14 July 2017.
4. Ciftci ON (2012) Supercritical Fluid Technology: Application to Food Processing. J Food
Process Technol 3:e105.
5. M. Kandiah & M. Spiro. “Extraction of ginger rhizome: kinetic studies with supercritical
carbon dioxide” : International Journal of Food Science and Technology (1990) 25,328-338.
6. B. S.A.B. Vieira De Melo, G.M.N. Costa, R. Garau, A. Casula And B. Pittau. “ Supercritical
Co2 Extraction Of Essential Oils From Thymus Vulgaris” Braz. J. Chem. Eng. Vol.17 N.3 São
Paulo Sept. 2000
7. C. Gopalan Etal“supercritical Carbon Dioxide Extraction Of Turmeric (Curcuma Longa)”. J
Agric Food Chem 2000 Jun;48(6):2189-92.
30
REFERENCES 27

31. 8. D. Mira, B & Blasco, M & Berna, A & Subirats, Sebastian. (1999). Supercritical Co2
Extraction Of Essential Oil From Orange Peel. Effect Of Operation Conditions On The
Extract Composition. The Journal Of Supercritical Fluids.
9. Taylor SL, King JW, Richard JL, Greer JI (1993) J Agric Food Chem 41:910Ð91349.
10. Boenke A, van Egmond HP, Wagsta¤e PJ (1993) Fresenius J Anal Chem 345:224Ð22650
11. Engelhardt H, Haas P (1993) J Chromatogr Sci 31:13Ð19
12. Mustafa I. Selim, Saleh H. El-Sharkawy, and William J. Popendorf, Supercritical Fluid
Extraction of Fumonisin B1 from Grain Dust, J. Agric. Food Chem., Vol. 44, No. 10, 1996.
13. Multiresidue analysis of pesticides in tea by supercritical fluid extraction (SFE) and GC-MS,
Shokuhin Eiseigaku Zasshi. 2012;53(3):139-45.
14. Nam KS, Kapila S, Yanders AF, Puri RK (1990) Chemosphere 20:873-880
15. SFE–SFC–MS for the Analysis of Pesticide Residues in Food Products, Gesa Schad, The
Column, Jul 09, 2015, Volume 11, Issue 12.
16. Cleland SL, Olson LK, Caruso JA, Carey JM (1994) J Anal At Spectrom 9:975-979.
17. Holak W (1995) J AOAC Int 78:1124-1125.
18. Market, trends and applications of phytoextracts produced by supercritical CO2, M. Klasik, N.
Igl, A. Wuzik, DEUTSCHE GESELLSCHAFT FÜR QUALITÄTSFORSCHUNG.
31
REFERENCES 28

32. THANK YOU !