Novel Separation Techniques in Oil/Fats, Fatty acids and By products viz, sterols, tocopherols etc. Chromatographic techniques, urea inclusion and exclusion, distillation, fractionation, crystallization etc

1. Novel Separation Techniques for Isolation
of Fatty Acids and Oil By-Products
National Conference on
“Newer Oleo-chemicals: Production & Industrial Application”
Organised By
Centre of Excellence
on
‘Applied Research, Training & Education in Lipid Science’
Department of Oil & Paint Technology,
Harcourt Butler Technological Institute, Kanpur
January, 10-11, 2015
Sadanand Patel & Sanjay Kr Singh
Department of Oil & Paint Technology
HBTI, Kanpur
sanjayhbti19@gmail.com

2. Contents
Conclusion
By products
Enzymatic & Urea Complexion
Counter Current Chromatography
Need & Fundamentals

3. Need???
Potential health benefits
of bioactive compounds
Challenge of
refineries
Profitability of
processing operations
.
.

4. Separation
Density
Boiling Point
Melting Point
Centrifugal
Separation
Distillation
Crystallization
Principle of Separations
Molecular Properties
Bulk Properties
 Melting Point
 Boiling Point
 Density
 Position & Nature of Bonds
 Packing Structure
 Molecular Dimensions

5. Liquid
Chromatography
Packing
Materials Type
Adsorption
Ion Exchange
Gel Filtration
Non Packing
Materials Type
Centrifugal
Field
Gravitational
Preparative Liquid Chromatography

6. Counter Current Chromatography
This technique includes:
 Liquid-liquid partition,
 Countercurrent distribution of solute mixture
between two immiscible liquid phases,
In the most distinct variants of CPC, one liquid phase remains
stationary while the second solvent phase passes through the
stationary phase solvent.

7. Animation

9. Animation
Rotar
(Instrumentation)

10.  Cost Effective: 10-20%
 Efficient: 99.5 – 99.8%
 Faster: 2-3 times
 Flexible
 Easy Scale Up
One single separation /
purification technique
adapted to samples ranging
from mg to several kg.
Bousquet et al*.
EPA and DHA from micro algal oil used heptane as the stationary
phase and acetonitrile/ water (3% v/v) as the mobile phase.
*O. Bousquet, N. Sellier, and F. L. Goffic, Chrotographia, 39, 40–44 (1994).
Advantages of CPC/CCC & Application

11. Enzymatic Separations
Long and highly unsaturated
fatty acids show high
resistance for lipases at attack
on ester bonds due to large
stearic hindrance and thus
method is widely used in
separation of  3-PUFA from
marine oils.
Bottino et al.* have illustrated the
mechanism of resistance of lipases
toward long-chain o3-PUFA in
marine oils.
*N. R. Bottino, G. A. Vandenberg, and R. Reiser, Lipids, 2, 489–493 (1967)

12. Pure Urea crystallizes in
tetragonal structure with
channels of 5.67 A° diameter
Crystal channel diameter: 5.6 A0
Fatty acid equivalent diameter: 6.5 – 13.5 A0
Urea complexation
In presence of long
straight-chain molecules,
it crystallizes in hexagonal
structure with inner
channels of 8–12 A°
diameter
Source: A. E. Smith, Acta Cryst., 5, 224–235

13. TAGs + Alc. KOH
Saponifiable
Unsaponifiable
Almost pure FFAs
and Subjected to
Urea Complexation
FFAs
+
Alcohol
Urea
UIC
Two fraction are developed and then UIC fraction is
evaporated to separate urea and fatty acids
Step: 1
Step: 2

14. Urea fractionation has been most frequently used for the
purification of fatty acids from fish oil [1] and from several vegetal
oils like, blackcurrant oil [2] borage oil [3]; rapeseed oil [4].
Sources:
1. Ratnayake, WMN et al., Fat Sci Technol, 90, 381, 1988.
2. Traitler H et al., J. Am Oil Chem Soc, 65, 755, 1988.
3. Shimada Y et al., J Am Oil Chem Soc, 75, 1539, 1988.
4. Hayes DG et al., J Am Oil Chem Soc, 75, 1403, 1998.
Advantages & Applications
Crystallization is technically not feasible to separate high unsaturated
fatty acids from moderately high unsaturated fatty acids. e.g.: C18:3 &
C18:5 can't easily separated as they have negative melting point, in
such cases UIC is robust tool.

15. By - products
Component Sunflower Cottonseed Soybean
Unsaponifiables, % 39 42 33
Tocopherols, % 9.3 11.4 11.1
a-tocopherol, % 5.7 6.3 0.9
Sterols, % 18 20 18
Stigmasterol, % 2.9 0.3 4.4
 Gums
 Deodorizer distillate (DOD)
 Waxes
 Soap Stock
Deodorizer distillate (DOD)

16. Tocols from Deodorizer distillate (DOD)
 Tocols = Tocopherols + Totrienols
 A combination of molecular distillation (MD), ethanol
fractionation, chemical alcoholysis, and ion-exchange
chromatography
 MD may not produce a high purity tocopherol because very
similar molecular weights of Tocols and Sterols
 By Ethanol fractionation purity can be enhanced as sterols are
insoluble, whereas Tocopherols are soluble in ethanol.

17. Esterification
of alcohols
Series of
Distillation
FAME
Tocols
Esters
Residues
Barnicki et al. *
Patented a solvent less process, comprised of an esterification
reaction to reduce the volatility of alcohols followed by a series of
distillation steps, where components boiling higher and lower than
tocopherols are separated from tocopherols.
*S. D. Barnicki, C. E. Sumner, Jr., and H. C. Williams, U.S. Patent 5,512,691, 1996.

18. Sterols are polycyclic alcohols,
Unsaponifiable fraction & quality
depends on temperature, duration,
quantity of stripping steam, and
the extent of vacuum
Phytosterols from DOD
•Either by
hydrolysis or
trans-
esterification
Breaking of
ester bonds
•Re-esterification of
Phytosterols occurs
during methyl ester
Trans-
esterification •Phytosterols
content up to 50%
by removing
FAMEs
Distillation

19.  Instead of Distillation we can go for Crystallization
(physical), solvent extraction (chemical), or crystallization
with additives via adduct formation and separation
(physicochemical)
 Organic solvents or solvent mixtures composed of low- and
high-polarity solvents and water are used for crystallization
of Phytosterols

20.  Quality & Quantity of feed and final product are the major
parameters for selection of isolation techniques
 Present day demand for a variety of fatty acids, including long-
chain polyunsaturated fatty acids (PUFA), may require a
combination of different processes and further steps of
separation
 Bulk properties as well as molecular properties both should be
combined to isolate the product within the specifications
 Good amount of experimental work & processes feasibility
analysis is required in order implement novel techniques on
industrial scale
 CCC/CPC techniques can be used for isolation of oleo-chemicals
at commercial scale
Conclusion

21. Carbon
No.
Chloroform Benzene Cyclohexane Acetone
Ethanol
95%
Acetic
acid
Methanol
10 3260 3980 3420 4070 4400 5670 5100
12 830 936 680 605 912 818 1200
14 325 292 215 159 189 102 173
16 151 73 65 53.8 49.3 21.4 37
18 60 24.6 24 15.4 11.3 1.2 1
Solubility
Volatility
Component Mol. Wt. Rel. Volatility
Fatty Acids 280 2.5
Squalene 411 5
Tocopherols 410 1
Sterols 415 0.6
Sterols esters 675 0.04
TAGs 885 small

22. Systematic Name Trivial Name
Shorthand
designation
Molecular
wt.
Melting point
(0
C)
Dodecanoic Lauric 12:00 200.3 44.2
Tetradecanoic Myristic 14:00 228.4 53.9
Hexadecanoic Palmitic 16:00 256.4 63.1
Heptadecanoic Margaric
(Daturic)
17:00 270.4 61.3
Octadecanoic Stearic 18:00 284.4 69.6
Eicosanoic Arachidic 20:00 312.5 75.3
Docosanoic Behenic 22:00 340.5 79.9
Tetracosanoic Lignoceric 24:00 368.6 84.2
Hexacosanoic Cerotic 26:00 396.7 88
Melting Point (M.Wt.)

23. Systematic Name Trivial Name
Shorthand
designation
Molecular
wt.
Melting point
(°C)
Cis-6-
octadecenoic
Petroselinic 18:1 (n-12) 282.4 30
Cis-9-
octadecenoic
Oleic 18:1 (n-9) 282.4 16.2
Tr-9-octadecenoic Elaidic tr18:1 (n-9) 282.4 43.7
Cis-11-
octadecenoic
Vaccenic
(Asclepic)
18:1 (n-7) 282.4 39
9,12-
octadecadienoic
Linoleic Acid
18:2(n-6) 280.4 -5
6,9,12-
octadecatrienoic
G-linolenic
Acid
18:3(n-6) 278.4
Melting Point (Unsaturation)