Toward the Discovery of the Tri-acylglycerol Composition of Milk Fat
Graduation report
Towards the Discovery of the Tri-acylglycerol
Composition of Milk Fat
N.J.L.C. Das
Bio-organic Chemistry
Stratingh Institute for Chemistry
2012 – 2013
, Toward the Discovery of the Tri-acylglycerol Composition of Milk Fat
Graduation report
Towards the Discovery of the Tri-acylglycerol
Composition of Milk Fat
Name: N. Das
Educational institute: University of Groningen
Department of Bio-organic Chemistry
Stratingh Institute for Chemistry
Nijenborgh 7, 9747 AG Groningen
Mentor: Prof. Dr. Ir. A.J. Minnaard
Tutor: P. Fodran
Industrial collaborator: FrieslandCampina
Mentor: Dr. W. Kloek
Educational institute: Avans Hogeschool
Academie voor de Technologie van Gezondheid en Milieu (ATGM)
Education: Chemistry
Specialisation: Organic and Polymer Chemistry
Teacher: Dr. E.W. Dijk
Period: 16-01-2013 – 14-06-2013
, Toward the Discovery of the Tri-acylglycerol Composition of Milk Fat
Summary
The identification and quantification of a single component in a complex mixture can be a daunting
task. In the field of lipid analysis, this is a regularly encountered challenge. Within a particular class of
lipids, for example tri-acyl glycerols (TAGs), di-acyl glycerols (DAGs), or phospholipids, the members
differ often little in their physical properties. This makes separation, in order to allow identification of
a single compound, even with advanced techniques often very difficult or impossible. Milk fat is a
very complex mixture of lipids. Bovine milk fat predominantly consists of TAGs (>98%). The analysis
of the quantity and identity of the constituting, individual fatty acids (FA) of the fat is very well
possible upon hydrolysis. However, the identification of the individual TAGs is extremely difficult.
The outline of the project was to synthesize a library of TAGs via a stereo-specific synthetic route.
Previous work from the Minnaard group has proven that the Co[R,R-Salen] catalyst (scheme 1; left) is
successful in the selective formation of the 1,3-DAG out of a glycidyl ester without the occurring of
migration to the 1,2-DAG. A library of 26 TAGs has now been synthesized in three steps in overall
yields of 80 – 90%. For an even more efficient approach, we aimed for a parallel synthesis. A resin
bound catalyst was necessary for the parallel synthesis, which could be easily filtered off after
completion of the reaction to afford pure products after removal of the solvent.
Scheme 1: (Left): Structure formula of Co[R,R-Salen] catalyst. (Right): Resin bound Co[R,R-Salen] catalyst
The resin bound Co[R,R-Salen] catalyst (scheme 1; right) was synthesized in three steps out of a
hydroxymethyl resin. To be able to attach the Salen ligand to the resin, a linker with a functional
group was introduced. The desired Salen ligand could then be bounded with a trans-esterification to
afford the desired Salen ligand on the resin and the reaction was able to monitor. Cobalt was
inserted to afford the desired catalyst on resin with a loading of 0.2 mmol/g of Co. The Co[R,R-Salen]
catalyst was used in 1 mol% in the ring opening of epoxides. Full conversion was achieved after 6
days in CH2Cl2, compared to 10 h of the homogeneous Co[R,R-Salen] catalyst in the ring opening
without solvent.
The second goal of this graduation project was to separate (regio)-isomers of the synthesized TAGs
by HPLC. These results could be compared to HPLC results of milk fat fractions, in which the
composition of TAGs in milk fat could be determined. Separation of a mixture of three TAGs with
different TCNs by HPLC with optimized conditions has been achieved. For regio-isomers and mixtures
of four or more TAGs which do not differ much in chain length, separation has not been achieved or
has not been studied well enough. The optimized conditions are with a runtime of 30 minutes:
- Mobile Phase: 100% n-heptane
- Flow: 0,5 ml/min
- Inj. Vol. : 5 µl
- Column: Chiracel OB-H
- Detector: Electron Light Scattering Detector (ELSD)
, Toward the Discovery of the Tri-acylglycerol Composition of Milk Fat
List of abbreviations
BnBr Benzylbromide
bs (NMR-Spectrum) Broad singlet
DAG Di-acyl glycerol
DCC N,N’-Dicyclohexylcarbodiimide
DIPEA N,N-Diisopropylethylamine
DMF N,N-Dimethylformamide
dd (NMR-Spectrum) Doublet of doublets
EtOAc Ethyl acetate
Eq Equivalent
FA Fatty acid
HRMS High Resolution Mass Spectrometry
J (NMR-Spectrum) Couplings constant / Hz
m (NMR-Spectrum) Multiplet
MHz Megahertz
MUFAs Mono-unsaturated Fatty Acids
NMR Nuclear Magnetic Resonance
PUFAs Poly-unsaturated Fatty Acids
ppm Parts per million
RT Room temperature
s (NMR-Spectrum) Singlet
SFAs Saturated Fatty Acids
SFC Supercritical Fluid Chromatography
SN Stereospecific numbering
t (NMR-Spectrum) Triplet
TAG Tri-acyl glycerol
TBAF Tetra-n-butylammonium fluoride
TBHQ tert-Butylhydroquinone
TCN Total Carbon Number
TIPSCl Triisopropyl chloride
δ Chemical shift (ppm)
Graduation report
Towards the Discovery of the Tri-acylglycerol
Composition of Milk Fat
N.J.L.C. Das
Bio-organic Chemistry
Stratingh Institute for Chemistry
2012 – 2013
, Toward the Discovery of the Tri-acylglycerol Composition of Milk Fat
Graduation report
Towards the Discovery of the Tri-acylglycerol
Composition of Milk Fat
Name: N. Das
Educational institute: University of Groningen
Department of Bio-organic Chemistry
Stratingh Institute for Chemistry
Nijenborgh 7, 9747 AG Groningen
Mentor: Prof. Dr. Ir. A.J. Minnaard
Tutor: P. Fodran
Industrial collaborator: FrieslandCampina
Mentor: Dr. W. Kloek
Educational institute: Avans Hogeschool
Academie voor de Technologie van Gezondheid en Milieu (ATGM)
Education: Chemistry
Specialisation: Organic and Polymer Chemistry
Teacher: Dr. E.W. Dijk
Period: 16-01-2013 – 14-06-2013
, Toward the Discovery of the Tri-acylglycerol Composition of Milk Fat
Summary
The identification and quantification of a single component in a complex mixture can be a daunting
task. In the field of lipid analysis, this is a regularly encountered challenge. Within a particular class of
lipids, for example tri-acyl glycerols (TAGs), di-acyl glycerols (DAGs), or phospholipids, the members
differ often little in their physical properties. This makes separation, in order to allow identification of
a single compound, even with advanced techniques often very difficult or impossible. Milk fat is a
very complex mixture of lipids. Bovine milk fat predominantly consists of TAGs (>98%). The analysis
of the quantity and identity of the constituting, individual fatty acids (FA) of the fat is very well
possible upon hydrolysis. However, the identification of the individual TAGs is extremely difficult.
The outline of the project was to synthesize a library of TAGs via a stereo-specific synthetic route.
Previous work from the Minnaard group has proven that the Co[R,R-Salen] catalyst (scheme 1; left) is
successful in the selective formation of the 1,3-DAG out of a glycidyl ester without the occurring of
migration to the 1,2-DAG. A library of 26 TAGs has now been synthesized in three steps in overall
yields of 80 – 90%. For an even more efficient approach, we aimed for a parallel synthesis. A resin
bound catalyst was necessary for the parallel synthesis, which could be easily filtered off after
completion of the reaction to afford pure products after removal of the solvent.
Scheme 1: (Left): Structure formula of Co[R,R-Salen] catalyst. (Right): Resin bound Co[R,R-Salen] catalyst
The resin bound Co[R,R-Salen] catalyst (scheme 1; right) was synthesized in three steps out of a
hydroxymethyl resin. To be able to attach the Salen ligand to the resin, a linker with a functional
group was introduced. The desired Salen ligand could then be bounded with a trans-esterification to
afford the desired Salen ligand on the resin and the reaction was able to monitor. Cobalt was
inserted to afford the desired catalyst on resin with a loading of 0.2 mmol/g of Co. The Co[R,R-Salen]
catalyst was used in 1 mol% in the ring opening of epoxides. Full conversion was achieved after 6
days in CH2Cl2, compared to 10 h of the homogeneous Co[R,R-Salen] catalyst in the ring opening
without solvent.
The second goal of this graduation project was to separate (regio)-isomers of the synthesized TAGs
by HPLC. These results could be compared to HPLC results of milk fat fractions, in which the
composition of TAGs in milk fat could be determined. Separation of a mixture of three TAGs with
different TCNs by HPLC with optimized conditions has been achieved. For regio-isomers and mixtures
of four or more TAGs which do not differ much in chain length, separation has not been achieved or
has not been studied well enough. The optimized conditions are with a runtime of 30 minutes:
- Mobile Phase: 100% n-heptane
- Flow: 0,5 ml/min
- Inj. Vol. : 5 µl
- Column: Chiracel OB-H
- Detector: Electron Light Scattering Detector (ELSD)
, Toward the Discovery of the Tri-acylglycerol Composition of Milk Fat
List of abbreviations
BnBr Benzylbromide
bs (NMR-Spectrum) Broad singlet
DAG Di-acyl glycerol
DCC N,N’-Dicyclohexylcarbodiimide
DIPEA N,N-Diisopropylethylamine
DMF N,N-Dimethylformamide
dd (NMR-Spectrum) Doublet of doublets
EtOAc Ethyl acetate
Eq Equivalent
FA Fatty acid
HRMS High Resolution Mass Spectrometry
J (NMR-Spectrum) Couplings constant / Hz
m (NMR-Spectrum) Multiplet
MHz Megahertz
MUFAs Mono-unsaturated Fatty Acids
NMR Nuclear Magnetic Resonance
PUFAs Poly-unsaturated Fatty Acids
ppm Parts per million
RT Room temperature
s (NMR-Spectrum) Singlet
SFAs Saturated Fatty Acids
SFC Supercritical Fluid Chromatography
SN Stereospecific numbering
t (NMR-Spectrum) Triplet
TAG Tri-acyl glycerol
TBAF Tetra-n-butylammonium fluoride
TBHQ tert-Butylhydroquinone
TCN Total Carbon Number
TIPSCl Triisopropyl chloride
δ Chemical shift (ppm)
