characterization of non- endcapped polymeric ods column ... · 1 characterization of non-endcapped...
TRANSCRIPT
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Characterization of non-endcapped polymeric ODS
column for the separation of triacylglycerol positional isomers
Gotoh, N1, Matsumoto, Y1, Yuji, H1, Nagai, T2, Mizobe, H2, Ichioka, K2, Kuroda, I3, Wada, S1
1 Tokyo University of Marine Science and Technology, Minato-ku, Tokyo, Japan
2 Tsukishima Foods Industry Co. Ltd., Edogawa-ku, Tokyo, Japan3 GL Sciences Inc., Iruma-shi, Saitama, Japan
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TAG comprises one glycerol and three fatty acids and all the fatty acids are esterified with alcohol groups in the glycerol.
O
CH2-O-C-RA
CH2-O-C-RC
RBC-O-CH
O
O
sn-1 () position
TAG structure
B
C
A
Triacylglycerol(TAG)
sn-2 () position
sn-3 () position
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TAG positional isomer (TAG-PI)
They are distinguished as TAG-PIs even though they consist of the same fatty acids combination.
C
A
B
B
C
A
A
B
C
A
B
A
A
A
B
ABC type TAG molecular species
AAB type TAG molecular species
ABC CAB BCA AAB ABA
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Analysis of TAG-PI
Ag+ column
Reverse phase column
Separation of TAG-PI ○ ×Endurance × ○
Repeatability × ○
Recently, we reported that non-endcapped polymeric ODS column could separate pair of TAG-PI.
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We examined the resolution of several kinds of TAG-PI pairs using a recycle HPLC/APCI-MS system equipped with non-endcapped polymeric ODS columns to understand the separation mechanism.
Object
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UV-Visdetector
APCI/MSPolymeric ODS column
Injector Mobile phase
Column oven
Recycle valvePump
Recycle released
What is recycle system?
Recycle
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Capillary voltage 4.0kVCone voltage 40VHeater temperature 400oCFull Scan +Full Scan –SIR(Selected Ion Recording)
SIR 1. TG+H+
2. TG+NH4+
3. DG+H+
4. DG+H+
Column: Intersil ODS-P(5mm, 250mm×4.6mm I.D.)x2(GL Sciences Inc., Saitama, Japan)
Mobile phase: Acetonitrile/2-Propanol = 6:4 (v/v)Flow rate: 0.8mL/minDetectors:UV-Vis (210nm)
APCI-MS (Waters Alliance ZMD LC/MS System, Waters Corporation, Milford, MA )
Conditions
Column temperatures and recycle times were not fixed and changed for the best separation of individual TAG-PI pair.
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TAG-PI standards used in this study (1)(Tsukishima Foods Industry Co. Ltd.)
1. C8C10C8/C8C8C10 2. C8C12C8/C8C8C12
3. C8C14C8/C8C8C14 4. C8C16C8/C8C8C16
5. C8C18C8/C8C8C18 6. C8DC8/C8C8D
COOH
Palmitic acid (C16) Stearic acid (C18)
COOH
Docosahexaenoic acid(DHA:D)
COOH
COOH
Lauric acid (C12) Myristic acid (C14)
COOHCOOH
Caprylic acid (C8) Capric acid (C10)
COOH
C8
C18
C8
C8
C8
C18
:C8C18C8
Example)
:C8C8C18
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C8C8C10 + C8C10C8
C8C8C12 + C8C12C8
C8C8C14 + C8C14C8
C8C8C16 + C8C16C8
C8C8C18 + C8C18C8
C8C8D + C8DC8
Time (min)
Time (min)
Time (min)
Time (min)
Time (min)
Time (min)
Rel
ativ
e pe
akst
reng
th (%
)R
elat
ive
peak
stre
ngth
(%)
Rel
ativ
e pe
akst
reng
th (%
)R
elat
ive
peak
stre
ngth
(%)
Rel
ativ
e pe
akst
reng
th (%
)R
elat
ive
peak
stre
ngth
(%)
C8C14C8 C8C8C14
C8C16C8 C8C8C16
C8C18C8 C8C8C18
at 18oC
at 18oC
at 18oC
at 22oC
at 30oC
at 18oC
Comparison of the resolution of TAG-PI pairs containing two C8s on chromatograms.
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TAG-PI standards used in this study (2)(Tsukishima Foods Industry Co. Ltd.)
1. DC10D/DDC10 2. DC12D/DDC123. DC14D/DDC14 4. DC16D/DDC165. DC18D/DDC18
D
C18
D
D
D
C18
:DC18D
例)
:DDC18
COOH
Palmitic acid (C16) Stearic acid (C18)
COOH
Docosahexaenoic acid(DHA:D)
COOH
COOH
Lauric acid (C12) Myristic acid (C14)
COOH
Capric acid (C10)
COOH
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DDC10 + DC10D
DDC12 + DC12D
DDC14 + DC14D
DDC16 + DC16D
DDC18 + DC18D
Time (min)
Time (min)
Time (min)
Time (min)
Time (min)
Rel
ativ
e pe
akst
reng
th (%
)R
elat
ive
peak
stre
ngth
(%)
Rel
ativ
e pe
akst
reng
th (%
)R
elat
ive
peak
stre
ngth
(%)
Rel
ativ
e pe
akst
reng
th (%
)
DC12D DDC12
DC14D DDC14
DC16D DDC16
DC18D DDC18
at 18oC
at 18oC
at 18oC
at 22oC
at 30oC
Comparison of the resolution of TAG-PI pairs containing two Ds on chromatograms.
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Resolution was affected by the length of the saturated fatty acid chain. A 14-C length was needed for complete separation of TAG-PI pairs without the need for recycle runs under this analysis condition. On the other hand, TAG-PI pairs consisting of two C8s and a C10 or two C8s and a D were not separated, even by several recycle runs. The same tendency was observed in TAG-PI pairs containing two Ds in place of the two C8.
The ODS groups of a polymeric ODS stationary phase are arranged more closely than those of other kinds of ODS stationary phases, and the space between the ODS groups in a polymeric ODS phase is therefore narrower than that in a monomeric ODS phase. Saturated fatty acid has a linear structure and might deeply penetrate the space, thereby more strongly interacting with the polymeric ODS group than an unsaturated fatty acid, which has a nonlinear structure. Resolution was also affected by the length of the saturated fatty acid chain.
TAG-PI containing oleic acid (possesses a cis-type double bond) or elaidic acid (possesses a trans-type double bond) was devoted to the same system to investigate above idea.
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Elaidic acid (trans-C18:1:El)
COOH
Oleic acid (cis-C18:1:O)
COOH
Nor
mal
se
para
tion
Rec
ycle
1
Rec
ycle
2
Rec
ycle
3
Rec
ycle
4
Rec
ycle
5
Rec
ycle
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Rec
ycle
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Rec
ycle
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Rec
ycle
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Rec
ycle
10
Rec
ycle
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Rec
ycle
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Nor
mal
se
para
tion
Rec
ycle
1
Rec
ycle
2
Rec
ycle
3
Rec
ycle
4
Rec
ycle
5
Rec
ycle
6
Rec
ycle
7
Rec
ycle
8
Rec
ycle
9
Rec
ycle
10
Rec
ycle
11
Rec
ycle
13
Rec
ycle
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Time (min)
Rel
ativ
e pe
akst
reng
th (%
)
Time (min)
Rel
ativ
e pe
akst
reng
th (%
)
-DDEl + -DElD
-DDO + -DOD
Docosahexaenoic acid(DHA:D)
COOH
Comparison of the resolution of pairs of TAG-PI with either two Ds and One O or two Ds and one El on recycling chromatograms.
Sepa
rate
d!
Sepa
rate
d!
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The different double bond structure affects the whole molecular structure of the fatty acid, i.e., O is a nonlinear structure but El is a linear structure with a small gap caused by the trans-type double bond. O could probably not penetrate the polymeric ODS stationary phase because of the bent structure, whereas El could slightly penetrate the polymeric ODS stationary phase on which the structural differences of the TAG-PI were recognized. The structure of TAG-PI comprising two Ds and one C18 is similar to that of TAG-PI comprising two Ds and one El, because both El and C18 consist of a linear 18-carbon chain. The space in the polymeric ODS stationary phase is very narrow and El might not be able to sufficiently penetrate the space to produce two peaks on the chromatogram. These findings and explanations support our idea already shown
ODSODSODS
ODSODS
polymeric ODS stationary phase
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Saturated fatty acid in TAG-PI has a linear structure and might deeply penetrate the space of polymeric ODS stationary phase, thereby more strongly interacting with the ODS group than an unsaturated fatty acid, which has a nonlinear structure. The stationary phase might recognize differences in the binding positions of the saturated fatty acids on the glycerol backbone in TAG-PI to separate TAG-PI pairs.
Conclusion