1

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

2

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

3

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.

5

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

6

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

6

Rec

ycle

7

Rec

ycle

8

Rec

ycle

9

Rec

ycle

10

Rec

ycle

11

Rec

ycle

12

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

12

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