Improved Analysis of Fatty Acids by Capillary GC columns based on Ionic Liquids

•F. Michel, L.M. Sidisky, M.D. Buchanan,

•G.A. Baney, Y. Ni,J.L. Desorcie, and K.K. Stenerson

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Introduction

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O

CH3

OH

O

CH3

OH

O

CH3

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Estimation: replacing of trans-fats by plant oils may prevent up to 100.000 premature fatal incidents per year (http://www.hsph.harvard.edu/reviews/transfats.html)

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Introduction

•Fatty acids are typically separated by GC as their methyl

esters (FAME)

•Capillary column choice for FAME analysis will depend

upon the information required from the analysis.

• Non-polar columns are routinely used to provide results on the amount of saturated versus unsaturated fatty acids.

• Polyethylene glycol based columns are used to provide carbon chain length & degree of saturation resolution.

• The analysis of cis and trans FAMEs, along with resolving their positional isomers, requires the use of highly polar biscyanopropyl polysiloxane columns [1].

[1] AOCS Method Ce 1h-05, “Determination of cis-, trans-, Saturated, Monounsaturated and Polyunsaturated Fatty Acids in Vegetable or Non-ruminant Animal Oils and Fats by Capillary GLC” AOCS Official Methods (2005) American Oil Chemists Society.

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Fatty Acid / FAME ChemistryFAME Structures

Why use derivatization?

• The active carboxyl group has a much stronger interaction with the stationary phase than the remainder of the analyte

• Neutralizing the carboxyl group interactions allows slight differences in interaction strengths resulting from saturation level and double bond orientation to be observed

•Derivatization via an esterification reaction

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Short chain fatty acids are often analyzed in their free form using GC.

Long chain fatty acids are often analyzed as FAMEs using GC.

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Derivatization of Fatty Acids

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6� Click on „Technical Literature“

Technical Literature on Derivatization

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The limit of classic method for FAME analysisExtracted fat from margarine

20 30 40 50Time (min)

35.0 36.0 37.0 38.0

Time (min)

16:0 18:0

18:1

18:2

18:3

6t 9t10t

11t 12t

13t6c7c

9c

10c11c 12c13c

Peaks from 18:1 trans and cis isomers overlap

Overlap of trans/cis monounsaturated octadecenoic fatty acids on the GC chromatogram of a biscyanopropyl siloxane phase

SPE approach to overcome co-elution of cis- andtrans-FAMEs

•Use of silver-ion loaded SPE material for separation of cis-

/trans-FAMEs

• W. Christie: Ag-ion SPE for separation of FAMEs according to their degree of saturation in 1989

• Japanese research group (Food Analysis): Custom made Ag-ion SPE cartridge for separation of cis-/trans isomer up to trienes in 2005

How does it work?

•Silver ions immobilized onto

SCX SPE phase

•The FAMEs sample extract is

passed through the cartridge

•Ag ions interact with the

double-bonds of the FAMEs

•The more double bonds, the greater the retention

•Cis-isomers retain stronger than trans-isomers

How Is It Used?

•Fatty acids (FA) extracted from food sample

•FA converted to FAMEs using BF3

•FAMEs are extracted into hexane

•Hexane sample applied to Discovery Ag-ION SPE cartridge

•FAMEs separated using different mixtures of

hexane:acetone to extract from cartridge

•Fractions analyzed by GC-MS (SP-2560 capillary column).

Discovery Ag-ION Protocol

Fractionation of the standard FAME mixture

Standard sample, total FAMEs at 1 mg/ml

6 8 10 12 14 16 18 20

Time (min)

Standard Mixture

16:0 18:0

18:118:2 18:3

14:0

18:1 cSPE fraction 2

7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.0

Time (min)

Hexane: Acetone 90:10

18:1 t

6 8 10 12 14 16 18 20

Time (min)

SPE fraction1Hexane: Acetone 96:4

6 8 10 12 14 16 18 20

Time (min)

SPE fraction 3 Acetone

6 8 10 12 14 16 18 20

Time (min)

SPE fraction 4 Acetonitrile

GC Results of Cis/Trans Fractionation of Potato Chips

column: SP-2560, 75 m x 0.18 mm I.D., 0.14 µm (23348-U)

oven: 180 °°°°C, isothermalinj.: 220 °°°°C

det.: FID, 220 °°°°Ccarrier gas: hydrogen, 40 cm/sec. at 180 °°°°C

injection: 0.5 µL, 100:1 splitliner: 4 mm I.D., split, cup design

Discovery Ag-Ion SPE

•Technical Report T406062 (IRV)

• 54225-U

–750 mg/6 mL SPE Tube

• 54226-U

–750 mg/1 mL Rezorian™

Improvements in the GC Analysis of FAMEs

17

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IntroductionStructure of Polysiloxane and PEG Polymer Phase

•Drawbacks

• Active hydroxyl (-OH) groups at the polymer termini allow a back-biting reaction

– Resulting in phase degradation

– Contributing to column bleed

• Chemistry modifications are limited to pendent group changes

• PEG/Wax: Limited to 280 °C maximum temperatureR = methyl, phenyl, fluoropropyl, and/or cyanopropyl (least polar to most polar).

x,y = percentage in the overall polymer composition.

HO Si O Si O H

R1 R3

R2 R4

x y

HO CH2 CH2 O CH2 CH2 O CH2 CH2 OH

n

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Definition, Early Literature, and Use of Ionic Liquids

•Ionic liquids are a class of solvents with low melting points

that consist of organic cations associated with (inorganic or

organic) anions

•Ethyl ammonium nitrate (C2H5NH3+)(NO3

-), which has a

melting point of 12 oC, was described in 1914

• P. Walden, Bull. Acad. Imper. Sci. (St. Petersburg) 1800 (1914)

•Today, they are used as solvents, electrically conducting

fluids, and sealants

Ionic Liquids

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Water

Ionic Liquid

CHCl3

Source: Prof. Jared Anderson,

University of Toledo, USA

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Ionic Liquids in GC

•Several properties make ionic liquids desirable as GC

stationary phases

• Very low volatility…

– should result in columns with lower bleed and longer life

• Remain in the liquid state over a wide temperature range…

– should result in columns with extended temperature ranges

• No active hydroxyl (-OH) groups at their termini…

– should result in columns resistant to damage from moisture/oxygen

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Ionic Liquids in GC

•Several properties make ionic liquids desirable as GC

stationary phases

• Are inherently highly polar…

– should result in columns with higher polarity that have lower elution temperatures plus an increased selectivity for polarizable analytes

• Have the broadest range of physical-chemical solvation interactions of any solvent…

– should result in columns with unique selectivity

• High viscosity…

– should be easy to coat columns

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Example Structure of an Ionic Liquid Phase

• Numerous combinations of cations, anions and linkers are possible allowing for “tailored” selectivity or application

– Dicationic (shown) or polycationic

– Cations (imidazolium, phosphonium, pyrrolidinium, …), anions (NTf2-, triflat, tetrafluoroborate, hexafluorophosphate, …), and linkages (alkanes, polyethylenglycols, different lengths, …) can be changed

– Pendant groups can be added to cations and/or linkages

Phase used to make the SLB-IL100

1,9-di(3-vinylimidazolium) nonane bis(trifluoromethyl) sulfonyl imide

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GC Column Polarity ScaleVisual Representation

Above the scale: positions/maximum temperatures of non-ionic liquid columns.

Below the scale: positions/maximum temperatures of Supelco ionic liquid columns.

Non-Polar

Intermediate Polar

Polar Highly Polar Extremely Polar

0 65 1003210

Ionic LiquidPhases

Non-IonicLiquid Phases

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Selectivity: Test Mix Peak IDs and Conditions

•Peak IDs (listed in boiling point order)

• Toluene

• Ethylbenzene

• p-Xylene

• Isopropylbenzene (Cumene)

• Cyclohexanone

• 1,2,4-Trimethylbenzene

• 1,2,4,5-Tetramethylbenzene

• n-Tridecane (C13)

•Conditions

• columns: 30 m x 0.25 mm I.D., 0.20 µm

• oven: 110 °C• inj.: 250 °C• det.: FID, 250 °C• carrier gas: helium, 26 cm/sec

• injection: 1.0 µL, 100:1 split

• sample: each analyte at various concentrations in isooctane

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Selectivity: Test Mix on Ionic Liquid Columns30 m Columns, 110 °°°°C Isothermal

2.0 3.0 4.0 5.0Time (min)

2.0 3.0 4.0 5.0Time (min)

2.0 3.0 4.0 5.0Time (min)

2.0 3.0 4.0 5.0Time (min)

2.0 3.0 4.0 5.0Time (min)

2.0 3.0 4.0 5.0Time (min)

8

8

8

8

8

8 5

5

5

5

5

5SLB-IL59

SLB-IL61

SLB-IL76

SLB-IL82

SLB-IL100

SLB-IL111

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Rapeseed Oil FAME Peak IDs and Conditions

•Peak IDs and composition

• Myristic (C14:0) @ 1.0%

• Palmitic (C16:0) @ 4.0%

• Stearic (C18:0) @ 3.0%

• Oleic (C18:1n9c) @ 60.0%

• Linoleic (C18:2) @ 12.0%

• Linolenic (C18:3) @ 5.0%

• Arachidic (C20:0) @ 3.0%

• cis-11-Eicosenoic (C20:1) @ 1.0%

• Behenic (C22:0) @ 3.0%

• Erucic (C22:1) @ 5.0%

• Lignoceric (C24:0) @ 3.0%

•Conditions

• columns: 30 m x 0.25 mm x 0.20 µm

• oven: 180 °C isothermal

• inj.: 250 °C• det.: FID, 250 °C• carrier gas: helium, 25 cm/sec

• injection: 1.0 µL, split 100:1

• liner: 4 mm I.D., split type, cup design

• sample: Rapeseed oil FAME mix (O7756-1AMP) diluted to 10 mg/mL in methylene chloride

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0 10 20

Time (min)

Rapeseed Oil FAMEs on Ionic Liquid Columns30 m Columns, 180 °°°°C Isothermal

0 10 20Time (min)

0 10 20Time (min)

0 10 20Time (min)

0 10 20

Time (min)

0 10 20Time (min)

6

6

6

6

6

7

7

7

7

7

8

8

8

8

8

7

8

6

SLB-IL59

SLB-IL61

SLB-IL76

SLB-IL82

SLB-IL100

SLB-IL111

6: C18:37: C20:08: C20:1

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Partially Hydrogenated Vegetable Oil (PHVO) FAMEs

SP-2560 SLB-IL111

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Partially Hydrogenated Vegetable Oil (PHVO) FAMEs

•When comparing FAME isomers with the same degree of

unsaturation, the SLB-IL111 provides increased retention of cis

isomers relative to trans isomers with the double bond at the

same location

•The SLB-IL111 was able to provide resolution of C18:1∆9c

from C18:1∆15t, a separation not possible with the SP-2560.

•The SLB-IL111 offered improved resolution of some isomers

that cannot be completely resolved with the SP-2560 either

• C18:1∆10t from C18:1∆11t

• The pair C18:1∆13t/C18:1∆14t from other isomers

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CLA FAMEs in Ruminant Fats

•The SLB-IL111 is able to resolve C18:2∆9c,11t from

C18:2∆7t,9c – the two most abundant conjugated linoleic acid (CLA) isomers found in ruminant fats [5]

• No other column can resolve these

•SLB-IL111 as complementary column (to biscyanopropyl polysiloxane) for complete analysis of cis- and trans-fat

[5] P. Delmonte, A.-R.F. Kia, J.K.G. Kramer, M.M. Mossoba, L. Sidisky, J.I. Rader, J. Chrom. A, 1218 (2011) 545

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To Learn More…

•Visit the Ionic Liquid GC column landing page (sigma-aldrich.com/il-gc) at the Sigma-Aldrich web site

•Download or request “Supelco Ionic Liquid GC Columns:

Applications”

•Download or request “Supelco Ionic Liquid GC Columns:

Bibliography”

sigma-aldrich.com/il-gc

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Summary

• Ag-Ion SPE provides a separation of cis and trans FAMEs

– Allow for a complete separation of the critical mono-unsaturated C18 FAMEs

– Correct calculation of amount of cis/trans fatty acids

– Alternative approach to using SLB-IL111 and biscyanopropylpolysiloxane phases (e.g. SP-2560) as complementary column

• SLB-IL111 as complementary column to biscyanopropyl

polysiloxane phases allows the complete analysis of cis and

trans fatty acids

–Separation of several isomers that could not be resolved before

–Further publications on FAME analysis on Ionic Liquid GC [6]

[6] F. Destaillats, M. Guitard, C. Cruz-Hernandez , J. Chrom. A, 1218 (2011) 9384

Fatty Acid/FAME Application Guide (T408126, KUK)

•24-page brochure

•Includes columns, standards, and other

products for the:

• Analysis of free fatty acids

• Derivatization of fatty acids to FAMEs

• SPE fractionation of FAMEs

• Analysis of FAMEs by boiling point elution

• Analysis of FAMEs by degree of unsaturation

• Analysis of omega 3 and omega 6 FAMEs

• Analysis of cis/trans FAME isomers

•Also includes:

• A list of non-Supelco references

• A list of our product literature 36

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Dziękuję za uwagę!

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Prof. Dan Armstrong, University of Texas at ArlingtonProf. Luigi Mondello, University of Messina, ItalyDr. Pierluigi Delmonte, US FDA