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Separation of fatty acid methyl esters by GC-online hydrogenation x GC. Pierluigi Delmonte, Ali Reza Fardin-Kia, and Jeanne I. Rader Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ac302707z • Publication Date (Web): 20 Dec 2012 Downloaded from http://pubs.acs.org on December 24, 2012
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Analytical Chemistry
1 1 2 3 4 5 6
Separation of fatty acid methyl esters by GC-online hydrogenation × GC.
Pierluigi Delmonte*, Ali Reza Fardin-Kia, Jeanne I. Rader
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Office of Regulatory Science, Center for Food Safety and Applied Nutrition, Food and Drug Administration, College Park, MD, USA.
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Running title: Separation of fatty acid methyl esters by GC-online hydrogenation x GC.
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Key words:
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SLB-IL111, GC×GC, fish oil, PUFA, fatty acids, hydrogenation, gas chromatography.
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*Corresponding author:
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Pierluigi Delmonte, Ph.D.
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HFS-717
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US Food and Drug Administration
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5100 Paint Branch Pkwy
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College Park, MD, 20740
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Phone: 301-436-1779
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Fax: 301-436-2622
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[email protected] 27 28
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ABSTRACT
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The separation of fatty acid methyl esters (FAME) provided by a 200 m x 0.25 mm SLB-
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IL111 capillary column is enhanced by adding a second dimension of separation (2D) in
33
a GC×GC design. Rather than employing two GC columns of different polarities or using
34
different elution temperatures, the separation in the two dimensional space is achieved by
35
altering the chemical structure of selected analytes between the two dimensions of
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separation. A capillary tube coated with palladium is added between the first dimension
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of separation (1D) column and the cryogenic modulator, providing the reduction of
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unsaturated FAMEs to their fully saturated forms. The 2D separation is achieved using a
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2.5 m x 0.10 mm SLB-IL111 capillary column and separates FAMEs based solely on
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their carbon skeleton. The two dimensional separation can be easily interpreted based on
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the principle that all the saturated FAMEs lie on a straight diagonal line bisecting the
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separation plane, while the FAMEs with the same carbon skeleton but differing in the
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number, geometric configuration or position of double bonds lie on lines parallel to the
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1D time axis. This technique allows the separation of trans fatty acids and
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polyunsaturated FAs (PUFA) in a single experiment and eliminates the overlap between
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PUFAs with different chain lengths. To our knowledge, this the first example of GC×GC
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in which a chemical change is instituted between the two dimensions in order to alter the
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relative retentions of components and identify unsaturated FAMEs.
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INTRODUCTION
52 53
Oils and fats extracted from products of marine origin such as fish and algae contain a
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large number of saturated, monounsaturated and polyunsaturated fatty acids1,2. Gas
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chromatography using mid and high polarity capillary columns was shown to provide
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only a partial separation of the FAMEs prepared from these oils and fats1,2. Santercole et
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al.1 recently compared the separations of menhaden oil FAMEs using a mid polarity GC
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column (polyethylene glycol phase, PEG) and a high polarity column (100% cyanopropyl
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siloxane phase, CPS). The CPS column used by the authors provided a more detailed
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separation of positional and geometric isomers of unsaturated fatty acids compared to the
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PEG counterpart but none of the sets of chromatographic conditions evaluated could
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separate all of the FAMEs contained in the samples investigated1.
63 64
About a decade ago, Armstrong et al. showed that room temperature ionic liquids (RTIL)
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possess the volatility, viscosity, solubility and polarity proprieties required for use as
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stationary phases for capillary GC3,4. Used as stationary phases in GC, they showed dual
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nature retention selectivity, separating polar molecules as a polar stationary phase and
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non polar molecules as a non polar stationary phase3,4,5. The development of
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dicationic6,7,8, trigonal tricationic9, and cross-linked10 RTILs led to the novel IL capillary
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columns currently used for the separation of FAMEs11-14. Capillary columns coated with
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RTILs are increasingly used for GC×GC separations due to their unique selectivity and
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thermal stability15-27. The use of a column coated with an RTIL
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(trihexyl(tetradecyl)phosphonium bis(trifluoromethane)sulfonamide) for GC×GC
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separations was first tested by Seeley et al, using standard mixtures of organic
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compounds with a wide range of functional groups15. Used for 1D or 2D, RTIL columns
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have been used successfully for the separation of phosphorous–oxygen (P–O) containing
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compounds16, FAMEs17-19, PCBs20, 21, perfume allergens22, pesticides in water23,
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petroleum fractions containing nitrogen and sulphur compunds24,25. A GC×GC×GC
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application for the separation of phosphorous–oxygen (P–O) containing compounds in
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diesel samples has also been reported26. The use of RTIL columns for GC separations has
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recently been reviewed in greater detail by Ragonese et al 27.
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Delmonte et al. tested the novel SLB-IL111 ionic liquid column and showed that the
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higher polarity of its stationary phase provides a higher selectivity for FAMEs based on
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the number, geometric configuration and position of double bonds than other phases used
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for FAME analysis12. Based on these findings, the authors developed a procedure capable
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of separating most FAMEs prepared from milk fat using a 200 m x 0.25 mm SLB-IL111
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capillary column13. Among other improvements, this methodology allowed the separation
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of almost all positional/geometric isomers of 18:1 and 18:2 milk fat FAMEs. However,
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certain FAMEs differing in chain length and number of double bonds still co-eluted. All
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of the trans-18:1 FAMEs up to t15-18:1 eluted before cis 9-18:1. The biologically active
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trans vaccenic acid (t11-18:1) was clearly separated from other trans-18:1, and in the
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conjugated linoleic acid region (CLA) t7,c9- was separated from c9,t11-18:213.
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Comprehensive two-dimensional gas chromatography can provide a more detailed
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separation of FAMEs by combining the selectivity of two different gas chromatographic
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separations 28. The orthogonality between the two separations is commonly achieved by
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employing GC columns of different polarities, different elution temperatures for 1D and
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2D, or a combination of both. Several combinations of experimental conditions have been
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developed targeting FAMEs prepared from different lipid sources and biodiesel blends17-
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19, 29-37
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lengths, and are often preferred for the 1D separation. Highly polar capillary columns
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provide selectivity based on the number, geometric configuration and position of double
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bonds and are preferred for the 2D separation. Gu et al.17 tested several combinations of
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capillary columns for the GC×GC separation of FAMEs prepared from samples of marine
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origin. The authors obtained a group-type GC×GC separation of FAMEs with different
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carbon numbers and number of unsaturations by using an apolar polydimethyl siloxane
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capillary column for 1D combined with a polar ionic liquid column for 2D. Tranchida et
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al.18 separated FAMEs prepared from menhaden oil using an SLB-IL5ms for 1D
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combined with an SP2560 for 2D. These platforms could separate a large number of
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PUFAs and divide them into groups based on their carbon number but provided limited
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separation based on the geometric configuration and position of double bonds17,18.
. Non polar capillary columns separate FAMEs based primarily on their chain
113 114
Online hydrogenation of FAMEs using a capillary fused silica tube coated with Pt or Pd
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before or after GC separation was used in the early stages of lipid research38-40.
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Schomburg et al.38 prepared a 300 mm x 0.35 mm I.D. capillary reactor by dynamic or
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static coating, loading Pt or Pd on the capillary tube as an acetyl-acetonate salt dissolved
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in MeCl2. The salt was successively dried on the inner surface of the tube, and
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decomposed into metallic Pt or Pd by heating at 200 °C in a mild flow of carrier gas. The
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efficiency of the Pt capillary reactor was tested by analyzing several types of unsaturated
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compounds, including unsaturated FAMEs. The authors recommended using Pd over Pt
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for the reduction of double bonds in FAMEs because the stronger reactivity of the latter
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might cause the reduction of the ester group. Le Quere et al.40 characterized unsaturated
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cyclic fatty acid monomers using a post column 60 cm x 0.32 mm capillary tube coated
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with Pd prior to the MS detector.
126 127
This study describes the development of a novel approach to the GC×GC separation of
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FAMEs in which the analytes undergo chemical reduction between 1D and 2D, and 2D
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separates the products of the reaction. Unsaturated FAMEs (U-FAME) are reduced to
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their fully saturated form by passing them through a capillary tube coated with Pd
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(reactor) in the presence of H2 as the carrier gas. The GC×GC separation is achieved by
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using two capillary columns coated with the same highly polar stationary phase, and
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maintained at the same temperature. To our knowledge, this is the first GC×GC
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application in which the two dimensional separation is achieved by modifying the
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chemical structure of the analytes instead of by applying different chromatographic
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conditions.
137 138 139
MATERIALS AND METHODS
140 141
Fatty acid methyl ester reference mixture GLC 463 and other FAMEs reference materials
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used for identification purposes were purchased from Nu-chek Prep, Inc. (Elysian, MN).
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Menhaden oil and Pd acetyl acetonate were purchased from Sigma Aldrich (St. Louis,
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MO). The FAMEs prepared from human colon adenocarcinoma cells HT-29 were kindly
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provided by Christian Degen of University of Jena (Germany). The origin and
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preparation of this sample are described in a previous study41.
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Capillary reactor preparation. Fifty milligrams of Pd acetyl acetonate were weighed in
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a 10 ml volumetric flask and dissolved with MeCl2. The solution was loaded into a 4 m x
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0.15 mm deactivated fused silica capillary tube using a 1 ml glass syringe, allowing
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several drops to exit from the end side. The capillary tube was dried for 2 days under
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vacuum with an end plugged with silicone vacuum grease and then maintained for 2
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hours at 200 °C in the GC oven under a mild flow of hydrogen. If the crystallization and
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thermal decomposition of the acetyl acetonate salt produce a homogeneous layer of Pd,
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the expected thickness is 5 nm. A piece of uncoated silica capillary tube was placed
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between the injector port and the Pd reactor to avoid contamination. The capillary tube
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coated with metallic Pd was cut in 50 cm portions to be used as capillary reactors.
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GC×GC conditions. Separations were achieved with an Agilent 7890A gas
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chromatograph (Agilent Tech., Wilmington, DE) combined with a Zoex ZX2 dual stage
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cryogenic modulator (Houston, TX). The injection port of the gas chromatograph was
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connected to a 1034 kPa electronic pressure control module and hydrogen was used as
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the carrier gas. The fluidic path of the system is shown in Figure 1. A Supelco SLB-
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IL111 capillary column (200 m x 0.25 mm I.D., 0.2 µm coating, Bellefonte, PA) was
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used for 1D separation and the same capillary column but with different dimensions (2.75
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m x 0.10 mm I.D., 0.08 µm coating) was used for the 2D separation. The cryogenic
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modulator first loop consisted of a 2 m x 0.10 mm deactivated capillary tube and the
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modulation (first stage) was set at 25 cm from the beginning of the tube. The second
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stage of modulation was achieved using part of the 2D column and the modulation spot
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was set at 25 cm from the column front end leaving 2.50 m of 2D column for separation.
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The 50 cm x 0.15 mm Pd capillary reactor was placed between the 1D column and the
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modulator. The timing of the modulation was set to 3.5 s, the temperature of the hot jet
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was set at 350 °C with a pulse of 350 ms. The column oven temperature was set to 170
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°C, and the injector port at 300 °C. Chromatography was acquired in constant pressure
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mode at 690 kPa and the split flow was set to 125.69 ml/min. The FID was maintained at
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250 °C and fed with 30 ml/min of nitrogen make up gas, 400 ml/min air, and 25 ml/min
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of hydrogen. All capillary connections were made with Ultimate unions (Agilent
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Technologies).
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GC×GC conditions, temperature program. The separation was achieved using the
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same instrumental configuration used for isothermal separation, with the exception of the
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Analytical Chemistry
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100 m x 0.25 mm SLB-IL111 used for 1D. The elution temperature was set to 130 °C per
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23 minutes, then increased 1.3 °C/min to 220 °C. The inlet pressure was maintained at
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483 kPa per 23 minutes, and then raised at 1.514 kPa/min until 588 kPa. The modulation
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time was set to 2 s, and the chromatograms were adjusted by shifting the 2D phase -1.8 s.
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GC-MS identification of FAMEs. Identities of FAMEs were confirmed or achieved by
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mono-dimensional GC-MS using a Micromass GCT TOF mass spectrometer
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(Manchester, UK) coupled with an Agilent 6890N GC. The separation was achieved
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using a 100 m x 0.25 mm SLB-IL111 capillary column maintained at 170°C and H2
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carrier gas at the pressure of 80 kPa. Experiments were carried out in CI+ mode using
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isobutane as the ionization gas. The source was maintained at 150°C, the electron energy
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was 70 eV, the emission current 250 µA, and the CI gas flow was set to 10. The
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molecular ion of FAMEs identified the chain length and the number of unsaturations.
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RESULTS AND DISCUSSION
198 199
The current preferred approach for quantitation of FAMEs is mono-dimensional GC
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using the longest and most polar capillary columns available. Methods for the
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quantitation of trans fatty acids and PUFAs require the separation of FAMEs based on
202
the number and geometric configuration of their double bonds. Rather than enhancing
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these separations by adding a second dimension of separation, most GC×GC methods use
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a low polarity column for 1D, thus separating FAMEs based primarily on their chain
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length, combined with a short polar 2D column providing limited separation based on the
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number, position and geometry of double bonds.
207 208
In this study, we selected for 1D separation the same chromatographic conditions we
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prefer for mono-dimensional GC analyses: a highly polar 200 m x 0.25 mm SLB-IL111
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column maintained at 170°C. After being separated by the 1D column, the U-FAMEs are
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reduced to their carbon skeleton by the capillary reactor and the products of reaction are
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separated using a 2.5 m x 0.10 mm SLB-IL111 maintained at the same temperature as
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1D. This GC×GC system requires a very simple hardware configuration: the two
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capillary columns are maintained in the same oven and there is no need for supplying
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extra carrier gas at the mid-point before the 2D column, as in the case of using larger
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diameter columns for 2D separation (i.e., flow modulation).
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Fatty acid methyl esters prepared from samples of marine origin are characterized by a
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high sample dimensionality, primarily derived from the presence of double bonds on their
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alkyl chains42. The number, position (individual, or cumulative using n-x nomenclature)
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and geometric configuration of double bonds generate a number of sample dimensions
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higher than the number of separation dimensions that can be combined in a single
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chromatographic system. The hydrogenation of double bonds reduces the dimensionality
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of the sample solely to chain length, or to the chain length and the position of the methyl
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group substituent if iso- and anteiso-FAMEs are also considered. While the 1D column
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provides a complex peak distribution by retaining FAMEs based on all sample
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dimensions, the 2D provides an ordinate separation based only on one sample dimension
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(or two, including iso-/anteiso- FAMEs). The result is a GC×GC separation in which
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FAMEs are eluting from 1D in a “disordered” manner, and are then “ordered” by 2D into
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lines based on their chain length dimension. If the desired separation is limited to straight
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chain C8-C26 FAMEs, a theoretical 2D peak capacity of 19 can be sufficient to separate
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all the products of hydrogenation. The low peak capacity required for performing the 2D
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separation allows the rapid modulation time of 3.5 s.
234 235
The highly polar column used for 2D was selected based on its lower retention for long
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chain saturated FAMEs relative to short/medium chain saturated FAMEs, in comparison
237
with lower polarity columns. It also allowed the use of a single GC oven, thus
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simplifying the hardware configuration. The disadvantage of this selection is the lower
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solubility of the FAMEs in the highly polar liquid phase, resulting in a lower loading
240
capacity. Capillary reactors were prepared by loading either 0.5 or 1.0% (w/v) Pd acetyl
241
acetonate in MeCl2 onto the capillary tube. Capillary reactors coated using the 1.0%
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solution as short as 5 cm were observed to provide quantitative reduction of U-FAMEs.
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The 50 cm version prepared using the 0.5% solution proved to be less dependent on the
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Analytical Chemistry
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uniformity of the Pd coating layer and was preferred for this study. Combinations of
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excessive Pd load and/or length of the capillary reactor, such as use of a capillary of at
246
least 1 cm prepared using a 5% Pd acetyl acetonate solution, were observed to generate
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1D peak tailing and the degradation of FAMEs. This was recognized by the appearance
248
of peaks in proximity to the 2D unretained band.
249 250
Figure 2 shows the separation of the FAMEs contained in reference mixture GLC 463.
251
The two dimensional separation can be easily interpreted using two basic rules: 1) all the
252
saturated FAMEs lie on a straight diagonal line bisecting the separation plane, as
253
expected from the lack of orthogonality between the 1D and 2D chromatographic
254
conditions, and 2) the FAMEs with the same carbon skeleton but differing in the
255
number/geometric configuration/position of double bonds lie on lines parallel to the 1D
256
time axis, based on the fact that the reduction converted them into the same compound.
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The result is a two dimensional separation in which the 1D separation shows limited loss
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of resolution compared to its mono-dimensional equivalent, as shown by the fact that the
259
18:1 and 20:1 FAMEs contained in this reference mixture are still separated from each
260
other. The most critical co-elutions affecting the same separation by mono-dimensional
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GC are instead resolved by 2D: the n:1 FAMEs are separated from the saturated (n+1):0
262
(eg, 18:1 vs. 19:0), the 18:2 FAMEs are separated from the 20:0 and 19:1, and the 18:3
263
FAMEs are separated from the 20:1 and 21:0.
264 265
The pressure of 690 kPa applied to the injection port provided an average linear velocity
266
of 17 cm/s for 1D and 230 cm/s for 2D, based on the retention times of hexane, an un-
267
retained compound. Two dimension chromatograms were adjusted by shifting the 2D
268
phase by the estimated holdup time, 1.1 s. The 0 s time of the chromatogram corresponds
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to 1.1 s of separation, while 3.5 s correspond of 1.1 s of the subsequent modulation cycle.
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The 3.5 s modulation time allowed the separation of FAMEs with up to 23 carbons in a
271
single modulation cycle, while FAMEs with 24 or more carbons if present elute during
272
the subsequent modulation cycle. If the sample does not contain FAMEs with more than
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21 carbons, as in the case of several vegetable oils, the 2D modulation time can be
274
reduced to 2 s. The combination of the long 1D column (200 m) and the 3.5 s modulation
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time allowed a modulation ratio (MR) of at least 3 for FAMEs with at least 10 carbons.
276
The isothermal and constant flow elution conditions applied in this study caused broad
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late eluting peaks, and the last eluted FAME (22:6) showed an MR of 15 or more
278
according to its load on the column.
279 280
Figure 3 shows the separation of FAMEs prepared from menhaden oil. Identification of
281
FAMEs was achieved by combining information obtained from the separations of
282
available reference materials, fractionation using silver ion chromatography13, GC-MS
283
analysis, and available literature1,2,12,13. The FAMEs prepared from menhaden oil were
284
fractionated by silver ion chromatography into saturated FAMEs, trans-monounsaturated
285
FAMEs (MUFA), cis-MUFAs, and PUFAs as previously described13. The FAMEs in the
286
saturated FAMEs fraction were identified by comparison with available reference
287
material and separations reported in the literature1,2,12,13.The FAMEs isolated in the cis-
288
and trans-MUFAs fractions were identified using previously prepared and characterized
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reference mixtures12,13,43,44. The identification of the FAMEs in the PUFA fraction was
290
achieved by integrating identifications reported in the literature1,2,13 using different
291
separation columns with GC-MS data obtained in this study. The molecular ion of PUFA
292
indicated the n:x structure of the FAME, and, for each n:x series, the location of double
293
bonds was obtained by comparison with identifications reported in the literature1,2. The
294
separation of PUFA up to 22:6 requires 165 min, and FAMEs with different chain lengths
295
are separated by 2D on different lines. The separation on the two dimensional space
296
shows that several C16, C18 and C20 U-FAMEs require a second dimension of
297
separation in order to be resolved. Valuable information provided by this separation
298
technique is the identification of the FAME carbon skeleton, including the presence of
299
branches. Branched chain n:x FAMEs (i.e. iso and anteiso isomers) are eluted by 2D on
300
lines located between the C(n-1) and Cn FAME. If branched chain FAMEs with
301
unsaturations are present, the 2D retention time indicates the iso or anteiso structure and
302
the 1D retention time provides information regarding the number and geometric
303
configuration of double bonds. Obtaining the same information utilizing other analytical
304
techniques including mass spectrometry might be challenging, especially if the targeted
305
FAME is present in low concentration and/or it co-elutes with other FAMEs.
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Analytical Chemistry
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Figure 4 shows the separation of the FAMEs prepared from menhaden oil from 14:0 to
308
18:3. The two dimensional separation of the 16:1 and 17:0 FAME isomers provides a
309
dramatic improvement over the most refined separation obtained by mono-dimensional
310
GC: the 16:1 isomers are separated by 1D along the C16 line, while the 17:0 branch
311
chain isomers are eluted on the saturated FAME diagonal line. The x1 compound
312
analyzed by mass spectrometry showed a skeleton constituted of 17 carbons and one
313
unsaturation or a ring. The 2D retention time indicates that this FAME is reduced by Pd
314
because it elutes below the diagonal line bisecting the plane, and it is not an n-/iso-
315
/anteiso-17:1 FAME because its 2D retention time is different. The 17:1 and 16:2
316
FAMEs are separated on different lines and, as in the case of several other co-elutions,
317
they could be resolved only by taking advantage of the 2D separation. Of particular
318
interest was the identification of the 16:4 FAME coeluting with c9,c12-18:2 in 1D, of the
319
16:4 FAME co-eluting with c11-20:1 and 21:0, and the 16:2 FAME co-eluting with c5-
320
18:1 in the middle of the 18:1 cluster. The separation described here not only provides a
321
unique separation capability but it also allows a simple identification of the separated
322
FAMEs.
323 324
The application presented in this study is expected to be suitable for the separation of
325
FAMEs prepared from complex samples of various origins. Figure 5 shows the
326
separation of FAMEs prepared from human colon adenocarcinoma cells HT-29 incubated
327
with conjugated fatty acids. More details regarding the composition and the biological
328
significance of this sample can be found in the original study41. In this separation, the
329
diagonal line of saturated FAMEs bisecting the plane is accompanied by at least two
330
similar other lines, probably not belonging to FAMEs. No attempt was made to identify
331
these compounds. The identification of the FAMEs contained in this sample is
332
challenging, but the model of interpretation described in this study simplifies the
333
identification of most FAMEs based on the 2D lines on which they are eluted. A single
334
dimension of separation can provide a very limited resolution of the FAMEs prepared
335
from this sample, while the chromatographic system described here can separate these
336
FAMEs from each others and from other interfering compounds. As an example, while
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12 337
the C16 PUFAs are separated in the two dimension plane from other compounds
338
contained in this sample, their separation and identification by mono-dimensional GC is a
339
very challenging task.
340 341
The GC×GC separation of marine oil FAMEs can be further optimized by applying an
342
elution temperature program. Figure 6 shows the separation of FAMEs prepared from
343
menhaden oil using a 100 m x 0.25 mm SLB-IL111 for 1D and a temperature program
344
from 130°C to 220°C. The chromatogram is adjusted by shifting the phase 1.8 s. The
345
saturated FAMEs with 14 or more carbons are eluted in an almost straight line parallel to
346
the time axis, and, for each chain length, the unsaturated FAMEs are eluted on crescents.
347
The temperature elution program provided an optimized utilization of the separation
348
space, shortened the separation to 90 minutes, and allowed the reduction of the
349
modulation time to 2 s. The GC×GC separation shown in Figure 6 is characterized by a
350
reversed pattern compared with the separations provided by the system constituted by a
351
non-polar column for 1D and a polar column for 2D17,18: for each chain length, the
352
saturated FAME elutes first at the top of the crescent and unsaturated FAMEs follow in
353
order of increasing number of double bonds. The result is still a group-type GC×GC
354
separation based on the FAME chain length and number of unsaturations, but the groups
355
are arranged in reversed 2D elution order.
356 357 358
CONCLUSION
359 360
The GC×GC chromatographic system described here provides an enhancement of our
361
preferred method for trans fatty acid separation by adding a second dimension of
362
separation capable of resolving most of the FAMEs not separated by 1D. The FAMEs
363
can be easily identified based on a simple interpretation model: saturated FAMEs lie on a
364
diagonal line bisecting the separation plane and FAMEs with the same carbon skeleton
365
lie on lines parallel to the 1D time axis. The chromatographic conditions described have
366
been shown to allow the separation of the FAMEs prepared from the most complex
367
samples, including fish oil and biological extracts. The proposed approach, reducing
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Analytical Chemistry
13 368
unsaturated FAMEs to their saturated form between the two dimensions of separation,
369
provided an enhanced separation compared to GC×GC systems based solely on the
370
orthogonality of the two separations. Reducing U-FAMEs prior to 2D separation also
371
allowed faster modulation times, because the reaction products (saturated FAME) show
372
significantly lower retention times when they are separated on highly polar columns. We
373
invite the users of GC×GC to consider whether or not the described chromatographic
374
system can be considered orthogonal, despite the fact that there is no orthogonality
375
between the separation columns selected for 1D and 2D.
376 377 378
AKNOWLEDGEMENTS
379 380
We are grateful to Dr. John Kramer for contributing to the identification of PUFA and to
381
Edward B. Ledford Jr. of Zoex Corporation for helpful discussions and guidance in the
382
optimization of the GC×GC instrumentation. We also thank Christian Degen (University
383
of Jena, Germany) for generously providing the FAMEs prepared from human colon
384
adenocarcinoma cells HT-29 incubated with conjugated fatty acids.
385 386 387
REFERENCES
388 389
(1) Santercole, V.; Delmonte, P.; Kramer, J.K. Lipids 2012, 47, 329-344.
390 391
(2) AOCS Official Method Ce 1i-07, AOCS Press, Urbana, IL, 2007.
392 393
(3) Armstrong, D.W.; He, L.; Liu, Y.S.; Anal. Chem. 1999, 71, 3873-3876.
394 395
(4) Anderson, J.L.; Armstrong, D.W. Anal. Chem. 2003, 75, 4851-4858.
396 397
(5) Anderson, J.L.; Ding, J.; Welton, T.; Armstrong, D.W. J. Am. Chem. Soc. 2002, 124,
398
14247-14254.
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Page 14 of 23
14 399 400
(6) Anderson, J.L.; Ding, R.; Ellern, A.; Armstrong, D.W.; J. Am. Chem. Soc. 2005, 127,
401
593-604.
402 403
(7) Huang, K.; Han, X.; Zhang, X.; Armstrong, D.W.; Anal. Bioanal. Chem. 2007, 389,
404
2265-2275.
405 406
(8) Breitbach, Z.S.; Armstrong, D.W.; Anal. Bioanal. Chem. 2008, 390, 1605-1617.
407 408
(9) Payagala, T.; Zhang, Y.; Wanigasekara, E.; Huang, K.; Breitbach, Z.S.; Sharma, P.S.;
409
Sidisky, L.M.; Armstrong, D.W. Anal. Chem. 2009, 81, 160-173.
410 411
(10) Anderson, J.L.; Armstrong, D.W. Anal. Chem. 2005, 77, 6453-6462.
412 413
(11) Ragonese, C.; Tranchida, P.Q.; Dugo, P.; Dugo, G.; Sidisky, L.M.; Robillard, M.V.;
414
Mondello, L.; Anal. Chem. 2009, 81, 5561-5568.
415 416
(12) Delmonte, P.; Fardin Kia, A.R.; Kramer, J.K.; Mossoba, M.M.; Sidisky, L.; Rader,
417
J.I. J. Chromatogr. A 2011, 1218, 545-554.
418 419
(13) Delmonte, P.; Fardin-Kia, A.R.; Kramer, J.K.; Mossoba, M.M.; Sidisky, L.;
420
Tyburczy, C.; Rader, J.I. J. Chromatogr. A 2012, 1233, 137-146.
421 422
(14) Destaillats, F.; Guitard, M.; Cruz-Hernandez, C. J. Chromatogr. A 2011, 1218,
423
9384-9389.
424 425
(15) Seeley, J.V ; Seeley, S.K. ; Libby, E.K. ; Breitbach, Z.S. ; Armstrong, D.W. Anal.
426
Bioanal. Chem. 2008, 390, 323-332.
427 428
(16) Reid, V.R. ; Crank, J.A. ; Armstrong, D.W. ; Synovec, R.E. J. Sep. Sci. 2008, 31,
429
3429-3436.
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Analytical Chemistry
15 430 431
(17) Gu, Q.; David, F.; Lynen, F.; Vanormelingen, P.; Vyverman, W.; Rumpel, K.; Xu,
432
G.; Sandra, P. J. Chromatogr. A 2011, 1218, 3056-3063.
433 434
(18) Tranchida, P.Q.; Franchina, F.A.; Dugo, P.; Mondello, L. J. Chromatogr. A 2012
435
1255, 171-176.
436 437
(19) Villegas, C.; Zhao, Y.; Curtis, J.M. J. Chromatogr. A 2010, 1217, 775-784.
438 439
(20) Zapadlo, M.; Krupcík, J.; Májek, P.; Armstrong, D.W.; Sandra, P. J. Chromatogr. A
440
2010, 1217, 5859-5867.
441 442
(21) Zapadlo, M.; Krupčík, J.; Kovalczuk, T.; Májek, P.; Spánik, I.; Armstrong, D.W.;
443
Sandra, P.; J. Chromatogr. A 2011, 1218, 746-751.
444 445
(22) Purcaro, G.; Tranchida, P.Q.; Ragonese, C.; Conte, L.; Dugo, P.; Dugo, G.;
446
Mondello, L. Anal. Chem. 2010, 82, 8583-8590.
447 448
(23) Purcaro, G.; Tranchida P.Q., Conte, L.; Obiedzińska, A.; Dugo, P.; Dugo, G.;
449
Mondello, L. J. Sep. Sci. 2011, 34, 2411-2417.
450 451
(24) Dutriez, T.; Borras, J.; Courtiade, M.; Thiébaut, D.; Dulot, H.; Bertoncini, F.;
452
Hennion, M.C. J. Chromatogr. A 2011, 1218, 3190-3199.
453 454
(25) Mahé, L.; Dutriez, T.; Courtiade, M.; Thiébaut, D.; Dulot. H.; Bertoncini, F. J.
455
Chromatogr. A 2011 1218, 534-544.
456 457
(26) Siegler, W.C. ; Crank, J.A. ; Armstrong, D.W. ; Synovec, R.E. J. Chromatogr. A
458
2010, 1217, 3144-3149.
459
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16 460
(27) Ragonese, C.; Sciarrone, D.; Tranchida, P.Q.; Dugo, P.; Mondello, L. J.
461
Chromatogr. A 2012, 1255, 130-144.
462 463
(28) Adahchour, M., Beens, J., Vreuls, R. J. J., and Brinkman, U. A. T. Trends Anal.
464
Chem. 2006, 25, 438– 454.
465 466
(29) Mondello, L.; Casilli, A.; Tranchida, P.Q.; Dugo, P.; Dugo, G. J. Chromatogr. A
467
2003, 1019, 187-196.
468 469
(30) Adahchour, M.; Jover, E.; Beens, J.; Vreuls, R.J.J.; Brinkman, U.A.T. J.
470
Chromatogr. A 2005, 1086, 128-134.
471 472
(31) de Geus, H.J.; Aidos, I.; de Boer, J.; Luten, J.B.; Brinkman, U.A.T. J. Chromatogr.
473
A 2001, 910, 95-103.
474 475
(32) Western, R.J.; Lau, S.S.G.; Mariott, P.J.; Nichols, P.D. Lipids 2002, 37, 715-724.
476 477
(33) Harynuk, J.; Vlaeminck, B.; Zaher, P.; Marriott, P.J. Anal. Bioanal. Chem. 2006,
478
386, 602-613.
479 480
(34) Seeley, J.V.; Seeley, S.K.; Libby, E.K.; McCurry, J.D. J. Chromatogr. Sci. 2007,
481
45, 650-656.
482 483
(35) Tiyapongpattana, W.; Wilairat, P.; Marriott, P.J. J. Sep. Sci. 2008, 31, 2640-2649.
484 485
(36) Adam, F.; Bertoncini, F.; Coupard, V.; Charon, N.; Thiebaut, D.; Espinat, D.;
486
Hennion, M.C. J. Chromatogr. A 2008, 1186, 236-244.
487 488
(37) De Koning, S.; Janssen, H.G.; Brinkman, U.A.T. LC-GC Eur. 2006, 18, 590-600.
489
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17 490
(38) Schomburg, G.; Hubinger, E.; Husmann, H.; Weeke, F. Chromatographia 1982, 16,
491
228-232.
492 493
(39) Henneberg, D.; Henrichs, U.; Schomburg, G. J. Chromatogr. 1975, 112, 343–352.
494 495
(40) LeQuere, J.L.; Semon, E.; Lanher, B.; Sebedio, J.L. Lipids 1989, 24, 347-350.
496 497
(41) Degen, C.; Ecker, J.; Piegholdt, S.; Liebisch, G.; Schmitz, G.; Jahreis, G. Biochim.
498
Biophys. Acta 2011, 1811, 1070-1080.
499 500
(42) Giddings, J.C. J. Chromatogr. A 1995, 703, 3-15.
501 502
(43) Delmonte, P.; Kia, A.R.; Hu, Q.; Rader, J.I. J. AOAC Int. 2009, 92, 1310-1326.
503 504 505
(44) Delmonte, P.; Hu, Q.; Kia, A.R.; Rader, J.I. J. Chromatogr. A 2008, 1214, 30-36.
506 507 508 509 510 511 512 513 514 515
C20 C19 C18 C17 C16 C15
C24
516
For TOC only
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Inj. Port
FID
690 kPa
GC oven 170 ºC Modulator SLB-IL111
200 m x 0.25 mm
2.50 m x 0.10 mm
1D (200 m)
Reducer
25 cm
SLB-IL111 25 cm
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 18 of 23
Fused silica 2 m x 0.10 mm
2D (0.25 + 2.50 m)
Figure 1. Scheme of GC×GC apparatus.
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2.50
c1322:1
22:0
22:2 n-6
22:3 n-3
22:4 n-6
22:5 n-3
22:6 n-3
2.00
21:0 20:1
20:2 n-6
c5 c8 c11
1.50
20:3 n-6
20:0
18:1*
17:0 16:0
t9 c9
18:2
t9,t12
18:0
n-3
n-6
18:3 n-6
n-3
c10-17:1 16:1
0.50
24:0 15:0 14:0
20:5 n-3
20:4 n-6
c719:1
19:0 1.00
2D, 2.5 m x 0.10 mm SLB-IL111, seconds
c10-15:1
c15-24:1
c9-14:1 13:0 12-13:1 12:0 11-12:1 11:0 10-11:1 10:0 0.00
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Analytical Chemistry
18.96
30.64
42.31
53.98
65.65
77.32
88.99
100.66
112.33
124.00
135.67
147.34
1D, 200 m x 0.25 mm SLB-IL111, minutes
Figure 2. GC×GC separation of the FAME contained in reference mixture GLC 463 (Nu-chek Prep., Inc.). Instrumental configuration as shown in Figure 1. *In order of elution, t9-, t11-, c6-, c9- and c11-18:1.
ACS Paragon Plus Environment
159.01
Analytical Chemistry
c11 22:1 c13 c15
22:0
2.50 1.50 1.00 2.00 0.50 2D, 2.5 m x 0.10 mm SLB-IL111, seconds
22:5 n-6 22:4 n-3
22:4 n-6
22:5 n-3
22:6 n-3
21:5 n-3 21:0 20:2
20:1
NMI n-6
20:0
20:3 n-6 20:4 n-4 n3
20:5 n-3
n-3
19:1
19:0 18:0
n-6
18:1 18:2
18:3
n-3 n-6 n-4
18:4 n-6
n-3
n-1
17:1 17:0 16:0
c15-24:1
16:1 16:2 16:3 16:4
24:0
15:0 14:1 14:0
0.00
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 20 of 23
18.96
30.64
42.31
53.98
65.65
77.32
88.99
100.66
112.33
124.00
135.67
147.34
159.01
1D, 200 m x 0.25 mm SLB-IL111, minutes
Figure 3. GC×GC separation of the FAME prepared from a menhaden fish oil sample. Instrument configuration as shown in Figure 1. NMI, non-methylene interrupted.
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20:1 1.60
20:0
c7 c9
c11 c13 c15
1.40 1.20 1.00 0.60 0.80 2D, 2.5 m x 0.10 mm SLB-IL111, seconds
c1119:1 19:0 c9
18:1 c11
t8 c7 t6 c5 /t9
18:0
18:2 x2
c5, c11
c13 c14
c8, n-6 c11
18:3 n-4
n-6
n-4
n-3
i-18:0
17:1
17:0 y2 i-17:0
16:0
c5
y1 i-16:0
15:0
c7 c9
ai-15:0 i-15:0
0.40
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Analytical Chemistry
14:0
c7
24.69
c9
26.90
t6
ai17:0
c7
c9
17:2
c11 c13
16:3
n-6 16:2 n-4 +NMI?
16:1 c9 x1 t6 t9 c7 c11 -12
n-4
n-3
n-3
16:4
n-1
15:1
14:1
29.13
31.34
33.56
35.78
38.00
40.21
42.44
44.66
46.88
49.00
51.31
53.54
1D, 200 m x 0.25 mm SLB-IL111, minutes
Figure 4. Partial GC×GC separation from C14 to C20 of the FAME prepared from a menhaden oil sample. Instrument configuration as shown in Figure 1. Y1, pristanic acid methyl ester; y2, phytanic acid methyl ester; x1, FAME with 17 carbons and one unsaturation; x2, FAME with 19 carbons and one unsaturation NMI, nonmethylene interrupted.
ACS Paragon Plus Environment
55.75
Analytical Chemistry
3.50 23:1
23:0 3.00
22:4 n-6 22:1
22:0
2.50 2.00 1.50 0.50 1.00 2D, 2.5 m x 0.10 mm SLB-IL111, seconds
22:5 n-3
22:5 22:4 n-6 n-3
22:6 n-3
21:5 n-3 21:1
21:0
20:3 20:1
20:2
n-6
20:0 19:0
16:0
20:4 n-6
20:4 n-3
20:5 n-3
19:1
18:1 18:0
n-3
18:2
17:1 16:3 16:1 16:2
18:3 n-6
18:2 18:4 n-3 conj. n-3 n-1
16:4
24:0
24:1
14:0
0.00
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 22 of 23
18.97
30.64
42.31
53.98
65.65
77.32
88.99
100.66
112.33
124.00
135.67
147.34
159.01
170.68
1D, 200 m x 0.25 mm SLB-IL111, minutes
Figure 5. GC×GC separation of the FAME prepared from human colon adenocarcinoma cells HT-29 incubated with conjugated fatty acids. Instrument configuration as shown in Figure 1.
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2.00 y2
y1
1.75
24:0 16:0
1.50 14:0
1.00 0.75 0.50 1.25 2D, 2.5 m x 0.10 mm SLB-IL111, seconds
i-16:0 ai15:0 i- 15:0 15:0
i17:0
ai17:0
i- 18:0 18:0
22:0
17:0
20:0
19:0
21:0
c15 c5
i14:0
t6 c7 c7
14:1
16:1
t6 c9
c9
c915:1
c11
x1
c9
13:0
c11
c5
17:1
c7 c9 18:1 c11 19:1
c11 c13
c13 n-6 +NMI?
16:2
n-4
c9 c7
20:1
24:1
22:1
c17
c13
c11 c13
c15
c15
c13 x2
NMI
c14/c15 c5, c11 c8, c11
17:2
NMI
20:2
n-4
n-6 n-4 n-6 n-6
18:3 n-4
n-4 16:3 n-3 n-3
22:2
n-6
n-6
18:2
12:0
0.25
16:4 n-1
n-3 18:4 n-6 n-3 n-1
20:3
n-4 n3 20:4 n-6 20:4 n-3
22:4 n-6 22:5 n-6 22:4 22:5 n-3 n-3 20:5 n-3 21:5
22:6 n-3
18:5 n-3
0.00
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Analytical Chemistry
9.00
15.67
22.33
29.00
35.67
42.33
49.00
55.67
62.33
59.00
75.67
82.33
89.00
1D, 100 m x 0.25 mm SLB-IL111, minutes
Figure 6. GC×GC separation of the FAME prepared from a menhaden fish oil sample. Instrument configuration as shown in Figure 1, with modifications: 1D column 100 m x 0. 25 mm SLB-IL111, and elution temperature program. Y1, pristanic acid methyl ester; y2, phytanic acid methyl ester; x1, FAME with 17 carbons and one unsaturation; x2, FAME with 19 carbons and one unsaturation; NMI, non-methylene interrupted.
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