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    ANALYSES OF FATANALYSES OF FAT--SOLUSOLUBLE VITAMINS, CAROTENOIDS AND LIPIDS BY BLE VITAMINS, CAROTENOIDS AND LIPIDS BY

    SUPERCRITICAL FLUID CHROMATOGRAPHY WITH SUBSUPERCRITICAL FLUID CHROMATOGRAPHY WITH SUB--22µµM PARTICLE M PARTICLE

    COLUMNSCOLUMNS

    Dominic Roberts1, Jinchuan Yang2, Rui Chen2, Michael Jones2, Giorgis Isaac2 1 Waters Corporation, Manchester, United Kingdom; 2 Waters Corporation, Milford, MA, USA.

    INTRODUCTION

    UltraPerformance Convergence Chromatography

    (UPC2)TM is a separation technique that uses

    compressed carbon dioxide as the primary mobile

    phase. It takes advantage of sub-two micron particle

    chromatography columns and advanced

    chromatography system design to achieve fast and reproducible separation with high efficiencies and

    unique selectivity. It also generates much lower

    solvent wastes as compared to liquid chromatography

    (LC). These improvements lead to new interest in

    applying this technology to various industrial analytical areas, especially those areas where normal-

    phase (NP) LC has been commonly used, such as fat-

    soluble vitamins (FSV), carotenoids, and lipids. NPLC

    of these compounds suffers long runtime, slow

    equilibration and poor reproducibility. Preliminary studies of using UPC2 for the separation of fat-soluble

    vitamins (FSV), carotenoids and lipids are presented

    here to illustrate the performance of UPC2 technology

    in these important analysis areas.

    FREE FATTY ACIDS SEPARATION Instrumentation Waters ACQUITY UPC2 System with SYNAPT G2 MS controlled by MassLynx

    Chromatographic Conditions:

    Column: ACQUITY UPC2 HSS C18 SB

    Co-solvent: MeOH with 2 g/L ammonium formate Gradient: 1 to 10% over 5 minutes

    Flow: 2.5 mL/min Temperature: 600C

    Pressure: 1885 psi Injection Vol: 0.5 µL

    Make-up flow: 0.2 mL/min of 0.1% formic acid

    FFA: C8 to C24

    Sample Concentration: 0.25 mg/mL

    EDIBLE OILS SEPARATION Instrumentation

    Waters ACQUITY UPC2 System with ACQUITY UPC2 PDA Detector and Xevo G2 QTof MS controlled by MassLynx.

    Chromatographic conditions

    Column: ACQUITY UPC2 HSS C18 SB (3.0x150 mm, 1.8 µm) Mobile phase A: Compressed CO2

    Mobile phase B: ACN

    ABPR: 1500 psi Sample diluent: Chloroform

    Flow rate: 1.0mL/min Column temperature: 20oC

    Inj. Vol.: 1 µL PDA: 210nm, Ref 400-500nm

    FREE FATTY ACIDS AND EDIBLE OILS

    Figure 4. UV chromatograms of edible oils by UPC2 on a single UPC2 HSS C18 SB column. Run time: 22 minutes. Peaks were identified based on

    high/low energy accurate mass spectra in MSE function.

    Figure 2. Separation of free fatty acids (C8 to C24) by UPC2 and QTOF MS (ESI– mode).

    Figure 3. Separation of FFA (C8-C34) in algae extract using UPC2 HSS C18 SB (1.8 µm) column. The co-solvent gradient is shown in

    Figure 3. The FFA elute before 2 min.

    C16:0

    C28:0

    C34:0

    5% - 20% MeOH in 10 min

    2.8 min

    2.0 min

    References

    1. E. Klesper, A.H. Corwin, D.A. Turner, J. Org. Chem. 27 (1962) 700.

    2. Packed column SFC by T.A. Berger, The Royal Society of Chemistry 1995, Cambridge, UK

    3. Food Analysis by HPLC, 2nd Ed. L. M. L. Nollet edit, Marcel Dekker 2000, New York, USA

    4. R. Chen, J. Yang, J. McCauley, Waters Application Note, Lit. Code: 720004551en. 2013.

    5. M.D. Jones, G. Isaac, G. Astarita, A. Aubin, J. Shockcor, V. Shulaev, C. Legido-Quigley, and N. Smith, Waters Application Note Lit. Code: 720004579en. 2013

    Structures of FSV and carotenoids standards.

    INSTRUMENTATION:

    Waters ACQUITYTM UPC2 System equipped with a UPC2 PDA detector.

    The system is controlled by Empower III.

    Chromatographic conditions:

    Mobile phase A: Compressed CO2

    Mobile phase B: Acetonitrile Flow rate: 1 mL/min

    Column: ACQUITY UPLC HSS C18 (3.0 x 100 mm, 1.8 µm)

    Backpressure: 2500 psi Temperature: 30 °C

    Sample diluent: MTBE Inj. Vol.: 1 µL

    PDA scan range: 210-600 nm

    SAMPLES:

    Figure 1. Simultaneous separation of FSV and carotenoids standards by UPC2 with PDA detection in a single run (Chromatogram on the Left) and their UV spectra

    (210-600nm) in the order of their elution time (on the right). The spectra from the top to the bottom: vitamin A acetate, E acetate, K2, K1, vitamin E, D2, vitamin

    A palmitate, lycopene, and beta-carotene.

    Table 1. Repeatability results (RSD) for retention and UV peak area for nine FSV and carotenes used in Figure 1 (n=6)

    FAT-SOLUBLE VITAMINS AND CAROTENOIDS ANALYSIS

    Time B

    (min) %

    0 2

    2 2

    2.5 20

    3.5 20

    3.75 2

    4 2

    Data courtesy of Davy Guillarme, Jean-Luc Veuthey LCAP, University of Geneva, Switzerland

    Diagram 1

    UltraPerformance Convergence

    Chromatography is the result of significant

    technological advance-ments in Supercritical

    Fluid Chromatography that finally enable this

    technique to become a reliable and robust

    analytical tool.

    Gradient:

    Time %B

    0 3

    2 3

    17 70

    22 70

    CONCLUSION

    UPC2 provides

    Fast separation

    Unique selectivity

    High separation efficiency

    Low solvent usage

    Simplified sample preparation

    All these results in significant improvement in

    analysis throughput and savings in operational

    cost.

    DISCUSSION

    Performance of UPC2

    Low viscosity and high diffusivity of supercritical CO2, low particle size (sub-2 micron) of column render high separation efficiency, fast analysis with less back pressure (Diagram 1)

    Separation of FSV and carotenoids

    NPLC separation of these compounds suffers long runtime (about

    30min), slow equilibration and poor reproducibility. RPLC separation of these compounds has potential sample carryover issue and requires more stringent sample clean-up to remove fat and other hydrophobic materials.

    UPC2 provides fast, reliable, and simultaneous separation of multiple analytes in a single run. (Figure 1 and Table 1)

    Separation of free fatty acids (FFA)

    Separation of FFA can be carried out by GC after derivatisation. Derivatisation is time consuming and has a risk of re-arrangement of FFA.

    Also, for high carbon FA, their low volatility may cause inaccurate quantification. UPC2 does not require derivatisation, and provides fast separation of FFA from short to long chains. (Figure 2, and 3)

    Separation of triacylglycerols (TAG) in edible oils

    UPC2 provides faster and more efficient separation of TAGs than HPLC

    does and generates much less solvent waste. (Figure 4)


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