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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)
Recommended