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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)