Preparative SFC/SFE with Switching System and MS Detector

Download PDF May 9, 2018

Introduction

Fig. 1 SFE/PrepSFC-MS Switching System

A mass spectrometer not only provides mass information that is crucial for purification chemists but also delivers increased sensitivity and selectivity relative to conventional chromatography detectors. In recent years it has been used as the method of selective detection for target analytes and identification of impurities in the development of pharmaceuticals and in a variety of other industries.

Supercritical fluid extraction (SFE) enables fast and efficient extraction using a supercritical fluid that has the specific characteristics of high diffusivity, permeability, and solubility.  SFE with supercritical CO2 has advantages of easy post extraction handling, lower solvent costs, and automation by device control.

We developed a switching system that has both SFE and Preparative SFC/MS capabilities.  This presentation outlines the extraction of caffeine from coffee beans and the subsequent purification and re-analysis of the fractions.

SFEPrepSFC-MS
CO2 Flow Rate20 mL/minSFCAlcyon SFC SIL
(20 mmI.D. x 250 mmL, 5μm)
EntrainerCH3OH/H2O (39/1) (Sample No.1)
C2H5OH/H2O (39/1) (Sample No.2)
ColumnCO2/Methanol Gradient
Entrainer Flow Rate0 mL/min (0 ~ 60 min)→1 mL/min (60 ~ 90 min)
→5 mL/min (90 ~ 120 min)
→0 mL/min (120 ~ 150 min)
EluentCO2/Methanol Gradient
80/20 (0 min)→60/40 (4 min)→
60/40 (5 min)→80/20 (5.1 min)
1cycle 10 min
Extraction Temp40˚CFlow Rate30 mL/min
Extraction VesselEV-2 (10 mL)Column Temp.40˚C
Pressure10 MPa (0 ~ 30 min)→25 MPa (30 ~ 120 min)
→10 MPa (120 ~ 150 min)
Wavelength270 nm
Wavelength270 nmPressure10MPa
SampleGreen Coffee Beans 4 wet gInjection Volume500 μL
SampleGreen Coffee Beans Extraction Solution
MS
Ion SourceESI (+)
ModeSIM
m/z195.2
Make Up SolventMethanol
Make Up Solvent Flow Rate1 mL/min
Splitter
Flow Rate30 mL/min
Split Ratio1000 : 1

Experimental

Apparatus

Fig. 2 Flow Diagram Above: SFE, Below: PrepSFC-MS

Figure 2 shows the JASCO switching system of SFE/Prep SFC with MS detector used in this experiment. Figure 2 also shows a schematic diagram of this system. The column oven equipped with 10 position-11 port valves enables the switching of several extraction vessels, analytical scale columns, and semi-prep scale columns without re-plumbing.


Figure 3 shows the cyclone separator (CS-87)  used on the open-bed fraction collector. It significantly increases the fraction recovery. Figure 4 shows the extraction vessel (EV-2) used for SFE.

Fig. 4 Extraction vessel (EV-2)
Fig. 3 Cyclone Separator (CS-87)

 

 

 


Conditions for SFE and Prep SFC-MS

The SFE procedure was performed by PEEM (Programmed Extraction Elution Method) that enhances extraction efficiency and selectivity with changing the flow rate of an entrainer and back pressure in stages. We used mixtures of methanol/water and ethanol/water as entrainers.

In Prep SFC, we used a silica column (20 mmI.D.), and the separation was performed by gradient elution of CO2 and methanol at 30 mL/min total flow rate. The MS detector ion source was ESI-positive, and measurement mode was selected ion monitoring (SIM) at 195.2 m/z.

 

Sample Preparation

Fig. 5 Coffee beans Left: Dry, Right: Wet

Procedure of sample pretreatment of green coffee beans:

Green coffee beans

Soak them in  water at room temperature overnight  in order to extract caffeine efficiently (Fig. 5).

Take them out of water, and wipe with laboratory tissue paper.

Load 4g of this sample into an extraction vessel.

Results

SFE

Fig. 6 Extraction Result of Sample 1

Figure 6 shows the extraction result of sample 1. The extracts were collected in 4 vials at each extraction step (extraction 1 to 4). Table 1 shows the loading of green coffee beans into vessels, the composition of entrainers, and the collection volumes.

 

 

 

Table 1 Sample Loading, Entrainer Composition and Fraction Volume

Sample No.12
Sample (wet g)4.094.07
Sample (dry g)2.182.17
EntrainerMethanol/Water (39/1)Ethanol/Water (39/1)
Collection Volume (mL)
Extraction 12735.5
Extraction 23937.5
Extraction 36667.5
Extraction 4178184

Prep SFC-MS

Fig. 7 Chromatograms of Extraction 1 to 4 from Sample 1, Peak 1: Caffeine

Figure 7 shows the chromatograms of each extraction (1 to 4) from sample 1. Table 2 shows the total extraction amount in each extraction, calculated from the peak area of caffeine standard solution (100 ppm, 500 µL). As shown in this table, high extraction efficiency was achieved for sample 1 using a mixture of methanol and water as an entrainer.

 

 

 

Table 2 Extraction Amount

Caffeine STD
Concentration (µg/mL)100
Injection Volume (mL)0.5
Injection Amount (µg)50
Peak Area (µV • sec)406959
Extraction No.Extraction Amount per Coffee Beans 2.18 g (µg)
5583
Sample 11234
Peak Area (µV • sec)293049
8029912777618218
Amount per 1 Injection (µg/0.5 mL)36.09.915.72.2
Collection Volume (mL)273966178
Total Extraction Amount (µg)19447702072797
Extraction No.Extraction Amount per Coffee Beans 2.17 g (µg)
2663
Sample 21234
Peak Area (µV • sec)131308340673480013295
Amount per 1 Injection (µg/0.5 mL)16.34.24.31.7
Collection Volume (mL)35.537.567.5184
Total Extraction Amount (µg)1156317583607

 

Fig. 8 Collection Result of Extraction 1 from Sample 1

Figure 8 shows the collection results of the caffeine peak in extraction 1 from sample 1, triggered based on the MS signal (CH1: 270 nm, CH2: m/z 195.2). MS triggering is effective for the fraction collection of non-chromophoric compounds. ChromNAV with fraction collector management program (chromatography software) supports automatic fractions with advanced fraction algorithms for peak collection, using time, threshold, and slope, and can also be programmed to use signals simultaneously from multiple detectors.

 

 

Purity Determination of Extracts by HPLC-PDA

Fig. 9 Chromatograms of Caffeine
Peak 1: Caffeine

We determined the purity of extracts collected in SFE by HPLC-PDA. Figure 9 shows the chromatograms of caffeine standard solution and concentrated extracts (mixture of extraction 1 to 4) from sample 1.  Figure 10 shows the comparison of caffeine spectra between standard solution and sample solution. A high correlation coefficient was obtained between them (> 0.9995). Figure 11 and Table 3 show the results of peak purity. 98.8% of the peak was determined high-purity caffeine.

 

SystemConditions
PumpPU-4180ColumnInertSustain C18
(4.6 mmI.D. x 150 mmL, 3 μm)
Pump OptionLPG Unit, DG UnitEluentCH3CN/H2O (20/80)
AutosamplerAS-4150Flow Rate0.9 mL/min
Column OvenCO-4060Column Temp40 ˚C
DetectorMD-4010Wavelength274 nm
Injection Volume10 μL
Fig. 10 Spectrum Correlation Coefficient

Sample Preparation

Fig. 11 Peak purity

 

Evaporate the extracts (extraction 1 to 4) from sample 1 to dryness.

Dissolve in 3 mL of methanol.

Inject to HPLC.

 

 

Table 3 Peak Purity

Spectrum CorrelationProportion (%)
>0.99998.84
0.90< 0.9991.05
<0.900.12
Correlation coefficients between the peak top spectrum and spectra at all data points within the peak are calculated and presented in different colors according to the degrees of correlation.

Conclusion

  • We achieved the extraction and fraction collection of caffeine from green coffee beans using a switching system SFE/Prep SFC-MS.
  • The combination of PDA and MS detectors enables the detection and fraction collection of various compounds with or without a chromophore.
  • ChromNAV software provides automatic and accurate fractions with advanced fraction algorithms, and useful functions (ex. fraction simulation, graphical display of collection monitoring and results).

References

Presented at SFC 2017 Rockville, MD

Akitaka Tearada1, Satoe Iijima1, DJ Tognarelli2, John Burchell2,  Yasuyo Sato1, Miki Kuwajima1
1JASCO Corporation, 2967-5 Ishikawa-machi, Hachioji, Tokyo 192-8537
2JASCO Incorporated, 28600 Mary’s Court, Easton, MD 21601
E-mail: satoe.iijima@jasco.co.jp

About the Author

Ms Iijima is a member of the chromatography separations team at JASCO Corporation main applications laboratory in Tokyo Japan.