Comparing SPE to Batch LC for Stevia Purification

The main sweetening compounds in Stevia rebaudiana are Steviol glycosides. As they have up to 400 times higher sweetening power compared to sucrose or glucose, they are frequently employed as natural sugar substitutes. The plant extracts must be purified to enable commercial usage. In this work, for improvement of overall purity due to reduction of matrix contamination, preparative online SPE (solid phase extraction) was investigated.

Introduction

Research has been performed for several years to find calorie-free sugar substitutes that possess the same properties and taste as classic sugar. These substitutes are particularly important in the diets necessary for diabetics, and increasingly as part of the so-called "low-carb" movement. A popular substitute is “Stevia”, which is a mixture of steviol glycosides that are isolated from the plant Stevia rebaudiana [1].

The main compound of interest is the steviol glycoside rebaudioside A, as it is the less bitter and sweetest compound of the extract. Typically, Stevia products contain a mixture of rebaudioside A and stevioside. The development of a purification technique with a high yield of rebaudioside A, high throughput, and minimal stevioside impurities, would enhance the economic output of Stevia production.

Results

A gradient technique for analysis of steviol glycosides was transferred to an isocratic technique (VFD0170), for purification of rebaudioside A and stevioside from stevia leaves. The final technique was up-scaled with the KNAUER up-scale converter [2] to an ID 20 mm column of the same length as the analytical column, increasing the flow rate from 1.2 mL/min to 22 mL/min.

Sample injections of up to 2 mL still exhibited a slight separation of the stevioside and rebaudioside A peaks (Fig. 1). The matrix peak (1-5 min) notably increased (Fig. 1, green). Large sample matrix can affect the separation abilities negatively, and wear off the main column.  Hence, elimination of matrix before purification is desirable.

An online-SPE technique was developed using a short preparative column in front of the main column. 10 mL of sample was loaded, the matrix washed away and the target compounds were then automatically injected on the main column (Fig. 2). When comparing the chromatograms of the classical batch process (Fig. 1) and the online-SPE process (Fig. 2) it is established that the automated SPE process decreased the matrix significantly.

The fraction analysis showed that only a small part of the overlapping peak contained almost pure rebaudioside A; fractions 3-5 approx. 15 mL with >90% rebaudioside A and <10% stevioside (Fig. 3, B). The later fractions exhibited large quantities of stevioside but also rebaudioside A still (Fig. 3, C). The results illustrated that purification of highly pure rebaudioside A is viable by introducing an additional online-SPE step, yet, yield is sacrificed.

Methods and Materials

The AZURA Preparative HPLC system was made up of AZURA P 2.1L 100 mL sst pump with ternary LPG module, AZURA UVD2.1L detector with 3 mm, 2 µl flow cell,  a 6 port 2 position 1/16 ” sst injection valve, an AZURA assistant module with a 6 port multi-position 1/8 ” sst valve (solvent selection), a Labocol vario-4000 fraction collector and a P 4.1S 50 ml sst feed pump.

The final purification technique was divided into two phases: SPE loading and target purification.

SPE Loading

1) Conditioning 1.5 min with 20 mL/min 100% ACN;

2) Re-equilibration 2.5 min with 20 mL/min 20/80 ACN/H20;

3) Sample loading 1 min 5 mL/min

4) Washing 6.5 min with 20 mL/min; target purification: 20 min with 22 mL/min 30/70 ACN/H2O; at 210 nm and 25 °C.

Fraction analysis was carried out using an AZURA analytical HPLC system.

Conclusion

A preparative HPLC method for use in the purification of the most preferred steviol glycoside rebaudioside A from dried stevia leaves was examined. Throughout the technique development, an automatic online-SPE method was determined, significantly decreasing the matrix in the sample.

This should protect the main column from contamination and increases the loading with the main compounds. Yet, the two components stevioside and rebaudioside A are coeluting and under tested conditions, a clean separation is not possible. Pure rebaudioside A can be purified but with low yield.

Overload experiments on preparative column, 200 µL (red), 500 µL (blue), 2000 µL (green); 1) rebaudioside A, 2) stevioside, blue bars – matrix, 25 °C, 22 ml/min.

Figure 1. Overload experiments on preparative column, 200 µL (red), 500 µL (blue), 2000 µL (green); 1) rebaudioside A, 2) stevioside, blue bars – matrix, 25 °C, 22 ml/min.

Preparative online SPE, 10 mL loading; 1) rebaudioside A, 2) stevioside, blue bars – matrix, 25 °C, 22 mL/min.

Figure 2. Preparative online SPE, 10 mL loading; 1) rebaudioside A, 2) stevioside, blue bars – matrix, 25 °C, 22 mL/min.

Fraction analysis of preparative online-SPE purification (Fig.2) of rebaudioside A (1) and stevioside (2); A) fractionation of target peak, 5 mL fractions B) F3 (blue), F4 (red), F5 (green), F6 (light blue); C) F7 (red dashed), F10 (blue dashed), F12 (green sashed), F15 (light blue dashed).

Figure 3. Fraction analysis of preparative online-SPE purification (Fig.2) of rebaudioside A (1) and stevioside (2); A) fractionation of target peak, 5 mL fractions B) F3 (blue), F4 (red), F5 (green), F6 (light blue); C) F7 (red dashed), F10 (blue dashed), F12 (green sashed), F15 (light blue dashed).

References

[1] “Stevia Leaf to Stevia Sweetener: Exploring Its Science, Benefits, and Future Potential” P. Samuel, K. T. Ayoob, B. A. Magnuson, et al. J Nutr, Volume 148, Issue 7, 1 July 2018, Pages 1186S–1205S

[2] Scale up converter: https://www.knauer.net/en/knauer-scaleup-converter/p14082

Additional Results

Sample loading on SPE column, 10 mL sample, step 1 - conditioning, step 2 - re-equilibration, step 3 - sample loeading, step 4 - washing

Figure 1A. Sample loading on SPE column, 10 mL sample, step 1 - conditioning, step 2 - re-equilibration, step 3 - sample loeading, step 4 - washing

Additional Materials and Methods

Table A1. Method parameters (preparative online-SPE)

.
Eluent A 100% ACN        
Eluent B 20%/80% ACN/H2O        
Sample Concentrated stevia extract
Step Flow rate Time (min) % A % B Sample (%)
Conditioning 20 mL/min 1.5 100 0 0
Re-equilibration 20 mL/min 2.5 0 100 0
Sample loading 5 mL/min 2 0 0 100
Washing 20 mL/min 9.5 0 100 0
Run temperature 25 °C Run time 15.5 min    
Injection volume 10 mL Injection mode Feed pump    
Detection wavelength 210 nm Data rate 2 Hz    
    Time constant 0.05 s    

 

Table A2. System configuration & data

Instrument Description Article No.
Pump AZURA P 2.1L, 100 mL, SST
AZURA ternary module for P 2.1L
APE20KA
AZZ00AB
Detector AZURA UVD 2.1L ADA01XA
Assistant Left: 6 Mpos,1/8””,sst
Middle:6Port2Pos,1/16”,sst
Right:P4.1S, 50 ml,sst
AYEKEABR
Flow cell 3 mm, 2 µL; 1/16” A4069
Column Eurospher II 100-10 C18 250x4.6 mm
Eurospher II 100-10 C18 250x20 mm
Eurospher II 100-5 C18 30x20 mm
25VE181E2N
25PE181E2N
03PE181E2J
Fraction collector Labocol Vario-4000 A591022
Software PurityChrom5 Basic A2650

 

Table A2. Method parameters (preparative method)

.
Eluent A 30%/70% ACN/H20    
Eluent B -    
Gradient isocratic
Flow rate 22 mL/min System pressure 80 bar
Run temperature 25 °C Run time 20 min
Injection volume From above Injection mode -
Detection wavelength 210 nm Data rate 2 Hz
    Time constant 0.05 s

 

About KNAUER Wissenschaftliche Geräte GmbH

KNAUER Wissenschaftliche Geräte GmbH is a middle-sized company that has been developing, manufacturing and distributing laboratory instruments around the world since 1962.

With more than 130 employees, Knauer is one of the well-established manufacturers of HPLC instruments, SMB systems, and osmometers. Product portfolio includes extremely compact HPLC solutions, UHPLC systems for high-resolution analysis, preparative HPLC instruments, process LC equipment for the purification of substances in the kilogram scale, autosamplers, column thermostats, degassers, detectors, dosing pumps, eluent mixers, flowmeters, LC columns, and accessories and spare parts, amongst others.

The source of Knauer’s success is numerous world’s firsts that have won more than 20 awards for innovation.


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Last updated: Oct 3, 2019 at 8:55 AM

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