Mikael E.
Fridén
a,
Firas
Jumaah
b,
Christer
Gustavsson
c,
Martin
Enmark
c,
Torgny
Fornstedt
c,
Charlotta
Turner
b,
Per J. R.
Sjöberg
a and
Jörgen
Samuelsson
*c
aUppsala University, Department of Chemistry – BMC, Analytical Chemistry, P.O. Box 599, 75124 Uppsala, Sweden
bLund University, Department of Chemistry, Centre for Analysis and Synthesis, P.O. Box 124, 22100 Lund, Sweden
cKarlstad University, Department of Engineering and Chemical Sciences, SE-651 88 Karlstad, Sweden. E-mail: jorgen.samuelsson@kau.se; Fax: +46 547001460; Tel: +46 547001620
First published on 20th August 2015
Betulin from birch bark was extracted using two principally different extraction methodologies – classical Reflux Boiling (RB) and Pressurized Liquid Extraction (PLE). The extraction methods were analyzed based on both recovery and purity as well as for RB industrial feasibility. The purity and recovery for the different extraction methods were analyzed using High Performance Liquid Chromatography (HPLC) coupled with three different detection principles: Diode Array Detection (DAD), Mass Spectrometry (MS) and Charged Aerosol Detection (CAD). The chromatographic purity was determined by all detections whereas the DAD was used also for complementary gravimetric calculations of the purity of the extracts. The MS detection (in MS and MS/MS modes) was mainly used to characterize the impurities. Two steps to increase the purity of RB extracts were evaluated – pre-boiling the bark in water and precipitation by adding water to the extract. Finally, the methods were compared in terms of amounts of betulin produced and solvent consumed. The RB method including a precipitation step produced the highest purity of betulin. However, results indicate that PLE using three cycles with the precipitation step gives similar purities as for RB. The PLE method produced up to 1.6 times higher amount of extract compared to the RB method. However, the solvent consumption (liter solvent per gram product) for PLE was around 4.5 times higher as compared to the classical RB. PLE performed with only one extraction cycle gave results more similar to RB with 1.2 times higher yield and 1.4 times higher solvent consumption. The RB process was investigated on an industrial scale using a model approach and several important key-factors could be identified. The most energy demanding step was the recycling of extraction solvent which motivates that solvent consumption should be kept low and calculations show a great putative energy reduction by decreasing the ethanol concentration used in the RB process to lower than 90%.
There are numerous studies exploring different extraction techniques for betulin in birch bark, including pressurized liquid extraction (PLE),14 supercritical fluid extraction (SFE),15 microwave assisted extraction (MAE),16 and classical reflux boiling (RB) or leaching.17 While PLE and MAE require special equipment, the increased temperature and pressure usually utilized is believed to allow for more exhaustive extraction of the target compounds as compared to classical RB. On the contrary, SFE is known as a more selective extraction technique.18 None of the published studies have calculated the energy usage necessary to compare different methodologies in terms of betulin production. Furthermore, only a few studies actually did purify betulin from the extract, and if they did, the method for determination of the purity varied largely, resulting in non-comparable or even erroneous results. For more information about the chemistry and general processing of birch bark, the reader is referred to the comprehensive review by P. Krasutsky.19
The required purity of the extracted target compound depends on the intended use of the final product. Plant extracts intended for use in medical products are regulated e.g. by the Food and Drug Administration in the USA and European Medicines Agency in the EU.20,21 Should the same extract be used as a chemical of technical quality, it does not have the same stringent requirements. In fact, “technical quality” does not seem to be a well-defined term; descriptions like “reasonable quality”22 and “do not have an established standard set for quality and impurity levels”23 are used by some major suppliers.
The aim of this study is to find a suitable strategy for producing betulin with an appropriate purity and quality while considering environmental aspects such as solvent and energy consumption. This involves first a careful analytical evaluation of promising and environmentally sustainable extraction principles (RB and PLE). Secondly, utilizing different detection principles such as mass spectrometry (MS), UV/vis spectroscopy (using a diode array detector, DAD) and charged aerosol detector (CAD) coupled with HPLC to determine the purity and recovery of the extraction methods. Finally, the industrial feasibility of the most promising extraction technique is investigated in more detail by establishing mass and energy balances for industrial scale extraction, and solvent recovery processes based on reliable experimental data. Additionally, SFE was tested as a potential extraction method but was not pursued further due to poor performance, see ESI.†
Fig. 1 A schematic overview of how birch wood is processed and where our suggested extra process steps fit into the whole process. The investigated part of the process is marked with dashed lines. |
Fig. 2 (a) Chromatograms recorded with DAD 210 nm after different means of purifying the extract; no purification (upper black line, RB), pre-boiling in water (grey line, pre-RB) and precipitation (lower black line, RB-pc). For increased clarity RB has been off-set by 20 mAU, and pre-RB by 5 mAU. Betulin is labeled bet and betulinic acid ba. (b) Magnification of part of the chromatograms in (a), with RB off-set by 5 mAU and pre-RB by 3 mAU. Peaks are labeled in chronological order, according to the retention times in ESI Table S7.† |
Extraction method | Chromatographic purity, DAD (%) | Chromatographic purity, CAD (%) | Chromatographic purity, TIC (%) | Gravimetric purity (%) | Extracted amount (mg betulin per g bark) | Solvent consumption (l solvent per g betulin) |
---|---|---|---|---|---|---|
n.d., not determined. | ||||||
Reflux boiling (n), ethanol | 72.9 ± 7.1 | 80.8 ± 0.5 | 39.5 ± 2.9 | 33.9 ± 3.8 | 51.6 ± 4.1 | 0.194 ± 0.015 |
Reflux boiling (n), acetone | 76.1 ± 2.7 | 80.0 ± 2.0 | 32.3 ± 2.2 | 33.9 ± 3.4 | 44.6 ± 3.6 | 0.224 ± 0.018 |
Reflux boiling (2), ethanol | 85.3 ± 3.6 | 87.1 ± 0.4 | 28.6 ± 3.6 | 60.9 ± 1.7 | 47.5 ± 7.5 | 0.210 ± 0.033 |
Reflux boiling (2), acetone | 86.5 ± 2.6 | 86.5 ± 3.5 | 36.2 ± 1.6 | 59.3 ± 4.3 | 44.5 ± 1.7 | 0.225 ± 0.009 |
Reflux boiling (3), ethanol | 78.7 ± 2.0 | 85.1 ± 1.5 | 33.5 ± 3.8 | 38.2 ± 3.6 | 48.3 ± 3.2 | 0.207 ± 0.014 |
Reflux boiling (3), acetone | 80.6 ± 2.0 | 86.8 ± 0.1 | 43.2 ± 3.6 | 46.5 ± 9.5 | 45.2 ± 2.1 | 0.221 ± 0.010 |
Reflux boiling (b), ethanol | 87.6 ± 2.8 | 86.4 ± 0.1 | 30.8 ± 4.4 | 62.8 ± 8.6 | 48.6 ± 9.4 | 0.206 ± 0.040 |
Reflux boiling (b), acetone | 85.6 ± 3.8 | 90.0 ± 0.7 | 39.6 ± 2.2 | 60.1 ± 1.8 | 45.1 ± 2.3 | 0.222 ± 0.011 |
PLE | 72.6 ± 4.0 | 77.9 ± 1.7 | 34.8 ± 7.2 | 17.0 ± 3.1 | 76.7 ± 8.1 | 0.980 ± 0.106 |
PLE (1st cycle) | 73.3 ± 8.0 | 82.0 ± 1.5 | 57.5 ± 7.3 | 25.4 ± 4.0 | 62.5 ± 9.8 | 0.254 ± 0.040 |
PLE (p, 1st cycle) | 66.5 ± 3.0 | n.d. | n.d. | 47.4 ± 0.3 | 58.8 ± 0.4 | 0.287 ± 0.002 |
In the ESI† we have detailed the data even more: the total ion chromatograms (TICs) and extracted ion chromatograms (XICs), can be found in Fig. S1† with peak data in Table S3† (RB extracts), Fig. S2† with peak data in Table S4† (PLE) and Fig. S3† with peak data in Table S5† (SFE). Triterpenes previously reported in birch bark are for example betulinic aldehyde, betulone, betulonic acid, betulonic aldehyde and lupeol and β-amyrin.3,19 Among the impurities are compounds showing similar fragmentation pattern as betulin, betulinic acid and the triterpenes mentioned.24 For example peak 17 is assigned as betulonic acid, however the substituent (230 Da, at m/z 685) was only observed in one replicate. Furthermore, two peaks show signals matching betulin with substituents: peaks 14 (betulin with a 26 Da substituent, m/z 469) and 19 (betulin with a 75 Da substituent, m/z 518). Peak 19 also shows a signal which probably is a water adduct (m/z 461) or from the loss of parts of the substituent mentioned above. Detailed MS and MS/MS data of betulin, betulinic acid and some of the major impurities along with possible identities are shown in ESI, Table S6.†
Purities determined by gravimetric analysis (see the procedure in the ESI†) gave lower values than those obtained both by DAD and CAD, indicating that there could be several impurities not detected by the detectors, see Table 2. Many times this is observed because the contaminations are unsuitable for one or more of the detectors, such as lack of a chromophore for DAD or poor ionization for MS, or because they occur at levels too low to give a response which would be integrated (see ESI†). Table 2 shows that the precipitation step results in the highest purity, both for RB and PLE. In order to investigate where the observed extra contaminations in the gravimetric analysis originate from, the precipitate from the RB was dissolved in ethanol, filtered and precipitated again. The analysis of this sample showed to have similar purity both for gravimetric and DAD. This clearly indicates that insoluble material, which was also observed in the filter (probably dust and cellulose), are present in the first precipitate. Because CAD, DAD and MS methods are based on chromatographic separations these contaminations are lost in the sample preparation filtration steps, and as a consequence the estimated purity is overestimated. To improve the process, a finer filter after the leaching step was used (102 Double Ring filter paper). The gravimetric purity with the additional filtration step without water pre-boiling was determined to be 60.2% and 63.1% after the precipitation step. These gravimetric purities are similar to the ones observed with birch bark pre-boiled in water after the precipitation step, see Table 2. Fourier transform infrared spectroscopy (FTIR) analysis showed that the spectral library similarity against wood decreases and similarity against betulin increases with process steps and filtration followed by precipitation had the same quality using birch bark with or without the pre-boiling step. In Fig. S4† photos of dried betulin process fluids or precipitates are presented for morphological comparison.
When the RB extracts purified by precipitation are compared before and after the precipitation step the gravimetric purity is significantly increased while only a slight (not statistically significant) decrease in the extracted amount occurs, based on analysis of variance (ANOVA) (data not shown). This indicates that the purification should be performed on the other extracts as well. ANOVA (data not shown) also suggests that the additional pre-boiling step affects neither the extracted amount nor the final purity to a notable extent (comparing extracts purified only by precipitation to extracts purified by both pre-boiling and precipitation). As a consequence we draw the conclusion that the pre-boiling step could be omitted from the final process without a reduction in purity, assuming precipitation is performed.
The greatest extracted amount was obtained by PLE using three cycles, reaching a total of 79 mg per g bark, see Table 2. However, the amount of betulin produced is approximately 1.6 times more than RB but with a solvent consumption (liter solvent per gram product) of 4.5 times higher. The value obtained for each cycle could be of interest if a thorough analysis of the economic feasibility for scaling up should be investigated, as it would then be possible to determine if only one or two cycles should be used which would reduce solvent consumption. For instance, using only one extraction cycle in PLE and introducing a precipitation step resulted in the pure product (Fig. S5†) with 1.2 times higher yield and 1.4 times higher solvent consumption compared to RB. However, RB still outperforms PLE in terms of purity of the product, see Table 2. Both additional steps in RB (pre-boiling and precipitation) result in increased purity, with the exception for MS detection (which was mainly used for characterization of impurities as discussed above). None of these additional steps result in a significant loss of betulin so combining this with the data in ESI Table S7,† showing that many impurities are extracted by all three techniques, the potential of increasing the purity of PLE extract is apparent as can be seen in ESI Fig. S5.†
Modelling assumptions | |
---|---|
Actual data from Gruvön Mill | |
Birch bark production (tons per year dry solids) | 50000 |
Bark dry content (%) | 50 |
Biomass boiler design capacity (MWth) | 90 |
Steam turbine | |
Admission data (bara/°C) | 58/470 |
Low pressure steam (turbine outlet) (bara) | 4.2 |
Biomass moisture content (%) | 50 |
Assumed data | |
Biomass boiler flue gas temperature (°C) | 170 |
O2 in boiler flue gases (vol% wet) | 6 |
Steam turbine | |
Isentropic efficiency | 0.80 |
Atmospheric distillation | |
No of ideal stages | 30 |
Feed stage | 25 |
Bottom ethanol concentration (% weight) | 0.1 |
Calculated results | |
Betulin production (tons per year) | 2500 |
Distillation reboiler duty (MW) | 49.4 |
Distillation condenser duty (MW) | 41.1 |
Change in mill power production (MW) | 13.3 |
Change in mill fuel consumption (wet tons per annum) | 259000 |
The main results from the Chemcad simulations are shown in Table 3. As can be seen, adding the betulin extraction process to the mill entails significant new mass and energy streams. With the simulated process configuration, the total mill low-pressure steam demand increases to 93 tons per h, corresponding to 56 MW of additional heat usage. The combustion of the dried birch bark in the biomass boiler instead of the present wet bark only generates 4.5 tons per h additional steam. In order to generate the remaining 89 tons per h steam, the biomass consumption is increased by 9.0 kg s−1. The additional steam usage results in an additional power production of approximately 13.3 MW.
From an industrialization perspective, the large-scale betulin production outlined in this study could be considered viable from a mass handling perspective. However, for the actual mill the concept would be troublesome due to the high energy consumption, especially for ethanol recovery. An additional steam production of almost 90 tons per h cannot be provided without very significant investments in new boiler capacity. In order to identify a more feasible process concept the issue of the main heat consumer, the distillation reboiler, should be addressed. When, for example, the ethanol concentration from the distillation column increases from 90 to 95 wt%, the energy requirement increases from approx. 30 MW to 100 MW.
As shown above the major cost in the process is the recycling of ethanol. Extraction with a lower ethanol concentration than close to the azeotropic ethanol water mixture (95 vol%), used in this study would be clearly beneficial from an energy consumption point of view and should be tested in future work. Furthermore, a modified process configuration utilizing evaporation of the extract prior to the precipitation might be a feasible method to reduce the distillation load and should be verified in further laboratory trials. Finally, the potential for heat integration between streams within such a modified betulin process as well as the mill's secondary heat streams should be further investigated.
For the analysis methods LC-MS grade methanol and acetonitrile from Fisher Scientific (Västra Frölunda, Sweden) and water from Sigma Aldrich Chemie GmbH (Schnelldorf, Germany) were used respectively. Betulin, betulinic acid and progesterone (all ≥98% purity) were obtained from Sigma Aldrich Chemie GmbH and NaNO3 (≥99.5% purity) from Merck (Darmstadt, Germany).
The optimized RB process was selected as a model for industrial calculations for 50000 tons of birch bark. The RB method is suitable for scale-up and a number of potential process modifications have been identified that would significantly improve the feasibility for large-scale purification. Among others it could be demonstrated that ethanol concentration from the distillation column <90% results in drastically decreasing energy consumption. A further techno-economical study is planned in which process parameters and heat integration will be optimized. Extraction tests will be carried out in order to verify that betulin yield and purity can be maintained. If successful, the proposed extraction and purification process can be used as a valorization process in the forest industry.
Footnote |
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5gc00519a |
This journal is © The Royal Society of Chemistry 2016 |