Application Note. Purification of ω-3-fatty acids from fish oil using MCSGP. Summary. Countercurrent chromatography, multicolumn.

Transcription

1 Application Note Purification of ω-3-fatty acids from fish oil using MCSGP Category Matrix Method Keywords Analytes ID Countercurrent chromatography, multicolumn HPLC ω -3-fatty acids MCSGP MCSGP, countercurrent chromatography, ω-3-fatty acids, fish oil, Omacor, Lovaza TM Eicosapentaenoic acid(epa), docosahexaenoic acid(dha) VFD0128N_C_E Summary This application note describes the purification of ω-3-fatty acids made from fish oil using multicolumn countercurrent solvent gradient purification (MCSGP). The KNAUER Contichrom system offered in a cooperation of KNAUER and ChromaCon is the only available system on the market that can be used in MCSGP mode for difficult separations. MCSGP is a countercurrent chromatographic process suited for three-fraction chromatographic separations and able to perform linear gradients. 1 Two columns are used in the process which is superior to batch chromatography in terms of buffer consumption, yield, purity, and productivity due to the countercurrent movement of the liquid and the solid phase. The main ω-3-fatty acids in fish oil are eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). 2 Their chromatographic separation is very challenging since the components correlate in their molecular structures and therefore have similar adsorptive properties. Using preparative batch chromatography they elute very closely and overlap at least partially leading to very low purities and/or yields for the fatty acid of interest. This trade-off between yield and purity can be resolved by MCSGP.

2 Introduction ω-3-fatty polyunsaturated acids have become increasingly popular in dietary supplements and the global demand for highly purity ω-3-fatty acids in clinical applications has also been rising rapidly in the last years. 2 Especially the consumption of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) has been shown to reduce the risk of cardiovascular disease. 3 The starting substance for ω-fatty acids from fish oil is the muscle oil of fatty fish species. In natural fish oil ω-3-fatty acids are existent as triglycerides that are isolated by distillation. From these triglycerides ω-3-fatty acids are usually prepared as ethyl esters by esterification and subsequent physicochemical purification processes including short-path distillation, urea fractionation, and preparative chromatography. 4 Traditional methods for isolating fatty acids, however, generally require multiple, cumbersome steps to obtain high purity products. 2 In this application note the purification of esterified EPA (EPA-EE) from pretreated fish oil is presented. The main impurity is esterified DHA (DHA-EE). Figure 1 shows the molecule structure of free EPA and DHA. Both acids have their first double bond at the 3 rd carbon atom identifying them as ω-3-fatty acids. There are only minor differences in structure making a chromatographic separation difficult. By using MCSGP such complicated separation problems can be resolved in a single process step only. Eicosapentaenoic acid (EPA): Docosahexaenoic acid (DHA): Fig. 1: Molecular structure of EPA and DHA Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), the major fatty acids in fish oil. Experimental sample preparation Preparative method for transfer to MCSGP As feed, the pretreated esterified Ethyl all cis-5, 8, 11, 14, 17-eicosapentaenoate (stabilized with Tocopherols) from fish oil (TCI Deutschland GmbH) was used. The feed purity is > 65%. Prior to use, the oil was diluted with pure ethanol (gradient grade) in a ratio 1:4 and filtered through a 0.22 µm syringe filter. Before starting a MCSGP process, a reference batch chromatography run under reversedphase conditions has to be performed. As food grade solvents are needed for the purification only the use of ethanol can be considered for the chromatography step. 5. The following conditions were used: Column Eurospher II C18 H, 120 x 8 mm Buffer A 75 % Ethanol, 25 % Water % acetic acid Buffer B 100 % Ethanol % acetic acid Gradient Time [min] % A % B Flow rate 2 ml/min Injection volume 100 µl Column temperature Detection Run time ambient UV at 215 nm 29 min VFD0128N_C_E Page 2 of 7

3 Fig. 2: Batch chromatogram of the feed mixture The main peak (1) is the target compound, the esterified eicosapentaenoic acid (EPA-EE). The main impurity (2) is the esterified docosahexaenoic acid (DHA-EE). No baseline separation between the EPA-EE and the DHA-EE is achieved. For practical purposes such a separation means a purity-yield trade-off. The target compound can be purified either with high purity (elution window on the left side) leading to a low yield, as the polluted product in the side-fractions is wasted. If the elution window on the right side is chosen, the target compound is recovered with high yield but with relatively low purity, as there is some polluted product in the overlapping areas of the peaks. Fig. 3: Yield-purity trade-off for difficult separations Difficult separations with no clear baseline separation typically lead to a trade-off between yield and purity. Highly purified products can only be found in a small elution window (left graph). A wider elution window (right graph) results in a higher yield but also leads to less purity, since the product is polluted with impurities from the side fractions (neighbouring peaks). VFD0128N_C_E Page 3 of 7

4 Therefore, the generic problem in the chromatographic purification of chemically similar compounds without baseline separation can be simplified as shown in the chromatogram in Figure 4. The chromatogram can be divided into five fractions: 1= weak adsorbing impurities 2= product contaminated by weak adsorbing impurities 3= product 4= product contaminated with strong adsorbing impurities 5= strong adsorbing impurities. The aim of an ideal purification process is to collect fraction 3, to drain fractions 1 and 5, and to recover the product contained in fractions 2 and 4. Fig. 4: Principle of the MCSGP Process Schematic chromatogram of the generic chromatographic purification problem: 1: weak adsorbing, early eluting impurities; 2 and 4: recycling fractions; 3: product; 5: strong adsorbing, late eluting impurities. VFD0128N_C_E Page 4 of 7

5 Automatic transfer from Batch to MCSGP The definition of the fractions 1 5 and therefore the operating parameters of the MCSGP process are based on the preparative batch chromatogram in Figure 2. For the MCSGP process the same columns are used which are used for the batch experiments. The transfer from the results obtained in a batch process to a MCSGP method can be carried out automatically using the Chrom IQ operating software of the Contichrom system. For the MCSGP process the two columns are interconnected to recycle fractions 2 and 4 from one column to the other. They switch to batch mode to elute the impurity fractions 1 and 5 and the pure product fraction 3. MCSGP is a semi-continuous process. During one MCSGP cycle primarily the first column is loaded with feed and eluted under the same conditions as defined for the batch run in Figure 1. In the meantime the second column is cleaned, regenerated, and loaded with the recycling fractions from the first column. Then the second column is loaded with feed and eluted. In the meantime the first column is cleaned, regenerated, and loaded with the recycling fractions from the first column. Figure 5 shows the chromatogram for ten serial MCSGP cycles for one of the two columns. During the run, the defined waste and product areas are eluted and the overlapping areas are recycled in the process. Because of the recycling of the overlapping areas for the first 2 4 cycles a slight offset of the concentration profile can be observed. After that time, a stationary concentration profile of the process is reached inside. The purified product can be continuously withdrawn from the process. To learn more about the MCSGP principle and the method transfer from a batch run to a MCSGP process please click here: systems/prep_smb/brochures/how_to_convert_batch_into_mcsgp_knauer.pdf Fig. 5: Chromatograms of the first ten cycles of the MCSGP process for the first column. During one MCSGP cycle the two columns are loaded one after another with feed and the recycling fractions and are cleaned and regenerated. The purified product was analyzed offline using an AZURA compact HPLC system. The following conditions were used for the analytical HPLC: Column Buffer A Gradient Flow rate Eurospher II C18 H, 120 x 8 mm 87 % Methanol, 13 % Water % acetic acid isocratic 4 ml/min Injection volume 10 µl Column temperature Detection Run time ambient UV at 215 nm 50 min VFD0128N_C_E Page 5 of 7

6 Figure 6 shows the analytical chromatogram of the product fraction compared to that of the unpurified feed mixture. A clear purification effect can be seen. Only the EPA-EE peak is found in the product fraction. Fig. 6: Chromatograms of the unpurified feed and the purified product The unpurified feed compared to the purified product with a single EPA-EE peak. Conclusion The Contichrom system in MCSGP mode can be used to purify esterified eicosapentaenoic acid made from fish oil under reversed-phase conditions. Preparative batch conditions led to a poor separation only. Using MCSGP for such difficult purifications the target compounds can be separated with high yield and purity making the process much more efficient than normal batch chromatography. References 1. Müller-Späth, T., Aumann, L., Melter, L., Ströhlein, G., Morbidelli, M.: Biotechnology and Bioengineering, Vol. 100(6), (2008). 2. Müller-Späth, T., Ulmer, N., Aumann, L., Ströhlein, G., Bavand, M., Hendriks, L., de Kruif, J., Throsby, M., Bakker X., BioProcess International, Vol. 11(5), (2013). 3. Wang, C., Harris, W.S., Chung, M., Lichtenstein, A.H., Balk, E.M., Kupelnich, B., Jordan, H.S., Lau, J.: Am. J. Clin. Nutr., Vol. 84, 5-17 (2006). 4. Breivik, H., Haraldsson, G., Kristinsson, B.: Journal of the American Oil Chemists Society, Vol. 74(11), Krzynowek, J., D Entremont, L., Panunzio, L.J., Maney, R.S.: SST, 12th Annual Confrence, (1987). Authors Dr. Friederike Sander, Columns and Applications Department, KNAUER. VFD0128N_C_E Page 6 of 7

7 Physical properties of recommended columns Stationary phase Eurospher II C18 H USP code L1 Pore size 100 Å Pore volume 0.8 ml/g Specific surface area 320 m 2 /g Particle size 10 µm Form spherical Surface area 320 m 2 /g % C 17 Endcapping yes Dimensions 120 x 8 mm Order number 11GE185E2N Recommended instrumentation The purifcation was performed on the KNAUER Contichrom system in MCSGP mode. Description Order No. Contichrom Lab-10 System C Contichrom Prep-100 System C The analytical testing of the purified product was performed on an isocratic AZURA HPLC system equipped with degasser, manual injection valve, and UV detector. The AZURA line of instruments has been designed to provide reliable and highly adaptable HPLC solutions. Its extensibility makes AZURA HPLC a future-proof investment. Description ASM 2.1L with 2 channel degasser, pump 4.1 S unit with pressure transducer and 10 ml pump head, UVD 2.1S variable single wavelength UV/VIS detector AZURA Eluent Tray E 2.1L for up to 6x1000 ml bottles Analytical flow cell UV, 10 mm path length, 10 µl volume, 1.1 mm ID, 1/16", stainless steel Manual 6-port/3-channel injection valve, stainless steel, 1/16 including accessories PC with Chromatography software Order No. AYIABACA AZC00 A4061XB A1357 upon request Contact information Wissenschaftliche Gerätebau Tel: Dr. Ing. Herbert Knauer GmbH Fax: Hegauer Weg 38 info@knauer.net Berlin, Germany Internet: ChromaCon AG Tel: Technoparkstraße 1 sales@chromacon.ch 8005 Zürich, Switzerland Internet: VFD0128N_C_E Page 7 of 7