Resumo do Pôster:
Many phenolic compounds found in plants have been shown to exhibit analgesic, antimicrobial, anti-inflammatory, antioxidant, antipyretic, and immuno-modulatory properties. As scavengers of reactive oxygen species, these compounds have shown promise as therapeutic drugs in the treatment and management of cardiovascular diseases, neurodegenerative diseases, and cancer. Rapid and simple isolation of these bioactive compounds present in natural products is critical to the advancement of new medical treatments and therapies1. Vanillin, known best as the principal flavor component of vanilla extract, has also been shown to exhibit some of these bioactive properties2. Using a combination of Flash and prep HPLC capabilities of the same system, vanillin was successfully isolated from vanilla extract.
To provide an efficient method for purifying vanillin from all-natural vanilla extract.
Vanillin purification was achieved using a two-step process, combining the flash and prep capabilities of the same instrument. With the system first configured for flash injection, 1 mL of all-natural vanilla extract was injected onto a 12 g SiliaSep flash column (Silicyle). Using a 25 mL/min water-methanol step gradient, the vanillin-containing fraction was collected by threshold using Abs254 settings, with additional wavelength monitoring available with the four channel diode array detector. The vanillin-containing fraction was concentrated for a single prep injection and the system was easily reconfigured for prep chromatography with a single valve switch. Using a 5 mL full-loop injection on the prep side of the PLC system, the sample was further purified by injection onto a C18 prep HPLC column (Phenomenex Luna 5u C18(2), 100 A, Axia-packed). The vanillin-containing fraction was eluted at ~5 min using a 20 mL/min water-methanol gradient. Peak purity was subsequently confirmed by TLC using a water/methanol development system.
4. Results and Discussion
Vanillin was successfully isolated from vanilla extract through a combination of the flash and prep HPLC on the same platform. Flash chromatography was successful in cleaning up the extract sample prior to injection on the preparative HPLC column. A vanillin-containing fraction was collected approximately 1.2 minutes after injection, leaving pigmented impurities bound to the flash silica column. The collected fraction was then concentrated for injection onto the prep column. With a simple valve switch, the concentrated flash fraction was further purified on the prep HPLC column; the identity of the vanillin-containing fraction was verified by TLC. The use of both flash and prep HPLC provides crude clean-up and more refined purification on the same instrument, saving time and money. The initial flash chromatography step provided crude clean-up of the sample, removing impurities before subsequent injection on a prep HPLC column. This combination of flash and prep HPLC enabled rapid purification of a much greater sample volume that could be achieved by prep HPLC alone, with the initial bulk flash purification protecting the prep HPLC column integrity upon subsequent injection. Full-loop injection of the concentrated collected flash fraction provided additional sample purification using the prep HPLC system configuration. The four wavelength capability of the diode array detector provided thorough monitoring of the purification process.
Vanillin isolation from vanilla extract was easily achieved using the combination of the flash and prep HPLC offered by the same platform used. The ability to change between these two purification modes with a single valve switch enables rapid and easy utilization of both configurations individually or in combination with one another, saving time, money, and valuable laboratory space, while increasing potential sample recovery. With the single platform combination of flash and prep HPLC capabilities, natural product purification is a quick and easy process, able to achieve the combination of bulk purification provided by flash and the refined purification possible with prep HPLC.
- Mradu, G., Saumyakanti, S.; Sohini, M.; Arup, M. International Journal of Pharmacognosy and Phytochemical Research 2012; 4(3); 162-167.
- Kuma, R.; Sharma, P.K.; Mishra, P.S. International Journal of PharmTech Research 2012; 4(1); 266-279.