ACQUISITION OF A PREPARATIVE LC TO ENHANCE RESEARCH ON SUBCRITICAL WATER EXTRACTION OF PHYTOCHEMICALS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: NRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 0207058
• Grant No.: 2006-35503-17238
• Proposal No.: 2006-00998
• Project Start Date: Jul 15, 2006
• Project End Date: Jul 14, 2007
• Grant Year: 2006
• Program Code: [71.1]- (N/A)

Recipient Organization
UNIVERSITY OF ARKANSAS
(N/A)
FAYETTEVILLE,AR 72703

Performing Department
BIOLOGICAL & AGRICULTURAL ENGINEERING

Non Technical Summary
Phytochemical extracts are used to fortify foods such as soups and juices, improving their quality and enhancing their competitiveness in global markets. These phytochemical streams may be produced by extracting the targeted biomass with organic solvents. It is possible to replace the use of organic solvents with subcritical water (water at temperatures > 100 oC). There are numerous advantages of using water as an extraction solvent: less costly to purchase; no need to be recovered; less costly to dispose of; and, residual trace of extraction solvent in the extract is not toxic. Because the use of subcritical water poses some disadvantages, we propose acquiring a preparative liquid chromatography instrument to better characterize the process. This proposal is for the acquisition of a preparative liquid chromatography (LC) instrument (Waters PrepLCr 150 ml system). This instrument will be useful to characterize the subcritical water extraction process by examining the advantages and the pitfalls of varying extraction methods. Through this, process engineering can be designed such that pitfalls are minimized.

Animal Health Component

(N/A)

Research Effort Categories

Basic

100%

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
501	2220	2020	100%

Knowledge Area
501 - New and Improved Food Processing Technologies;

Subject Of Investigation
2220 - Medicinal crops, non-narcotic;

Field Of Science
2020 - Engineering;

Keywords

preparative high pressure liquid chromatography

water extraction

byproduct generation

toxicity

phytochemicals

Goals / Objectives
Subcritical water extraction of phytochemicals is exciting and innovative processing technology because it extracts without organic solvents. There are numerous advantages of using water: less costly to purchase; no need to be recovered; less costly to dispose of; and, residual trace of extraction solvent in the extract is not toxic. However, in developing innovative processes, one always encounters limits to the technology. Recent work is showing that with subcritical water extraction comes the generation of byproducts. This proposal is for the acquisition of a preparative liquid chromatography (LC) instrument (brand named: Waters PrepLC 150 ml system). Acquisition of the preparative LC will allow us to fractionate the peaks obtained during water extraction and characterize them in terms of their chemistry, biological activity and toxicity. Better understanding of the generated byproducts would allow for optimum subcritical water extraction process development. Improving food quality and value is essential in meeting the needs of the consumer and enhancing the competitiveness in global markets. Enriching foods with proven bioactive compounds, which often are phytochemicals, can result in the manufacturing of foods that prevent the onset of age-related illnesses, such as diabetes type II and atherosclerosis. The long term goals of this proposal are to formulate ingredients based on the knowledge of chemical interactions for better functionality of foods; develop new and improved technologies to produce better foods; and, produce foods with optimum level of health components. Blending phytochemical-rich extracts into food preparations is a way to ensure that optimum levels of desired phytochemicals are present.

Project Methods
The current research program is centered on the extraction of phytochemicals and their ability to aid in certain disease, namely atherosclerosis. Concomitantly, the possibility of phytochemical extraction using benign solvents, such as water, is also being pursued. As for any novel process, operating parameters must be fully understood before proceeding to scale-up. Recent work is showing that at water temperatures above 140 degrees C, co-product (or "byproducts") occur. These byproducts need to be characterized in terms of their chemical identity, their health benefits and their toxicity. Unfortunately, these byproducts cannot be purchased and copious quantities are needed for chemical and biological characterization. The acquisition of a preparative liquid chromatography (LC) instrument will enable such collection. With sufficient quantities of the byproducts in hand, it will then become possible to characterize them. With this information in hand, it will become possible to adjust the processing parameters to reduce the amount of byproduct generation.

Progress 07/15/06 to 07/14/07

Outputs
This equipment grant was used to acquire a preparative instrument to use for the separation and fractionation of compounds. This instrument is critical to our research program. In a nutshell, we prepare plant extracts, containing a multitude of compounds, using subcritical water technology. To obtain mg to gram quantities of these purified compounds, the crude extracts need to be separated and fractionated. Often these plant-derived compounds cannot be purchased and must be generated in-house. This is where this preparative instrument is critical as it is enabling us to generate these mg to gram quantities of the characterized compounds. Originally, our grant was written with the intention of purchasing a preparative liquid chromatography (LC) instrument (Waters PrepLC 150 ml system). Because the Waters PrepLC instrument consumes large quantities of solvent and requires expensive columns, we investigated other options. Our laboratory has chosen a Centrifugal partition chromatography (CPC) instrument as our scale-up separations tool. CPC is a unique separations tool based on the principles of countercurrent chromatography. CPC is based on the use of two immiscible liquids, and does not need a column to maintain its stationary phase. The separation mechanism is based on the different migration velocities of the phases caused by the difference in their partitioning between the two phases. A CPC consists of a series of polychlorotrifluoroethylene engraved channels that hold the liquid stationary phase. Spinning the rotor creates a centrifugal field that holds the stationary phase in place so that the mobile phase can flow through it. An indicator of how a compound is distributed between the stationary and mobile phases is the partition coefficient, K. For a high-quality separation, a K value between 0.5 and 2.5 is acceptable and a K value closest to unity is desired. The CPC system was purchased from Kromaton Technologies (Angers, France) and has a 200 mL rotor. The instrument has been purchased and installed during the summer of 2007. The CPC is assembled with a Waters 510 (1-20 mL/min) pump, a Dell Computer and Reflect Scientific UV/Vis variable wavelength detector. The CPC is currently the pivotal instrument of a PhD dissertation. Ms. Abigail Engelberth, a PhD graduate student, is using the instrument in three distinct projects. The first project is centered on the separation of flavonolignans from milk thistle (Silybum marianum) as these compounds are individually evaluated for their anti-cancer properties. The second project is devoted to the generation of mg-g quantities of saponins from mimosa (Albizia julibrissin) as these compounds show some anti-protozoa potential. The third project is aimed at purifying and characterizing hemicellulose-derived oligomers. An NSF Energy for Sustainability proposal [Characterization and quantification of hemicellulose, monomers, oligomers and by-products from xylan during biomass pretreatment], which heavily relies on the CPC, has been submitted.

Impacts
This instrument will give us the flexibility to prepare mg to gram quantities of compounds that are not usually commercially available. This instrument also serves as the work horse for our proposal [Characterization and quantification of hemicellulose, monomers, oligomers and by-products from xylan during biomass pretreatment], which has recently been submitted to NSF.

Publications

• Engelberth A, Carrier DJ, Clausen E. 2007. Separation of milk thistle (Silybum marianum) silymarins using centrifugal partition chromatography. Poster presentation at the American Society of Pharmacognosy. Portland, ME