堆積物中のバイオ マーカーの抽出溶媒抽出を加速

Accelerated solvent extraction is a fast, efficient, and high-throughput technique used to separate organic biomarkers from large numbers of geological sediment samples.

Traditionally, extraction methods such as sonication or Soxhlet are used, however, the drawback with these protocols is that they are too slow to process enough material for detailed paleoclimate reconstruction. A newer technique, the Accelerated Solvent Extraction, or ASE method, was developed with efficiency and high throughput in mind.

The ASE method uses a combination of high temperatures and high pressure to extract samples and can carry out multiple samples in a single, relatively fast preparation run.

This video is the third in a series detailing how to extract biomarkers from sediment. It will cover the procedure and provide insight into merits of ASE over sonication or Soxhlet extraction.

In accelerated solvent extraction, samples are loaded into steel cells that are then loaded onto a carousel. Collection vials for each sample cell are also loaded onto a separate carousel. The instrument loads a sample cell into an internal oven. Solvent is pumped from a solvent bottle through a series of valves until a sufficient pressure is reached.

This pressure is held for an amount of time dictated by the sample and analyte. Then, the solvent is flushed from the sample cell through a steel line into the corresponding collection vial. The process can be repeated a number of times. The temperature, pressure, and duration can all be customized for the sample.

The high temperature used increases the kinetics of the extraction while the high pressure keeps the solvent from volatizing. The collection vial now contains a total lipid extract, and what's left in the sample cell is called a residue. It comprises non-organic material as well as organic material that is not solvent-extractable, called kerogen.

Now that we are familiar with the principles behind accelerated solvent extraction, let's take a look at how this is carried out in the laboratory.

After collecting samples of interest; freeze-dry, homogenize, and decontaminate as demonstrated in another video in this series.

Once all samples have been prepared, assemble a cell for each one to be extracted, and one extra for a blank. To do this, screw an end cap onto the cell body. Using solvent-rinsed tweezers, place a combusted glass fiber filter on top of each one. Slowly and gently press the filter down with the plunger.

Label the cell bodies by number for each sample, and label the blank separately. Place a combusted weighing tin onto a laboratory scale and tare. Rinse a spatula with solvent, then use it to transfer 5 to 10 g of sample to the weighing tin, and record the mass.

Transfer all the material in the weighing tin to its corresponding cell. Place another glass fiber filter on top, and gently tamp until it reaches the top of the sample.

Add a dispersant, such as diatomaceous earth or sand, until it is almost full, being careful to avoid getting any debris into the cell body threads. Cap the top of the cell with another end cap. Repeat these steps for each sample and the blank.

Label each collection vial with the number of a corresponding sample cell or blank, and cap with a vial cap. Place each cell into a numbered slot on the upper ASE tray. Set the parameters for the extraction method using the keypad on the ASE to extract at 100 °C and 1,200 psi. Extract each sample 3 times with a static hold of 10 min and flush the cell body with 50% of its total volume between static holds.

Next, ensure that the solvent bottle contains enough to extract all of the samples. Rinse the instrument lines 3 times before starting the run. Finally, press start.

Remove the vial from the ASE. Now that the biomarkers have been extracted, they must be purified before analysis.

Accelerated Solvent Extraction is a versatile technique, which can be utilized for a variety of applications, some of which are explored here.

Accelerated solvent extraction can also be used on other types of sample, including food. Residue analysis to test for pesticide contamination is frequently carried out in regulatory and industrial facilities to ascertain the safety and quality of food products like fruit or vegetables. ASE can be used to extract organochlorine pesticides from food samples, and determine the types or levels of residue present in the produce. This information can then be used to establish whether produce is fit for human or animal consumption. For instance, dieldrin should be found within 0 to 0.1 parts per million on food, depending on the product.

Food nutritional components can also be extracted using ASE. For example, products like chocolate, which may have high gravimetric fat content, can be extracted. Using ASE with petroleum ether as a solvent, fat can be separated from chocolate samples and subjected to quantitative analysis to determine an accurate percentage fat content per known quantity of chocolate. Using this information, regulatory bodies can verify claims made by chocolate manufacturers, or manufacturers can obtain information to create accurate food labels.

You've just watched JoVE's introduction to the extraction of lipid biomarkers using accelerated solvent extraction, or ASE. Methods for further processing and analysis can be found in subsequent videos.

Thanks for watching!