Isolation Cholesterol from Gallstones via Extraction and Recrystallization

I. Introduction

A. Objective

The purpose of this experiment is to isolate cholesterol from gallstones via the techniques of extraction and recrystallization. The gallstones will first be dissolved in 2-butanone heated in a hot sand bath. The solution will then be transferred to a micro column using a pipet to filter out the bilirubin, which is the primary impurity of gallstones. The remaining 2-butanone will be removed and the crude cholesterol left over will be recrystallized by dissolving it with methanol and centrifuging it in a Craig tube.

B. Materials and Safety

C. Experimental Procedure

A sample of crushed gallstones weighing about 100 mg will be obtained and weighed. The crushed gallstones will then be placed in a 10 x 100 nm reaction tube along with a boiling stick and 1.5 mL of 2-butanone. This mixture will be gently heated in a hot sand bath. In a second reaction tube also containing a boiling stick, 1.0 mL of 2-butanone will be heated until it is boiling. When the gallstones have disintegrated and the cholesterol has dissolved in the first reaction tube, it will be filtered through a micro column. The micro column will be prepared from a Pasteur pipet packed with a loose wad of cotton, 2 mm of sand, 3-4 mm of anhydrous magnesium sulfate, 1 cm of anhydrous sodium sulfate, 1 cm of Norit RO 0.8 activated carbon pellets, and a very small piece of cotton. The micro column will be clamped in a vertical position and will have a weighed and cleaned 10 x 100 mm reaction tube beneath it.

The solution in the original reaction tube will be transferred into the micro column using a warm Pasteur pipet, which is warmed by immersing it in the hot vapors of the boiling 2-butanone. The 1.0 mL of boiling 2-butanone will be used to wash out the original reaction tube and pipet into the micro column. The filtered hot solution of 2-butanone solution will then be warmed in a sand bath and the 2-butanone will be removed under a gentle stream of nitrogen. The crude cholesterol will be scraped from the reaction tube onto a previously tared piece of a creased, glazed weighing paper. After obtaining the weight of the crude cholesterol, it will be placed into a Craig tube. To recrystallize the cholesterol, it will be first dissolved in the minimum amount of hot methanol. The solution will then be allowed to slowly cool to room temperature, and it will then be cooled in an ice bath. The crystalline cholesterol will be isolated via centrifugation. The cholesterol will then be allowed to air dry. It will then be weighed and its melting point will be determined.

II. Experiment and Results

A. Data

First, a sample of crushed gallstones weighing 0.131 g was obtained. Next, a micro column was prepared using a Pasteur pipet packed with a loose wad of cotton, about 2 mm of sand, about 3 to 4 mm of anhydrous magnesium sulfate, about 1 cm of anhydrous sodium sulfate, about 1 cm of Norit RO 0.8 activated carbon pellets, and a small piece of cotton. One prepared, the micro column was clamped vertically on a ring stand. The crushed gallstones were then placed in a clean glass centrifuge tube along with a boiling stick and 1.5 mL of 2-butanone. The solution was gently heated in a sand bath, along with a separate centrifuge tube containing about 1.0 mL of 2-butanone. One the gallstones dissolved, the solution was transferred to the micro column using a warm pipet. The pipet was warmed by immersing it into the hot vapors of the second tube containing boiling 2-butanone. The reaction tube was rinsed with the hot 2-butanone and the remaining solution was filtered in the micro column.

The filtrate was collected in a clean centrifuge tube. The 2-butanone was removed from this tube under a stream of nitrogen and the remaining cholesterol was allowed to air dry for a week. A minimum amount of methanol was added to the cholesterol and the tube was heated in a sand bath along with a boiling stick until the cholesterol dissolved. The solution was then allowed to cool to room temperature and was then put into an ice bath to cool more. The crystalline cholesterol formed was isolated via centrifugation. The crystals were removed from the tube using a spatula onto filter paper and the crystals were allowed to air dry for a week.

III. Conclusions

Without knowing my final weight and melting point of the crystalline cholesterol, I can only discuss possible sources of error during the procedure. When preparing the micro column, if the wrong amount of any of the materials was added, the bilirubin may not have filtered out. This make the amount of cholesterol recovered be less than expected because the cholesterol may not have crystallized if the bilirubin was still in the solution. If the filtered solution was cooled too fast, that may have prevented the formation of crystalline cholesterol, too.