Nicholas Hoyt

Organic Chemistry

10/19/17

Isolation and Purification of Piperine

Food is quite literally the spice of life. But what makes food taste like food? Is it in the

way it is prepared, or is it something else? Akin to most of the things in and out of the culinary

realm, the basis of taste lies within the chemical makeup. In this lab, we carried out an

experiment that outlined the isolation of piperine, the natural molecule that gives black pepper its

distinctive taste. This experiment delves into the process of isolating a natural compound from its

constituents, as this process has many real-life applications. As noted in our lab manual, the

benefit of carrying out such a procedure is such that many medicinally-valuable molecules are

derived and studied with this methodology; such as Taxol, a natural compound first isolated from

the pacific yew tree’s bark. Through the thorough study of Taxol, it has been found to be one of

the most effective treatments for several types of cancer. Such methods of isolating a compound

from its source include liquid chromatography, which was the method that was used in this

experiment.

The purpose of this lab is to introduce and familiarize ourselves with the practice of

isolating a specific molecule from the rest of its natural compound. To do this, we had to

determine if piperine was a polar or nonpolar molecule (See Figures 1a and 1b below) to choose

a solvent that would draw out the piperine from the black pepper. With the determination of

piperine’s polarity, my hypothesis was that due to the two polar ends of the molecule and the

non-polar chain in the middle, a solution containing polar and non-polar molecules would be the
ideal solution to draw out and to purify the piperine from its crude form to a pure substance, as it

would draw upon the respective polar and non-polar regions.

Polar

N
N
O
O
O Polar
O
O

O Non-polar

Figure 1a: Piperine Structure Figure 1b: Polarity regions of piperine

With that in mind, this experiment included two types of analytical techniques and one

purification technique: thin-layer chromatography analysis (TLC), melting point analysis, and

purification via liquid chromatography. TLC analysis was to confirm which eluents to use for the

liquid chromatography to separate the piperine from the impurities, in which the Rf value should

be around 0.3. The eluents consisted of a mix of two of three of the following compounds:

hexanes (hex), dichloromethane (DCM), and ethyl acetate (EtoAc) (See Figures 2a, 2b, and 2c

below). The reason for the testing of this is to see which ratios of the three compounds produced

the best eluent that was close to the preset Rf value, eventually to be used for the liquid

chromatography purification process. Liquid chromatography is like TLC analysis in which you

separate out the piperine and its impurities, but on a larger scale. In liquid chromatography you

load a column with silica gel, eluent, and crude piperine solution with guaiazulene (blue dye).

After loading a silica gel column with crude piperine/dye solution, you add more eluent and

collect fractions of the liquid coming out of the column in small test tubes.
 Cl
O

H
Cl
O H

Figure 2a: Hexane Figure 2b: Ethyl acetate Figure 2c:

Dichloromethane

Results:

Table 1: Masses of starting material, theoretical yield of piperine, experimental yield of piperine,

melting points, and percent yield. Calculations can be found on page 10 & 12 of lab notebook.

Compound Mass (g) Theoretical Experimental Percent Melting

Yield of Yield of Yield of Point (°C)

Piperine (g) Piperine(g) Piperine

Black pepper 30.071 1.50 --- --- ---
Crude Piperine --- --- .498 ± 33% 98-125
Pure Piperine --- --- .0542 ± 3.76% 114-129
 Table 2: Rf values from pooled class data from TLC analysis (EtoAc, D

25% 50% 75% 25% 50% 75% 25% EtoAc 50% EtoAc 75% EtoAc
EtoAc, EtoAc EtoAc DCM DCM DCM 75% DCM 50% DCM 25% DCM
75% Hex 50% Hex 25% Hex 75% Hex 50% Hex 25% Hex
0.15 0.4 0.67 0 0.02 0.1 0.6 --- 0.679

0.15 0.4 0.7 0 --- 0.1 --- --- 0.70

0.1 0.34 0.71 --- --- 0.143 --- --- 0.5

0.1 0.47 0.5 --- --- 0.14 --- --- ---

0.131 0.33 0.725 --- --- 0.17 --- --- ---

--- 0.34 0.7125 --- --- --- --- --- ---

Figure 3: TLC plates of collected fractions from liquid chromatography w/ Rf values

Rf = 0.40 Rf = 0.40 Rf = 0.40

C 1 3 5 7 9 11 13 15 17 19 21 23 25

*All the spots eluted to the same position, which is why only one Rf value is shown on the plates

Discussion:

As stated before, my hypothesis was that a solution containing polar and non-polar

molecules would be the best solution to isolate and purify piperine by attracting its polar and

non-polar regions. As seen in Table 2, the best solution consisted of half polar, half non-polar
molecules to draw piperine away from its impurities. It was at this point that I realized that my

hypothesis was incorrect in that the non-polar constituents of the eluent would also attract the

non-polar regions of the piperine molecule (see Table 2). Aside from that discrepancy, it was the

50% EtoAc. 50% hexane solution that got the most consistent Rf values around 0.3, as set by the

professor. The choice of a solution consisting of half EtoAc-half hexane is also supported by the

structures of the molecules themselves (see post lab #1, pg. 16), and the reason why the

combination of these two molecules work is because of how the polar solution pulls on the

piperine and its constituent polar regions. Although, the mixture ratios of the EtoAc and hexane

solutions is a huge factor in how the liquid chromatography. Due to piperine’s being a polar

molecule, having too much of a polar-based eluent would have attracted the piperine too fast,

and would’ve caused some issues in the collection of the pure piperine (post lab #3, pg. 16).

Now, while the eluent predicted and chosen were accurate, the actual purification of the

piperine resulted in a very low yield. Despite the low yield, the piperine that we did extract was

pure. This was seen in the melting point analysis. Looking at Table 1, the melting points of the

crude piperine vs. the pure piperine differed by almost 20°C, meaning that the piperine we did

recover was separated from its impurities through liquid chromatography. Another few other

possibilities as to the low yield could have come from three steps in the experiment. The first

being the initial separation of the piperine from the ground black pepper. When pouring out the

crude piperine solution, much was left behind in the round bottom flask as it clung to the black

pepper. The second is that when conducting the liquid chromatography, the second round of

eluent was not the EtoAc-hexane solution, but the dichloromethane solution.

This caused a portion of the crude piperine solution to remain on top of the silica column

since the non-polar dichloromethane had little pull on piperine. I posit that the top, observed with
a yellow tinge is where some of the piperine resided. The third is something not written in the lab

notebook, which was that during the fraction collecting, we discarded tubes with the

dichloromethane solution as we thought that there would be no piperine present in said fractions.

If there was indeed piperine in the fractions discarded, that would account for the good portion of

piperine lost, and explains the irregular spacing in spots and fractions as seen in Figure 3 above.

Conclusion:

In summary, the correct eluent was chosen despite a slight discrepancy in my hypothesis,

and regardless of the low yield during the initial isolation of piperine and the low yield during

liquid chromatography portion of the experiment, the piperine collected was ultimately separated

from some of its impurities as seen in the TLC and melting point analyses. To improve the

results of this experiment, a stricter adherence and closer look to the procedure given in the lab

manual would be beneficial to a greater yield in piperine.