EXPT 1.

COLUMN CHROMATOGRAPHY (ISOLATION OF LYCOPENE AND -CAROTENE)

Bea Lorraine S. Acosta and Izza May L. Tomas
Locker No. 82

Chemistry Department, Xavier University - Ateneo de Cagayan, Cagayan de Oro City

Date Performed: June 22, 29, 2017
Date Submitted: July 5, 2017

ABSTRACT

Lycopene is the compound responsible for the red pigment in ripe tomatoes and -carotene is
responsible for the orange pigment. The two compounds were extracted from tomato paste via solid-
liquid extraction method. Lycopene and -carotene were isolated via column chromatography. Hexane
was used to elute -carotene while 10% acetone in hexane was used for lycopene. The spectrum of
lycopene was analyzed by UV-Vis and was isomerized with iodine in hexane. The isomerized lycopene
underwent spectral analysis to determine the percentage of all-trans lycopene in the sample. The
percent all-trans lycopene in the sample before and after isomerization was reported to be -36.0% and -
36.4%.

INTRODUCTION

Lycopene is a phytochemical that is partially responsible for the red color of ripe tomatoes,
while -carotene is responsible for the orange color of tomatoes (1, 2). Lycopene and -carotene are
isomers; they both have a molecular formula of C40H56; they are classified as carotenoids. Carotenoids
are organic compounds that are responsible for the bright red, yellow and orange hues in plants (3).
They are a class pigments that are synthesized by plants, algae, and photosynthetic bacteria (4).
Chromatographic techniques have been employed to isolate such compounds from plants.
Chromatography, as a separation of method, distributes the components of the mixture or
sample between two phases: the stationary and the mobile phases (5). The mobile phase may be a gas
or liquid that can dissolve the sample while the stationary phase is fixed and may be an adsorbent
column or paper. The mobile phase, along with the components of the sample, will travel through the
immiscible stationary phase (6). The components with high affinity for or more soluble to the stationary
phase will move slower and take longer to travel than the components with high affinity for or more
soluble to the mobile phase (6, 7).
Column chromatography is a solid-liquid technique where the solid is the stationary phase and
the liquid is the mobile phase (8). This type of chromatography has the same method of separation as
the thin layer chromatography but instead of a thin layer, the stationary phase was packed on a vertical
column (9). The stationary phase or adsorbent is first selected; the most common adsorbents are silica
and alumina (10). Both silica and alumina are polar adsorbents but retain different molecules. Alumina is
basic and retains acidic compounds while silica, less polar than alumina, is acidic and retains basic
compounds (11). The mobile phase or solvent is selected based on the nature of both adsorbent and
solvent (8). The rate of separation of the components of a sample is dependent on the adsorbent
activity and polarity of the solvent (8). Generally, a nonpolar solvent passes through the column first
and then followed by a polar solvent. The nonpolar compounds will dissolve in the nonpolar solvent
while the polar compounds adsorb to the polar stationary phase. Once the nonpolar compound is
collected, a polar solvent will then pass through the column to dissolve and collect the polar compounds
(11).
Spectroscopy is the study of the interaction of all forms of electromagnetic radiation with
matter. The interaction could give rise to electron excitations, molecular vibrations, or nuclear spin
orientations (12). Absorption spectroscopy is a type of spectroscopy that refers to techniques that
measure the sample's absorbance to the radiation. An application of absorption spectroscopy is the
ultraviolet-visible spectroscopy or UV-Vis. UV-Vis refers to absorption or reflectance spectroscopy in the
ultraviolet-visible spectral region. UV-Vis uses light in the visible and adjacent ranges. The color of the
chemicals or samples involved directly affect the absorption or reflectance in the visible range (13).
The objectives of this experiment was to properly prepare the column through dry packing
method, to prepare the sample through extraction of pigments from tomato paste, to separate the
sample through column chromatography, and to analyze the spectra of lycopene and its isomer (from
isomerization with iodine).

MATERIALS AND METHOD

In preparing the column, cotton was placed at the bottom of the column. Sand was poured
using a dry funnel and the column was tapped gently so that the sand was uniform and level. 5.5 grams
of dry absorbent (alumina) was added, column was tapped again to dislodge air bubbles. When the
absorbent had settled into a compact column, 1cm layer of sand was added at the top of the absorbent
to prevent disturbance of the surface as solvent was added.
In preparation of the sample, pigments were extracted from tomato paste. 5 grams of tomato
paste and 7-ml of acetone was added to a test tube. The mixture was stirred for several minutes until it
was no longer gummy. Using a Buchner Funnel, all the mixture was filtered. Filtrate was discarded and
solid residue was returned to the test tube and 5ml of CH2Cl2 was added to effect extraction. The
mixture was shaken and filtered. The extraction was repeated with 2-3 additional 5-ml potions of CH2Cl2.
The solution was dried by adding calcium chloride pellets and then filtered to remove the pellets. The
remaining solution was placed in the hood to evaporate the solvent until about 0.5ml remains.
In separation of the sample by column chromatography, all supplies were obtained first before
chromatography had started. In separate 30-ml test tubes, 20-ml each of hexane, 10% acetone in
hexane and methylene chloride was placed. Four 30-ml test tubes and four transfer pipet and an
aspirator were needed. The concentrated tomato paste extract was placed onto the column. During
elution, column should not run dry. Using the transfer pipet, 2-ml hexane was immediately added and as
the pigment solution was drained into the sand, few drops of hexane was used to rinse, and filled the
space above the column with more hexane and eluting had started. Adding hexane continued until first
color eluted. The colorless eluate was discarded. If there was no movement of pigments on the column,
next solvent was used.
 In spectral analysis and isomerization of lycopene, the lycopene eluate was diluted with 10%
acetone in hexane and transferred to a sample cell and filled with about full of the diluted lycopene
solution. The mixture was swirled. Water was used in the reference cell. The spectrum was recorded. To
isomerize the lycopene, a drop of iodine was added into the sample cell used in the preceding step, the
cell was returned to the sample beam at 475nm, and then the absorbance was monitored.

RESULTS AND DISCCUSION

Extraction of Lycopene
The extraction method, solid-liquid extraction, was an effective method for isolating lycopene
from tomato paste. The tomato paste was first extracted with acetone to remove moisture or water
from the paste. It was then extracted 3-4 times with dichloromethane; the extracts were combined and
dried over calcium chloride pellets. The remaining extract was then left in the hood and allowed the
solvent to evaporate to concentrate the extract.

Column Chromatography
Column chromatography was the separation method used for the separation of lycopene and -
carotene from the tomato paste extract. In column chromatography, generally, the least polar solvent is
eluted first followed by the more polar solvent. In the experiment, hexane was the least polar solvent.
Acetone was more polar than hexane but when mixed together their polarity was slightly higher than
that of hexane alone. Between lycopene and -carotene, the former is more polar because it is highly
conjugated than the latter. During elution, -carotene was first eluted with hexane as the eluent,
followed by lycopene with 10% acetone in hexane as its solvent. The colorless eluates collected were
discarded.

Table 1. The results obtained from column chromatography.

Solvent Color of eluate
Hexane Clear Red orange
10% acetone in hexane Clear Yellow
CH2Cl2 Clear Light yellow

UV-Vis Spectrum and Isomerization of Lycopene

The spectrum of the collected eluates were recorded at ~400-600nm. The known absorptive
maximum (max) of lycopene is 471nm. The obtained max of lycopene was 474nm.

The lycopene present in tomato is an all-trans form of lycopene. When subjected to heat, light,
or certain chemicals, the compound is converted to its 13-cis isomer. The lycopene was isomerized with
0.025% iodine in hexane. The % all-trans-lycopene present in the sample before and after isomerization
was calculated to be -36.0% and -36.4%.
 Figure 1. Line structure of all-trans lycopene.

(a) (b)

Figure2. UV-Vis spectra of lycopene (a) before and (b) after isomerization with iodine.

Error Analysis
In the experiment, the isomerization of lycopene was not successful since it yielded negative
values of the % all-trans-lycopene. Possible sources of error were that the sample analyzed was not
lycopene; and the amount of iodine added was not enough to isomerize lycopene

CONCLUSION

This experiment aimed to properly prepare a column for column chromatography, to prepare
the samples through extraction of pigments from tomato paste, to separate the sample through column
chromatography, and to analyze the spectra of lycopene and its isomer (from isomerization with iodine).
The column was successfully prepared; the stationary phase used was alumina and the mobile phases
were hexane and 10% acetone in hexane. The samples were properly extracted by using the appropriate
extracting solvents. The spectrum of lycopene was analyzed by UV-Vis. Lycopene was isomerized with
iodine in hexane. The isomerized lycopene underwent spectral analysis to determine the percentage of
all-trans lycopene in the sample. The percent all-trans lycopene in the sample before and after
isomerization was reported to be -36.0% and -36.4%, respectively.

REFERENCES

(1) Brown, W.; Iverson, B.; Anslyn, E.; Foote, C. Organic chemistry; 7th ed.; Cengage Learning, 2013; p.
210.
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2016).

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Answers to Questions:

1. a. if excessive solvent is used when adding the sample to the column, instead of a tight, concentrated
band, a broad, diluted band will form. This could cause overlaps resulting to a less efficient separation.
b. if a cracks develop in the column, the efficiency of the separation my decrease.

2.

Ans.
4. a.

b. 13-cis lycopene is more stable because it has less steric hindrance.

5. a.

b.

6.