Lucinda Shotts

F Band Organic Chemistry

May 9, 2017

An Investigation of Various Conjugated Compounds Through Absorption

Spectroscopy

Lucinda Shotts, Liam Springer and Wiley Turner

Packer Collegiate Institute, ​170 Joralemon St, Brooklyn, NY 11201

Received by Alice. Lurain, May 9, 2017; E-mail: ​lushotts@packer.edu

Abstract
The purpose of this investigation was to extract and then separate the multiple

different molecules within spinach in order to then examine those molecules through

light spectroscopy in order to observe the relationship between conjugation and light

absorption. Spinach was ground with hexane and acetone, filtered, then pushed through

a chromatography column which help to separate the different molecule within the

spinach. The different molecules absorbed the wavelengths we thought they would. The

green chlorophylls absorbed in the red and purple wavelengths and the yellow carotene

absorbed in the blue and purple wavelength regions. We were able to connect

conjugation to absorption. The yellow carotene the less conjugated molecule adsorbed

lower wavelengths while the chlorophyll absorbed higher wavelengths.

Introduction
One might think that the origins of organic chemistry come from farming or health

but in fact it was the dye industry that started this. Vibrant colors were found in plants,

sea creatures, beetles, and other natural sources. These extracted forms were not

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super effective, they faded and weren’t as vibrant as the plants they came from. In

some sense organic chemistry was born through the discovery of synthesis of these

dyes. Dyes in general are usually conjugated compounds.

Ultraviolet-visible spectroscopy is an incredibly useful tool for studying the

structure and more specifically the conjugation of a molecule. As conjugation is more

populous within a molecule the energy is lowered within the molecule. Therefore

electrons are more likely to go into an excited state, resulting in molecules that can

absorb in the visible region. Spinach pigments are highly conjugated molecules that can

absorb in the visible region. The green pigments in spinach are called chlorophylls and

the yellow pigments are referred to as carotenoids. Many of these pigmented molecules

are used in the photosynthesis process. Among the chlorophyll group, chlorophyll a and

b are most common. They are very similar except that a methyl side-chain in chlorophyll

a is substituted with a CHO group in chlorophyll b. Carotenoids are part of the

hydrocarbon class, carotenes and their oxygenated derivatives, xanthophylls.

Conjugated bonds are double bonds alternated with single bonds. In a double

bond, the two bonds within that bond are known as sigma and pi. In the sigma bond, the

electrons are right between the atoms, in the pie bond, the electrons are located just

above and below the sigma bond that is already there. In this state, the molecule

requires less energy in order for those electrons in the pi bond to become excited. In

this delocalized state, the excited electrons absorb light causing us to perceive them as

a colored.

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 In our experiments we used thin layer chromatography. Chromatography is a

process that includes two phases: a stationary phase and a mobile phase (eluent) that

flows past the stationary. A clear example is a piece of paper in water. Paper being the

stationary phase and water being the mobile phase. If you touch a piece of paper in

water, the water travels up the paper, like a paper towel absorbs water (same idea). If

you put something on that piece of paper when it is dipped in the water, depending on

its polarity it will either travel up the paper with the water (something more polar) or stay

stationary/move less (something non polar). Different substances will act differently with

the mobile and stationary phases giving different information.

Thin Layer Chromatography or TLC is a process used to: check the purity of a

substance, separate substances in a mixture, monitor the progress of a reaction and to

identify substances. For our purposes we used for our stationary phase a thin sheet of

glass coated in a thin layer of alumina or silica and hexane/acetone solution. We put a

spots of the extracted substance at the bottom of the plate and examined how it moved

up the plates after the eluant had reached about a centimeter below the top of the plate.

The ratio of the length the spot moved from the origin over the length between the origin

(where the spots of substance are originally placed) and the solvent front (the place in

which the solvent rose to) is called a retention value or Rf Value. This technique was

used to show just how many different substances were in the spinach.

The most complex method we used in the data collection was spectroscopy.

Spectroscopy is the study of the absorption of electromagnetic radiation by matter. For

our purposes, the technique was used to determine the conjugation in different

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substance from within the spinach. Light was projected through a diluted extracted

sample and the amount of light that reached to the other side gave an indication about

the conjugation in a given sample.

Results and Discussion

TLC Plate Data

Spot placement (from highest to lowest) RF Value

1 .833

2 .523

3 .420

4 .387

5 .359

6 .327

7 .220

8 .107

Figure 1. TLC plate photo and calculated rf values.

If figure 1, it shown that there are many different molecules that are soluble in

hexane and acetone in spinach. There a multiple chlorophylls, carotenoids and more.

The separation carried out with the chromatography column didn’t separate every

different molecule but it did separate the carotenoids from the chlorophylls. Even then

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there was contamination between fractions. This TLC plate shows that the process

carried out to separate the different molecules within the spinach was good but not

great. When separating with the chromatographic tube, we were really only able to

separate the compounds into two respective groups.

Fraction numbers Solvent Wavelength (nm)

1 hexane 410.8 nm (peak #1)

659.1 nm (peak #2)

2 hexane 407.0 nm (peak #1)

659.0 nm (peak #2)

3 hexane 410.8 nm (peak #1)

659.0 nm (peak #2)

4 hexane 408.2 nm (peal #1)

407.0 nm (peak #2)

5 acetone 425.8 nm (peak #1)

470.8 nm (peak #2)

6 acetone 422.0 nm (peak #1)

662.0 nm (peak #2)

7 acetone 403.2 nm (peak #1)

440.8 nm (peak #2)

8 acetone 403.2 nm (peak #1)

440.8 nm (peak #2)
Figure 2. Collected fraction absorption values from spectroscopy.

Because fractions 1, 2, and 3 all had similar peak values (see figure 2), meaning

they all absorbed the same wavelengths it is safe to assume that they all had the same

substance in them. The fractions were green and absorbed red( 620–750 nm) and violet

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(380–450 nm) wavelengths, so it safe to assume that these fractions contained

chlorophylls. The substance reacted in a way that we had predicted, because of the

molecules conjugation it absorbed the light from the spectrophotometer. Once acetone

was added, we expected to be collecting carotene. This substance was more yellow.

Fractions 5, 7, and 8 all seemed to have carotene in them as they absorbed blue

wavelengths, from 450–495 (see figure 2). Fraction six is an outlier among these, it

absorbed light in the red region just like the chlorophylls did. It is safe to assume there

may have been some contamination in this fraction.

There were a couple of sources of error within the experiment. The first was

when carrying out the chromatography process, the tube was not held completely

vertical. It was initially tilted which may have been what caused some of the

contamination in the sixth fraction (see figure 2). In the future the tube could be held by

a ring stand apparatus insuring that it would stay completely vertical. Also when

collecting fractions, it is possible that there was contamination between each one and

we didn’t start or stop early enough when collecting certain portions. We could also just

take more fractions if we wanted to to ensure at least one without contamination.

In the future it would be interesting to use more solvents. There may be other

conjugated compounds in the spinach we don’t know about because they aren’t soluble

in hexane or acetone. It would also be interesting to explore other vibrant vegetables to

see if there are other different colored compounds that are easily extracted.

Conclusion
In this investigation, molecules from spinach were extracted and separated. They

were then examined with light spectroscopy in order to observe the relationship

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between conjugation and light absorption. Spinach was ground with hexane and

acetone, filtered, then pushed through a chromatography column to help to separate the

different molecule within the spinach. The different molecules absorbed the

wavelengths we thought they would. The green chlorophylls absorbed in the red and

purple wavelengths and the yellow carotene absorbed in the blue and purple

wavelength regions. We were able to connect conjugation to absorption. The

conjugated bonds were easily excited and therefore absorbed light.

Experimental Methods
First, 1.0 g of fresh spinach leaves were collected. The leaves were torn into

small pieces then placed in a mortar. Then, 3 mL of acetone and 3 mL of hexane were

added to the spinach in the mortar and the mixture was ground for 3–5 minutes. A

filtering pipet was then created by putting a cotton plug in a disposable Pasteur pipet.

The green solution was then filtered into a clean test tube. The solution in the test tube

was washed by adding 2 mL of saturated aqueous sodium chloride solution. A stopper

was placed on the test tube so that it could then be shaken vigorously. The stopper was

removed to vent. The stopper was then placed back on the test tube and the process

was repeated. After washing, the test tube was placed in the test tube rack to rest and

allow the mixture to separate.

Next, a filtering pipet was created, using a disposable Pasteur pipet and cotton

plug. The pipet was filled half-way with solid sodium sulfate. (Weighing paper was used

to fill the pipet similarly to funnel.) The final traces of water were removed by treating the

organic solution with a drying agent. The green organic layer was filtered into a clean

test tube. Removing the aqueous layer was avoided.A 70°C hot water bath was

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prepared in a 250 mL beaker. The temperature was monitored using a Wide-Range

Temperature Probe or thermometer. The solvent was evaporated to approximately

0.2–0.3 mL by placing the test tube in the hot water bath. A boiling stone was added to

the test tube to prevent the solution from bumping.

For thin layer chromatography, 10 mL of a 3:7 acetone/hexane solution was

prepared by mixing 3 mL of acetone with 7 mL of hexane in a small beaker. A TLC

development chamber was prepared using a 400 mL beaker and watch glasse. A piece

of filter paper was placed against the side of the beaker. The filter paper was placed to

help saturate the beaker with solvent vapors. The beaker was filled with 510 mL of 3:7

acetone/hexane solution and covered with the watch glass.The TLC plate was prepared

and handled carefully so that the absorbent didn’t flake off. Using a pencil (NOT an ink

pen), a light line was drawn across the plate, approximately 1 cm from the bottom.

Across this line, the location where the sample will be spotted was marked, making sure

it was not too close to the edge of the plate. The sample was loaded onto the plate by

dipping one end of a capillary tube into the spinach extract solution (capillary action

drew the liquid into the tube) and lightly touching the end of the tube to the mark. The

mark was then blown on to evaporate the solvent, then the end of the tube was then

lightly placed on the same spot again. The spot was (1-2 mm in diameter). Then, the

TLC plate was placed in the developing chamber and covered with the watch glass. The

solvent level wasn’t above the spot on the plate or the sample would have dissolved into

the solvent. When the solvent had risen to within 1 cm from the top of the plate, the

plate was removed from the chamber and a pencil was used to gently draw a line to

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mark the position of the final solvent front. After the plate dried, the tlc plate was

observed under a UV lamp. The spots were lightly outlide with a pencil the RF values

were calculated for each spot.

For column chromatography, a pipet column was assembled in the fume hood. A

small piece of cotton was put into the bottom of the pipet followed by 0.5 cm of sand

and enough silica to fill the pipet about 2/3 full. The side of the pipette was gently

tapped for one minute to pack the silica. Another 0.5 cm was added on top of the pipet.

An 100 mL waste beaker was placed under the pipet column 23 mL of hexane was

eluted through the column. Another 23 mL of hexane was eluted. During the process

the column was kept damp at all times, solvent was replenished as needed. The column

was loaded and the separation was carried out. Once the solvent had drained to just

above the silica, the green pigment solution was pipetted evenly onto the column. The

solution was allowed to adsorb onto the silica. A yellow band appeared that began to

separate from the green band. Hexane was continually added that allowed the solvent

to move down through the column. When the yellow pigment began exiting the column,

a fraction was collected in a clean test tube. Once the yellow fraction was collected, the

solvent was changed by adding acetone. Acetone was continually added as the green

pigment moved down column. The clear portion was collected in the waste beaker.

When the green pigment began to exit the column, a fraction was collected in a

separate clean test tube. Not all of the pigments were removed from the column.

To start the UV-vis spectroscopy, a SpectroVis Plus Spectrophotometer was

plugged into a computer, Logger Pro was opened, and “New” was chosen from the File

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menu. The Spectrophotometer was calibrated. A blank was prepared by filling a glass

cuvette with hexane. The blank cuvette was placed in the Spectrophotometer.

“Calibrate” was chosen from the “Experiment” menu of Logger Pro. When the warm up

period was complete, “Finish Calibration” was selected. Next, “OK” was selected. The

wavelengths of maximum absorbance were determined for the peaks in the yellow

solution. The blank cuvette was then emptied, the cuvette was filled about 3/4 full with

the yellow solution, and placed in the Spectrophotometer. The data collection was

initiated. A full spectrum graph of the solution was displayed. The data collected was

terminated. The wavelengths of maximum absorbance for each peak and prominent

shoulders were identified. Next, “Examine” was and from the “Analyze” menu. The

cursor was moved to find the wavelengths of each peak and its shoulders. The run was

saved, then the cuvette was removed from the Spectrophotometer and the solution was

disposed of in the organic waste container. The process was repeated to determine the

wavelengths of maximum absorbance for the peaks in the green solution. This time,

acetone was used as the blank for calibration.

References
Lurain, A. (2017, May 9. Unsaturated Hydrocarbons (Alkenes,Alkynes and Aromatics).
Lecture presented in Packer Collegiate Institute, Brooklyn.

Lurain, A. Investigating Conjugated Compounds by Absorption Spectroscopy. (2016,

2017). Retrieved April 24, 2017, from Alice Lurain.

Signature: ________________________________________ Date: _______________

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