Experiment 4

Analysis of Plant Pigments

The purpose of this experiment is to extract, separate, and identify various plant pigments.

I. Lipids

Lipids are water-insoluble substances found in cells. Lipids have several functions. They
are structural components of membranes, they store metabolic fuel, and they are protective
components of cell walls and skin. Other compounds classified as lipids include some
vitamins, pigments, and hormones.

Lipids are chemically diverse and many different classes of lipids exist, including fatty
acids, triglycerides, phospholipids, glycolipids, sphingolipids, steroids, and terpenes. Plant
pigments also have lipid-like properties. The yellow pigments (carotenes and xanthophylls)
and the green pigments (chlorophylls) are unsaturated polyalcohols and hydrocarbons.
Typical pigments found in plants include chlorophyll a, chlorophyll b, $-carotene, lutein,
neoxanthin, violaxanthin, and phaeophytin.

II. Extraction and separation of lipids

Lipids can be difficult to handle experimentally because of their insolubility in water and
because they can be labile in oxygen and light. In general, lipids can be extracted from
tissue by using organic solvents. Non-lipid contaminants can be removed from the organic
phase by washing with an aqueous salt solution. This produces a solution of lipids in
organic solvent. This solution can now be analyzed in several ways.

One good method for separating different lipids is thin-layer chromatography. TLC is very
similar to paper chromatography, except that instead of using paper, a plastic sheet coated
with a thin layer of silica, cellulose, etc. is used. After spotting the lipids at one end of the
TLC sheet, the sheet is placed in a chromatography tank with a solvent which moves up the
thin-layer by capillary action. The mobile phase consists of the non-polar solvent
components which move up the thin-layer. The stationary phase consists of the polar
solvent components that bind to the thin-layer or the solvent that was used to make the thin-
layer and is still present within the thin-layer. Lipids move with Rf values that depend
upon a particular lipids relative solubility in the mobile and stationary phases.

Another method for separating lipids is adsorption chromatography. Here a column is

packed with a solid adsorbent (alumina, silica, etc.) The lipids are added at the top of the
column, and then the column is developed by adding a solvent at the top. As the solvent
runs down the column, it carries the lipids with it. How fast a particular lipid runs down the
column depends on its relative affinity for the adsorbent vs. the solvent.

III. HPLC

An alternate method for separating lipids, including pigments, is high performance liquid
chromatography (HPLC). HPLC is a relatively new technique that is actually an extension
of traditional column liquid chromatographic methods such as adsorption chromatography
and gel filtration. In HPLC, a column is filled with a solid support, and some liquid (buffer,
organic solvent, etc.) is run through the column. Different molecules can be separated,
analyzed, and identified depending upon their interaction with the solid support and the
liquid. The advantages of HPLC (and its differences compared with traditional column
chromatography) are several. First, the solid stationary phase consists of very small
particles, creating a large amount of surface area. This results in better separations and
resolution than can be achieved with classical column chromatographic methods. Second,
the solid support can withstand very high pressures (up to 5000 psi.), and running the
column under high pressure speeds up elution and makes HPLC a rapid technique. Other
advantages include the ability to re-use the column a large number of times, improved
reproducibility between runs, and automated equipment.

The basic components of an HPLC system are shown in the link below. A solvent
reservoir holds the liquid phase. The solvent is drawn through a filter to remove any
particulate matter, and then through a pump which provides a constant, reproducible flow
of solvent through the column. An injection port is used to introduce the sample into the
column, and the column itself is generally made of stainless steel or glass-Teflon tubing,
with a diameter of 2-30 mm and a length of 5-100 cm, packed with the stationary phase.
An oven surrounding the column provides a constant temperature, if required. After the
column is a detector. Photometric detectors monitor UV or visible absorbance as
compounds elute from the column. Also used are fluorescent detectors and differential
refractometers (which monitor refractive index). The detector is connected to a recorder or
computer for data acquisition. Following the detector is a fraction collector for collecting
samples.

HPLC: User's guide (go over this link to review all concepts related to HPLC)
 Stationary phases often consist of small porous particles with an inert solid core covered
with a thin porous outer shell of silica, alumina, copolymers, or silica with bonded
substituent groups. Alternatively, microporous particles are made entirely of silica,
alumina, etc. and allow for greater resolution. The most common supports have particles of
4-10 m in size. Mobile phases must be of high purity and include a wide variety of
organic solvents, solvent mixtures, and aqueous solutions. Materials are available to run
adsorption chromatography, gel filtration, and other types of column chromatography as
HPLC, each producing separations according to the theoretical basis of the type of
chromatography being performed. Normal phase chromatography uses a polar solid
support and a nonpolar mobile phase solvent. Reversed-phase HPLC employs a non-polar
solid support with a more polar solvent and is better for polar biomolecules.

The separated compounds eluting from the column are described by their retention time
(tr), which is the time necessary for elution of a particular compound. The recorder
produces an elution profile of the compounds. If a mixture of three compounds is run on an
HPLC column, a typical profile would be that shown in the link above, where each
substances tr could be measured from its peak. Most HPLC systems are equipped with a
computer for data handling, including quantifying data by peak integration.

HPLC works best with very clean samples, so crude samples must be pre-treated to remove
salts, metal ions, detergents, and particulates. The concentration of the sample must be
properly adjusted, and conditions must be optimized, including type of support, type of
solvent, flow rate, etc. When performed correctly, HPLC is a powerful tool for
biomolecule separation. Very small samples can be analyzed or, with a bigger column,
large amounts of a biomolecule can be isolated.

IV. Procedures

A. Extraction

Two grams of spinach leaves, with the veins removed, are cut up and placed in 100 mL of
boiling water for two minutes. The water is then cooled and discarded. The leaves are then
extracted for about ten minutes with 25 mL of 90 percent methanol / 10 percent diethyl
ether. The extraction should consist of gentle swirling to avoid generating small bits of
leaves. Pour the organic mixture into a separatory funnel. Add 25 mL of 70 percent
methanol/30 percent diethyl ether to the leaves and repeat the extraction. Combine the
organic mixtures in the separatory funnel. To the extract, add 10 mL of petroleum ether and
25 mL of saturated aqueous NaCl solution. Shake to mix thoroughly and discard the lower
layer, saving the green organic phase. Wash the pigments twice with 25 mL of water. Then
place the organic phase in a small beaker and concentrate to 1 mL using a stream of
nitrogen in the dark.

B. Thin layer chromatography

Format a cellulose TLC plate using soft pencil and only the very mildest of pressure.
Make a short (0.5 - 1.0 cm) horizontal mark on both sides of the plate 1 cm up from the
bottom to mark the origin. There should be 3.0+ cm left free in the middle of the plate.

To pre-elute the plate, place 50 mL of acetone: petroleum ether (1:1, v/v) into a clean TLC
developing chamber, insert the TLC plate which has already been formatted, cover, and let
develop until the solvent front is about cm from the top. Remove the plate and gently
swing it in the air until it is dry. Touch only the top of the plate and lay it down only onto a
piece of aluminum foil. The presence of fats, oils and greases from the human body, and
other contamination, leads to smearing of the chromatogram and thus gives a poor
separation.

Load the sample onto the TLC plate using a capillary tube. Insert the capillary into the
extract and allow the green solution to rise up in the tube. Using your finger, as you do
with a pipet, stopper the tube and withdraw it from the solution. Spot the origin by
barely touching the plate in the area between the origin marks with the capillary tube.
Done properly, only a very small amount of solution will be pulled out of the tube and onto
the cellulose of the plate. It is necessary to touch the plate and remove the tube rapidly in
order to keep the size of the loading spot to a minimum. Repeat this across the plate to
form a series of overlapping spots; these will begin to resemble a small streak. Let the first
loading dry and repeat several times until your origin is relatively dark green. This loads
enough pigment onto the TLC plate such that separated components can be visualized.

To develop the chromatogram, empty the TLC chamber into an Organic Solvent waste
container and refill with 50 mL acetone: petroleum ether (1:9, v/v) as the developing
solvent. Place your TLC plate into the TLC tank, cover and let the separation develop. Try
to keep the tank and the pigments out of bright light as much as possible. Light, in the
presence of molecular oxygen (O2), degrades these pigments (photooxidation). Stop the
TLC run when the pigments have separated. Air dry the chromatogram. Draw a picture of
your TLC plate, indicating the size and color of the spots.

To recover the pigments, prepare 6 TLC spot removers using Pasteur pipets and glass
wool. Fold a small amount of glass wool, insert it into the top (large end) of the Pasteur
pipet and push down about 0.5 cm, leaving the remaining fluffy excess sticking up. Trim
off the excess, forming a small glass plug in the pipet. Push the formed plug down to the
tip constriction using a piece of wire, but do not force - just snug!

Label the 6 TLC spot removers #1-6. Prepare 6 small test tubes by putting them in a rack
and labelling them #1-6.

Begin with the dark yellow spot at the top, which will be #1. Hook up a piece of vacuum
tubing to the vacuum, insert the tip (pointed end) of a Pasteur pipet spot remover into the
vacuum tubing, and using the larger open end of the pipet scrape the pigmented material off
the TLC plate slowly. You will note that the material is vacuumed into the pipet and is
stopped by the glass wool plug. Perform this vacuuming in such a way as to collect the
majority of each pigment. Do not collect material from any overlapping areas. Invert the
pipet so that the glass wool plug is down, remove from the vacuum tubing and place (tip
down) into the correspondingly labelled test tube. Repeat for spots #2-6.

To elute the pigment from the cellulose, raise the spot remover about 2 cm up from the
bottom of the test tube and rinse the inside of the pipet twice with 1.0 mL of 100% acetone.
Discard the spot remover in the broken glass receptacle. Repeat for each of the 6 spots.

C. HPLC

The HPLC system for this experiment has the following features. First, it uses a column in
which the solid support consists of silica beads (10 m) linked to a hydrocarbon chain of 18
carbons, creating octadecylsilane (ODS). This makes the column material non-polar,
producing reversed phase HPLC. Second, the instrument employs a single pump and
therefore a single solvent system, unlike more complicated HPLC systems with multiple
pumps that can use multiple solvents and solvent gradients. The solvent used here is one
part acetonitrile: water (9:1) and one part ethyl acetate. Third, the detector is a dual
wavelength detector that can monitor absorbance of the pigments coming off the column at
two different wavelengths simultaneously. The detector is set for 410 nm (to detect the
gray phaeophytin) and 440nm (to detect the green and yellow pigments). Fourth, sample
loading is accomplished by using an autoinjector that automatically loads the sample onto
the column.

To run the pigment sample, first evaporate the sample in petroleum ether almost to dryness
under nitrogen. Add some 100% acetone and evaporate again. Dissolve pigments in 90%
aqueous acetone. Draw some of the sample into a syringe and filter through a 0.45 micron
filter. Expel the sample into a clean glass vial. Place the sample in the autosampler. With
the help of the instructor, program the HPLC to load the sample. As the column runs, data
will be collected and displayed.

V For the lab report

1. Include a one paragraph introduction.

2. Include all procedures.

3. Results - Include all data

1. Draw a picture of your chromatogram showing the pigment bands you observed. Draw
a picture of your column showing the bands you observed.

2. Include the HPLC chromatogram.

3. Include the absorbance spectra of your samples.

4. Include the known absorbance spectra of the various pigments.

D. Discussion

1. By comparing the spectrum of each of your pigment bands to the known spectra,
determine what pigment or pigments are present in each pigment band obtained in the
different techniques. Be sure to explain how you made this determination by citing
absorption peaks, band widths, color, polarity, chemical structure, etc.

2. Explain the reasons for any poor separations that occured.

3. Compare the methods as to speed, resolution, sample size, etc.

VI. Questions for the lab exam

1. Why are the spinach leaves placed in boiling water?

2. Why is the organic phase washed with a salt solution?

3. Why is the concentration done in the dark under nitrogen?

4. What will be the result if the TLC chromatogram is overloaded?

What if it is underloaded?

5. Why does HPLC give better resolution of the pigments compared to other methods?
6. Why does the pigment with the largest Rf value in TLC have the largest retention time
in reversed-phase HPLC?