Using Thin-Layer chromatography to

identify chlorophyll pigments in spinach

Chromatography is a technique used to separate and identify different
pigments present in leaves, such as chlorophylls and Xanthophylls. This
techbique allows scientists to understand the function of these pigments
in detail.
White powder in the chromatography paper called silicane it acts as an adsorbent of the
components of the mixture being separated differently based on their interactions with the silica
surface

Risk assessment-
Glassware breakage: Handling glassware during the chromatography process which could lead to
risk of breakage leading to cuts or wounds.

Safety measure-Lab coats, gloves and safety goggles

Method-
1)cut the select leaves into small pieces.

2) place the leaf pieces and approximately 3cm (cubed) of extraction solvent in the mortar grind
until a runny paste is formed.
3)Using a dropping pipette to measure chromatography solvent into the boiling point

4)l place a clean sheet of ordinary paper on the beaker and using gloves put the piece of
chromatography paper on this.

5) Using a pencil, draw a faint origin line across the chromatography paper approximately 1cm
from the bottom.

6) Using a pencil, draw a faint cross on the orgin line in the middle of one of the columns.

7)Hold onto the chromatography paper up against the boiling tube and mark a line where the
bung is.

8) using the capillary technique, spot the leaf extract onto the cross, ensuring that this point is
above the level of the solvent.
9)carefully pin the top of the chromatography paper to the bottom of the bung and gently lower
and paper, so it’s in the solvent at the bottom of the boiling tube nut does not touch the sides.

10)you should see the solvent front rising evenly over the origin line and on up the paper.

Chromatography separates compounds based on how they interact with the stationary and mobile
phases. The compounds that stickmore to the stationary phase tend to travek slower and don’t go
as far. The mobile phase travel faster and farther.

There are several factors which influence the separation of mixtures in chromatography which
are stationary Phase – the choice of stationary phase such as silica gel or a specific column
material affects the interactions between the compounds and the stationary phases have varying
affinities for different compounds.

Mobile phase Composition: The mobile phase including the solvent type and concentration
including the solvent type.
Here are the many errors of the experiment-
Too much propane added to extraction to dilute the spinach in the beaker. Use boiling tube to
measure how much propane to use.

The extraction drop onto the wrong end of the baseline

Used a smaller beaker for the chromatography paper . Use a large volume of beaker so the
chromatogrpahy paper would fit and create a larger volume.
Diagram (up)

Rf= distance from start to center of substance

spot/distance from start to solvent front
4.65/5.9=0.7
Use paper chromatography to identify components of ink
Method for ink chromatography-
Using a measuring cylinder measure 10cm (cubed) of solvent.

Pour solvent into chromatography beaker immediately cover it with a slide

Place a sheet of chromatography paper on a clean piece of paper and draw a small dot of ink on a
dot of ink on a strip of filter paper. Then, dip the bottom of the paper strip into a solvent into
water without letting the ink touch the solvent.

As the solvent travels up the paper, it’ll carry the ink with it separating the pigments in the ink.

Once the solvent has traveled near the top of the paper, take it out and let it dry. You’ll see
different colored bands where the pigment had separated.

After the paper strip dries you can measure the Rf Value

X4 black inks used from different companies of the inks

-waterman intense

-black cartridges

-WH smith

3 colors that make black are red, blue, and yellow

The different separation of the ink identifies different seperations

The polarity of the solvent affects how well it can dissolve the ink pigments and how quickly it
moves up the paper.

The paper type affects how quickly the solvent travels and how well it interacts with the ink's
pigments.
Errors following- Contaminated equipment, contamination of glassware (chromatography
beaker) which can affect the separation.

Uneven spotting the ink applied to the paper strip is too large and lead to distorted separation
patterns

The chromatography paper wasn’t fully dried before placing into solvent which led to effecting
accuracy of the separation.

Diagram

RF Value= Distance from start to center of substance spot/

distance from start to solvent front
Rf=5.3/6=0.8
In conclusion chromatography is a powerful technique for
separating pigments present in leaves. Both ink and leaf
chromatography offer valuability for scientific research