Experiment # 9:

Chromatography
Separation
Group 9: Olivarez, Oliveros, Osiana
 1. INTRODUCTION
- What is Chromatography?

Topics 2. EXPERIMENT 1
-DATA AND RESULTS

Covered 3. EXPERIMENT 2
-DATA AND RESULTS

4. GUIDE QUESTIONS
INTRODUCTION

What is Chromatography?
Mikhail Semenovich Tsvet
HE INVENTED CHROMATOGRAPHY IN 1906. HE WAS
A BOTANIST, AND THE INVENTION OF
CHROMATOGRAPHY HAPPENED WHILE HE WAS
WORKING IN WARSAW, POLAND, WHICH WAS AT
THE TIME PART OF THE RUSSIAN EMPIRE.

IT WAS TSVET’S PASSION FOR BOTANY AND DESIRE

TO UNDERSTAND PLANT PIGMENTS WHICH LED
HIM TO DEVISE A SEPARATION TECHNIQUE KNOWN
AS CHROMATOGRAPHY.
Chromatography
IS AN IMPORTANT BIOPHYSICAL TECHNIQUE THAT ENABLES
THE SEPARATION, IDENTIFICATION, AND PURIFICATION OF
THE COMPONENTS OF A MIXTURE FOR QUALITATIVE AND
QUANTITATIVE ANALYSIS.

IT IS A PROCESS OF SEPARATING COMPONENTS OF A

MIXTURE. IT IS BASED ON THE PRINCIPLE WHERE
MOLECULES IN MIXTURE APPLIED ONTO THE SURFACE OR
INTO THE SOLID, AND FLUID STATIONARY PHASE (STABLE
PHASE) IS SEPARATING FROM EACH OTHER WHILE MOVING
WITH THE AID OF A MOBILE PHASE.
The Four Primary Different types of
chromatography

Gas chromatography
High-performance liquid chromatography
Thin-layer chromatography
Paper chromatography
PAPER
CHROMATOGRAPHY
is an inexpensive and powerful analytical technique, which requires a piece of
paper or strips serving as an adsorbent in the stationary phase across which a
particular solution is allowed to pass.

In paper chromatography separation of component is distributed between

phases of liquid. Here, one phase of liquid is water that is held amidst the pores of
filter paper and the other liquid is the mobile phase that travels along with the
filter paper. Separation of the mixture is the result that is obtained from the
differences in the affinities towards the water and mobile phase when travelling
under capillary action between the pores of the filter paper.
Calculating the Rate of Flow:
The ratio of the rate of movement of the solute to the rate of movement of
the developing solvent is called the RF (retardation factor) value or rate of
flow. The ratio can be expressed in terms of the distances moved by each.
EXPERIMENT 1
Separation of Components from a
Mixture of Red and Blue Inks
 OBJECTIVES

1. differentiate between a substance and a mixture by

chromatographic
separation;

.
1. identify the number of components of each given mixture
by paper chromatography.

.
 MATERIALS
scissors mixture of
ruler (isopropyl alcohol and water)
pencil mixture of
empty jar with string (red and blue ink)
chromatography paper
ruler
PROCEDURE:

Separation of Components from a

Mixture of Red and Blue Inks
RESULT
GUIDE QUESTIONS
EXPERIMENT 1
Separation of Components from a Mixture of
Red and Blue Inks
GUIDE QUESTIONS
1. Is the sample spot a mixture or a
substance? Why do you say so?

A pure substance consists only of one

element or one compound. A mixture
consists of two or more different
substances, not chemically joined together.
In the experiment conducted, the sample
spot is a mixture since we mixed 2
different substances which are the red
and blue ink before putting it into the filter
paper (paper towel).
GUIDE QUESTIONS
2. What is the RF value of the spot with the
deepest color? (Show your computation)
The spot that has the deepest color is the
color blue
GUIDE QUESTIONS
3. Draw the resulting chromatogram with
the color results.
EXPERIMENT 2
Plant Pigment Chromatography
 MATERIALS
mortar and pestle chromatography paper
leaves tape
scissors mixture of
ruler (isopropyl alcohol and acetone)
pencil transparent jar or glass
PROCEDURE:

Plant Pigment Chromatography

RESULT
GUIDE QUESTIONS
EXPERIMENT 2
Plant Pigment Chromatography
GUIDE QUESTIONS
1.Illustrate the results of the chromatogram
on the diagram.
GUIDE QUESTIONS

2. Why are two solvents used in the

process?

Different pigments will be soluble in one

solvent but not another. Better separation
of pigment bands will result if a
combination of solvents is used.
GUIDE QUESTIONS

3. Justify the separation of the bands

Bands that travel the greatest distance are

more soluble in the solvent than those that
traveled less distance.
GUIDE QUESTIONS
4. What is the RF value of the spot with the
deepest color? (Show your computation)

The spot that has the deepest color is the

pigment number 4:
CONCLUSION:
Through the use of chromatographic separation, we were able
to distinguish between a substance and a mixture in this
experiment. Also, we learned how to use paper
chromatography to count the quantity of components in each
mixture. In the first experiment, we counted the components of
the two samples and the total number of colors that resulted
from them. In the second experiment, we found that the bands
that travel the largest distance are more soluble in the solvent
than the bands that travel the shortest distance.
Thank you!
REFERENCES:
https://www.ncbi.nlm.nih.gov/pmc
/articles/PMC5206469/#:~:text=C
hromatography%20is%20based%
20on%20the,aid%20of%20a%20m
obile%20phase
https://studymind.co.uk/notes/chr
omatography-and-rf-values/
https://www.ncbi.nlm.nih.gov/pmc
/articles/PMC5206469/#:~:text=C
hromatography%20is%20based%
20on%20the,aid%20of%20a%20m
obile%20phase