CHM_____ Organic Chemistry __ Laboratory

___ Quarter SY__________

Chromatography

Domingo, Gerald C.1

Student (s), Subject/Section, School of Chemical, Biological and Material Engineering and Science, Mapua University

ABSTRACT

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statement to introduce your report. State your main purpose or objective and hypothesis. Describe the important points of your
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measurements). Summarize the main results numerically and qualitatively (include standard errors and p values as required).
Summarize the major points from the discussion/conclusion. Focus on the points that directly relate to your
hypothesis/question. For each type of information, use the same tense as in each corresponding section (i.e., past tense for
methods and results, present tense for theory and conclusions).

Keywords: albumin, casein, invertase, Bradford Assay, Warburg-Christian Assay, Benedict’s reagent

INTRODUCTION

Chromatography is an analytical technique commonly used
in separating a mixture into its individual component.
The mixture is dissolved in a fluid called the mobile phase,
which carries it through a structure holding another material
called the stationary phase. The various constituents of the
mixture travel at different speeds, causing them to
separate. The separation is based on differential
partitioning between the mobile and stationary phases.
Subtle differences in a compound's partition coefficient
result in differential retention on the stationary phase and
thus affect the separation. (McMurry, 2011).
Retention factor is defined as the distance a liquid
compound travels up a chromatography plate. The
chromatography paper is the stationary phase and the
liquid compound is the mobile phase; the liquid carries the
sample solutions along the paper. When a liquid travels up
the paper, it separates, allowing it to analyze the different
components of the liquid solution. All compounds have a
specific retention factor value for every specific solvent, and
retention factor values are used to compare unknown
samples with known compounds.
Moreover, retention factor is affected by the adsorption and
solubility of the analyte in relation with the stationary phase.
Differential affinities (strength of adhesion) of the various
components of the analyte towards the stationary and
mobile phase results in the differential separation of the

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 CHM_____ Organic Chemistry __ Laboratory
___ Quarter SY__________
components. Affinity, in turn, is dictated by two properties of
the molecule: ‘Adsorption’ and ‘Solubility’. (Khan Academy,
n.d.)
We can define adsorption as the property of how well a
component of the mixture sticks to the stationary phase,
while solubility is the property of how well a component of
the mixture dissolves in the mobile phase. Therefore,
● Higher the adsorption to the stationary phase, the
slower the molecules will move through the
column.
● Higher solubility in the mobile phase, the faster the
molecules will move through the column.
Adsorption and solubility of a certain molecule can be
manipulated by choosing the appropriate stationary phase
and mobile phase.
For paper chromatography, substances are distributed
between a stationary phase and a mobile phase. The
stationary phase is the water trapped between the cellulose
fibers of the paper. The mobile phase is a developing
solution that travels up the stationary phase, carrying the
samples with it. Components of the sample will separate
readily according to how strongly they adsorb onto the
stationary phase versus how readily they dissolve in the
mobile phase. (Wikipedia, 2019)
On the other hand, chromatography may be preparative or
analytical. The purpose of preparative chromatography is to
separate the components of a mixture for later use, and is
thus a form of purification. Analytical chromatography is
done normally with smaller amounts of material and is for
establishing the presence or measuring the relative
proportions of analytes in a mixture. The two are not
mutually exclusive.( Hos tettmann, 1998)
Term Definition
Mobile solvent moving through the column
phase
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Stationary substance that stays fixed inside the
phase column
Eluent fluid entering the column
Eluate fluid exiting the column (that is collected in
flasks)
Elution the process of washing out a compound
through a column using a suitable solvent
Analyte mixture whose individual components
must be separated and analyzed
Table 1. Terminologies associated with chromatography
For further knowledge, the figure above is given to show the
different terminologies associated with chromatography.
MATERIALS AND METHODS
This experiment (focuses on paper chromatography) focuses
on two matters: the stationary phase ; and the mobile phase.
The stationary phase is a chromatogram paper (8 x 22 cm)
divided equally into ten, 2 cm each of length. Each part
signifies a chemical subjected for experiment (methyl orange,
fluorescein, erythrosine, methylene blue and indigo carmine).
Refer to Figure 1 for the division of the chemicals at
chromatogram paper.
Figure 1. Orientation of the chemicals in chromatography
paper.
For mobile phase, prepare a 600 mL beaker with 5 mL of 5%
ethanol. Place the chromatography paper at the beaker
submerging the bottom part (about 3 cm) of it into the liquid
solution. Note that the spots of chemicals in the chromatogram
paper should not touch the liquid solution upon submerging.
Cover the beaker.
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 CHM_____ Organic Chemistry __ Laboratory
___ Quarter SY__________

Afterwards, observe the liquid solution until it reaches the top (

leave 1 cm space before it reaches the top).

RESULTS AND DISCUSSIONS

current theory.

CONCLUSIONS AND RECOMMENDATIONS

recommendations.

REFERENCES

 McMurry, John (2011). Organic chemistry:

with biological applications (2nd ed.). Belmont,
CA: Brooks/Cole. P. 395. ISBN
9780495391470.
 Hostettmann, K; Marston, A; Hostettmann, M
(1998). Preparative Chromatography
Techniques Applications in Natural Product
Isolation (Second ed.). Berlin, Heidelberg:
Springer Berlin Heidelberg. P. 50. ISBN
9783662036310.
 Wikipedia. (2019, September 12). Paper
chromatography. Retrieved October 7, 2019,
from
https://en.wikipedia.org/wiki/Paper_chromatog
raphy#Rƒ_value,_solutes,_and_solvents.

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