Volume 2. Issue 4.

Received: November 10, 2015

Published: November 25, 2015

Rf Value Determination through Chromatography

MARTINEZ, CHELSEA M.1, *, LEGARDE, JASMINE MARJORIE C.2, DERRAMAS, MICAELA ANGELA
T.3, CRUZ, KATHLIA D.4, *

Abstract: Paper chromatography is utilized to determine the components of a sample which are
separated based on their solubility in a particular solvent, hence, based on how far they travel along the
chromatography paper. This experiment aims to separate organic compounds, determine their R f values
and use these to identify an unknown sample. In the first part of the experiment, a common
chromatogram paper was used. Different spots of substances were applied to the paper and then rolled. It
was then placed in a beaker containing sufficient amount of solvent to allow chromatogram to develop. In
this way, the Rf values of the substances were determined. In the second part of the experiment, amino
acids were subjected. This time, TLC plate was used. Same principles worked on this part. Different
unknown samples were identified based on the obtained R f values. From this part of the experiment, it can
be inferred that compounds with low polarity have higher R f values than compounds with high polarity. It
can be concluded that the objectives of this experiment were efficiently met.
Keywords: Chromatography, TLC, Rf value, Ninhydrin, polarity

Student, CHM142L, School of ChE-CHM, Mapua Institue of Technology, Muralla St.,

Intramuros, Manila
4
Professor, School of ChE-CHM, Mapua Institue of Technology, Muralla St., Intramuros, Manila
*Corresponding author: cmmartinez@mymail.mapua.edu.ph
1

Organic Chemistry Laboratory Report

Introduction

composed of cellulose. This cellulose is in which

polar molecules coming from the water undergo

Separating mixtures of substances into their

respective components can be done using
chromatography. There are various forms of
chromatography, but they entirely work on a

typically comprising of a mixture of an organic

compound, liquid in form, and water.
The paper normally used in the process
comprises

similar principle.
These forms all possess a stationary phase and
a mobile phase. The stationary phase involves
most likely a solid form or a liquid sustained on a
solid. The mobile phase on the other hand
involves a gas form or a liquid form. Along the
stationary phase, flows the mobile phase which
then conveys the mixture components with it.
Each component of a mixture has varying travel

Cellulose

of

extremely
is

refined

considered

cellulose.
as

homopolysaccharide of glucose. It contains

numerous thousands of anhydro-glucose which
are units-linked through atoms of oxygen. The
paper displays poor capability to undergo ion
exchange and adsorption.
TLC (Thin Layer Cromatography) was also done
in the experiment. This type of chromatography
works on stronger solvents. It is also done faster

rates.
When it comes to paper chromatography, a
precisely even permeable paper is considered
as the stationary phase while an appropriate
solvent mixture or simply a liquid in form is
Paper chromatography has been proven to be
effective

in

than the former type of chromatography.

The fraction of the distance travelled by a
component to the distance travelled by the
solvent front, is called the Resolution front (R f)
value

considered as the mobile phase.

actually

adsorption. The solvent, being less polar, is

examining

chemical

or

the

Retention

value

for

that

component. Both distances are measured from

the point of the application of the mixture.

compounds. In this process, the solvent mixture

is dropped to a piece of chromatogram paper.
The side of the paper is immersed in a specific
solvent. By capillary action, the solvent is
eventually absorbed by the paper as it moves
up.
With changing degrees, the solvent carries the
different mixture components, subject on the
fondness

of

the

compound

to

undergo

adsorption on the paper against being conceded

along the solvent. The paper is specialized to be

Student, CHM142L, School of ChE-CHM, Mapua Institue of Technology, Muralla St.,

Intramuros, Manila
4
Professor, School of ChE-CHM, Mapua Institue of Technology, Muralla St.,
Intramuros, Manila
*Corresponding author: cmmartinez@mymail.mapua.edu.ph
1

Organic Chemistry Laboratory Report

Polar compounds do not travel that far because
they stick to the stationary phase more than
non-polar compounds.
Certain precautions must be followed in doing
the process. The paper must be handled only at
the edges to avoid fingerprints that may hinder
the elution. The chromatography chamber must
be tightly covered because the solvents in the
mobile phase are volatile that cause them to
evaporate. The edges of the paper must not
touch each other during the development of the
chromatogram because the eluent will sneak
and the spots will travel together. It may
eventually ruin the chromatogram. The spots
must be above the solvent level. Failure to do so
may cause the spots to just wash out from the
mobile phase instead of eluting up the stationary
phase. When the solvent front has reached the

Figure 1
The figure above shows how to compute for the
Rf value of the substance.
A specific compound is expected to travel the
similar distance along the stationary phase by a
particular

solvent

provided

that

further

experimental situations are retained constant.

Technically, each compound, be it a pigment, die
or some organic liquid, has a particular Rf value
for

every

particular

solvent

and

solvent

concentration. These specific values enables

easier identification because Rf values of the
unknown substance can be compared to that of

upper edge, it must be removed immediately, the

eluting solution will reach the top edge and all
the spots may reach it also, making them vague
to notice. The spots must also neither be too
light

nor

too

heavy

in

application.

Fair

application will do as long as it will be enough to

travel a certain distance. Sufficient separation
must also be observed from the spots. Too
narrow spaces may lead to mixing of substances
that will lead to erroneous results.

Experimental Section

the known compounds.

Materials

Relating it to the polarity of compounds, R f

600mL beaker, blower and chromatogram paper

Used:

Capillary

tube,

petridish,

values appears to be higher for non-polar

compounds or compounds with low polarity.

Student, CHM142L, School of ChE-CHM, Mapua Institue of Technology, Muralla St.,

Intramuros, Manila
4
Professor, School of ChE-CHM, Mapua Institue of Technology, Muralla St.,
Intramuros, Manila
*Corresponding author: cmmartinez@mymail.mapua.edu.ph
1

Organic Chemistry Laboratory Report

Reagents Used: Methyl orange, Fluorescein,

Results and Discussion

Conclusion

Erythrosine, Methylene blue, Indigo carmine,

50% ethanol and an unknown reagent
Using

Chromatogram

Paper:

First,

rectangular piece of chromatogram paper was

obtained. Using a pencil, a parallel line, 2cm
from its longer side, was drawn. It was then

In general, the adsorptivity of compounds

marked with ten dots, having equal space of

increases with increased polarity (i.e. the more

2cm from each other. These dots were spotted

polar the compound then the stronger it binds to

with different dyes using a capillary tube. Every

the adsorbent). The eluting power of solvents

spot of a dye, a blower was used to dry it. The

increases with polarity. Therefore, low polarity

paper was rolled, with its two edges connected

compounds can be eluted with low polarity

but not touching through staple wire. The 600mL

solvents,

beaker was filled with 5mL of 50% ethanol.

require solvents of higher polarity. The stronger

Inside the beaker, the rolled paper was placed

a compound is bound to the adsorbent , the

carefully, not touching its sides. The beaker was

slower it moves up the TLC plate. Non-polar

then covered with petridish until the dyes

compounds move up the plate most rapidly

reached the end of the line. The distance

(higher Rf value), whereas polar substances

traveled by the dyes were marked. The paper

travel up the TLC plate slowly or not at all (lower

was then dried. Other necessary measurements

Rf value).

were also done. The Rf values of the dyes were

calculated. The unknown was identified by its Rf

value.
Using TLC plate: A rectangular piece of TLC

while

higher

polarity

compounds

References

Bruice, P. (2013). Organic Chemistry. New York,

NY: Pearson Education.

plate was obtained. A parallel line to its shorter

Gocan, S. (2002). Stationary phases for thin-

edge was drawn. Again, equally spaced dots

layer chromatography. Journal of

were also drawn but this time, amino acids were

Chromatographic Science, 40(10), 538-549.

used to spot the dots and the beaker was filled

with different mixture of reagents. Since the

Clark, J. (2007). Paper Chromatography.

amino acids were colorless, spraying the TLC

Retrieved from:

plate with Ninhydrin made the colors visible

http://www.chemguide.co.uk/analysis/chromatog

enough. The same thing was done to obtain the

raphy/paper.html

Rf value of the unknown.

Student, CHM142L, School of ChE-CHM, Mapua Institue of Technology, Muralla St.,

Intramuros, Manila
4
Professor, School of ChE-CHM, Mapua Institue of Technology, Muralla St.,
Intramuros, Manila
*Corresponding author: cmmartinez@mymail.mapua.edu.ph
1

Organic Chemistry Laboratory Report

Pastro, D. J., John, C. R., & Miller, M. S. (1998).
Experiment and Techniques in Organic
Chemistry. New Jersey: Prentice Hall. Pg. 43-46

Figure 2 shows the visible travel of the

spots from the point of application as
the chromatogram develops.

Pavia, D. L., Lampman, G. M., Kriz, G. S., &

Engel, R. G. (1999). Organic Laboratory
Techniques: A Microscale Approach (3rd Ed.).
Harcourt College Publisher. Pg. 558-576.

Supporting Information

Figure 3. TLC Chromatography

As can be seen on the figure above,

the solvent goes up the TLC plate and
the substances subjected to the paper
were not visible.

Figure 2. Development of Chromatogram

Student, CHM142L, School of ChE-CHM, Mapua Institue of Technology, Muralla St.,

Intramuros, Manila
4
Professor, School of ChE-CHM, Mapua Institue of Technology, Muralla St.,
Intramuros, Manila
*Corresponding author: cmmartinez@mymail.mapua.edu.ph
1