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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quickly understand the purpose, methods, results and significance of your research without reading the entire paper.
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you can quickly determine whether the research report will be relevant to your topic. The material in the abstract is
written in the same order as that within the paper, and has the same emphasis. An effective abstract should include a
sentence or two summarizing the highlights from each of the sections: introduction (including purpose), methods,
results, and discussion. To reflect the content (especially results and conclusions) of the paper accurately, the abstract
should be written after the final draft of your paper is complete, although it is placed at the beginning of the paper. Begin
the abstract with a brief, but specific, background statement to introduce your report. State your main purpose or
objective and hypothesis. Describe the important points of your methodology (species/reagents/ingredients, the number
of subjects or samples, and techniques or instruments used to make 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 the paper. When a liquid travels up the paper, it separates,
in separating a mixture into its individual component. allowing it to analyze the different components of the liquid
solution. All compounds have a specific retention factor value
The mixture is dissolved in a fluid called the mobile phase, for every specific solvent, and retention factor values are
which carries it through a structure holding another material used to compare unknown samples with known compounds.
called the stationary phase. The various constituents of the
mixture travel at different speeds, causing them to separate. Moreover, retention factor is affected by the adsorption and
The separation is based on differential partitioning between solubility of the analyte in relation with the stationary phase.
the mobile and stationary phases. Subtle differences in a
compound's partition coefficient result in differential retention Differential affinities (strength of adhesion) of the various
on the stationary phase and thus affect the separation. components of the analyte towards the stationary and mobile
(McMurry, 2011). phase results in the differential separation of the
components. Affinity, in turn, is dictated by two properties of
Retention factor is defined as the distance a liquid compound the molecule: ‘Adsorption’ and ‘Solubility’. (Khan Academy,
travels up a chromatography plate. The chromatography n.d.)
paper is the stationary phase and the liquid compound is the
mobile phase; the liquid carries the sample solutions along We can define adsorption as the property of how well a
component of the mixture sticks to the stationary phase,

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 CHM_____ Organic Chemistry __ Laboratory
___ Quarter SY__________

while solubility is the property of how well a component of the Eluate fluid exiting the column (that is collected in
mixture dissolves in the mobile phase. Therefore, flasks)

● Higher the adsorption to the stationary phase, the Elution the process of washing out a compound
slower the molecules will move through the column. through a column using a suitable solvent

● Higher solubility in the mobile phase, the faster the Analyte mixture whose individual components must
molecules will move through the column. be separated and analyzed

Adsorption and solubility of a certain molecule can be Table 1. Terminologies associated with chromatography
manipulated by choosing the appropriate stationary phase
and mobile phase. For further knowledge, the figure above is given to show the
different terminologies associated with chromatography.
For paper chromatography, substances are distributed
between a stationary phase and a mobile phase. The MATERIALS AND METHODS
stationary phase is the water trapped between the cellulose
fibers of the paper. The mobile phase is a developing This experiment (focuses on paper chromatography) focuses
on two matters: the stationary phase ; and the mobile phase.
solution that travels up the stationary phase, carrying the
samples with it. Components of the sample will separate The stationary phase is a chromatogram paper (8 x 22 cm)
readily according to how strongly they adsorb onto the divided equally into ten, 2 cm each of length. Each part signifies
stationary phase versus how readily they dissolve in the a chemical subjected for experiment (methyl orange,
fluorescein, erythrosine, methylene blue and indigo carmine).
mobile phase. (Wikipedia, 2019)
Refer to Figure 1 for the division of the chemicals at
chromatogram paper.
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)
Figure 1. Orientation of the chemicals in chromatography
paper.

Term Definition
For mobile phase, prepare a 600 mL beaker with 5 mL of 5%
Mobile solvent moving through the column ethanol. Place the chromatography paper at the beaker
phase submerging the bottom part (about 3 cm) of it into the liquid
solution. Note that the spots of chemicals in the chromatogram
Stationary substance that stays fixed inside the paper should not touch the liquid solution upon submerging.
Cover the beaker.
phase column
Afterwards, observe the liquid solution until it reaches the top (
Eluent fluid entering the column leave 1 cm space before it reaches the top).

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 CHM_____ Organic Chemistry __ Laboratory
___ Quarter SY__________

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_ch
romatography#Rƒ_value,_solutes,_an
d_solvents.

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