FACULTY OF ENGINEERING TECHNOLOGY

DEPARTMENT OF CHEMICAL ENGINEERING TECHNOLOGY

BIOCHEMISTRY AND BIOMOLECULAR TECHNIQUE LABORATORY

LABORATORY REPORT

COURSE CODE BNN30104

EXPERIMENT NO. EXPERIMENT 5

EXPERIMENT TITLE BASIC PAPER CHROMATOGRAPHY

DATE 24/10/2018
GROUP NO. 7
LECTURER/ INSTRUCTOR/ 1) TS. DR. SITY AISHAH BINTI MANSUR
TUTOR 2) PN. MASAYU BINTI MASLAN
DATE OF REPORT 31/10/2018
SUBMISSION
ATTENDANCE/PARTICIPATION/DISPLINE /5%
INTRODUCTION: /5%
PROCEDURE: /5%
RESULTS & CALCULATIONS /15%
ANALYSIS /15%
DISTRIBUTION OF MARKS DISCUSSIONS: /20%
FOR LABORATORY REPORT: ADDITIONAL QUESTIONS: /15%
CONCLUSION: /10%
SUGGESTION & RECOMENDATIONS /5%
REFERENCES: /5%
TOTAL: /100%

EXAMINER COMMENTS: RECEIVED DATE AND STAMP:

 STUDENT CODE OF ETHICS

DEPARTMENT OF CHEMICAL ENGINEERING

TECHNOLOGY

FACULTY OF ENGINEERING TECHNOLOGY

I hereby declare that I have prepared this report with my own efforts. I also admit to not

accept or provide any assistance in preparing this report and anything that is in it is

true.

NAME SIGNATURE

NAME : MOHD NIZAM BIN JUMADI

MATRIC NO: AN150424

NAME : NUR AINAA ZUHAIRAH BINTI HEIRMAN

MATRIC NO : AN150008

NAME : NUR ISMALIS SHAFEQA BINTI ZULKARNAIN

MATRIC NO : DN160344
1.0 INTRODUCTION

Paper chromatography is a paper strips or sheets that use in this experiment and act as
the adsorbent stationary phase through which a solution flows. Paper chromatography is a paper
that used in experiments to separate soluble chemicals. This is because of their different rates of
migration across sheets of paper. This paper is very cheap to be use in order to conduct the
experiment but this paper also is very powerful analytical tool. It is requires in very small
amount of material. This paper is basically known to separate mixtures of soluble substances.
These are often coloured substances such as food colourings, inks, dyes or plant pigments.
It is necessary for the different chemicals in the solution for have different properties
such as molecule size or a different ability to dissolve in a solvent. The stationary phase will
absorb or slow down different components of the tested solution to different degrees creating
layers as the components of the solution are separated. Chromatography was invented by the
Russian botanist, Mikhail Tsvet. Chemists use this process to identify unknown substances by
separating them into the different molecules that make them up.
This a technique that we use to analyse mixtures solutes by exploiting differences in
their distribution between a stationary and a mobile phase. We need to know that this basic
paper chromatography is also a fundamental technique for the separation, detection,
identification and quantitation of chemical species. Chromatographic techniques can also be
classified according to whether they are being used for identification and measurement
(analytical, for microscale to trace quantities of analyte) or as a purification step (preparative,
for semi micro to macroscale quantities of analyte). Today, chromatographic techniques are
essential tools in areas such as chemistry, biology, medicine, forensic science, manufacturing,
and the environment, and are arguably the most widely used of any family of analytical
methods.
The principle working of paper chromatography is that the mixture is spotted onto the
paper chromatography. Then dried and the solvent is allowed to flow along the sheet by
capillary attraction. By doing observation in few minutes we can see the motion of solvent that
going slowly move to the top through the paper, in this paper obviously the different
compounds of the mixture separate into different coloured spots. The paper is dried and the
position of different compounds is observed. The basic principle of the paper chromatography is
that the most soluble substances move further to the top on the filter paper than the least soluble
substances. Normally, different plant pigments can be separated by using the technique of paper
chromatography.
 Example of how to conduct the experiment.
After the experiment, the data height of motion soluble substances will be recorded.
Based on the record of data will be use to indicate the R f value. Rf values can be used to identify
unknown chemicals if they can be compared to a range of reference substances. The R f value is
always the same for a particular substance.
The Rf value of a spot is calculated using:
Rf =

Rf values vary from 0 (the substance is not attracted at all to the mobile phase) to 1 (the
substance is not attracted at all to the stationary phase).

Example of how to measure the height of motion soluble that found in this paper
chromatography.
2.0 PROCEDURE
IMPORTANT: For one piece of paper into one beaker of one solvent.

1. Paper chromatography is divided into six.

2. An origin lines measured 2.5cm are outlined above paper chromatography.
3. All samples are labelled SW1, SW2, SW3, SB1, SB2 and SB3.
4. 50 ml of distilled water is put into 3 beaker.
5. Each sample (sample 1, sample 2 and sample 3) is point on 3 chromatography paper.

Step of how to point the 3 sample on the paper chromatography

6. Three paper chromatography is inserted into beaker that containing 50ml distilled water
by affixing with a glass rod vertically.
7. 10 minutes has been set and colour changes are observed.
8. Reading data is recorded.
9. 50 ml butanol: acetic acid is put into 3 beakers.
10. Each sample (sample 1, sample 2 and sample 3) is point on 3 chromatography paper.
11. Three chromatography papers are inserted into beaker containing 50ml butanol: acetic
acid by affixing with glass rod vertically.
12. 10 minutes has been set and colour changes are observed.
13. Reading data is recorded.
3.0 RESULTS
a) Solvent: Distilled water

Sample Distance of Compound Retardation

Colour Distance on the
solvent on the Factor (Rf)
chromatography
chromatography
paper (cm)
paper (cm)
1 17.00 Purple 14.10 0.829
Blue 15.00 0.882
(Blue)
2 17.40 Green 14.60 0.839
Blue 16.50 0.948
(Green)
3 16.90 Orange 16.20 0.959
Purple 16.70 0.988
(Orange)

Table 1: Result obtained on chromatography paper for distilled water as solvent

b) Solvent: Butanol: acetic acid: water in ratio (4:1:5)

Sample Distance of Compound Retardation

Colour Distance on the
solvent on the Factor (Rf)
chromatography
chromatography
paper (cm)
paper (cm)
1 6.70 Purple 5.60 0.836
Blue 6.40 0.955
(Blue)
2 7.50 Green 5.30 0.707
Blue 7.30 0.973
(Green)
3 6.30 Orange 3.80 0.603
Purple 5.20 0.825
(Orange)
Blue 6.20 0.984

Table 2: Result obtained on chromatography paper for butanol as solvent

Calculation

Retardation Factor (Rf) =

Example:
Sample – 1 (Blue) Solvent – Distilled water
Compound colour – Purple
Distance travelled by the purple colour compound = 14.10 cm
Distance travelled by the distilled water = 17.00 cm

Rf = distance travelled by the compound

distance travelled by the solvent

Retardation Factor (Rf) = = 0.829

4.0 ANALYSIS
The experiment was held between two different types of solvent which are distilled
water and butanol. Three type of food colouring with different colours was used which blue
colourant sample represent as sample 1, green colourant represented as sample 2 and orange
colourant represented as sample 3. In all three sample, we analyse that the distance of colour
disperse further while using distilled water as solvent compare to butanol. We measure that the
distance of distilled water on the chromatography paper reaches up to 17.40 cm while the
farthest distance reaches by butanol on the chromatography paper was 7.50.
While taking distilled water as the solvent, we identify that for sample 1 (blue), the
distance for the solvent on the chromatography were 17.0 cm for sample 1, 17.40 cm for sample
2 and 16.90 cm for sample 3. Two colours present which are purple and blue on the first sample
after 10 minutes. Both having retardation factor of 0.829 and 0.882 respectively. Sample 2
formed two colours after 10 minutes which are green and blue, both having retardation value of
0.839 and 0.948 respectively.
For sample 3, two colours presence which are orange and purple with measurement of
retardation factor of 0.959 and 0.988 respectively. Whereas in taking butanol as the solvent, we
measure the distance for the solvent (butanol) on the chromatography are 6.70cm in sample 1
(blue), 7.50cm in sample 2 (green) and 6.30 in sample 3 (orange). After 10 minutes, sample
1(blue) produces two colours which are purple and blue which 0.836 and 0.955 retardation
factor each. In sample 2 (green), two colours also present which are green as well and blue with
retardation factor or 0.707 and 0.973 respectively. Lastly, for the last sample which is orange
colourant, after 10 minutes immersed in the butanol, it forming three colours which are orange,
purple and blue with 0.604,0.825 and 0.984 retardation factor respectively.
Figure 1: (from right) Sample 1(Blue), Sample 2(Green) and Sample 3(Orange)

Figure 2: Colour disperse on the chromatography paper after 10 minutes

 Distilled water (solvent)

Figure 3.1: The soluble molecules are moving Figure 3.2: The observation of change colours
to the top from Origin after 10 minutes

Butanol: acetic acid (solvent)

Figure 4.2: The observation of change colours after

Figure 4.1: The soluble molecules are moving to
10 minutes
the top from Origin
 5.0 DISCUSSIONS
Based on the results calculated above, the distance of solvent on the chromatography
paper from starting line in taking distilled water as the solvent shows the farthest distance
compare to butanol mixture. Sample 1 is blue colorant, sample 2 is green colorant and sample 3
is orange colorant. Taking distilled water as the solvent, resulting the distance of solvent on
chromatography paper for sample 1 is 17.00 cm, sample 2 is 17.40 cm and sample 3 is 16.90
cm.
For sample 1, the compound forms having two colours which are purple and blue. The
distance on the chromatography paper of purple colour from the starting line is 14.10 cm while
for blue colour is 15.00 cm. The retardation factor, R f for both colour are 0.829 and 0.882
respectively. The differences obtained for the distances from starting line and the R f for both
colours happen due to the compound content. The Rf for blue colour is higher compare to purple
as it has farthest distance than purple. As for the sample 2, the compounds form is having also
two colours which are green and blue. The distance on the chromatography paper of green
colour from the starting line is 14.60 cm while for blue colour is 16.50 cm. The retardation
factor, Rf for both colour are 0.839 and 0.948 respectively. The R f for green colour is lower
compare to blue as it has shortest distance than blue colorant. Lastly, taking distilled water as
solvent makes sample 3 having also two colours which are orange and purple. The distance on
the chromatography paper of orange colour from the starting line is 16.20 cm while for purple
colour is 16.70 cm. The retardation factor, R f for both colour are 0.959 and 0.988 respectively.
The Rf for purple colour is higher compare to orange as it has farthest distance than orange.
Butanol mixture is the mixture of butanol, acetic acid and water in ratio 4:1:5. Taking
butanol mixture as the solvents resulting in the distances of solvent on the chromatography
paper from starting line for sample 1 is 6.70 cm, sample 2, 7.50 cm and for sample 3 is 6.30 cm.
For sample 1, the compound forms having two colours which are purple and blue. The
distance on the chromatography paper of purple colour from the starting line is 5.60 cm while
for blue colour is 6.40 cm. The retardation factor, R f for both colour are 0.836 and 0.955
respectively. The Rf for blue colour is higher compare to purple as it has farthest distance than
purple. As for the sample 2, the compounds form is having also two colours which are green
and blue. The distance on the chromatography paper of green colour from the starting line is
5.30 cm while for blue colour is 7.30 cm. The retardation factor, R f for both colour are 0.707
and 0.973 respectively. The Rf for green colour is lower compare to blue as it has shortest
distance than blue colour. Lastly, butanol mixture as solvent makes sample 3 having three
colours which are orange, purple and blue. The distance on the chromatography paper of orange
colour from the starting line is 3.80 cm, for purple colour is 5.20 cm while for blue colour is
6.20 cm. The retardation factor, Rf for colours obtained are 0.603, 0.825 and 0.984 respectively.
The Rf for blue colour is higher compare to the other two colour as it has farthest distance.
After the separation is completed, individual compounds appear as spots separated
vertically. Each spot has a retardation factor, R f which is equal to distance travelled by the
compound over the distance travelled by the solvent. The R f value can be used to identify
compounds due to their uniqueness to each compound. When comparing two different
compounds under the same conditions, the compound with the larger R f value is less polar
because it does not stick to the stationary phase as long as the polar compound which would
have a lower Rf value. A high Rf value for example 0.80, would refer to a substance that is very
non-polar. Means that the substance move 80% of the entire distance the solvent travelled. A
low Rf value, for example 0.10 would refer to a substance that is very polar. Means that the
substance was only able to move 10% of the entire distance the solvent travelled.
Since the solvent carries the chemical up the plate, the particular solvent used will also
have substantial impact on the Rf value for a chemical. A solvent which has stronger interaction
for a particular chemical will more easily overcome any affinity of the chemical for the
absorbent layer, and move that chemical farther in a given period of time. When comparing two
different compounds run under the identical chromatography conditions, the compound with the
larger Rf is less polar because it interacts less strongly with the polar adsorbent on the TLC
plate. Mixtures of solvents can also have different effects depending on the proportion of each
solvent.
For this case, it proven by the butanol mixtures which give different R f value for each
sample. For this case distilled water is proved to be highly polar. So the colorant is stick to the
stationary phase rise fast and high as they stick well. Rf value and reproducibility can be
affected by a number of different factors such as layer thickness, moisture on the TLC,
temperature, depth of mobile phase, sample size and solvent parameters. The effects normally
cause an increase in Rf values. The temperature of the solvent and plate may make slight
changes, since, for example, the solvent can often better dissolve the chemicals it is transporting
at higher temperatures. The techniques of handling the sample to plate may also change the
retardation factor. Applying too much sample may result in large, diffuse bonds of chemical
moving up the plate, making it difficult to accurately measure the distance the chemical has
been transported.
6.0 ADDITIONAL QUESTIONS
1. Justify the reason(s) of covering the chamber with aluminium foil during
experiment.

It is important to cover the chamber to be sure that the solvent does not evaporate. An open
beaker will cause the solvent mixture to more quickly diverge from the intended mixture for
the chromatography, which will detract from reproducibility and likely harm the usefulness
of the separation. Covering the beaker will create a pseudo equilibrium of sorts inside the
beaker, which will yield better and more reproducible results.
2. Can you relate the principle of thin layer chromatography (TLC) in food industry?
Extensively explain.

Separation in food industry are used hundreds of applications, some of which include
detection of aflatoxins in foods, amino acids analysis, vitamins separations, vitamins in soft
drinks, profiling various food components, analysis of colorants and residues like residues in
vegetables, salads and meats, triglyceride determination, sugar content analysis, and
determination of various other organic compounds like pesticides and fungicides in drinking
water. Examples of testing using TLC are quantitative assay of betanine in red beet root dye
samples by dual wavelength absorption densitometry on cellulose layers. Synthetic dyes
were identified and quantified in wine, wine-containing, and non-alcoholic beverages and
spirits at a detection limit by silica gel and cellulose TLC. The effects of microwave heating
on the loss of vitamin B12 in foods were also studied by TLC.

3. Compare and contrast between paper and column chromatography.

The differences between paper chromatography and column chromatography are:

Paper Chromatography Column Chromatography

Criterions
Separation Chromatographic technique Use a column packed with a
use to separate compounds matrix that is used to
based on the liquid-liquid separate molecules mainly
adsorption and solubility of based on their size, affinity
the compound. It uses a or its charge.
cellulose paper as its
stationary phase.
Stationary Phase Paper made of nitrocellulose A column packed with
 of Whatman is used as the
stationary phase in paper
chromatography.
Mobile Phase Running solvent is the
mobile phase of the paper
chromatography.
Mechanisms Used for Paper chromatography is
Separation based on solid-liquid
absorption.
Elution Buffer Not required by paper
chromatography.
Detection Staining and by determining
the Retention factor.
suitable packing material is
used as the stationary phase
in the column
chromatography.
Wash buffer is the mobile
phase of the column
chromatography.
Column chromatography is
based on size exclusion,
charge and shape.
Required in column
chromatography.
Spectrophotometric
determination.

7.0 CONCLUSION
There are various type of methods of separation were designed in the application of
industries, and one of it was the paper chromatography which act as the simplest methods of
separation. Paper chromatography enabled us to identify the variety of component present in a
substances where in this experiment, separation of colour was done. The paper chromatography
enabled us to measure the purity of the substance as well as a pure substance should only have
single composition presence on the chromatography paper. Hence this methods are applicable in
detecting an early purity identification.
The distance on the chromatography paper determined the polarity of certain colour that
presence in the mixture. The higher the distance reaches, the higher the polarity of the colour.
There are various types of separation method, in order to obtain the most precise separation
result, it is important for us to be able to identify the best separation method depending on the
type of substance we examined.
The paper chromatography methods are best to be use in separating substance with
similar polarity such as amino acid. Paper chromatography could pose as the finalised methods
to obtain the most precise and accurate separation. Hence, we can conclude that the paper
chromatography method enable us to separate the component presence in the colorant which
usually use in food processing.
 8.0 SUGGESTIONS AND RECOMMENDATIONS

For this experiment, we would like to suggest few improvements that helping this
experiment getting better in future. This experiment is basically useful for our studies about
separation molecules that soluble in food.
Firstly, we agreed that the proper procedures need to be prepared. This is because basic
paper chromatography is about fundamental technique. Some equipment are not same to be use
in this experiment if all groups are conducting experiments based on the internet resources but
still the concept is same.
Next, we would like remind that when conducting this experiment. The paper
chromatography must be taking pictures due to observation of colour. This is because of we
want to record the measurement and even after 10 minutes the change colour still happen and
this will lead to error because of mix colour that make us did not know which level need to be
measured.
Besides that, we also recommend a precaution step which is the preparation of butanol :
acetic acid that need to be done in the fume hood in order to avoid the solution is vaporised
when doing the observation of changes colour.
Lastly but not least, the parallax error may be happen. In order to reduce this error. We
suggest that this experiment need to be done in many times to get an average value for all
sample. The repeated experiment will produce an accurate reading for all measurement taken
from observation changes of colour.
9.0 REFERENCES

1. Paper chromatography (n.d) retrieved October 27 2018 from

http://genchem.rutgers.edu/chrompap.html
2. amrita.olabs.edu.in, (2014). Paper Chromatography. Retrieved 27 October 2018 from
amrita.olabs.edu.in/?sub=79&brch=17&sim=124&cnt=1
3. Teresa Coppens. (15 June 2016) : what is paper chromatography and how does it work
retrieved October 27 2018 from
https://owlcation.com/stem/What-is-Paper-Chromatography-and-How-does-it-Work.
4. Separation and purification (n.d) retrieved from October 27 2018 from
https://www.bbc.com/bitesize/guides/zqc6w6f/revision/4
5. Paper chromatography (n.d) retrieved October 27 2018 from
https://www.britannica.com/science/paper-chromatography

6. A Comprehensive Working, Principles and Applications of Thin Layer Chromatography

by Bipin D Lade, Anita S Patil, Hariprasad M Paikrao, Ankit S Kale and Kushal K Hire.
Retrieved 26 October, 2018, from https://www.rjpbcs.com/pdf/2014_5(4)/[53].pdf
7. Thin-Layer Chromatography in Food and Agricultural Analysis by Joseph Sherma.
Retrieved 26 October, 2018, from
https://www.researchgate.net/profile/Dr_Alok_Nahata/post/Which_solvent_mixture_sho
uld_be_used_in_Thin_layer_chromatography_as_a_mobile_phase/attachment/59d61f16
79197b807797d805/AS
%3A282571435986947%401444381688724/download/TLC+METHODS+OF+ANALY
SIS.pdf
8. Manufacturing High Quality Chromatography Products since 1961. Retrieved 26
October, 2018, from https://www.milesscientific.com/applications-of-thin-layer-
chromatography/
10.0 APPENDIX