Final Research Paper Ciaramitaro Doerr Weidemann

Ciaramitaro-Doerr-A.
Weidemann
Testing the Effect of Porosity and Polarity on the Retention Factor of Red 40 FD&C
Food Dye Using Paper Chromatography
Reis Ciaramitaro / Kevin Doerr / Andy Weidemann
Macomb Mathematics Science Technology Center
Honors Chemistry
10C
Mrs. Hilliard/Mr. Supal/Mrs. Dewey
May 24, 2017
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Ciaramitaro-Doerr-A.Weidemann
Table of Contents
Introduction………………………………………………………………………...1
Review of Literature……………………………………………………………….3
Problem Statement………………………………………………………………..9
Experimental Design……………………………………………………………...10
Data and Observations………………………………………………………...…16
Data Analysis and Interpretation…………………………………………………21
Conclusion……………………………………………………………………….…29
Appendix A. Solution Preparation……………………………………………..…32
Appendix B. Retention Factor Sample Calculation……………………………..33
Works Cited…………………………………………………………………………34
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Introduction:
Before the early 20th century, there were limited ways to separate and
identify the components of a homogenous mixture. If a mixture was not able to
be separated by means of distillation, it did not have a chance of being separated
and identified. This remained true until 1903, when Mikhail Tswett separated the
pigments in a leaf, using a new method called chromatography (“History of
Chromatography”). The purpose of this experiment is to test the effect of different
types of qualitative paper and polarity of solvents on the retention factors of
FD&C Red 40 food dye.
A design of experiment was conducted over a span of one week to collect
the retention factors of each chromatogram. This was done by allocating highs
and lows to the factors, polarity and porosity. Once all the factors were
established, the trials were conducted and observed to calculate the Rf value of
each chromatogram. These Rf values were then averaged with its corresponding
factor and used to determine if the effects were significant. A design of
experiment was used to help the collection of data in an organized fashion and
limit variables by only focusing on few data points of collection.
The research was conducted to test the use of different solvent polarities
and porosity on the effect of the developed chromatogram to help indicate the
improved efficiency of a factor. Retention factor values are used in paper
chromatography to help identify compounds by the scientific community when
other forms of chromatography do not apply. Finding an improved method of
developing chromatograms in paper chromatography could be utilized in other
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fields of chromatography to refine the process of the identification of compounds.
For example, the use of specific mediums in relation to specific compound
counterparts may drastically enhance the benefits of an analysis by producing an
answer faster without the need of an excessive amount of other materials
because the less of the new medium is needed. Other applications may be the
deeper understanding of porosity and polarity on paper chromatography, so
improved processes can be used, and materials will not be wasted trying to find
the optimum combination of solvent and qualitative paper in an experiment.
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Review Of Literature:
Chromatography, or the separation and identification of the parts in a
homogenous mixture, has been used in innumerable scientific fields.
Homogenous mixtures, opposed to heterogeneous mixtures, have limited means
of separation, so many different forms of chromatography are required to analyze
certain substances that the mixture is composed of. Many forms of
chromatography have been developed throughout the last century in order to
evaluate specific substances depending on their characteristics. The most
commonly used forms of chromatography include gas chromatography and liquid
chromatography, although twelve total techniques exist.
Figure 1. Homogeneous and Heterogenous Mixtures
Figure 1 above shows the difference between Homogeneous and
Heterogeneous mixtures. This image was found on the article Chemistry for Kids
published by Ducksters. Heterogeneous mixtures are not uniform throughout and
can be separated easily. Homogeneous mixtures are uniform throughout and
have to be separated using different techniques.
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Chromatography and chromatographic concepts have been used since
the early 20th century. Founded by Russian botanist Mikhail Tswett in 1903,
chromatography has since evolved into a vital tool in laboratories worldwide.
Today, it is one of the most widely used techniques for many analytical and
preparative processes spanning many scientific fields.
Paper Chromatography, in analytical chemistry, is a technique for
separating dissolved substances by taking advantage of their different rates of
movement across segments of paper. It is inexpensive, but it is also a powerful
analytical tool that aids in separation and identification of components in a
mixture. It is relatively easy to use because it only needs a small amount of the
mixture to correctly separate and analyze (“Paper Chromatography”).
Forms of chromatography, including paper, column, thin-layer, and size
exclusion chromatography, use stationary and mobile phases to examine the
retention factor of substances. This means that these forms of chromatography
involve a liquid solvent carrying a stationary solvent, which is the mixture that is
being analyzed, with it. Although this method slightly changes with each type of
chromatography, the idea stays the same (“Principles of Chromatography, Khan
Academy”). The ratio of the distance travelled between the two is what is known
as the retention value.
The method of chromatography that will be used in this experiment will
look at the retention factors (Rf values for short) of certain substances. Every
compound has its own retention factor for the solvent used. This value is
determined using a simple calculation requiring the distance travelled by the
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solute and the distance travelled by the solvent front, the endpoint of the
movement of the solvent.
𝑅𝑒𝑡𝑒𝑛𝑡𝑖𝑜𝑛 𝐹𝑎𝑐𝑡𝑜𝑟
= 𝐷𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑙𝑒𝑑 𝑏𝑦 𝑠𝑜𝑙𝑢𝑡𝑒
÷ 𝐷𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑙𝑒𝑑 𝑏𝑦 𝑠𝑜𝑙𝑣𝑒𝑛𝑡 𝑓𝑟𝑜𝑛𝑡
Since these values are ratios, they will always be between 1 and 0 because the
solvent front will always travel farther. These calculations are very important
when identifying mixtures and the compounds that make them up.
(Chromatography - RF Values[MarZ Chemistry]”).
Figure 2. How Retention Values Are Calculated
Shown above is how an example retention value is calculated. The solvent
front travelled 2.8 cm from the origin while the compound front only travelled 2.1.
The ratio is then calculated to be 0.75.
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Figure 3. Paper and Column Chromatography Diagram
Figure 3 explains how a simple chromatogram is examined and analyzed.
Different pigments were used to show the relation of Rf values in different
substances. Notice that the dyes are separated because they traveled different
distances up the paper. The solvent front carried each component up the paper
before the components stopped moving and the solvent front carried on. This
resulted in the ingredients in the mixture being carried up different distances.
Compound B in the figure above will have a larger retention factor because it
travelled further up and stayed with the solvent front longer.
One major part of paper chromatography is polarity. If a substance is
polar, its charges are not properly distributed. Polarity will have an impact on how
far the solution moves up the paper. Non-polar molecules in the mixture that are
trying to separate will have little attraction towards water molecules attached to
the cellulose on the paper sheet, and will dissolve in the solvent. These
molecules will therefore travel a greater distance upwards on the paper carried
by the solvent and, they will have relatively high retention factor. Polar molecules
are quite different and will have a high attraction towards the water
molecules;They will not be as attracted to the non-polar molecules. They will
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therefore tend to dissolve in the thin layer of water around the cellulose fibers
much more than in the agitating solvent. They spend more time dissolved in the
stationary phase and less time in the mobile phase ,so they aren't going to travel
very fast up the paper (“Paper Chromatography”).
Paper chromatography uses qualitative paper to conduct a chromatogram.
This paper comes in many shapes and grades. This experiment will use strips of
instead of sheets or circles. Each grade of paper is slightly different from the
next. Grade 1 qualitative paper has a paper like feel and is smooth. Grade 3 is
rough and thicker. As the grade of paper increases, the paper gets more porous.
High porosity paper will have more small pores which allows the solvent and the
compound to grip better. The two fronts will travel farther if the paper has more
pores.
There have been many past studies using chromatography. As an
example, researchers have found a way to use gas chromatography to identify
fatty acids in bacteria. Using a machine called Sherlock Microbial Identification
System, they are able to analyze more samples and identify more fatty acids at a
very low cost (“Identification of Bacteria by Gas Chromatography of Cellular Fatty
Acids”). The experiment that will be conducted is similar to this. It will use paper
chromatography to identify different pigments. The difference between the two is
the type of chromatography used, and the substance that will be identified.
Another example is researchers using thin-layer chromatography to simplify the
approach to identify aerobic actinomycetes by using reference cultures to study
the discharge of waste material that indicates the decomposition of certain
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materials(“Applied Microbiology”). This experiment harnesses thin-layer
chromatography, very similar to the method used in the current experiment, but
the present experiment focuses on the simple identification of pigments rather
than chemical compounds which would allow the interpolation of data. These
different methods of identification show how chromatography is an essential tool
in the process of identifying unknown substances and their implications.
The experiment that will be explained in this paper will use similar
methods to the two labs described above. This experiment will use paper
chromatography (instead of gas or thin-layer in the labs above) to try to identify
different dyes in a solution, and it will talk about the concepts behind the
individual retention factors and any trends or effects that show up. The
experiment will also focus more upon why the solvents and qualitative paper
behave as they do to attempt to find a more efficient way to conduct paper
chromatography. Porosity and polarity will be observed in this experiment. Any
findings regarding paper chromatography will also be expanded upon in relation
to other chromatographic fields. Chromatography is a helpful tool that has many
real world applications. This lab is designed to show how useful it can be in
identifying parts in a mixtures that could not have been separated even over one
hundred years ago.
Problem Statement:
What effect will different combinations of polarity of the solvent and type of
filter have on the retention factor of a FD&C food dye.
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Hypothesis:
The highest polarity solvent, water, and the most porous filter paper, grade
3, will produce the highest accuracy retention factor value results because the
high polarity and the great porosity will allow the dye solution to move faster and
more efficiently along the filter paper and create more observable/accurate
results.
Data Measured:
The independent variable used was the specific FD&C food dyes which
will be Red 40, while the dependent variable measured was the Rf values of the
FD&C Red 40 dye. The Rf values can be defined as the distance ratio in
millimeters of the ionized solvent, the FD&C food dyes, compared to the distance
of the solutions across the selected chromatogram. The Rf value will be analyzed
using a two factor D.O.E. method. The two factors include the type of filter paper,
and the polarity of the solvent. The D.O.E. will determine which level of each
factor had the biggest effect on the retention factor.
Experimental Design:
Materials:
(0.5 g) FD&C Red 40 (7) Beaker, 600-mL
Water, Distilled or Deionized, 3.8-L (2) Graduated Cylinder, 25-mL
(36) Grade 1 Qualitative Paper (1) Graduated Cylinder, 100-mL
(54) Grade 2 Qualitative Paper (2) Ruler
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(36) Grade 3 Qualitative Paper (3) Capillary Tube

(7 mL) Ethyl Alcohol (95%), C2H6O (10 mL) Isopropyl Alcohol (70%), C3H7OH
(2) Scoopula
Procedure:
Safety: Isopropyl alcohol is a flammable liquid and it is slightly toxic when

inhaled or ingested. Use proper ventilation to reduce the buildup of fumes. The
FD&C dyes are slightly hazardous by ingestion, skin/eye contact, and inhalation.
Avoid contact with eyes, skin, and clothing. Wear safety goggles, chemical
resistant gloves, and a chemical-resistant apron. Wash hands with soap before
leaving the lab.
Pre-Lab:
1. See appendix A to make the solutions needed for this lab.
Lab:
1. Mark each 600 mL beaker according to which factors will be present in

which. For example, a beaker marked with +,+. There should be 3
standard beakers and 7 marked beakers in total.
2. Using the 25 mL graduated cylinder, pour 20 mL of the appropriate

solution into each corresponding beaker. (The first + or - corresponds to
the grade of paper while the second + or - corresponds to the solution).
3. Acquire 6 grade 1 strips, 9 grade 2 stips, and 6 grade 3 strips.
4. Using a ruler and a pencil, draw a faint line 15 mm from the bottom of the
paper across the width of the strip. Measure 9.5 mm from the edge and
place a dot on the line. This is the starting point for the sample. See figure
1.
5. Using the same ruler, measure 21 mm from the top of the strip and fold
across the width of the strip. This will allow the strip to hang on the lip of
the flask. See figure 1.
6. Repeat steps 3 and 4 for each strip of each grade of filter paper
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7. Using the capillary tube, spot the chromatography strip by placing it into
the dye mixture solution then touching the tip of the tube gently onto the
designated pencil dot. Allow the sample to dry. Note: This step is
necessary to increase the concentration of the sample but do not allow the
size of the spot to increase.
8. Repeat step 6 for each subsequent chromatography strip of each grade of

filter paper.
9. After the dye has completely dried (1 min), carefully hang 3 of the
corresponding chromatography strips into the beaker with the dye dot end
down. Do not get any solvent on the upper portion of the strip. The sample
spots must remain above the level of the solvent. If the solvent level is too
high, the samples will dilute into the solvent. (Try not to touch the strip to
the edge of the beaker when it is hanging). See figure 2.
10. Allow the the chromatogram to develop for 25 minutes. Record the
observations of the dye sample as the solvent travels up the paper and
the chromatogram develops. See figure 3.
11. Repeat steps 8 and 9 using the other chromatographic solvents for each
trial.
12. After the 25 minutes is up, carefully take out the paper strips.
13. Immediately, with a pencil, lightly draw a line to mark the distance
travelled by the solvent. This is the solvent front. Draw a line marking the
furthest the compound traveled. This is the compound front. (This will be
measured in millimeters).
14. Repeat step 12 for each chromatogram.
15. Record results into a data table that calculates the Rf Value for each
chromatogram. (Compound Front / Solvent Front).
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Diagrams:
Figure 4. Materials
The materials used in the experiment are shown above. The deionized
water was used as one of the solvents and also to mix the other solvents. The
other solvents consisted of the isopropyl alcohol and the ethyl alcohol. The
deionized water was also used to mix the Red 40 FD&C Food Dye. The test
tubes were used to measure out the solvents that were then placed into the
600mL beakers. Rulers were used to measure the three different grades of
qualitative paper. The capillary tubes were used to dot the paper with the food
dye. The 100 mL graduated was used primarily for mixing solutions.
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Figure 5. Dotted Grade 3 Strips
The above figures shows the line that was drawn 15mm from the bottom.
This line was then used as a reference to place the two dots of Red 40 FD&C
Dye. This figure also depicts the folds that were done 21 mm from the top of the
strip. These folds were tasked at 21 mm because it was a perfect balance for the
strips not to touch the side of the beaker and to have the perfect height into the
solvent.
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Figure 6. Chromatograms in Development
The above figure shows an example of Grade 3 qualitative paper strips
soaking in the solvent. This picture was taken a 25 minutes after being placed
into the beaker. Notice each dye is relatively at the same distance. A trend in
Grade 3 strips, depicted in this figure, is that the retention factor is higher than
the others. This figure supports this claim as the compound front is close to the
solvent front.
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Figure 7. Measuring the Chromatogram

Figure 7 shows an example of the measurements made on a
chromatogram right after it is removed from the beaker. The marks were quickly
placed to prevent incorrect data due to development after taking out the paper.
The dashes were then measured and taken as a ratio. This was labeled as the Rf
Value for the trial.
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Data and Observations:
Table 1
Compound Front Measurements
+,+ +,- -,+ -,- Stan 1 Stan 2 Stan 3
April DOE
28th 1 101 101 85 73 81 88 88
DOE
2 110 100 86 71 87 89 89
DOE
3 114 103 87 79 84 87 95
May DOE
1st 4 101 97 75 88 83 87 80
DOE
5 109 92 78 76 88 82 80
DOE
6 108 103 74 82 86 116 92
May DOE
3rd 7 100 99 94 81 79 75 81
DOE
8 100 94 91 82 80 82 79
DOE
9 99 100 86 76 86 76 84
Table 1 shows the gathered compound fronts from the trials performed.
The compound front is the total distance the dye reached.
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Table 2
Solvent Front Measurements
+,+ +,- -,+ -,- Stan 1 Stan 2 Stan 3
April DOE 1
103 102 88 77 99 90 88
28th
DOE 2
112 102 88 74 89 91 118
DOE 3
117 105 90 83 85 89 97
May DOE 4
103 99 84 97 85 90 85
1st
DOE 5
111 95 83 97 90 86 85
DOE 6
110 105 81 89 87 125 95
May DOE 7
103 101 96 84 80 84 81
3rd
DOE 8
102 97 93 84 82 84 81
DOE 9
102 102 97 79 87 77 85
Table 2 shows the gathered solvent fronts from the trials performed. The
solvent front is the distance the solvent moved.
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Table 3
Rf Value Calculations
+,+ +,- -,+ -,- Stan 1 Stan 2 Stan 3
April DOE 1
28th 0.9806 0.9902 0.9659 0.9481 0.8605 0.9778 1.0000
DOE 2
0.9821 0.9804 0.9773 0.9595 0.9775 0.9780 0.7542
DOE 3
0.9744 0.9810 0.9667 0.9518 0.9882 0.9775 0.9794
May DOE 4
1st 0.9806 0.9798 0.8929 0.9072 0.7953 0.9667 0.9412
DOE 5
0.9820 0.9684 0.9398 0.7835 0.9778 0.9535 N/A
DOE 6
0.9818 0.9810 0.9136 0.9213 0.9885 0.9280 0.9684
May DOE 7
3rd 0.9709 0.9802 0.9792 0.9643 N/A 0.8929 1.0000
DOE 8
0.9804 0.9691 0.9785 0.9762 0.9756 N/A 0.9753
DOE 9
0.9706 0.9804 0.8866 0.9620 0.9885 0.9870 0.9882
May Make-
4th Up 0.8182
0.9765
0.9875 0.9762 0.9412
Average
0.9781 0.9789 0.9445 0.9304 0.9643 0.9597 0.9498
Table 3 displays every Rf value calculated. All of the data points with a
line through them were corrupted data and redone on May 4th.
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Table 4
Observations
Observations
April DOE 1 - A standard 1 trial was removed from the usable data
28th due to it falling off the beaker edge
- The (+,+) dots were unevenly placed, but should not
have an inconsequential impact
- Other trials were normal
DOE 2 - Trials were normal
DOE 3 - The (+,+) dots were unevenly placed, but should not
have an inconsequential impact
May 1st DOE 4 - A standard 1 trial was removed from the usable data
due to it falling off the beaker edge
DOE 5 - A standard 3 trial was removed from the usable data

May 3rd DOE 7 - A standard 1 trial was removed from the usable data
DOE 8 - A standard 2 trial was removed from the usable data

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Observations
May 4th Make-Ups - All previous corrupt trials were repeated with success
Table 4 displays all observations conducted over this experiment. A lot of
the standards were corrupted and had to be redone as a result. Notice it says
that most trials were normal. This means that the chromatograms were
successfully completed and had no contact with the beaker wall. These trials
were not contaminated in any way. The trials that were contaminated were
redone later.
Data Analysis and Interpretation:
Table 5
Table of Factors
Factors (+) Values Standards (-) Values
Polarity of Solvent Water 2% Ethyl Alcohol 2% Isopropyl Alcohol
Porosity of Paper Grade 3 Grade 2 Grade 1
Table above shows the different factors used in the experiment. Porosity
and Polarity each had a high and a low level used in the D.O.E.
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Table 6
Average Rf Value For Each Factor
Porosity Porosity
(+) (-)
Polarity 0.9781 0.9445

(+)
Polarity 0.9789 0.9304

(-)
The table above shows the average retention factor for each level of the
D.O.E. Notice that the level with the highest average retention factor was the
highest porosity paper and the lowest polarity solvent.
Table 7
Rf Values Of All 27 Standards
27 Standards
Standard 0.8182 0.9775 0.9882 0.9765 0.9778 0.9885 0.9875 0.9756 0.9885
1
Standard 0.9778 0.9780 0.9775 0.9667 0.9535 0.9280 0.8929 0.9762 0.9870
2
Standard 1.0000 0.7542 0.9794 0.9412 0.9412 0.9684 1.0000 0.9753 0.9882
3
Table 7, above, displays every Rf value for each standard. Each
numbered standard has no correlation in any sort of calculation. The numbered
standards were just used for organization.
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Figure 8. 27 Plotted Standards
Figure 8 displays all 27 standards plotted. The range of standards was
calculated by subtracting the highest Rf value from the lowest. This number was
calculated to be 0.2458. This number is relatively high due to some outliers. The
Rf value for each standard is relatively constant except for a few outliers.
Standard 20, is extremely low with a Rf value of 0.7542. Another standard that
seems a bit low is the first standard with a Rf value of 0.8182. These low
numbers could have occurred for a multitude of different reasons. The most
plausible reason for this could be that the paper touched the side of the beaker
during the trial resulting in a lower Rf value. Standard 20 could significantly play a
role in whether the effects will be statistically significant or not by making the
range of standards significantly high.
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Table 8
Rf Averages of Paper Porosity
Porosity Of The Paper
Grade 1 Grade 3
(-) (+)
0.9304 0.9781
0.9445 0.9789
Avg = 0.9375 Avg = 0.9785
Effect Value = 0.0410
Table 8 above, shows the average data for the corresponding factor,
porosity of the paper. The averages for the high and low of porosity were taken
and then subtracted from each other to calculate the effect value. This number
will be used later on to determine if the porosity of the paper was significant to
the results drawn from this experiment.
Figure 9. The Effect of Paper Porosity On Rf Values
Figure 9 above, shows low and high averages on the effect of paper
porosity. On average, as porosity increases, the Rf value also increased by about
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0.0410. As previously stated, this number was calculated by subtracting the high
average from the low average.
Table 9
Rf Averages of Solvent Polarity
Polarity Of the Solvent
Isopropyl Water
Alcohol (+)
(-)
0.9304 0.9781
0.9789 0.9445
Avg = 0.9547 Avg = 0.9613
Effect Value = 0.0066
Table 9 shows the average data for the corresponding factor, polarity of
the solvent. The averages for the high and low of porosity were taken and then
subtracted from each other to calculate the effect value. This number will be used
later on to determine if the polarity of the solvent was significant to the results
drawn from this experiment.
Figure 10. The Effect of Solvent Polarity On Rf Values
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Figure 10 above, shows low and high averages on the effect of solvent
polarity. On average, as polarity increases, the Rf value also increased by about
0.0066. As previously stated, this number was calculated by subtracting the high
average from the low average.
Table 10
Interaction Effect of Porosity and Polarity
Porosity
- +
Polarity + 0.9445 0.9781
- 0.9304 0.9789
Table 10 above, shows both the corresponding Rf values with each factor
in this experiment.
Figure 11. Interaction Effect Graph of Porosity and Polarity
Figure 11 ,above, shows the interaction effect between the porosity of the
paper used and the polarity of the solvent used. Judging from the graph, there
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appears to be an interaction occurring between the two factors as they do
intersect. Another graph was also created to represent this data but it served no
use as this graph shows everything quite clearly.
0.9781 − 0.9445 0.9789 − 0.9304

𝐼𝑛𝑡𝑒𝑟𝑎𝑐𝑡𝑖𝑜𝑛 𝐸𝑓𝑓𝑒𝑐𝑡 = −
1 − (−1) 1 − (−1)
𝐼𝑛𝑡𝑒𝑟𝑎𝑐𝑡𝑖𝑜𝑛 𝐸𝑓𝑓𝑒𝑐𝑡 = −0.0075
Figure 12. Interaction Effect Calculation
The above figure shows how the interaction effect was calculated. This
number was calculated by finding the slopes of both lines and subtracting the low
slope from the high slope. The high slope was calculated to be 0.0168. This was
done by dividing the difference in y’s over the difference of x’s. The low slope
was calculated using the same method. This was calculated to be 0.0243. The
interaction effect was then calculated to be -0.0075 by subtracting the two.
Figure 13. Dot Plot of Effects
Figure 13, above, displays all the effect values on a line. The boundaries
shown, depict whether these effects are statistically significant. These fences
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were calculated by multiplying the range of standards by 2. If any effects were
outside of these boundaries they would be proclaimed statistically significant. As
shown above, none of the effects were statistically significant as they all fell
inside of the fences. Porosity had the most effect on the Rf values as it had a
significantly higher effect value than the others.
0.0410 0.0066 −0.0075

𝑌 = 0.9580 + 𝑃𝑜𝑟 + 𝑃𝑜𝑙 + 𝑃𝑜𝑙&𝑃𝑜𝑟
2 2 2
+ "𝑛𝑜𝑖𝑠𝑒"
Y = 0.9781
Figure 14. Prediction Equation
The above figure shows the prediction equation for this experiment. Y was
calculated to be 0.9781. This means that if this experiment were to be ran again,
this number could be calculated once again.
Y = 0.9580 + “noise”
Figure 15. Parsimonious Prediction Equation
The above figure shows the parsimonious prediction equation. All of the
variables in this equation would be deemed statistically significant. In this
experiment, nothing was deemed statistically significant. Therefore, “noise” is the
only variable in this equation.
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Conclusion:
The objective of this experiment was to find the effect of solvent polarity
and the porosity of qualitative paper had on the resulting retention factor in paper
chromatography. The findings could then be compared to other results in other
chromatographic fields of science. Using the highest polarity solvent and the
most porous grade of qualitative paper, respectively water and grade 3
qualitative paper, was expected to yield the highest and most significant retention
factor results, but was concluded to be rejected as the interaction between the
two factors was found to be insignificant in a design of experiment. This
hypothesis was also denied because the highest grade of paper and the lowest
polarity solvent yielded the highest effect.
The effect value of the porosity of the qualitative paper, 0.0410, the
polarity of the solvent, 0.0066, and their interaction, -0.0075, were all found to be
statistically insignificant because all of the effect values laid inside of the
boundaries of statistical significance. This boundary was found by multiplying the
range of standards by 2. The calculated effect values represent that there is a
very high probability that the effects occurred purely by chance alone.
The effect value of the porosity was expected to yield the highest effect
value because of the paper used. The type of paper used include Grade 1, 2,
and 3 Whatman Qualitative paper. Grade 3 is significantly more porous and rigid
than the other grades, which makes it more absorbent. When the porosity of the
paper is increased, the solvent has more holes to latch on to therefore resulting
in a speedier climb. The data collected supports the fact that the grade 3
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qualitative paper is more absorbent because it typically yielded a higher solvent
and compound front. Grade 1 Whatman paper is very weak at absorbing solvent
as it is very thin. It does not have an absorbing ability as strong as Grade 3.
Likewise with the porosity of paper, the polarity of the solvents will also
greatly affect the retention factor value. Non-polar molecules will tend to have
little attraction towards the cellulose of the qualitative paper and as a result, lose
its speed which causes the solvent front to retain its distance with the compound
front. The reasoning of this has to do with the adhesion of the solvent. Water has
very strong negative and positive molecular ends. As result, water likes to bond
to itself, also known as cohesiveness. When decreasing the polarity, the solvent
loses its “stickiness” and doesn’t adhere to the paper as well. These findings do
not support the proposed hypothesis, but do support the understanding of why
the highest porosity and lowest polarity had the highest retention factor.
The experiment performed was not flawless and can be improved in
multiple ways. As previously stated in the data and observations, multiple
chromatograms came into contact with the beaker wall. This caused the solvent
front to rush up the paper leaving the compound front behind. The
chromatograms that came into contact with the wall were not included in the data
because they are outliers. These trials were repeated later in the week. Another
oversight that may have altered the results would be the amount of dye placed
on the strip of qualitative paper. A capillary tube was used to place two to three
drops of dye on the paper. The capillary tube was very inconsistent at measuring
31
drops resulting in differing amounts of dye on the qualitative paper therefore
changing the retention factor for the chromatogram.
In order to expand upon the research of chromatography, new
experiments and statistical tests could be performed upon different modern
chromatographic methods, such as liquid or gas chromatography. The findings in
this research could be tested to see if it will impact the efficiency in similar
methods of dissociation or if certain solvents affect the separation of chemicals
more than others. Many industries could be affected by new advances in
chromatography such as its use in the medical field for the identification of
substances in the bloodstream, its use in the analysis of materials in forensics, or
even in the food industry to monitor levels of a specific chemical compound in
drinks. The efficiency and accuracy of chromatography greatly affects many
industries worldwide because it is the basis of the identification of almost any
material and should be seen as a point of study for the future of every scientific
field.
32
Appendix A
Pre-Lab:
1. To prepare the 100 mL of dye solution, add 0.5 g of the FD&C red 40 solid
dye to a separate beaker with 100mL of distilled or deionized water. Mix
thoroughly.
2. To cut the 150mm by 20mm paper strips out of the 150mm paper circles,
first take a properly cut grade 3 strip and place it ontop of the circle so that
it fits inside of it. Once in position, trace the paper strip onto the circle of
choice. Carefully cut out the paper strip using the guidelines that were
drawn. Repeat this until the sufficient amount of paper strips have been
acquired.
3. To prepare 350 mL of 2% isopropyl alcohol solution, measure 10 mL of

70% isopropyl alcohol solution using the 25 mL graduated cylinder. Pour
this into the 600 mL beaker. Measure out 340 mL of distilled or deionized
water using the 100mL graduated cylinder. After measuring each
appropriate amount, pour this into the 600mL beaker containing the 10 mL
of 70% isopropyl alcohol solution. Mix thoroughly.
4. To prepare 333 mL of 2% ethyl alcohol solution, measure 7 mL of 95%

ethyl alcohol solution using the 25 mL graduated cylinder. Pour this into
the 600 mL beaker. Measure out 326 mL of distilled or deionized water
using the 100mL graduated cylinder. After measuring each appropriate
amount, pour this into the 600mL beaker containing the 7 mL of 95% ethyl
alcohol solution. Mix thoroughly.
33
Appendix B: Calculating A Retention Factor
To calculate a retention factor, two numbers are needed. These values
are the distance travelled by the solvent front, and the distance travelled by the
compound front. The equation for calculating the retention factor is shown below.
A sample calculation is shown in figure X below.
𝐷𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑇𝑟𝑎𝑣𝑒𝑙𝑙𝑒𝑑 𝑏𝑦 𝐶𝑜𝑚𝑝𝑜𝑢𝑛𝑑 𝐹𝑟𝑜𝑛𝑡

𝑅𝑓 =
𝐷𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑇𝑟𝑎𝑣𝑒𝑙𝑙𝑒𝑑 𝑏𝑦 𝑆𝑜𝑙𝑣𝑒𝑛𝑡 𝐹𝑟𝑜𝑛𝑡
89
=93 y
=0.9567
Figure 16. Sample Equation for Calculating Retention Factor
Figure 16 shows a sample Rf value calculation where 89 is the distance
travelled by the compound front in millimeters and 93 is the distance travelled by
the solvent front in millimeters.
34
Works Cited
Buie, John. "Evolution of Chromatography Columns." Lab Manager. Lab

Manager, 4 May 2011. Web. 14 Mar. 2017.
"Chemistry for Kids." Ducksters Educational Site. N.p., n.d. Web. 19 Mar. 2017
"Chromatography - RF Values[MarZ Chemistry]." Chromatography - RF

Values[MarZ Chemistry]. N.p., n.d. Web. 17 Mar. 2017.
"Chromatography." Journal of Chromatography A 23 (1966): 184. Modern

Chemical Techniques. Unilever & The Royal Society of Chemistry. Web.
14 Mar. 2017.
"History of Chromatography." History of Chromatography. N.p., n.d. Web. 14

Mar. 2017.
"KhanClark, Jim. "Paper Chromatography." Paper Chromatography. Chem

Guide,
2007. Web. 14 Mar. 2017.
Giddings, J. Calvin, and Roy A. Keller. "Chromatography." Encyclopædia

Britannica. Encyclopædia Britannica, Inc., 17 Nov. 2016. Web. 14 Mar.
2017.
Academy." Khan Academy. Khan Academy, n.d. Web. 14 Mar. 2017.
"Martin, Archer John Porter." Complete Dictionary of Scientific Biography.

Encyclopedia.com, n.d. Web. 16 Mar. 2017.
Maurer, Hans H. "Liquid Chromatography–mass Spectrometry in Forensic and

Clinical Toxicology1." Liquid Chromatography–mass Spectrometry in
Forensic and Clinical Toxicology. Science Direct, 1998. Web. 14 Mar.
2017.
Marz-kreations. "Chromatography - RF Values[MarZ Chemistry]."

Chromatography - RF Values[MarZ Chemistry]. N.p., n.d. Web. 16 Mar.
2017
Miller, James M. "Chromatography." Google Books. Wiley, n.d. Web. 14 Mar.
35
2017.
Morris, Peter T. "Martin, Archer John Porter." Complete Dictionary of Scientific

Biography. Encyclopedia.com, 2008. Web. 15 Mar. 2017.
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Inc., 20 July 1998. Web. 14 Mar. 2017.
Piety, Nathaniel Z., Xiaoxi Yang, Julie Kanter, Seth M. Vignes, Alex George, and
Sergey S. Shevkoplyas. "Validation of a Low-Cost Paper-Based Screening
Test for Sickle Cell Anemia." PLOS ONE. Public Library of Science, 6 Jan.
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Sasser, Myron. Bacteria Identification- MIDI, Inc. (n.d.): n. pag. Identification of

Bacteria by Gas Chromatography of Cellular Fatty Acids. MIDI, May 1990.
Web. 14 Mar. 2017.
Staneck, Joseph L. "Joseph L. Staneck." Applied Microbiology. American Society

For Microbiology, 01 Aug. 1974. Web. 18 Mar. 2017.
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Ezine Articles, 26 June 2006. Web. 14 Mar. 2017.
36

Final Research Paper Ciaramitaro Doerr Weidemann

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Ciaramitaro-Doerr-A.

Reis Ciaramitaro / Kevin Doerr / Andy Weidemann

Macomb Mathematics Science Technology Center

Mrs. Hilliard/Mr. Supal/Mrs. Dewey

May 24, 2017

Data and Observations………………………………………………………...…16

Data Analysis and Interpretation…………………………………………………21

Appendix A. Solution Preparation……………………………………………..…32

Appendix B. Retention Factor Sample Calculation……………………………..33

identify the components of a homogenous mixture. If a mixture was not able to

be separated by means of distillation, it did not have a chance of being separated

pigments in a leaf, using a new method called chromatography (“History of

Chromatography”). The purpose of this experiment is to test the effect of different

types of qualitative paper and polarity of solvents on the retention factors of

FD&C Red 40 food dye.

A design of experiment was conducted over a span of one week to collect

factor and used to determine if the effects were significant. A design of

limit variables by only focusing on few data points of collection.

improved efficiency of a factor. Retention factor values are used in paper

chromatography to help identify compounds by the scientific community when

other forms of chromatography do not apply. Finding an improved method of

developing chromatograms in paper chromatography could be utilized in other

fields of chromatography to refine the process of the identification of compounds.

For example, the use of specific mediums in relation to specific compound

counterparts may drastically enhance the benefits of an analysis by producing an

answer faster without the need of an excessive amount of other materials

deeper understanding of porosity and polarity on paper chromatography, so

the optimum combination of solvent and qualitative paper in an experiment.

Chromatography, or the separation and identification of the parts in a

homogenous mixture, has been used in innumerable scientific fields.

Homogenous mixtures, opposed to heterogeneous mixtures, have limited means

of separation, so many different forms of chromatography are required to analyze

certain substances that the mixture is composed of. Many forms of

chromatography have been developed throughout the last century in order to

evaluate specific substances depending on their characteristics. The most

commonly used forms of chromatography include gas chromatography and liquid

chromatography, although twelve total techniques exist.

Figure 1. Homogeneous and Heterogenous Mixtures

Figure 1 above shows the difference between Homogeneous and

published by Ducksters. Heterogeneous mixtures are not uniform throughout and

can be separated easily. Homogeneous mixtures are uniform throughout and

have to be separated using different techniques.

Chromatography and chromatographic concepts have been used since

chromatography has since evolved into a vital tool in laboratories worldwide.

preparative processes spanning many scientific fields.

Paper Chromatography, in analytical chemistry, is a technique for

separating dissolved substances by taking advantage of their different rates of

movement across segments of paper. It is inexpensive, but it is also a powerful

analytical tool that aids in separation and identification of components in a

mixture to correctly separate and analyze (“Paper Chromatography”).

Forms of chromatography, including paper, column, thin-layer, and size

exclusion chromatography, use stationary and mobile phases to examine the

retention factor of substances. This means that these forms of chromatography

chromatography, the idea stays the same (“Principles of Chromatography, Khan

as the retention value.

The method of chromatography that will be used in this experiment will

determined using a simple calculation requiring the distance travelled by the

movement of the solvent.

= 𝐷𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑙𝑒𝑑 𝑏𝑦 𝑠𝑜𝑙𝑢𝑡𝑒

÷ 𝐷𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑙𝑒𝑑 𝑏𝑦 𝑠𝑜𝑙𝑣𝑒𝑛𝑡 𝑓𝑟𝑜𝑛𝑡

(Chromatography - RF Values[MarZ Chemistry]”).

Figure 2. How Retention Values Are Calculated

Shown above is how an example retention value is calculated. The solvent