Paper Chromatography Lab

Prepared for: Mrs. Freeman

September 6, 2013

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Introduction

The separation of mixtures is an important part of chemistry. One such method of

separation is called chromatography, which utilizes the solubility differences in substances (of a

mixture) in a given solvent. In its most basic form, chromatography is used to separate a mixture

into its components.

There are many different kinds of chromatography, including column chromatography,

high performance liquid chromatography, gas chromatography, thin layer chromatography and

paper chromatography. However, they all use the same principle: they all have a stationary phase

and a mobile phase. The mobile phase, which is a liquid or gas, flows through the stationary

phase (solid, or a liquid on a solid), and carries the components of the mixture with it. What

separates the components are the rate at which they move across the stationary phase and the

distance they travel. This travel (or lack thereof) is caused by an affinity to either the stationary

phase, the mobile phase, or both (General Chemistry Online, 2011)

This lab uses paper chromatography as opposed to other forms of chromatography. In

paper chromatography, the stationary phase is the chromatography paper, and the mobile phase

is a liquid solvent. A line is drawn almost at the end of the paper, and a drop of the mixture is put

on the line. The paper below the line is put into the solvent, as to allow the solvent to diffuse up

the paper. If the solvent is polar, the components of the mixture that are polar to some extent will

dissolve in the solvent and travel up the paper, as opposed to any other nonpolar components.

This is reversed when using a nonpolar solvent. In most cases, the components of the mixture

will have slightly different polarities, and will travel different differences up the paper (Clark,

2012)

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 After this point, an Rf value (distance travelled relative to the solvent) can be calculated

for each component. To obtain this, the distance travelled by the component is divided by the

distance travelled by the solvent. These can be used to compare the components of a known

mixture vs. an unknown mixture.

Purpose

In this lab, you will use 5 known acid base indicators and their Rf values to analyze two

unknown substances. Acid base indicators are usually colorful, which change colors according to

the acidity of the substances they are present with.

Methods

1. Place all strips of chromatography paper on flat, dry surface.

2. Mark a line with pencil 2 centimeters from the end of each strip.

3. Dip pipette into one of the acid base indicators and touch the tip to the filter paper (not

chromatography paper). The spot of liquid should be no more than 5 mm in diameter.

Repeat until application procedure has been mastered.

4. Dip pipette back into the indicator solution and place a spot in the middle of the line on

one of the strips of chromatography paper. Immediately label the paper as to identify

which indicator was placed on this strip.

5. Repeat step 4 for each of the remaining indicators and the two unknown samples. Use a

clean pipette for each substance.

6. Fill 4 flasks with 25 mL of Ethanol saturated with NH3 (solvent). Stopper each flask

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7. After the strips are dried, remove the stopper from one of the flasks and position two

strips on either side of the stopper.

8. Place the stopper and the strips so that the solvent touches the strip below the pencil line,

and so that the sample spots do not come in contact with the solvent.

9. Repeat steps 7 and 8 for the remaining strips.

10. Allow the solvent to diffuse up the strips until the solvent front (the highest point of

solvent saturation) is at least 9 cm above the pencil lines.

11. Remove the strips and place them on a dry sheet of paper. Allow them to dry for a few

seconds

12. Draw a pencil line on the solvent front of each strip.

13. Draw a point in the middle of the sample spot. If this is not possible because of the paper

still being wet, poke a small hole in the middle of the sample spot. Note: phenolphthalein

spot will become colorless quickly.

14. When the strips have completely dried, measure the distance the sample spot(s) is from

the pencil line for each strip. In addition, measure the distance from the solvent front to

the pencil line. Record this in data table.

15. Clean the station and wash hands.

16. Calculate the Rf values for each sample spot and analyze the unknown substances.

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Data

Table 1- Paper Chromatography of pH Indicators

Distance solvent moved Distance sample moved

Sample Rf Values
(cm) (cm)

Bromothymol blue 5.11 cm 4.62 cm .904

Congo Red 5.55 cm 0.00 cm 0.00

Methyl orange 6.00 cm 4.69 cm .782

Phenol red 5.09 cm 4.38 cm .861

Phenolphthalein 5.03 cm 4.28 cm .851

4.71 cm 3.61 cm .766

Unknown A
4.71 cm 0.00 cm 0.00

5.19 cm 4.08 .786

Unknown B
5.19 cm 4.98 cm .960

NOTE:

𝑚𝑖𝑔𝑟𝑎𝑡𝑖𝑜𝑛 𝑑𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑜𝑓 𝑠𝑢𝑏𝑠𝑡𝑎𝑛𝑐𝑒

ℛ𝒻 =
𝑚𝑖𝑔𝑟𝑎𝑡𝑖𝑜𝑛 𝑑𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑜𝑓 𝑠𝑜𝑙𝑣𝑒𝑛𝑡 𝑓𝑟𝑜𝑛𝑡

Analysis/Discussion

As the results indicate, Unknown A was composed of both Congo red (Rf = 0.00) and

Methyl orange (Rf = .782), and Unknown B was composed of Methyl orange (Rf = .782) and

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Bromothymol blue (Rf = .904). This is clear because Unknown A had two sample spots,

separated from the original sample spot, and the Rf values of both (Rf = .766, Rf = 0.00)

corresponded closely enough to only two of the indicators, Methyl orange (Rf = .782) and Congo

red (Rf =0.00), respectively. Similarly, the two Rf values of Unknown B (Rf = .786, Rf = .960

corresponded with the Rf values of both Methyl orange (Rf = .782) and Bromothymol blue (Rf

=.904), respectively. In addition to the quantitative observation, a qualitative observation can be

made as to the colors of the sample spots, which corresponded accordingly (matching the Rf

value result). While the Rf values were not exactly the same, they were close enough to create a

solid conclusion. Because of a time constraint, my lab partners and I were not able to keep the

strips of chromatography paper in the solvent until the specified 9 cm in the directions. Perhaps

if this had been completed, the Rf values might have been even closer.

Looking at the Rf values of all the different indicators, we can see that they all have very

different values. There are several reasons for this. Our solvent (ethanol saturated with NH3) was

extremely polar, and consequently, the more polar indicators moved farther up the strips. For

example, we can conclude that Congo red was completely non-polar, as it did not dissolve and

travel up the strip at all, while Bromothymol blue was the most polar, as it had the highest Rf of

.904. In addition, while the solvent front of all the strips were not uniform, this is insignificant.

Because an Rf value is essentially a ratio, and each indicator’s travel is rationally proportional to

the solvent travel, the Rf values are sound.

Conclusions

At the conclusion of the experiment, we can conclude that Unknown A was made up of Congo

red and Methyl orange, and Unknown B was composed of Methyl orange and Bromothymol blue

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References

Chromatography. (n.d.). General Chemistry Online. Retrieved September 5, 2013, from

antoine.frostburg.edu/chem/senese/101/matter/chromatography.shtml

Clark, J. (n.d.). paper chromatography. chemguide: helping you to understand Chemistry - Main

Menu. Retrieved September 5, 2013, from

http://www.chemguide.co.uk/analysis/chromatography/paper.html#top