Paper Chromatography Report

7/14/2019 Paper Chromatography Report

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ABSTRACT

Amino acids are found everywhere, from the cell

composition up to the proteins that build up our body. Paper

chromatography is a process where we can identify and separate

desired compounds. In order to study the components of a mixture, polarity was used as the main factor and supported by the pH. The

eluting solvent used was a mixture of isopropanol and water, this is

important since the ratio to front value which is used for comparison to identify an unknown compound is dependent on the

distance it travels during a given time. The water is the mobile

phase where in it doesn’t attract non-polar substances causing it torise up the cellulose paper while the interaction of polar substances

to the slightly less polar than water solvent; isopropanol slows its

movement upwards. Spotting of respective amino acids were done

and then introduced to the set-up containing the eluting solvent.There were two unknown spots that formed after doing the

procedures; the higher spot with the highest Rf value 0.58 isleucine while the lower spot of the two trials have an average Rf

value of 0.35 which is closest to glutamic acid/ glutamic acid. Theobtained Rf values are significantly the same that of the theoretical

ones.

INTRODUCTION

Separation is a vital process that can involve different activities. This exercise focuses on

the separation and identification of specific amino acids. Amino acids contain two functional

groups, an amino group (-NH2) and a carboxylic acid group (-COOH) (Timberlake, 2004). The

specific process of separating mixtures of compounds, specifically amino acids into individualcomponents is called chromatography.

All chromatographic methods depend on the distribution of the substances being

separated between two phases of the chromatographic system, a mobile phase and a stationary phase. The mobile phase consists of a liquid or gas that carries the sample through the solid or

liquid that forms the stationary phase. In partition chromatography, the compounds to be

separated partition themselves between a stationary liquid phase and a mobile liquid or gas phase. This partitioning occurs in the same way a solute partitions itself between two immiscible

solvents used for an extraction. The greater the attraction a compound has for the stationary

liquid phase the slower it travels through the chromatography column (Mohrig et. al., 2006).

In paper chromatography, the stationary phase is the water. The mobile phase is a suitableliquid solvent or mixture of solvents, in this case isopropyl alcohol was used (due to its polarity).

Some compounds in a mixture travel almost as far as the solvent does; some stay much closer tothe base line. The distance travelled relative to the solvent is a constant for a particular

compound as long as you keep everything else constant - the type of paper and the exact

composition of the solvent. The distance travelled relative to the solvent front is called the Rf value (Clark, 2007). Paper Chromatography is a liquid-liquid partition chromatography

(Pedersen and Myers 2011).



Compounds that are water soluble or that have the ability to hydrogen bond are more

soluble in the stationary phase and do not migrate as readily as less polar molecules.

According to Frostburg University, ninhydrin (1,2,3-Indantrione monohydrate, or

triketohydrindene hydrate) is often used to detect -amino acids and also free amino and

carboxylic acid groups on proteins and peptides. When about 0.5 mL of a 0.1% solution of ninhydrin is boiled for one or two minutes with a few mL of dilute amino acid or protein

solution, a blue color develops. A ninhydrin solution in ethanol or other volatile solvents is often

used as a developer for amino acids in paper chromatography or thin layer chromatography.

Figure 1. Ninhydrin, an organic compound.

The ratio to front value (Rf value) was mentioned earlier. Aside from the distances

measured to find the Rf value, the physical appearance should also be considered.

OBJECTIVES

To learn the techniques of and understand the principles behind paper chromatography. To apply paper chromatography in separating components of a mixture of amino acids. To identify the composition of the unknown solution by comparing the ratio to front

value with other characteristics of those standard.

MATERIALS AND METHODS

There were a few changes in the procedure made. In the laboratory manual the eluting

solvent was not a ratio of glacial acetic acid, n-butyl alcohol and water, instead a ratio of

isopropyl and water formed the solvent used. Another is in the spotting experiment; instead of 4

spots, 8 spots were made. For the materials used and the complete procedure refer to page 58 to

59 of the chemistry 31.1 laboratory manual.

PRESENTATION OF RESULTS

Table 1. Ratio of the eluting solvent used.

Solvent Ratio Volume (mL)

Isopropanol 4 16Water 1 4

Total= 5 20



Table 2. Distances formed by the spots in the chromatography paper .

Amino AcidDistance travelled by

sample spot from origin

(cm)

Distance travelled bysolvent from the origin

(cm)

o Threonine x 2.65 8.0

o Threonine y 2.95 8.0o Leucine 5.15 8.1

o Alanine 2.70 8.0

o Tyrosine 2.69 8.0

o Glutamic acid 2.35 8.1

Lower Spot Higher Spot

o Unknown x 2.20 4.7 8.1

o Unknown y 2.35 4.7 8.1

Ratio to Front Values: Formula used:

Legend: Colors of the amino acid in words are the color of their spots in figure 2.

Figure 2. A picture of the chromatography paper with spots of amino acids.

SOLVENT FRONT



DISCUSSION OF RESULTS

Amino acids are the building blocks of proteins. These organic compounds are studied in

the experiment. The paper chromatography set up was covered with aluminium foil. The reason

for covering the container is to make sure that the atmosphere in the beaker is saturated with

solvent vapour. Saturating the atmosphere in the beaker with vapour stops the solvent from

evaporating as it rises up the paper

Figuratively glutamic acid nonetheless is the first unknown amino acid; the purple spot is

the closest to the silver ones. Its acquired value is 0.29 which is so close that of the theoretical

value 0.30 the lower points of the two trials of the unknown has also a close value to the valuessaid earlier; 0.272 and 0.290. Physically the spots are alike the three were dark and spherical.

The second, the spot above the first unknown amino acid is leucine. The yellow spots have a

similar distance that of the blue spot. The value of leucine is 0.639 close enough to the

theoretical value which is 0.73 while the average value of the unknown is 0.580. Errors can beaccounted for such variations of values. Lack of time; the solvent front was not in its desired

position which is 5 mm away the top side. Another is the uneven plane where the beaker (the set-

up) was placed causing an unsystematic reading of the solvent front (it is seen in the values thatthere are 0.1 variations of the distance travelled by solvent) and of course human errors in

handling the chromatography paper.

Even if amino acids are called “acids” not all have an ac idic pH, this is another factor on

explaining the distance these compounds have travelled. All are neutral in nature except glutamic

acid. The eluting solvent is a little bit higher than 7 making it slighty basic. The neutral aminoacids tend to go up with the solvent while glutamic acid is attracted to the stationary phase and

do not migrate as readily as less polar molecules.

Non-polar amino acids will have little attraction for the water molecules attached to the

cellulose (the paper), and so will spend most of their time dissolved in the moving solvent.

Molecules of leucine (0.639 Rf computed) and alanine (0.338 Rf computed) therefore travelled along way up the paper carried by the solvent. They had relatively high Rf values. On the other

hand, polar molecules like threonine (0.350 ave. Rf computed) and tyrosine (0.336 Rf computed)

will have a high attraction for the water molecules and less for the isopropanol (slightly less polar than water). Because they spend more time dissolved in the stationary phase and less time

in the mobile phase, they aren't going to travel very fast up the paper. The least is the acid since

it was really attracted to the eluting solvent due to its polarity. The theoretical values and

polarities of said amino acids are shown below.

Sources: http://www.biotopics.co.uk/as/amino_acid_chromatography.html and Ehlmurst College

of Chemistry.

Amino Acid Theoretical Rf ValueAlanine 0.38

Glutamic Acid 0.30

Leucine 0.73Threonine 0.35

Tyrosine 0.45

Amino Acid PolarityAlanine Non-polar

Glutamic Acid Polar

Leucine Non-polar

Threonine Polar

Tyrosine Polar

Table 3. Theoretical values of studied amino acids. Table 2. Polarity of said amino acids.

http://www.biotopics.co.uk/as/amino_acid_chromatography.html

http://www.biotopics.co.uk/as/amino_acid_chromatography.html



CONCLUSION

Chromatography is a physical mean of separation. In paper chromatography we learned

to identify using the ratio to front values/ the retention factor. Accuracy depends on how the

distances are measured and therefore the center of gravity is a very important measure in finding

the Rf value. There were only two unknown spots. Qualitatively it can already be deduced thatthe unknowns are leucine and glutamic acid, but it is still a rough measure that’s why retention

factor was used for assessing efficiency of the process. There was a 0.059 difference between the

unknown y values and leucine which therefore supports the claim that the higher unkown spot isleucine. For the unknown x values with an average of 0.281 Rf value, the closest is glutamic acid

and is proven by the closeness to the acquired value; 0.290 and the theoretical value 0.30.

Tyrosine was a tailing spot and worse it was so thin thus finding the center of gravity was hard.

For threonine and alanine the spots were dark enough and spherical which was easy to measure.

Next time the set-up should stand longer, the students should wait until the solvent frontreaches 5mm from the top of the paper. Also spotting should be done well. The purity of the

developing/ eluting solvent is an important factor in the success of the experiment; it may

interfere in the right placement of the distances of the spots, its polarity should always beconsidered. Errors were still present in the duration of the activity which is limited to the ratio to

front value and physical appearance only there is no measuring of the amount of amino acid doneand the ratio of the components found in the unknown mixture, only identification and

separation.

ANSWERS TO SELECTED QUESTIONS

Prelaboratory Questions:

1.) Explain the effect of having a broad sample spot on the chromatogram.

Broad sample spots are caused by dropping a large amount of an amino acid in the paper.You will have an inaccurate center of gravity which affects the distance travelled by sample fromorigin which affects your ratio to front value. The broad sample might also overlap other spots

nearby so it is advisable to have at least 2 cm space in between spots.

2.) What problem will arise if the level of developing liquid is higher than the sample spot?

The sample will be washed away into the developing liquid which hinders it to move

upwards since there’s attraction between the eluting solvent and the amino acids . When it’s time

to spray ninhydrin you will not see definite spots (dark spots) that represent your amino acids. It

will affect your experiment thus you should conduct it again.

3.) How important is a good choice of solvent mixture to chromatographic analysis?

The distances of the amino acids from the respected origin depend on what solvent wasused. The pH and the polarity of the solvent to be used are important since the amino acids have

specific reactions for such solvents. The solvent mixture should have water and another miscible

compound. The greater the attraction a compound has for the stationary liquid phase the slower it

travels through the cellulose paper.



Postlaboratory Questions:

1.) Look up for the structures of amino acids involved in the experiment. Explain on structural

bases the order in which they are found in the chromatogram.

Figure 3. Amino acid structures retrieved from: Timberlake, K. C. (2004). Chemistry: Structures

of Life.

Glutamic acid is the most soluble in water so it has the lowest Rf value and you can see

it’s the closest to the origin. Threonine is polar and has a simpler structure making it more polar

than glutamic acid thus it is farther from the origin. The middle amino acid is alanine it is non-

polar and has a simpler structure than tyrosine which alycyclic (4th

from the bottom), even if it is

polar it has a lot of carbons making it more complex. Lastly at the top is leucine for beinghydrophobic and a non-polar compound having a lot more carbon than alanine. The lowest

amino acid which is the glutamic acid has a different pH than the others the explanation why it is

found beneath the others are stated earlier in the discussion.

2.) What variables must be included when reporting Rf values?

The distances are vital to report the ratio to front values. The solvent’s and the spot’sdistance from the origin should be specified. The physical appearance of the spots should also be

considered. The pH, also helps in determining the accuracy of one’s sample and of course the polarity.

3.) What is the difference between paper chromatography and thin-layer chromstography?

TLC has a solid stationary phase unlike the paper chromatography. Both of them have a

liquid mobile phase. Paper chrom. is a type of partition chromatography while TLC is under theadsorption chromatography where the compound travels depending on how tight they adsorb to

the stationary phase but still the factor that greatly affects movements of spots in the two

methods depend on polarity.



REFERENCES

Book Sources:

Mohrig, J. R., Hammond, C. N. and Schatz, P. F. (2006). Techniques in Organic Chemistry:

Miniscale, Standard Taper Microscale and Williamson Microscale. 2

nd

ed. New York. W.H. Freeman and company. Pp. 175-178.

Pedersen, S. F., and Myers, A. M. (2011). Undrstanding the Principles of Organic Chemistry: A

Laboratory Course. Canada. Brooks/Cole, Cengage Learning. P. 135.

Timberlake, K. C. (2004). Chemistry: Structures of Life. San Francisco, California. Pearson

Education Incorporated. Pp. 644-645.

Internet Sources:

http://www.marz-kreations.com/Chemistry/Chromatography/Dyes/RF-Values.html

http://antoine.frostburg.edu/chem/senese/101/organic/faq/amino-acid-test.shtml

http://www.elmhurst.edu/~chm/vchembook/561aminostructure.html

http://galileo.phys.virginia.edu/outreach/8thGradeSOL/ChromatographyFrm.htm

http://web.mit.edu/7.88j/www/restricted/Wolfenden-88.pdf

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3092165/

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3092165/











Exercise No. 4

Paper Chromatography1

Bayquen, Carla Louise B.

II-BSBGroup 2 Section EF-1L

Cartojano, Thea Danielle

Omalza, Frederick

Zailon, Zamzam

July 11, 2013

1A post laboratory report in partial fulfilment of the requirements in Organic ChemistryLaboratory under Prof. Joel Tolentino, 1

stsemester, AY 2013-2014.

Paper Chromatography Report

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