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 to rise 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 is leucine while the lower spot of the two trials have an average Rf value of 0.35 which is closest to glutamic acid/ glutamic acid. The obtained 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 individual components 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 suitable liquid 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 to the 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 16

Water 1 4Total= 5 20

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

Amino AcidDistance travelled by

sample spot from origin (cm)

Distance travelled by solvent from the origin

(cm)o Threonine x 2.65 8.0o Threonine y 2.95 8.0o Leucine 5.15 8.1o Alanine 2.70 8.0o Tyrosine 2.69 8.0o Glutamic acid 2.35 8.1

Lower Spot Higher Spoto Unknown x 2.20 4.7 8.1o Unknown y 2.35 4.7 8.1

Ratio to Front Values: Formula used: Rf=Distance travelled by sample spot ¿ t heorigin ¿

Distance travelled by solvent front ¿t he origin¿

Threonine x Rf=2.65cm8.0 cm

=0.331

Threonine y Rf=2.95cm8.0cm

=0.369

Leucine Rf=5.15cm8.1cm

=0.639

AlanineRf=2.7 0cm8.0cm

=0.338

Tyrosine Rf=2.69cm8.0cm

=0. 33 6

Glutamic Acid Rf=2.35cm8.1cm

=0. 290

Unknown x1 Rf=2.2cm8.1cm

=0.272

Unknown x 2Rf=2.35cm8.1cm

=0.290

Unknown y 1Rf= 4.7 0cm8.1cm

=0.580

Unknown y 2Rf=4.7 0cm8.1cm

=0.580

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.

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 values said 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 be accounted 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 that there 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 acidic 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 amino acids 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 a long way up the paper carried by the solvent. They had relatively high Rf values. On the other

SOLVENT FRONT

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.

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 that the 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 is leucine. 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 front reaches 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 be considered. 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 done and 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.

Amino Acid Theoretical Rf ValueAlanine 0.38Glutamic Acid

0.30

Leucine 0.73Threonine 0.35Tyrosine 0.45

Amino Acid PolarityAlanine Non-polarGlutamic Acid PolarLeucine Non-polarThreonine PolarTyrosine Polar

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 from origin 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 was used. 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 (4 th 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 being hydrophobic 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’s distance 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 the adsorption 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. 2nd 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. 4Paper Chromatography1

Bayquen, Carla Louise B.II-BSB

Group 2 Section EF-1L

Cartojano, Thea DanielleOmalza, FrederickZailon, Zamzam

July 11, 2013

1A post laboratory report in partial fulfilment of the requirements in Organic Chemistry Laboratory under Prof. Joel Tolentino, 1st semester, AY 2013-2014.