IDENTIFICATION OF SUGARS BY PAPER CHROMATOGRAPHY

-Activity III-

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A Report Presented to Dr. Rodina Rivera-Gorospe Professor-Biochemistry College of Medicine and Surgery Cagayan State University

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In Partial Fulfillment of The Requirements in Biochemistry-Laboratory First Semester, SY 2009-2010

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Presented by: Cruz, Prince Lloyd Isabelo III (Leader) Dickpus, Reicha Jan Dines, Janice (Secretary) Dumelod, Faye Shamaine Flores, Emmanuela Ghosh, Shyamali

PAPER CHROMATOGRAPHY I. INTRODUCTION Chromatography, in general, is a procedure used to separate and identify substances in a mixture based on the different mobilities of the components. All forms of chromatography work on the same principle. The sample to be examined (called the solute) is allowed to interact with two physically distinct entities a mobile phase and a stationary phase. The mobile phase, which may be a gas or liquid, moves the sample through a region containing the solid or liquid stationary phase (called the solvent). The stationary phase may be considered as having the ability to bind some types of solutes. The sample, which may contain one or many molecular components, comes into contact with the stationary phase. The components distribute themselves between the mobile and stationary phases. If some of the sample components are preferentially bound by the stationary phase, they spend more time in the stationary phase and, hence, are retarded in their movement through the chromatography system. Molecules that may show weak affinity for the stationary phase spend more time with the mobile phase and are more rapidly removed or eluted from the system. The many interactions that occur between solute molecules and the stationary phase bring about a separation of molecules because of different affinities for the stationary phase. The general process of moving a solute mixture through a chromatographic system is called development. Paper chromatography is one method for testing the purity of compounds and identifying substances. Paper chromatography is a useful technique because it is relatively quick and requires small quantities of material. In paper chromatography, substances are distributed between a stationary phase and a mobile phase. The stationary phase is usually a piece of high quality filter paper. The mobile phase is a developing solution (water or another solvent like alcohol) that travels up the stationary phase, carrying the samples with it. Components of the sample will separate on the stationary phase according to how strongly they adsorb to the stationary phase versus how much they dissolve in the mobile phase. There are two types of development involved in paper chromatography: ascending and descending. Ascending development refers to that in which the mobile phase move against gravity while descending development refers to which the mobile phase moves with gravity.

II. OBJECTIVES This experiment aimed to: 1. determine the unknown sugar based on the Rf values obtained 2. determine the principle of paper chromatography

II. METHODOLOGY A. Materials/Reagents 1. Standard solutions of: a. galactose b. glucose c. fructose

d. maltose e. lactose f. sucrose

- These are sugars to which the unknown solution will be compared to. 2. Unknown sugar solution - This is the substance to be identified in this experiment. Given unknown sugars vary per group. 3. Solvent system: butanol:ethanol:water - The solvent system is comprised of polar solvents (e.g. alcohols) which do mix (miscible) with water to serve as the mobile phase. 4. Aniline acid oxalate spray - It is a spraying reagent for filter paper chromatography of sugars which is soluble in moist butanol. It is used to make a specific coloured complex with the sugar in order to see the spots better. 5. Chromatography chamber/jar - A developing chamber should be one that can be sealed well. The chamber should be large enough to hold the paper that is to be developed. The chamber should be clean and dry before use. The mobile phase is added to the chamber so that it is about 2 cm deep. The reason for covering the container is to make sure that the atmosphere in the chamber is saturated with solvent vapour. Saturating the atmosphere in the beaker with vapour stops the solvent from evaporating as it rises up the paper. As the solvent slowly travels up the paper, the different components of the mixtures travel at different rates. 6. Filter paper - The paper is made of cellulose fibers, and cellulose is a polymer of the simple sugar, glucose.

- The key point about cellulose is that the polymer chains have -OH groups sticking out all around them.

- The complication arises because the cellulose fibres attract water vapour from the atmosphere as well as any water that was present when the paper was made, hence the paper as being cellulose fibers with a very thin layer of water molecules bound to the surface. It is the interaction with this water which is the most important effect during paper chromatography. Non-polar molecules in the mixture that are being tried to be separated will have little attraction for the water molecules attached to the cellulose, and so will spend most of their time dissolved in the moving solvent. Molecules like this will therefore travel a long way up the paper carried by the solvent. They will have relatively high Rf values. On the other hand, polar molecules will have a high attraction for the water molecules. They will therefore tend to dissolve in the thin layer of water around the cellulose fibers much more than in the moving solvent. Because they spend more time dissolved in the stationary phase and less time in the mobile phase, they are not going to travel very fast up the paper.

B. Procedure > Preparing the stationary phase 1. Measure about 2cm from edges of the filter paper (horizontal and vertical slides) and mark using a pencil. Draw a line across the paper connecting the marks. This will serve as the baseline where samples will be applied.

2. Divide the baseline such that 7 spots can be accommodated within the whole length. Allow about 3 cm distance between spots.

3. Mark each spot with pencil and label accordingly with the name of the standard sugars to be used. Leave one spot for your unknown. 4. Insert the filter paper between the pages of the book, such that the baseline mark is exposed. Place the book in an inclined position on the table taking care not to allow the edge of the paper to touch the table. > Spotting the samples 5. Using a capillary pipette, apply about 4 uL each of the unknown sugar solution and the standard sugar solution (roughly 240 ug sample) on corresponding spots on the paper. Application should be done 3 to 4 times of each spot, drying the spot in between applications. Make sure the spot is as small as possible without exceeding 3mm in diameter. Too big spots will make samples overlap and influence the movement of solids.

> Preparing the mobile phase 6. While drying the spots, after all sugar solutions have been spotted, introduce the solvent into the chromatography chamber or jar. Cover and allow the solvent to equilibrate for 5 minutes. > Developing the chromatogram 7. When the spots have dried, place the paper on the trough containing the solvent (or alternatively, roll the paper spots facing out, and staple the ends before placing in the chromatographic jar). The edge of the paper where the sugars have been spotted should be placed nearer the trough but the spots should not touch the solvent.

8. Allow the solvent to permeate through the paper until the solvent front has reached the pencil mark at the top of the paper. Mark the position of the solvent front (or allow chromatography to proceed for 4 hours taking care not to run the spots off the paper).

Height reached by the solvent: the solvent front

9. Remove the chromatogram and dry in air. > Identifying the spots 10. Spray with aniline acid oxalate thoroughly (but not dipping wet) and heat in an oven at 100 degrees Celsius until the spots become visible.

11. Trace the outlines of the colored spots with pencil and measure the distance (in cm) traveled by each sugar from the baseline to the center of each colored spot.

Measurement of the distance travelled by the solvent front starts from the baseline to its highest point, as shown in the figure below.

IV. RESULTS & DISCUSSION Following the steps above, the five groups of the Biochemistry class in Medicine 1 - section B came up with the following data:

Table 1. The Distance Travelled by the Different Sugars Per Group Sugar 1. Galactose 2. Glucose 3. Fructose 4. Maltose 5. Lactose 6. Sucrose 7. Unknown Distance Travelled by Sugars (cm) Group Group 2 Group 3 Group 4 Group 5 1 1.1 1.5 1.6 1.5 1.2 2.1 2.1 1.6 2.1 1.7 1.4 0.8 1.0 0.9 0.7 0.6 0.5 0.5 0.7 0.5 1.4 1.1 1.2 1.5 1.1 1.7 1.9 0.8 0.5 Range (cm) 1.1 1.6 1.6 2.1 0.7 1.4 0.5 0.7 1.1 1.5 Ave. (cm) 1.38 1.92 0.96 0.56 1.26

Table 2. Distance Travelled by Solvent Front Per Group Group No. 1 2 3 4 5 Distance Travelled by Solvent Front (cm) 14.7 14.7 15.3 15 14.7 Range (cm) Average (cm)

14.7 15.3

14.88

Rf Value
- is the distance travelled relative to the solvent. The Rf value for each spot should be calculated. Rf stands for "ratio of fronts" and is characteristic for any given compound. Hence, known Rf values can be compared to those of unknown substances to aid in their identifications. - For each compound, it can be worked out using the formula: Rf= Distance traveled from start to center of substance spot x100 Distance traveled from start to solvent front (Note: Rf values often depend on the temperature, solvent, and type of paper used in the experiment; the most effective way to identify a compound is to spot known substances next to unknown substances on the same chromatogram.) - If R value of a solution is zero, the solute remains in the stationary phase and thus it is immobile. If R value = 1 then the solute has no affinity for the stationary phase and travels with the solvent front.

Table 3. Calculated Rf Values of Sugars Per Group Calculated Rf Values of Sugars Sugar Group 1 Group 2 Group 3 Group 4 Group 5 1. Galactose 7.48 10.2 10.46 10 8.16 2. Glucose 14.29 14.28 10.46 14 11.56 3. Fructose 4. Maltose 9.52 5.44 6.54 6 4.76 5. Lactose 4.08 3.4 3.27 4.66 3.4 6. Sucrose 9.52 7.48 7.84 10 7.48 7. Unknown 11.56 12.92 5.33 3.4

Range 7.48-10.46 10.46-14.97 4.76-9.52 3.27-4.66 7.48-10

Ave. 9.26 12.92 6.45 3.76 8.46

ANSWERS TO QUESTIONS:

1. Q: Based on the Rf values obtained, which sugar is most likely your unknown? A: Based on the computed Rf values, we therefore conclude that unknown sugars are the following: Group 1 Maltose/Sucrose (?) Group 2 Glucose Group 3 Fructose Group 4 Maltose Group 5 Lactose 2. Q: In case the solvent ran off the paper, i.e., the solvent exceeded the solvent front, how will you compute for the Rf values. A: Rf = excess distance traveled by solute distance traveled by the solvents x 100 Distance traveled by the solution 3. Q: What is the principle behind the visualization of color spots on the chromatogram? A: The principle behind the visualization of color spots on the chromatogram involved the spraying of the chromatogram with aniline acid oxalate spray (which is a specific reagent for carbohydrates) then heating in the oven where the organic compounds (carbohydrate) gave the appearance of charred spots.