Chromatography Experiment Lab Report

Purpose:

The purpose of this experiment is to detect the various contents of a mixture and separate various substances using chromatography.

Introduction:

This experiment investigates the effect of different solvents on substances in a mixture using chromatography. Chromatography is based on the principle that it allows the separation of molecules in a mixture by moving them from a stationary phase to a mobile phase (Coskun, 2016). The stationary phase remains fixed, either as a liquid or solid, while the mobile phase carries the solvent (Asante, 2016).

The ability to isolate complex mixtures is crucial in various fields, such as the pharmaceutical industry, to ensure the safety of products for patients (Kisley and Landes, 2014).

Apparatus and Chemicals:

• 3x beaker
• 3x glass rod
• 3x bulldog clip
• 4x chromatography paper
• 4x capillary tube
• 4x teat pipette
• Pencil
• Pestle and mortar
• Glass tube

Protocol:

The following steps were followed to conduct the chromatography experiment:

1. The mortar was filled with cut-up leaves to a depth of 2cm and mixed with approximately 5-6 drops of ethanol from the teat pipette, along with a small amount of sand.
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2. The mixture was ground using the pestle for three minutes.
3. A marked line was drawn 3cm from the bottom of the chromatography paper using a pencil.
4. A small amount of solute from the mixture was transferred onto the center of the drawn line using a glass tube, ensuring the spot was small in size.
5. A concentrated spot was created by repeating this process five times, allowing the spot to dry each time.
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6. The chromatography paper was attached to the pencil.
7. A beaker was filled with ethanol up to 10cm³, and the paper was placed inside the ethanol, ensuring the solution remained below the spot.
8. Once the ethanol had soaked to near the top of the paper, the paper was removed, and a mark was created to indicate how high the ethanol had traveled. These results were recorded.
9. The procedure was repeated, replacing ethanol with isopropanol, propanol, and a mixture of all three solutions. All results were recorded.

Results:

* All results are to 2 decimal places.

The calculated Rf values for each solution are obtained using the formula: Rf = Spot Distance / Solvent Front

Discussion:

The results clearly demonstrate a relationship between the polarity of compounds and their Rf values. Higher Rf values indicate less polar compounds, while lower Rf values suggest non-polar compounds (Bele and Khale, 2011). Polar substances tend to dissolve more in polar solvents, while non-polar substances dissolve more in non-polar solvents (Ng and Kushairi, 2017).

In this experiment, the pigments in plant leaves, such as chlorophyll and carotenoids, are non-polar. When dipped in non-polar solvents like propanone, they exhibit the highest Rf values, indicating a stronger affinity for the non-polar solvent and spending more time in the mobile phase. Conversely, when dipped in ethanol, which is more polar, the pigments exhibit lower Rf values, suggesting less interaction with the solvent and more time in the stationary phase.

However, it's worth noting that isopropanol should have theoretically shown a higher Rf value than ethanol due to its higher polarity (osu.edu, 2022). The discrepancy in the results could be attributed to experimental errors, such as incomplete adsorption, which might explain the lower values. To minimize errors, it is essential to allow the paper to fully dissolve in the solvent to obtain more accurate readings.

Comparing the experimental Rf values with published data (Table 2), the pigments in the experiment can be identified. For example, an Rf value of approximately 0.45 corresponds to chlorophyll a when dipped in isopropanol. Additionally, an Rf value of 0.82, obtained when dipped in ethanol, corresponds to phaeophytin, while dipping in propanone (Rf = 0.89) suggests the presence of carotene (Rf = 0.95).

Table 2: Rf values of pigments (pearsons.com, 2016)

Questions:

1. Why did the separation of pigments in the leaf extract occur as it did? The more polar a compound is, the less it will travel as there is more affinity to the solvent, resulting in longer time in the stationary phase.
2. What would happen if we used hexane as a solvent? Hexane is less polar than the other solvents used, so it would be expected that the pigments would travel further up the paper due to their lower affinity for hexane, spending more time in the mobile phase.
3. Why are chlorophyll A and B green, and why do other species have different colors? Chlorophyll A absorbs red-orange light (long wavelengths), while chlorophyll B absorbs blue-purple light (short wavelengths). Both pigments reflect green light, giving plants their green color (Li et al., 2018). Other species absorb different colors of the spectrum, reflecting others, resulting in unique colors.
4. Why do plants have more than one pigment? Different pigments absorb light at different wavelengths, ensuring maximum energy absorption for photosynthesis across a broad spectrum of light (Kume et al., 2018).
5. Why do leaves tend to change colors in autumn? During autumn, chlorophyll is broken down to prevent phototoxicity, revealing the yellow pigments in the leaves. Some trees also produce red leaves due to the presence of anthocyanin pigments (Field et al., 2001).

COSHH (Control of Substances Hazardous to Health):

References:

Asante, O. (2016). Paper chromatography experiment report. Available at: https://www.longdom.org/proceedings/paper-chromatography-experiment-report-54241.html (Accessed April 13, 2022)

Coskun, A. (2016). Separation techniques: Chromatography. Available at: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5206469 (Accessed April 10, 2022)

Fisher Scientific. (2022). Available at: https://www.fishersci.co.uk/gb/en/home.html (Accessed March 23, 2022)

Harper College. (2022). Polarity of Molecules. Available at: http://dept.harpercollege.edu/chemistry/chm100/dgodambe/thedisk/chromwback4.htm (Accessed April 9, 2022)

Kisley, L., Landes, C.F. (2015). Molecular Approaches to Chromatography Using Single Molecule Spectroscopy. Available at: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4287835 (Accessed April 11, 2022)

Kume, A., Akitsu, T., Nasahara, K.N. (2018). Why is chlorophyll b only used in light-harvesting systems? Available at: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6459968 (Accessed April 15, 2022)