CHM256 || PAPER CHROMATOGRAPHY :- QUALITATIVE ANALYSIS OF PHOTOSYNTHETIC PIGMENTS IN SPINACH LEAVES

[00:00:05][music] >> Our group members are Nur Danissa binti Muhammad Hamdan, Nur Syazwa binti Saiful Akmar, Nurul Shahira binti Abdul Malik, Nurul Izzati Shamimi binti Ku Azham and Nur Alisha Aqila binti Rosli Zain.

[00:00:20]>> Chromatography is a fundamental laboratory technique used to separate [music] a complex mixture into its individual components so they can be analyzed, purified, and also identified. The magic behind this process utilizes on the interaction between two main elements.

[00:00:32]Number one, a fixed stationary [music] phase, which can be a solid or liquid.

[00:00:36]Number two, a moving mobile phase, which is either a liquid or a gas. As the mobile phase carries a sample mixture through the system, the different components travel at different speeds based on their chemical properties, allowing them to separate clearly for further analysis.

[00:00:50]>> [music] >> Moving on, let's look at the different types of chromatography, which broadly fall into two main categories. Number one, column chromatography. In column chromatography, components of a mixture are separated inside a tube or column based on how differently they absorb to the stationary phase, causing them to travel at a different speeds.

[00:01:07]>> [music] >> Number two, planar chromatography.

[00:01:09]Planar chromatography is a technique where the stationary phase is set up on a flat plane. This can be broken down further into paper chromatography, where the separation takes place on a specialized paper, [music] and also thin layer chromatography, where the stationary phase is applied as a thin layer onto a solid support plate while using a liquid mobile phase to drive the separation.

[00:01:29]Next, let's talk about the major advantages of chromatography, which really highlight why this technique is so widely used. First, it allows for multi-component separation, meaning it can separate many different parts of a complex mixture. Second, it offers high sensitivity [music] and precision, giving it the incredible ability to detect and also measure tiny amounts of substance even down to parts per billion.

[00:01:50]>> [music] >> Finally, its versatility is unmatched, as it works across many different sample types and can identify both what is in a mixture and exactly how much of it [music] is is there.

[00:02:02]While chromatography is an incredibly powerful tool, it does come with a few notable disadvantages. First, it [music] can involve high cost typically due to the expensive specialized instrumentation, high quality solvent, and also maintenance required for advanced system. Second, the process also can be quite time-consuming. This is because complex separation can take hours and preparing the sample often takes longer than running the actual test itself. Finally, we also have to consider sample destruction as certain types of detectors can actually burn or alter sample during analysis, making it impossible to recover afterwards.

[00:02:37]>> Next, we are diving into a process called the alchemy of photosynthesis. At its core, photosynthesis is the remarkable biological process where green plants, algae, and certain bacteria convert light energy into chemical energy to create food. As noted by Paul Barham in 2012, this foundational mechanism is what essentially fuels life on Earth by locking solar energy into usable organic form.

[00:02:58]But, how exactly does this transformation happen?

[00:03:02]Plants take in sunlight and use that solar power to completely transform carbon dioxide and water into glucose, which serves as their primary food sources while simultaneously releasing oxygen back into the atmosphere as a crucial byproduct. According to research by James Allen in 2026, this complex chemical magic does not just happen everywhere.

[00:03:24]It occurs primarily within specialized structures called chloroplasts inside the plant cells.

[00:03:30]If we look at the chemical equation displayed at the bottom of the slide, we can see this balance beautifully laid out, the reactants, the catalyst, and the product.

[00:03:38]The process starts on the left with six molecules of carbon dioxide combined with six molecules of water in the presence of sunlight and the green pigments chlorophyll, the reaction is driven forward to produce one molecule of glucose used for plant energy and growth alongside six molecules of oxygen, which is released into the air for us to breathe.

[00:03:57]Ultimately, it is a perfect natural factory cycle, turning basic elements into the very building blocks of life.

[00:04:04]Moving on to the internal machinery, let's analyze the key plant pigments found in spinach that drive and support photosynthesis.

[00:04:12]First, chlorophylls. According to Joyet and Emerit in 2025, these primary pigments absorb blue and red light while reflecting green light, which gives spinach its signature green color. Next, flavonoids. These function as antioxidants and water-soluble pigments within plant cells. In nature, they are generally responsible for producing vibrant red, purple, and blue hues.

[00:04:34]Finally is carotenoids.

[00:04:37]These are accessory pigments that assist chlorophylls by absorbing blue-green light. They produce vibrant yellow, orange, and red colors, though they are masked by chlorophylls in fresh spinach.

[00:04:48]For your information, to identify and separate these individual spinach pigments, we use paper chromatography and calculate the RF value or retention factor. As cited by Stewart Hert, the formula is simple, which is RF value is equals to distance traveled by solute divided by the the distance traveled by solvent. Because it is a ratio of two distance, the RF value has no units, and it must always be less than or equals to one.

[00:05:13]Looking at the chromatographic diagram on the right, distance D represents the total with distance traveled by the solvent from the application baseline up to the solvent front. Distance A and B track the movement of the pigment spot or dye zone measured from its edges relative to the origin.

[00:05:30]By comparing these calculated ratios to standard values, we can accurately identify each pigment [music] present in our sample.

[00:05:38]This is the standard RF value for photosynthetic pigments, and this standard value will be used as the reference for the result.

[00:05:46]The primary goal here is separate and identify the individual photosynthetic pigments found in spinach leaf by utilizing the paper chromatography technique we discussed earlier. By doing this, we will be able to see firsthand how a complex natural mixture can be broken down into its distinct individual components based on how they interact with our system.

[00:06:05]>> Continue with the experiment. Let's start with the material used in this paper chromatography experiment. The solvent used was acetone and ethyl acetate solvent. The sample used is spinach leaf.

[00:06:15]Go to various containers.

[00:06:16]The apparatus required were a couple of test tubes, chromatography chambers, mortar and pestle, wash glass and spatula. In addition, several tools such as scissors, pencil, thread, stapler, scale and filter paper strip were used for our experiment.

[00:06:29]Let's continue with the methodology. The experiment was begun with the preparation of the spinach extract.

[00:06:34]First, spinach leaf were washed and cut into small pieces. Next, the leaf were ground with 5 ml acetone to extract the pigment and the resulting extract was transferred into a watch glass. After that, the chromatography paper was prepared by drawing a 2 cm to 3 cm line on the bottom of the filter paper. The spinach extract was then applied onto the line using a capillary tube several times, allowing it to dry between each application to obtain concentrated pigment spots. Subsequently, the filter paper was placed in a chromatography chamber containing the ethyl acetate solvent, ensuring that the sample spot remained above the solvent level. Then, the solvent was allowed to move up the paper by capillary action, causing the pigment to separate based on their differential solubility and affinity.

[00:07:16]Once the solvent front reached approximately 3/4 of the paper, the strip was removed and allowed it to dry.

[00:07:22]And finally, the separated pigment bands were observed and recorded for further analysis.

[00:07:27]>> Now, let's talk about the result. To identify the photosynthetic pigments separated by the paper chromatography, RF value was calculated using the formula RF value equals to distance traveled by pigment divided by distance traveled by solvent. And then, the RF value compared with the standard RF value of known photosynthetic pigments.

[00:07:47]For the first band, which is band A, we have the distance traveled by pigment, which is 9.5 cm, divided by distance traveled by solvent, which is 10 cm, and we got the RF value 0.95.

[00:07:58]So, the RF value matches the standard RF value of carotene.

[00:08:03]Therefore, band A is identified as carotene. Next, we have band B.

[00:08:09]We have the distance traveled by pigment, which is 7.5 cm, divided with distance traveled by solvent, which is 10 cm, and we got the RF value 0.71.

[00:08:18]And this RF value matches the standard RF value of xanthophyll. Therefore, band B um is identified as xanthophyll.

[00:08:27]Next, we have band C. The distance traveled by pigment, 6.5 cm, is divided by distance traveled by solvent, which is 10 cm, then we have the RF value 0.65.

[00:08:38]The RF value matches the standard RF value of chlorophyll A. Therefore, band C is identified as chlorophyll A.

[00:08:45]And the last one is we have band D. And the distance traveled by pigment, 4.5 cm, divided with distance traveled by solvent, which is 10 cm, and we have the RF value 0.45.

[00:08:58]And the RF value matches the standard RF value of chlorophyll B. Therefore, band D is identified as chlorophyll B.

[00:09:08]>> [music] >> And as you can see, carotene has the highest RF value. This indicates that carotene is the least polar pigment, so it has the highest solubility and weaker attraction to the paper. Then, it moved the furthest with the solvent.

[00:09:22]And we also can see that chlorophyll B has the lowest RF value.

[00:09:28]This indicates that chlorophyll B has the lowest solubility and greater attraction to the paper. Then, it moved the shortest with the solvent.

[00:09:38]>> So, based on the qualitative analysis of photosynthetic pigments in spinach leaves experiment, it can be concluded that paper chromatography is a very effective technique for separating complex mixtures into individual components.

[00:09:52]By using the mixture of the ethyl acetate and acetone, this experiment successfully separated [music] the spinach leaf extract into four different pigments based on how well they dissolve and [music] interact with the paper surface.

[00:10:03]The result showed specific Rf values for each pigment, which carotene had the highest distance traveled at 0.98, followed by xanthophyll at 0.55, and chlorophyll A at 0.42, and chlorophyll B at 0.34, which is the lowest distance traveled.

[00:10:22]While chromatography offers high sensitivity and precision in identifying those uh photosynthetic pigments, the process also showed the importance of understanding chemical properties such as the time-consuming issue of a sample preparation.

[00:10:37]All in all, this experiment confirms that different plant pigments travel at different speeds, allowing a clear qualitative analysis of the biological components found within green plants.