CHM256 Video Presentation - Separation of Plant Pigments Through Chromatographic Analysis

[00:00:20]Welcome and hi everyone. Today we are exploring comeatography through a classic and colorful application. The separation of plant pigments. By definition, chromatography is an analytical technique that separates the component of a mixture based on how they interact with two different phases. In our plant pigment experiment, these two phases are first the stationary phase, the fixed phase, which is typically the chromatography paper or TLC plate. The second one is the model phase, the solvent or liquid developer that move up through the stationary phase. Our mechanism of separation relies on the fact that different plant pigments have unique chemical structure and polarities. Because of this, they travel up the paper at different rates based on their affinity for the solvent versus the paper. While chromatography has many common varants including paper, thin layer, gas, and liquid chromatography.

[00:01:12]The significance of this technique is massive. Its versatility, accuracy and efficiency make it a foundation method in modern laboratories allowing us to identify, purify and analyze complex biological mixture just like the pigment that power photosynthesis.

[00:01:28]Asalam alaikum. My name is No Ali Masarint Abdul Rahmed. Now I will presenting about theory of chromatography. Chromatography separates compound according to differences in polarity and solubility. In paper chromatography, the paper act as stationary face while the solvent act as the mobile face. When the solvent move up the paper by capillary action, the pigments travel at different rates.

[00:01:52]Nonpolar pigments are more soluble in the solvent and move further. While polar pigments interact more slowly with the paper and move a shorter distance.

[00:02:02]The retention factor RF value can be calculated using formula RF equal to distance travel by pigment to divide distance travel by solvent form.

[00:02:13]Different pigments have categoristic RF value that help in their identification.

[00:02:19]Now I will explain the methodology used in this experiment. One, fresh green leaf are crushed using a mortar and pastel. Two, a small amount of solvent ethanol is added to extract the pigments. Three, a spot of the extract is placed near the bottom of chromatography paper. Four, the paper is suspended in a chromatography chamber containing a suitable solvent mixture.

[00:02:47]Five, the solvent rise through the paper and separate the pigments. Six, after spiration is complete, the paper is removed and allowed to dry. Seven. The distance traveled by each pigment and the solvent front is measured to calculate RF value.

[00:03:08]This slide show the example of the result obtained from paper chromatography.

[00:03:14]Four pigments were successfully separate from the leaf extract. Kerotin, centoil, chlorophyll A and chlorophyll B. Kerotin appears orange and has the highest RA value of 0 95 meaning it traveled the furthest because it is the most soluble in the mobile phase. Centil is yellow with an RF value of 0.71 followed by chloro chlorophyll A is blue green with an RF value of 0.65.

[00:03:50]Chlorophyll B is yellow green with the lowest RF value of 0.45.

[00:03:56]So it's traveled at the shortest distance. Paper chromatography separated pigment according to their solubility and attraction to the stationary phase.

[00:04:08]RF is calculated by dividing pigment distance by solvent front.

[00:04:14]Now I will explain the different between the mobile face and the stationary face in paper chromatography. The mobile face is a face that move over or through the stationary face carrying the analyte mixture. It is also called as the eliting fluid. Its function is acts as the carrier for the mixture or sample to pass through the stationary phase. So it is known as the moving part. In this experiment, the mobile face is a liquid solvent. In contrast, the stationary face is a face that is fixed in place in a column or on a planer surface. It is a for solid used alert or coated with a stationary liquid face. Its function is to absorb the component being separated.

[00:05:09]So it is known as the static part. In this experiment, the stationary phase is in the form of gas or liquid that move over the stationary face because different pigment interact differently with the paper. They move at different speeds producing separate colored bands.

[00:05:31]This difference >> next I will talk about absorption versus partition of chromatography. In absorption chromatography separation is based on surface attraction which is it happen because compound stick to the surface of the stationary face for the movement. If the attraction is strong the compound will move slower. The example of absorption is thin layer chromatography and partition chromatography separation depends on how soluble the compound is between two faces for the movement. If the compound is more soluble in the solvent, it will move faster. The example of partition is paper chromatography. Overall, absorption is about surface attraction while partition is about solubility.

[00:06:18]Paper chromatography versus layer chromatography. Paper chromatography PC and thin layer chromatography TLC are both used to separate compounds, but they have some differences. Paper chromatography use paper as the stationary face and works based on partition. It is lower and cheap but give lower resolution. The sensitivity is less than thin layer chromatography.

[00:06:43]Also it was used in sample analysis like plant pigments. Thin layer chromatography use silica or aluminina and works best on absorption. It is faster and more sensitive and give better separation but it is slightly more expensive. It also more accurate in L analysis. In short, PC is simpler while TLC is more efficient and accurate.

[00:07:10]Now let's look at the two different ways we can set it up physically. Column and planner. In column chromatography, the stationary face is packed inside a glass or plastic tube. The mobile face flows through the column usually driven by gravity or a pressure. This is a powerful method for purification and isolation. For example, if you wanted to isolate a large amount of pure chloro field A from a left extract to use in a separate experiment, we would use a column chromatography. On the other hand, for our analysis of plant pigments, we are using pendography. In this method, the stationary face is spread on a flat surface such as paper or glass plate. Instead of flowing through a tube, the mobile face move across the surface via capillary reaction. Plinary chromatography is the standard choice for identification and purity analysis. It's what allows us to see those beautiful distinct bands of green, yellow, and orange on our paper strip, making it easy to identify each pigments present in the plant.

[00:08:08]Next, let's look at the chemical behavior of our chromatography system.

[00:08:12]We classify this into normal phase and reverse phase, which is entirely decided by polarity. essentially which face is hydrophilic and which is hydrophobic. In normal face chromatography, the stationary face is polar like standard filter paper or silica gel while the mobile face is non-polar because polar compounds get held back by the stationary face while non-polar compound race ahead with their solvent. When separating plant pigments in a normal face system, the highly non-polar pigments like yellow orange keratins will travel the fastest and furthest up the paper. Meanwhile, more polar pigments like food B will bind tightly to the paper and stay closer to the bottom. Reverb phase chromatography is the exact opposite. Here, the stationary face is non-polar and the mobile face is polar. This completely fits the illusion order. Polar compounds move the fastest while non-polar compounds are retained longer. Understanding this distinction is key because it tells us exactly how to read our result and identify which pigments is which based on how far they travel.

[00:09:14]The conclusion for this presentation is chromatography analysis is an effective technique for separating and identifying plant pigments. The method works by exploiting difference in polarity and solubility among pigments. Through chromatography, a pigment such as a chlorophyll a chloroid B and centto and carotin can be clearly separated and analyzed. This technical information about plant composition and has many application in scientific issues and chemical analysis.