Lab Report Column Chromatography

Which one of the statements concerning valence bond (VB) and molecular orbital (MO) bond theories is correct?

In the beginning of this experiment, our TA added water, salt, and 75/25 hexane/acetone to spinach leaves to a blender and blended the mixture to assume equal amounts for each group and to avoid erros if each student had to do the blending. The addition of the water to the mixture allowed the it to separate into a distinct organic layer after being run in a centrifuge, which was available to be collected at the top of the centrifuge. Salt reduces solubility, which will force the organic parts of the mixture (the desired pigments for example) to separate into the organic layer at the top. Lastly, 75/25 hexane/acetone is added because this is a moderately polar solvent and will useful for both the non-polar and polar pigments present within the spinach leaves. A mixed solution of hexanes and acetone must be used because acetone is very polar, while hexane in very non-polar, and the spinach leaves contain both non-polar and polar pigments in them that are important in the extraction and for further analysis. The mixture was placed in the centrifuge so the solids in the mixture (mostly cellulose) could be separated from the liquids into separate distinct layers for further extraction and testing. In the tube, the organic substances separated into the top layer, whereas the water layer remains at the bottom of the tube below the solid layer made up of mainly cellulose.

Abstract: The purpose of this lab is to separate and identify pigments and other molecules within plant cells by a process called chromatography. We will also be measuring the rate of photosynthesis in isolated chloroplasts. Beta carotene, the most abundant carotene in plants, is carried along near the solvent front because it is very soluble in the solvent being used and because it forms no hydrogen bonds with cellulose. Xanthophyll is found further from the solvent font because it is less soluble in the solvent and has been slowed down by hydrogen bonding to the cellulose. Chlorophylls contain oxygen and nitrogen and are bound more tightly to the paper than the other pigments.

Pigments extracted from different greens have different polarities and may be different colors. Mixed pigments can be separated using chromatography paper. Chromatography paper is able to separate mixed pigments due to their polarity and solubility. Pigments of chlorophyll a, chlorophyll b and beta carotene will be separated on chromatography paper because each has its own polarity and solubility, which results in different distance traveled up the paper. Beta carotene is non-polar so it travels the highest distance, followed by chlorophyll a. Chlorophyll b is the most polar; therefore, it travels the shortest distance. The separated pigments on the chromatography paper can be eluted in acetone and absorbance spectrum is

Two pieces of chromatography paper are trimmed so they are exactly 20.5 cm by 11 cm. The paper is handled by its edges only to avoid contamination from finger oils.

In thin-layer chromatography a liquid is pumped across a bed of particles. The liquid that is pumped across is called the mobile phase and the particles are the stationary phase. A mixture of the molecules that will be separated is put into the mobile phase. Thin-layer chromatography tells you/helps you determine the number of compounds in a mixture, the purity of a compound, and the identity of compounds if you have examples to pull information from. Thin-layer chromatography is used to separate nonvolatile mixtures. The dye that was the most polar was the color red and pink which was Rhodamine B and the least polar was the light pink color which was Sudan IV. Our first TLC plate had five

     In liquid chromatography, the separator is called the column and consists in most cases of a tube filled with porous material called the stationary phase. A liquid, called the mobile phase, flows through the tube between the particles of stationary phase material. A liquid sample is taken from a mixture to be analyzed and introduced to a part of the system that is at elevated pressure. The sample is then transported to a separator by the flow in the system.

The purpose of this experiment was to take spinach leaves and extract the chlorophyll and carotenoid pigments by using acetone as the solvent. The chlorophyll and carotenoid pigments were extracted by using column chromography and alumina was used as the solvent. Solvents of different polarities were used, starting with the least polar, to extract the certain components from the leaves. They were then analyzed by using thin- layer chromatography.

After wearing the gloves we obtained a chromatography vial from professor and label it with my and my peer initials. We dried up the chromatography vial in fume hood and added 1 ml of chromatography solvent to the vial. Then we took a chromatography strip and measure it 1.5 cm with ruler from one end of the strip and drew a line with pencil we cut two small pieces below the pencil line to form a pointed end. We applied spinach on the strip using quarter to rub the spinach leaf on the line that we drew on the strip and put it into the chromatography vial and placed that in fume hood. We observed as the solvent was moving up the chromatography strip by capillary action. When the solvent was reached approximately 1 cm from the top of the strip then we removed the cap from the vial. We took out the strip from the vial using forceps and marked up the location of the solvent front because it evaporates quickly. We measure out the distance as well as the pigment in order to find out the rf value. Moreover we compared rf values to the one in reference list in order to identify the

Leaf litter was collected on a hike through the Christopher Creek area in Payson, Arizona. Small amounts of this biomass will be burned at a time. The resulting aerosols will be collected through a drier vent attached to a high volume air sampler. Burn aerosols will be collected on hydrochloric acid-cleaned cellulose filters. Acid-cleaned cellulose filters are commonly used when performing trace metal analysis on aerosols because they dissolve easily in concentrated acid and are cost effective. Filter cleaning and sample preparation will follow the procedure outlined in Mead, et al. (2013). [1]

For the Plant Pigments I and II- Pigment Extraction lab, the objective was to find a solution that separated pigments and allowed them to travel up the chromatograph. The results of the experiment found that a 1:5 Acetone to Hexane ratio was the best solvent to use. In order to execute the lab we had to roll a penny over the spinach, which would in turn extract the spinach pigment. Even though this is how the experiment was carried, it was also a limitation (and will affect the outcome of the experiment) to the experiment since the same amount of pigment was not always rolled on to the chromatograph. Not all of the 1:5 solutions produced similar results even though they had the same solvent ratio (as seen in Table 1 and Figure 2). There were

It is possible to separate these pigments from each other using a technique called paper chromatography. In this process, plant tissue extract is applied to a piece of chromatography paper. “A solvent is allowed to travel up the paper, and if the pigment is soluble in the solvent, it will be carried along with it.” (Benya, 2009) Different pigments have different affinities for the solvents or polarity and will travel at different rates. Chlorophyll, anthyocyanins, and carotenoids are typically non-polar.

The next step was to place the strip of chromatography paper on a paper towel. Then dip a capillary tube into the plant pigment extract (spinach pigment extract) provided by the teacher. The tube will fill on its own. We applied the extract to the pencil line on the paper, blew the strip dry, and repeated it three to four times until the line on the paper is a dark

Chromatography Investigation Chromatography is a highly regarded technique used to separate the components of a mixture. It is based on the principle that each component possesses a unique affinity for a stationary phase and a mobile phase. The components that are more inclined to enter the mobile phase will migrate further on the chromatogram and distinguish themselves from the other components. The type of solvent used in chromatography is known to directly affect the separation of the mixture. In this experiment, thin-layer and column chromatography will be utilized to separate the numerous chlorophyll and carotenoid pigments of a spinach extract.

Gel-Filtration Chromatography is a commonly used method used in order purify a protein from a mixture, by means of separations. Different biomolecules differ in size, or their molecular weight. In the gel matrix inside the chromatography column, there are gel beads which are porous to allow certain sized molecules to enter. The molecules that are able to enter the pores of the gel, are held in stationary phase and will elute from the column later on, these are usually smaller, to medium sized molecules. Larger molecules that are not able to fit in the pores will elute out of the column first, they are involved in mobile phase where they just go straight through the column without interacting with the gel beads. Smaller molecules will have a higher elution volume, while the larger molecules will have a lower elution volume. The volume to elute the protein is inversely proportional to the molecules size.