Analysis of Plant Pigments with Liquid Chromatography and Spectrometric Detection
The experiment is aimed at coming up with a solution of knowing on how to separate color pigments in a mixture of some solvent. An appropriate solvent is put into use and then some conclusion is drawn from the observations made. The following could be the
- The extract becomes darker progressively due to the fact that the spinach solution becomes more concentrated with the subsequent extractions of the acetone solution. It is observed that the extracts starts at around 10ml of the organic solvent, but as the experiment proceeds, the volume becomes 2.0ml which was almost as the same volume as that of the original extract. It is also observed that some of the pigments are taken out of the initial volume of the extract, and so from this observation, the color of the sample and the sample itself were less diluted with each other.
- The ratio of the peak area of the lutein/zeaxanthin peak over the signal of the violaxanthin peak is given by: 51.863/40.757=1.272.This ratio is believed to be similar to the ratio of the concentrations of the two sample pigments that were found in the plants pigments as was reported in table 1 which was given by: (40%+5%)/35%=1.2861. it is evident that the calculations of the pigment abundance was absolutely correct with very minute error records. This shows that the values that were obtained were to some extent correct as can be seen from the range of the calculations of the ratio.
- The two can also be separated like in the case of the chlorophyll (a and b) since they have almost the same content in terms of the pigment concentrations that will facilitate the process of the chromatography when carried out under the same condition and arrangement.
- When given a red-colored plant without a LC column, it would be easy to tell by carrying out an extraction experiment by grinding the sample and then placing it in an appropriate solvent. A solution of Sodium chloride can be added to the mixture then a layer would be formed between the two solutions. Since flavonoids are believed to be polar and carotenoids on the other hand is said to be nonpolar, the two will be separated as the pigments in the extract. From the knowledge of separation, the flavonoids will be at the more polar aqueous layer, whereas the nonpolar lycopene will settle at the more nonpolar organic layer. The layer that has the strongest red color shows whether the color was due to lycopene or a flavonoid. If the aqueous layer is a stronger red, the red color is due to a flavonoid, but if the organic layer is a stronger red, the color is due to lycopene.
- This was because it had the value that ranges between the values that were used in the calculation of gradient value. This helped in attaining the retention volume of the curve. It was also clear that the molecular mass of the pigments is between the values used in the calculations of the gradient or the retention volume.
- The best way of experiencing is by setting the spectrophotometer value to 650 nm since only chlorophylls a and b have abs peaks near that scope and will be able to be visible enough.
- This is because the other pigments' absorption peak is believed to be close to 450 nm and is closer than that of chlorophyll a. More importantly, besides chlorophyll a, not even one of the other pigments' closest absorption peak to 450 nm is further than 13 nm from 450, but chlorophyll a's closest absorption area is about double that distance away. Being farther away from its absorbance peak is the main reason that the peak for chlorophyll a is so much smaller than the rest of the pigments observed.
- The fluid phases will be added in the inverse order for it to be more effectively removing the pigments. Given that the stationary phase would be polar in nature, the mobile phases would have to go from the least to the most polar to remove the pigments faster and in the correct order. Then the paints would turn up on the chromatogram in the inverse order of the experiment since the nonpolar compounds will automatically be eluted in the first phase of the experiment.
- The pigments spots may overlap in the process and give a wrong impression of the results on the paper chromatograph and make the observations more difficult to make and come up with appropriate solution at the end.
Cramer, Friedrich. Paper Chromatography. London: Macmillan, 1955. Print.
Hais, I M, and Karel Macek. Paper Chromatography, a Comprehensive Treatise. Prague: Pub. House of the Czech. Acad, 1963. Print.