Introduction
Photosynthesis is a metabolic process that converts light energy to chemical energy in the chloroplasts of plants. Chemical energy is captured in ATP and NADPH, which are reduced to CO2 and sugars such as glucose. Light energy is captured by the primary photosynthetic pigments: chlorophylls a and b, and accessory pigments: xanthophylls and carotenes. (1)
Chlorophyll a and chlorophyll b both have a porphyrin ring and a long phytol chain. The alternating bonds of the porphyrin ring make chlorophyll very efficient at absorbing light. They also determine the shape of the chlorophyll’s absorption spectrum: they absorb strongly in the red and blue light regions and transmit in the green, which is why plants are green. (1). The phytol chain of chlorophylls does not contribute a lot to the absorption spectrum. The accessory pigments or carotenoids include the carotene, -carotene and xanthophylls: lutein and violaxanthin. Accessory pigments have long isoprenoid chains, the carotenes are composed of hydrogen and carbon while xanthophylls also have oxygen. (1) Accessory pigments absorb blue wavelengths of light and transmit orange and yellow. The presence of carotenoids broadens the range of wavelengths of light that can be used in photosynthesis. (1)
Paper chromatography is an analytical technique used to separate components of a mixture; thin layer chromatography (TLC) is used to separate mixtures according to their polarity. There are two phases in TLC: a stationary phase and a mobile phase. The stationary phase is the paper while the mobile phase is the moving solvent, which travels up the stationary phase by a process called capillary action. In the mobile phase, solutes separate because they dissolve to different extents. In TLC, there are two opposing forces: the driving force which is due to the movement of the solvent carrying solute up the paper, and the resisting force which is the dissolution of solute. The interaction of these forces is what causes separation (1). Separation of the components is measured by the Rf value:
Spectrophotometry is another analytical technique used to measure, quantitatively how much light a substance absorbs a particular wavelength. The beer-lambert equation can be used to determine the concentration of a substance with a known absorbance:
A is the absorbance, is the absorption coefficient (cm2/mg), c is the concentration (mg/cm3) and l is the path length in cuvette (1cm). (2)
In this experiment, we performed thin layer chromatography to separate the photosynthetic pigments found in the leaves of Spinaca oleracea, more commonly known as spinach. After eluting individual pigments from the TLC strips, we will use spectrophotometry to determine the absorption spectra for each, allowing us to determine which pigments absorb which wavelengths of light. (1) For the two major photosynthetic pigments, chlorophyll a and chlorophyll b, we performed a quantitative analysis to determine the concentration of each in the eluted sample. After finding the concentration we calculated the ratio [chl a]/ [chl b], which is a value characteristic for each plant species. (1)
Material and Methods
Preparation of the chromatography Jar. We obtained a 7.5 cm wide strip of Whatman 3MM chromatography paper. Under a hood, the prepared solvent (120 ml toluene: 80 ml acetone) was added to a height of approximately 2 cm, after which the jar was covered.
Preparation of the Leaf Extract. We obtained 3 g of fresh spinach leaves lacking major veins. We cut the leaves into small pieces and placed them in a chilled morter containing 15 ml ice-cold acetone and a sprinkling of clean sand, we grinded the leaves using a pestle for about a minute. After grinding, we transferred the spinach pulp and liquid to a 50-ml stoppered test tube and shook it vigorously for 10 seconds. Then we refrigerated it for 10 minutes so that the spinach pulp would settle. After the pulp settled, we transferred the dark upper layer (containing pigments) portion of the extract to a small test tube using a Pasteur pipet.
Preparative Paper Chromatography. Using a pencil and ruler, we drew a line about 3 cm from the bottom, across the width of the chromatography paper. We filled a capillary tube by immersing the tip in the extract and used it to streak the pigment extract along the line 10 times. We allowed each application to dry before making the following. Once completed, we placed the streaked chromatography paper in the jar and allowed the chromatogram to develop in the dark for 30 minutes. When we noticed a separation, we removed the chromatogram from the jar and marked the location of the origin, solvent front, and center of each band. We also recorded the color and pigment of each band.
Elution and Spectrophotometry. We allowed the spectrophotometer to warm up for 5-6 minutes. We calibrated it and set the wavelength to 400 nm. We labeled 5 cuvettes: reference blank, chl b, chl a, xanthophylls, and -carotene, Afterwards, we used forceps to scrape pigment from the chromatogram and placed them in their corresponding cuvette. Violaxanthin and lutein were placed in the cuvette labeled xanthophyll. We added 4.0 ml acetone to each cuvette and allowed the pigments to elute for 5 minutes, swirling occasionally. We inverted the tubes to mix the contents. We measured the absorbance of each sample at the wavelengths listed on the data sheet. The spectrophotometer was adjusted for the reference blank before reading the absorbance at each wavelength, the absorbances were then recorded.
Results
We wished to identify the pigments in spinach leaves, so we grinded a portion of spinach in acetone and toluene. We streaked a thin layer chromatography paper with our spinach sample and allowed the chromatogram to develop for 30 minutes. (picture 1) The TLC displayed some tear dropping, where the chlorophylls did not separate properly. – carotene is expected to form a band above the chlorophylls but it was no present in our TLC. In order from top to bottom: chlorophyll a, chlorophyll b, lutein, violaxanthin. The origin is at 3 cm and the solvent front is at 12 cm, these two measurements were used to calculate the Rf value.
To measure the Rf for the pigments of each band we used the ruler to measure distance from origin to center of each solute band divided by the distance from the origin to the solvent front. (Table 1) Chlorophyll a Rf = 0.91, chlorophyll b was 0.83, lutein was 0.71, and violaxanthin was 0.59. Because chlorophyll a traveled the farthest, it had the highest Rf value, and was the most nonpolar. Violaxanthin travelled the least, and had the lowest Rf value, it was also the most polar. There was minor difficulty in measuring the Rf values for the chlorophylls due to poor separation.
To see the absorption spectra of each pigment, we eluted each pigment band into its own cuvette and measured each absorbance at different wavelengths using a spectrophotometer (table 2) The pigment absorbances from wavelengths 400 –700 nm was measured. Lutein and violaxanthin pigments were combined into one cuvette and labelled xanthophylls, their combined absorbance was measured. Chlorophyll a absorbed most strongly at 470 nm and 660—663 nm with an absorbance of 0.085. The wavelength for blue light is 450—495 nm, and the wavelength for red light is 620—750 nm. Chlorophyll b absorbed most from 400 –460 nm (wavelengths of blue light) with absorbances ranging from 0.18- 0.07. It also had an increased absorbance of 0.5 at 660-663 nm, which is the wavelengths of red light. The xanthophylls had a high absorbance of 0.05 -0.04 at 400 –440 nm. The wavelength of violet light is 380 –450 nm, xanthophyll’s absorbance corresponded to violet light. From 500 –700 nm, the xanthophylls showed no absorbance. (Figure 1)
Using the absorbance data, we were able to calculate the concentration of chlorophylls in our sample. We know the absorption coefficients of chlorophyll a is 663 = 80.17 cm2/mg and chlorophyll b is 645 = 50.93. Using the Beer-Lambert equation, A= cl, where A is the absorbance, is the absorption coefficient (cm2/mg), c is the concentration (mg/cm3) and l is the path length in cuvette (1cm). We calculated the concentrations of chlorophyll a to be 0.00106 mg/ml and chlorophyll b to be 0.000589 mg/ml. We also calculated the ratio of [chl a] / [chl b], by dividing the concentration of chl a by chl b, the ratio was 1.80.
Picture 1: TLC Plate after separation. TLC displayed some tear dropping. – carotene is absent. Chl a and chl b did not separate. In order from top to bottom: chlorophyll a, chlorophyll b, lutein, violaxanthin. Chlorophyll a is the first light green pigment, chlorophyll b is the darker green pigment directly underneath. The two lighter yellow bands under violaxanthin are accessory pigments. Rf values were calculated by using the ruler to measure distance from origin to center of each solute band and dividing that by the distance from the origin to the solvent front.
Pigment
Distance from origin to solute band (mm)
Distance origin to solvent front (mm)
Rf
Chlorophyll a
8.2
9
0.911
Chlorophyll b
7.5
9
0.833
Lutein
6.4
9
0.711
Violaxanthin
5.3
9
0.588
Table 1: Rf values for spinach pigments. Distances calculated by dividing distance travelled by solute by distance traveled by the solvent from the origin. Distance travelled by solute was calculated by measuring from the origin to the center of each band. Distance traveled by solvent taken by measuring distance from origin to the solvent front. Chlorophyll a traveled the farthest distance from the origin while violaxanthin travelled the least.
Wavelength (nm)
Chl a
Chl b
Xanthophylls
400
0.31
0.18
0.05
410
0.27
0.14
0.04
420
0.19
0.11
0.04
430
0.15
0.07
0.04
440
0.13
0.07
0.04
450
0.13
0.07
0.035
460
0.12
0.07
0.03
470
0.085
0.04
0.02
480
0.06
0.03
0.01
490
0.05
0.03
0.005
500
0.045
0.02
0.00
520
0.035
0.01
0.00
540
0.033
0.01
0.00
560
0.025
0.012
0.00
580
0.03
0.01
0.00
600
0.031
0.015
0.00
620
0.03
0.015
0.00
640
0.05
0.03
0.00
645
0.06
0.03
0.00
660
0.085
0.05
0.00
663
0.085
0.05
0.00
680
0.03
0.015
0.00
700
0.01
0.00
0.00
Table 2: Absorbance data for Chl a, Chl b, and Xanthophylls. Chl a had peak absorbance at 470 nm and 660—663 nm, falling under blue light and red light, respectively. Chl b’s absorbance was strongest from 400- 460 nm with increased absorbance also at 660-630 nm, the wavelength corresponding to blue light and red light. Xanthophylls’ peak absorbance was at 400-440 nm falling under violet and blue light, from 500—700 xanthophylls showed no absorbance.
Figure 1: Absorbance for Photosynthetic Pigments. Chl a and Cl b had increased absorbances in wavelengths of blue or red light. Xanthophylls, lutein and violaxanthin displayed some absorbance in the violet-blue wavelength of light and showed little to no absorbances in the remaining wavelengths. Absorbance in the range between 500 – 640 nm remained low. All three pigments absorbed in blue wavelength of light and they absorbed strongest in the 400-nm range.
Discussion
Thin layer chromatography was used to separate the photosynthetic pigments found in spinach leaves based on their polarity. After eluting individual pigments from the TLC strips, we used spectrophotometry to determine the absorption spectra for each, allowing us to determine which pigments absorb which wavelengths of light.
Chlorophylls are expected to absorb light strongly in the red and blue regions of a visible light spectrum and transmit in green. Accessory pigments are expected to absorb blue wavelengths while transmitting orange and yellow. In our experiment, chlorophyll a had a peak absorption at 470 nm and 660-663 nm, which fall under blue wavelength and red wavelength, respectively. Chlorophyll b had a peak absorbance from 400 -460 nm, in the blue wavelength. Xanthophyll had a peak absorption from 400 –440 nm which is in violet light. The data for chlorophyll is consistent with our prediction of its absorbance. The xanthphylls, however, displayed absorbance was wavelengths less than we predicted. Instead of absorbing blue light, they absorbed violet light but considering accessory pigments serve to broaden the range of wavelengths utilizable by chloroplasts for photosynthesis, this data is still viable.
In our TLC plate, the order of pigments from top to bottom is chl a, chl b, lutein, and violaxanthin. The solvent used was toluene, a nonpolar solvent which served as our mobile phase. Our stationary phase consisted of silica gel, a polar substance. The pigments separated based on polarity, since chlorophyll a was at the top, it was the most nonpolar, it rose with the toluene. Violaxanthin was the was the lowest on the strip, making it the most polar. Normally, there should be a band above chl a for -carotene. However, there was no band in our sample, possible due to bleaching or poor separation of pigment. The band of chlorophyll a had poor separation from chlorophyll b, perhaps enough time had not elapsed to allow pigments to fully separate this may have affected the absorption spectra for each pigment as well as their concentration determination and ratios.
The Rf values for each eluted pigment was calculated: for chlorophyll a Rf = 0.91, chlorophyll b was 0.83, lutein was 0.71, and violaxanthin was 0.59. Using the Beer-Lambert equation we were able to use the absorbance to calculate the concentration of chlorophylls in our sample: chlorophyll a was 0.00106 mg/ml and chlorophyll b was 0.000589 mg/ml. The ratio of [cl a]/ [cl b] is a value characteristic of plants, our ratio was calculated to be 1.80.
When comparing our relative ratio of [cl a]/ [cl b] to previous studies, there ratio was not consistent with our own. In one study, the ratio of [cl a]/ [cl b] was determined to be 2.42-3.10 (3) while ours was found to be 1.80. The previous experiment was conducted in 1970, so perhaps the spinach during that time period was grown differently or produced from a different area than our own. The ratios may vary due to differences in sun exposure for the spinach leaves tested. Both chlorophyll a and chlorophyll b function in light harvesting but only chl a is capable of energy processing. (4) In strong light, there is a higher capacity for energy processing by spinach leaves because of an increase in photons. In low light, it’s a better investment for leaf resources to harvest the light instead of process energy from it. As a result, in low light, the relative amount of chl b will increase and the ratio of [cl a]/ [cl b] will decrease. (4) The spinach used in our experiment was possibly subjected to low light, while the spinach from the previous experiment was grown in abundant light, explaining the difference in ratio. It is also possible that our results are more accurate due to several technological advancements since that time, our spectrophotometer may have given a more accurate absorbance than the one used previously. In both experiments, however, chl a had a higher concentration than chl b. Perhaps, for further studies, the effect of light exposure on spinach pigment abundance should be researched, not only for chlorophylls but for accessory pigments a well.
References
1. Bergman, A. 2001. "Laboratory Investigations in Cell and Molecular Biology". John Wiley & Sons, New York. 4th edition, ISBN: 0-471-20133-2, pp. 152-153
2. Fath, Karl “Chromatography of Photosynthetic Pigments” Laboratory 2
3. Black, C. C., and B. C. Mayne. “P700 Activity and Chlorophyll Content of Plants with Different Photosynthetic Carbon Dioxide Fixation Cycles.” Plant Physiology, vol. 45, no. 6, Jan. 1970, pp. 738–741., doi:10.1104/pp.45.6.738.
4. Atwell, Brian J., Paul E. Kriedemann, and Colin G. N. Turnbull. "1.2.2 Chlorophyll Absorption and Photosynthetic Action Spectra." Plants in Action: Adaptation in Nature, Performance in Cultivation. 1st ed. N.p.: Macmillan Education Australia, 1999. N. pag. Web.