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A spectrophotometer is an instrument that measures the quantitative amounts of light of different wavelengths absorbed and transmitted by a pigment solution (Mitchell, Reece). The spectrophotometer includes a light bulb, a reflector, and a detector. When a sample is in place and the chamber lid is closed, light from the light bulb passes through it. The detector measures the amount of light transmitted. The white light from the bulb is separated into different wavelengths by reflecting the beam off of a diffraction grating, forming a spectrum. A mirror is used to control the angle at which the light is reflected, so light of a single wavelength can be passed through the sample. A pigment solution (such as spinach and acetone) is exposed to different wavelengths of light to determine the points of the highest absorbance and transmittance percentages. (Hickey, Mary Kay). For this experiment, a Bausch and Lomb Spectrophotometer was used. Often objects appear colored because of their absorption of light within regions of the visible spectrum. The color of light we can see is composed of the wavelengths that the object did not absorb. Photosynthesis in plants takes place in organelles called
chloroplasts. Chloroplasts contain a number of coloured compounds, known as pigments, in their thylakoids, where light dependent reactions occur(Mitchelsl, Reece). Spinach leaves contain chlorophyll a and b and b -carotene as major pigments as well as smaller amounts of other pigments such as xanthophylls. The two forms of chlorophyll are identical except that a methyl group in a is replaced by an aldehyde in b. In terrestrial plants, the most important pigments are chlorophyll a (blue-green), chlorophyll b (yellow-green), xanthophyll (yellow) and carotene (orange-yellow).(Pavia, D.L.) Each pigment has a characteristic absorption spectrum, showing which wavelengths of visible light it absorbs best. (Campbell, Neil) In this experiment, by observing the transmittance percentage and absorbance of light when shone at spinach at different wavelengths, we will determine which wavelengths are best absorbed by spinach.
Figure 1: Labelled Diagram of a Bausch and Lomb Spectrophotometer. ( Chemical Instrumentation Laboratories )
To determine the % of absorption and transmission of light from spinach leafs at different wavelengths of energy.
The wavelength with the greatest transmission % will be 550 nm because the spinach leaf reflects green light (hence, why plants are green). It is expected that the highest absorption % will be between 420-450 nm and 650-700 nm. The reason for this is because Plants absorb blue light and red light the best, and 650-700 nm is the range in which photosystem 1 and 2 absorb light to being the light dependant reactions.
* 6.1 mL Spinach solution
* 2 Cuvettes
* Room temperature
* Concentration of spinach
* Amount of spinach
* Type of spectrophotometer
* Transmittance percentage
* Absorption Percentage
* Wavelength of light
1) The instrument was turned on by rotating the amplifier control (c) in a clock-wise direction. 5 minutes were allowed for the instrument to warm up.
2) A blank cuvette was placed in the sample compartment and the guide mark was aligned on the cuvette at the front of the sample
compartment. The lid was closed. It was often checked that the meter needle reads 100% Transmittance when the blank cuvette was in the sample compartment with the lid closed.
3) The amplifier control was adjusted with the sample...
References: 1. Campbell Neil, Reece Jane, Mitchell Lawrence, 1996. BIOLOGY-Fifth
2. Buchanan, B.B., Gruissem, W., and Jones, R.L.
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