Spectrometry of Plant Pigment Extract

Laboratory Time Allotment: 1.5 hour

Components of the Exercise:

A. Prior to the laboratory period:

1. Read the "Experimental Method" section below.
2. Write the cryptic title "Spectrometry" at the top of a fresh page in your laboratory notebook.
3. Below the cryptic title, write a concise protocol for your investigation. Be sure to use numbered steps, put each step on a new line, and have only one action in each step.
4. Write the cryptic title in the Table of Contents in the front of your laboratory notebook. Indicate in the Table of Contents the page on which your record of that investigation starts. Leave three blank lines after the cryptic title for later insertion of a descriptive title

   B. During the laboratory period:

5. Observe the ethanol extract of chloroplast pigments with the spectrometer, recording the significant data in you laboratory notebook as explained in the Experimental Method below.
6. Observe the demonstration of the fluorescence of that some extract and record your observations.
7. Date and sign the pages of your laboratory notebook where you data are recorded and get your instructor to sign as a witness.

   C. Following the laboratory period:

8. In your laboratory notebook plot a transmission spectrum of the pigment extract, plotting per cent transmittance on the ordinate and wavelength on the abscissa. You may plot it directly in your notebook or on a piece of graph paper which you glue into your notebook. Be sure to use glue. Cellophane tape doesn't last, and staples tear through three or four pages ahead and behind when you close your notebook.
9. Calculate the absorbance of the observed extract at each wavelength which marks a change in per cent transmittance and plot an absorption spectrum for that extract in your notebook. Use the logarithmic scale shown on the last page of these directions.
10. Write a brief "Conclusions" section in your notebook, summarizing what you can conclude about the usefulness of the various wavelengths in the process of photosynthesis. Base your conclusions SOLELY on the evidence of your experiment.
11. Read about fluorescence in a physics book and write in your "Conclusions" a brief explanation of why light going straight through the pigment extract in the fluorescence demonstration appears green while the light an the other three sides appears dark red. Remember that a very strong light of one color can keep your eye from detecting another color with a much weaker intensity.

Learning Objectives:

On completion of this exercise you should be able to:

1. Define in your own words, pronounce correctly, and use correctly in sentences of your own composing, the following terms:
2. Define from memory, give the symbols for, and interconvert the following units: micron, millimicron, micrometer, nanometer, Angstrom.
3. Write from memory the equation for the relationship between per cent transmittance and absorbance.
4. Apply that equation to convert per cent transmittance to absorbance and vice versa.
5. Use a grating spectrometer to obtain the data necessary and plot the approximate transmittance spectrum of a given solution and plot it in standard scientific format.
6. Use those same data to plot the approximate absorbance spectrum of that same solution in standard scientific format.
7. Diagram the components of a spectrophotometer and briefly explain how it works.
8. Explain how the precision of visual estimation of per cent transmittance compares to the precision obtained with a typical spectrophotometer and why there is such a difference.
9. Explain why we estimate transmittance visually in this class instead of using a spectrophotometer.

Notes on Terminology:

Experimental Method:

In this investigation you will observe the absorption of light by pigments extracted with boiling ethanol from the chloroplasts of leaves. Light is passed through the extract (solution of pigments) and then passed through a grating spectrometer. The spectrometer diffracts the light passing through the pigment extract to form a spectrum which you can see when you look through the eyepiece.

If you compare that spectrum with the spectrum that results when the same light is passed through the spectrometer without passing through the pigment extract, you can see that certain colors of light are absorbed by the pigment extract.

The differences between the two spectra are due to absorption by the pigment extract, and the questions we want to answer are:

• What wavelengths are absorbed by the pigment?
• What is the approximate amount of absorbance?

In your chemistry classes you may have obtained such data with a spectrophotometer, which gave you readings in absorbance units. However, many students in the past have demonstrated that they did not have the faintest idea of what was going on inside the spectrophotometer nor of what it was really measuring, so in this class we do it visually. The precision of the measurements is miserable, because the human eye is a very poor instrument for judging relative amplitudes. But experience has shown that far more students will understand what is going on and what is being measured if we do it this way.

Observe the demonstration spectrometer set up with an ethanol extract of chloroplast pigments. Adjust the flask of pigment extract so that its surface bisects the slit at the left rear of the spectrometer, making two spectra appear in the eyepiece, the upper one form light which passes above the surface of the pigment extract and the lower one from light which passes through the pigment extract. Record in your notebook how the sample is set up in relation to the spectrometer. (A diagram is best for this.)

For the purposes of this exercise you should compare the upper and lower spectra every 10 nanometers within the visible spectrum (400 nm through 700 nm). At each wavelength which marks a change in per cent transmittance, estimate what per cent of the light of that wavelength is transmitted through the pigment. There will be some entire ranges of wavelengths in which the per cent transmittance will be zero, some in which it is 100 and some in which it will be some intermediate value. You will probably find it useful to tip the spectrometer slightly up or down to see the upper or the lower spectrum more clearly.

Later you will need to convert these per cent transmittance values into absorbance units, but absorbance units are logarithmic and can not realistically be estimated by eye. Therefore, the best approach is to estimate per cent transmittance and then calculate the conversion to absorbance units afterward.

Pay special attention to recording the wavelengths at which there is a conspicuous increase or decrease in the per cent transmittance. Your eye is reliable to only the nearest 25% transmittance, so do not try to estimate any closer than that. After the laboratory period you can calculate the absorbance and plot you absorption spectrum.

Observe also the fluorescence demonstration. Make a sketch of the apparatus and the direction of viewing relative to the direction of the light path. Record what you observe.

Theoretical Background:

Most of the terminology in Learning Objective number 1 should have been learned in chemistry and physics classes. Absorbance, transmittance, and per cent transmittance are usually not encountered until more advanced chemistry courses so they are explained below. If the other terms are not familiar to you, you should study the definitions in the first pages of these directions and also look them up in books on physics, analytic chemistry, microbiology, or biochemistry, and in an unabridged dictionary or a scientific and technical dictionary, such as the ones in the BAT Lab, the college library, and the Biology 1B lab.

Two of the units of measurement in Learning Objective number 2 are out of date, having been changed decades ago by an international commission on the metric system, now renamed "Systeme Internationale" (or S.I. for short). But there are still a few old fossils around who refuse to change, and there are still some very useful reference books around which were printed before those changes were made, so unless you enjoy being confused, you need to learn both sets of units. Note the similarities of the units and their abbreviations, and memorize the differences.

Table

A spectroscope is a device which allows a narrow beam of light to enter it and pass through either a prism or a diffraction grating to disperse the light into a spectrum. A spectrometer is a spectroscope with some method of indicating the wavelengths or of selecting a narrow band of wavelengths to pass through a specimen. A spectrophotometer adds an electrical or electronic photometer to the system to measure light intensity much more accurately than the human eye can judge it and display it on a meter or – on the more expensive models – print it out on a graph.

The investigation involves a grating spectrometer. The light passes through an adjustable slit and then through a diffraction grating in the eyepiece. The light is dispersed into a spectrum, which is projected onto the retina of the eye, giving the observer the impression that the spectrum is inside the instrument, although it is purely a virtual image. The instrument also projects an image of a wavelength scale into the eye, superimposing it on the spectrum so the observer can read what wavelength each color corresponds to. Most light sources provide a fairly complete spectrum with virtually all of the wavelengths present. If a solution is place between the light source and the spectrometer, only those wavelengths transmitted by the solution will be seen. Wavelengths which are absorbed by the solution will be either significantly dimmer or absent, leaving a black space in the spectrum..

Diagram

Transmittance means exactly what it implies. It is a measure of the light that is transmitted. It is the decimal fraction of the incident light which is transmitted through a sample of solution. Per cent transmittance is simply the expression of that fraction as a per cent instead of a decimal fraction. In spectrometry, we are concerned primarily with the differences in transmittance of different wavelengths. For example, if a given solution absorbs half of the light of a given wavelength and allows the other half of that light to be transmitted through the solution, then its transmittance for that wavelength is 0.5 and its per cent transmittance for that wavelength is 50%.

Absorbance is the opposite of transmittance; that is, it is a measure of the amount of light of a particular wavelength that is absorbed by the sample solution and therefore not transmitted. Absorbency is measured in absorbance units, which are related to transmittance inversely and logarithmically, as indicated in the equation below. (In older reference books you may see the term "optical density" and "optical density units". You may also see in chemistry books the term "extinction". Both of these mean the same as absorbance, and the units are identical to absorbance units.)

Math Formulas

Thus, a solution with 50% transmittance of a certain wavelength as an absorbance at that wavelength of:

In case you are wondering why anyone would invent a logarithmic unit, the reason is that absorbance is directly proportional to the concentration of the solute in the solution, and therefore, absorbance units are extremely useful for determining concentrations and changes in concentrations.

An Absorption Spectrum is the portion of the spectrum that is absorbed by a given substance. The term is also applied to a graph of the that spectrum, with absorbance plotted against wavelength. In a chemistry class you may have used an expensive spectrophotometer which automatically measured the absorbencies at numerous wavelengths and plotted the graph for you. In this exercise you are asked to make your best estimate by eye of the per cent transmittance and then, after class, convert those estimates to absorbance units and plot them against wavelength to obtain an absorption spectrum graph.

Remember that a graph is a form of communication, and communication is always clearer when a standard format is followed. In science, the standard format is that the independent variable is plotted on the horizontal axis (= X-axis or abscissa) and the dependent variable is plotted on the vertical axis (= Y-axis or ordinate). The independent variable is most easily remembered as the one that you can be independent about, In this case, if you do not feel like observing the per cent transmittance at 550 nm, you can express your independence by observing it at 520 nm are 560 nm. The wavelengths, therefore, is your independent variable. The per cent transmittance you observe, however, is dependent upon the wavelength you choose, so per cent transmittance is the dependent variable, and so is the absorbance you calculate from it.

Your transmission spectrum should be plotted as indicated below.

Transmission Spectrum

GRAPH

After plotting your transmission spectrum you need to calculate the absorbance at each wavelength where there is a significant change and plot a rough absorbance spectrum on a graph like the one below.

Be aware that the range of absorbance units is from zero (at 100% transmittance ) to infinity (at 0 % transmittance). (If you use your electronic calculator to calculate the base 10 logarithm of zero, it will probably give you an answer of ERROR. Few calculators will give you the correct answer of "Infinity".

It is customary to space the absorbance units out logarithmically on the Y-axis, so that an absorbance of 2 is 99% of the way to infinity, 1 is 90% of the way, and so forth.

Absorption Spectrum

GRAPH.

Return To Schedule

---

Updated 9/1/99 Miko mmalacho@ccsf.cc.ca.us

---