In cell physiology the spectrophotometer is the most important single instrument, especially the ultraviolet spectrophotometer. Ultraviolet measurements on proteins and nucleic acids are very sensitive and can be made without destroying the sample.

The percent transmittance of a sample is not directly related to the concentration of the sample, so measurements are usually made in units of absorbance. Absorbance is also called Optical density, and is related to % transmittance by Beer' s Law:

Where T is the transmittance, or the amount of light passing through the sample, L is the length of the path that the light must pass through in the solution (almost always one centimeter), C is the concentration of the solute that is absorbing the light, and K is a constant for any given solute at any particular wavelength. The equation above simply says that the sample will transmit "dT" less light if the concentration of the sample is increased by a small amount dC. "dT" is made equal to "dC" by the constant -K, which is negative because as the concentration increases, the amount of light that is transmitted through the sample decreases. In addition, dT is proportional to the length of the light path; since if the light path is twice as long only half as much light will get through. Finally, dT is proportional to the amount of light passing through the sample (T) since if twice as much light passes through the sample, twice as much light will be absorbed.

These terms can be rearranged to

and integrated:

The absorbance is defined as A = -log T, and. is extremely useful because A normally is proportional to the concentration. Thus by measuring the absorbance, the concentration of an unknown solution can be determined. One should keep in mind, however, that sometimes Beer's Law is not followed. For instance at high concentrations there can be interactions between molecules, such as "stacking" of hydrophobic molecules, that can affect the absorbance. Another source of error at high concentrations is stray light within the spectrophotometer. For these reasons, an empirical standard curve relating the concentration to the absorbance should be constructed for the assay of any substance, and readings are generally most accurate within the range of 0.1 to 1.0 absorbance unit.

The design of a spectrophotometer.

All spectrophotometers share the same fundamental design. First there is light source, which may be either a high intensity tungsten lamp for visible wavelengths or a hydrogen lamp for ultraviolet. Curved mirrors and lenses concentrate a beam of light from this source. Next the light is separated into different wavelengths, either by means of a prism or a diffraction grating. The prism or grating can be turned to select a specific wavelength, and the exit slit permits only this wavelength to pass on through the machine. The light beam may next pass through a filter to help remove contaminating wavelengths of light, for instance that may arise from second-order diffraction patterns in the diffraction grating. The light beam of intensity, I, next passes through a cuvette containing the sample, where some of the light may be absorbed, (the blank cuvette containing only solvent should decrease the light intensity a minimum amount, I₀, while the solvent + sample cuvette should decrease the light in proportion to sample concentration, I₁) and finally it strikes a phototube that measures the intensity of the beam. These phototubes are often very sensitive, and can be damaged if they are exposed to high intensity room 1ights. Most spectrophotometers have built-in shutters to protect the phototube when the sample compartment is opened.

The light beam can be observed when visible light is being used. For example, turn on a Spectronic 20 by giving the lower left knob 1/2 turn. to. the 'right. Set the wavelength scale to 550 nm, using the knob on the top of the machine. Drop a strip of paper about 4 cm long (= ~l 1/2") into the bottom of a Spec 20 cuvette. Insert the cuvette through the door on the top of the machine, pushing it down firmly. One should feel a click as the shutter opens inside the machine. Looking into the cuvette from above, the beam of light should now be visible where it strikes the piece of paper; the cuvette may be turned to get a better view, if necessary. Vary the wavelength setting and observe the effect.

This procedure can also be followed with the other spectrophotometers, and is often advisable in order to be certain exactly where the light beam passes through the cuvette.

Preparing the cuvettes for use.

Spectronic 20 and 21: The sample holders (cuvettes) for the Spectronic 20 are special round tubes l3 x 100 mm. Please be careful not to scratch them, nor to mix them with ordinary test tubes. The cuvettes should be filled about 1/2 full, one containing a blank solution consisting of only the solvent (including any salts or buffers), and another containing the sample dissolved in the same solvent. When you are through with the cuvettes, rinse them out with deionized water, dry the outside, and leave them upside down in a test tube rack.

Quartz cuvettes: Ordinary glass does not transmit ultraviolet, and so in the ultraviolet spectrophotometers quartz must be used for the optics and cuvettes. Everything is exquisitely expensive. Please work slowly and concentrate on what you are doing in order to minimize the chance of an accident.

Nothing should ever touch the optical windows of the quartz cuvettes except Kimwipes and cotton swabs used for cleaning. Handle them by the ground glass sides, and be careful not to scratch the optical surfaces with inserting or removing a sample. When not in use, the cuvettes should be stored in 0.5% sodium dodecyl sulfate, a strong detergent that does not absorb ultraviolet light. Before use, rinse the cuvettes in deionized water and then drain them upside down on a paper towel. The outside can be dried with a Kimwipe. Fill the cuvettes about 3/4 full. One cuvette should contain the blank solution and a second cuvette should contain the sample, as described above.

When you are through making your measurements, rinse out the cuvettes and return them to the detergent solution being certain that the cuvettes are completely immersed and standing right side up, not resting on their optical faces.

Using the spectrophotometers.

The basic procedure is the. same for all spectrophotometers:

1. turn it on and allow about 5 minutes for it to warm up,
2. select the wavelength, which is usually given in nm (=10^-9 meter),
3. with the light beam blocked, set the left end of the scale to 0% Transmittance,
4. insert the blank cuvette and adjust the meter to read 100% Transmittance (= 0 Absorbance), and
5. insert the sample and read the Absorbance.

If the wavelength is changed, most spectrophotometers must be restandardized to 100% Transmittance using the blank cuvette.

Specific Instructions for particular spectrophotometers

Spectronic 20. Use this machine for measurements in the visible light range (400-700 nm ).

1. Switch it on by turning the front left knob 1/2 turn to the right.
2. Select the wavelength using the knob on the top of the machine,
3. Adjust the 0% Transmittance with no cuvette in the machine and with the sample compartment closed. Use the front left knob to set the needle to the left end on the scale.
4. Insert the Blank cuvette into the machine. The cuvette insignia should face the index mark on the front of the sample holder, and the tube should be pushed straight down to be certain of properly seating it in the "V" shaped guide groove. With the Blank in place, adjust the right front knob to obtain 100% transmittance.
5. Remove the blank and insert another cuvette containing the sample. Read the absorbance off the meter. (Note: the absorbance reads from right to left, using a logarithmic scale.):

Spectronic 21:  A next generation Spectronic spectrophotometer, very similar to the Spec-20 but with UV capabilities in some models.

Spectronic Genesys 5. A UV/Visible Light Spectrophotometer. It features multiple sample handling and has a more complicated interface than the Spec 20/21 Series.
If this machine is to be used, considerable time must be taken in reading the Operation Section of the available manual.

Perkin-Elmer. Use this machine for ultraviolet measurements (220-400 nm).

1. Turn the upper right knob to "ON" and also turn on the Deuterium Power Supply.
2. Select the wavelength by turning the right front knob to the right. The filter holder inside the sample compartment should be in the "Open" position.
3. Insert square quartz cuvettes containing the Blank and experimental samples into the cuvette carrier. The carrier is then placed precisely into its holder within the sample compartment with the numbers facing, to the left. The cuvettes are remotely positioned inside the machine with the rod that extends from the front of the spectrophotometer; move this rod in and out with the sample chamber open in order to learn how. to position each cuvette in the light beam.
4. To' adjust 0% transmittance set the uppermost knob .to "Meter". With the sample compartment open (which closes a shutter inside the machine), adjust the meter to 0% transmittance using the knob marked "zero Adj."
5. Position the Blank cuvette into the beam and close the sample compartment. Adjust the front left knob to obtain 100% transmittance.
6. Position a cuvette containing a sample into the beam and read the absorbance from the meter.
   

The Measurement of Absorption Spectra.

The class. should be divided into groups and each group should determine at least one Visible. and one UV absorption spectrum. In each case, you must carefully decide upon the most appropriate Blank.

1. Using the Spectronic 20/21 measure the absorption spectrum of either 5 ug/ml methylene blue dissolved in water or 10 ug/ml of neutral red dissolved in water. Measure the absorbance from 400 to 700 nm at intervals of 25 nm.
2. Using a UV spectrophotometer, measure the ultraviolet absorption spectrum of 20 ug/ml DNA dissolved in 1 mM sodium citrate, pH 8, or the absorbtion spectrum of 1 mg/ml bovine serum albumin dissolved in water. Measure the absorbance at intervals of 10 nm from 300 nm down to the lowest wavelength at which you can set the Blank on the spectrophotometer.

For both your spectra, make graphs of the absorbance vs. the wavelength. (Note: the experimental variable (the wavelength) goes on the X-axis and the data obtained (the absorbance) is plotted on the Y-axis.

Determination of a Standard Curve.

Each group should determine one standard curve for either methylene blue or neutral red using the Spectronic 20 or 21. From a stock solution of 10 ug/ml methylene blue or 20 ug/ml neutral red, make up the following dilution series. Ask the lab instructor how to pipette, if you have forgotten. Calculate the final concentrations of dye in each sample.

 Tube     Dye solution     Water

  1         0.0             4
  2         0.5             3.5
  3         1.0             3.0
  4         2.0             2.0
  5         3.0             1.0
  6         4.0             0.0

Measure the absorbance of the methylene blue solutions at 660 nm, or the neutral red solutions at 530 nm. Prepare a graph of the absorbance vs. the concentration of the dye, thoughtfully deciding which parameter should be plotted on the X-axis and which on the Y-axis.

The laboratory instructor will give you a solution of your dye in unknown concentration. Measure its absorbance and determine its concentration from your standard curve.

Questions.

What are the possible sources of a nonlinear relationship between concentration and absorbance?

Preparations of enzyme or nucleic acids are often described by giving the ratios of the absorbance at 230 nm 260 nm, and 280 nm. Why? For the mathematically inclined: if you have an unknown mixture of a protein and a nucleic acid, how can you determine the concentration of each by measuring the absorbances at 260 nm and at 280 nm?