Spectrophotometry——2
OPERATION OF B&L SPEC. 20
The most commonly encountered spectrophotometer is one manufactured by Bausch and Lomb and known as the Spec 20. The 20 refers to the band size of light that it is capable of producing. If the instrument is adjust to a wavelength of 730, for example, it actually transmits light from 720 nm to 740 nm. Thus, it is not as precise or refined as instruments designed for research purposes where the wavelength may be controlled to a fraction of a nanometer. It is, however, the standard workhorse instrument found in nearly every lab.
Turn on the spectrophotometer and allow 10 minutes for warm up of the instrument before use.
Adjust the wavelength to that specificied for the procedure you are using.
Be sure the cover is closed on the cuvette holder and use the left knob on the front panel to adjust the dark current such that the meter is reading 0 transmittance. At this point, you are simply adjusting the internal electronics of the instrument to blank out any residual currents. This adjusts the lower limit of measurements. It establishes that no light is equivalent to 0 transmittance or infinite absorbance.
Insert a clean cuvette containing the blank into the holder. Be sure that the tube is clean, free of finger prints and that the painted line marker on the tube is aligned with the mark on the tube holder. Close the top of the tube holder. The blank for this exercise is the solution containing no dopachrome, but all other chemicals. The amount of solution placed in the cuvette is not important, but is usually about 5 ml. It should approximately reach the bottom of the logo printed on the side of the cuvette.
Adjust the meter to read 100% transmittance, using the right knob on the front of the instrument. This adjusts the instrument to read the upper limit of the measurements and establishes that your blank will give a reading of 100% transmittance (0 absorbance).
Remove the blank from the instrument and recheck that your 0 transmittance value has not changed. If it does, wait a few minutes for the instrument to stabilize and redo steps 1-5. Periodically throughout the exercise, check that calibration of the instrument is stable by re-inserting the blank and checking that the 0 and 100% T values are maintained.
To read a sample, simply insert a cuvette holding your test solution and close the cover. Read the transmittance value directly on the scale.
Record the % transmittance of your solution, remove the test tube cuvette and continue to read and record any other solutions you may have.
It is possible to read the absorbance directly, but with an analog meter (as opposed to a digital read out), absorbance estimations are less accurate and more difficult than reading transmittance. Absorbance can be easily calculated from the transmittance value. Be sure that you note which value you measure!
ABSORPTION SPECTRUM:
Analysis of pigments often requires a slightly different use of a spectrophotometer. In the use of the instrument for determination of concentration (Beer-Lambert Law), the wavelength was pre-set and left at a single value throughout the use of the instrument. This value is often given by the procedure being employed, but can be determined by an analysis of the absorption of a solution as the wavelength is varied.
The easiest means of accomplishing this is to use either a dual beam spectrophotometer or a computer controlled instrument. In either event, the baseline must be continously re-read as the wavelength is altered.
To use a single beam spectrophotometer (such as the Spec 20), the machine is zeroed first, the wavelength is set, the blank is adjusted and then the sample is inserted and read. The wavelength is then adjusted up or down by some determined interval, the zero is checked, the blank re-inserted and adjusted, and the sample re-inserted and read. This procedure continues until all wavelengths to be scanned have been read.
In this procedure, the sample remains the same, but the wavelength is adjusted. Compounds have differing absorbtion coefficients for each wavelength. Thus, each time the wavelength is altered, the instrument must be recalibrated.
A dual beam spectrophotometer divides the light into two paths. One beam is used to pass through a blank, while the remaining beam passes through the sample. Thus, the machine can monitor the difference between the two as the wavelength is altered. These instruments usually come with a motor driven mechanism for altering the wavelength, or scanning the sample.
The newer version of this procedure is the use of an instrument which scans a blank, and places the digitalized information in its computer memory. It then rescans a sample and compares the information from the sample scan to the information obtained from the blank scan. Since the information is digitalized (as opposed to an analog meter reading), manipulation of the data is possible. These instruments usually have direct ports for connection to personal computers, and often have built in temperature controls as well. This latter option would allow measurement of hanges in absorbtion due to temperature changes (known as hyperchromicity). These in turn can be used to monitor viscosity changes, which is related to the degree of molecular polymerization with the sample. For instruments with this capability, the voltage meter scale has given way to a CRT display, complete with graphics and built in functions for statistical analysis.
A temperature controlled UV spectrophotometer capable of reading several samples at pre-programmed time intervals is invaluable for enzyme kinetic analysis. An example of this type of instrument is the Beckman DU-70.
SPECIFIC PROCEDURES:
For routine use, substances to be monitored by spectrophotometry are often reacted with dyes to form a complex that is of another color, usually one easily read within the visible light range, and with precision by an instrument such as the Spec 20.
EXERCISE G.1 BRADFORD PROTEIN ASSAY
MATERIALS
Lyophilized bovine plasma gamma globulin or bovine serum albumin (BSA)
Coomasie Brilliant Blue 1
0.15 M NaCl
Spectrophotometer and tubes
Micropipettes
PROCEDURE (STANDARD ASSAY, 20-150 µ g protein; 200-1500 µ g/ml)
Prepare a series of protein standards using BSA diluted with 0.15 M NaCl to final concentrations of 0 (blank = NaCl only), 250, 500, 750 and 1500 µ g BSA/ml. Also prepare serial dilutions of the unknown sample to me measured.
Add 100 µ l of each of the above to a separate test tube (or spectrophotometer tube if using a Spec 20).
Add 5.0 ml of Coomasie Blue to each tube and mix by vortex, or inversion.
Adjust the spectrophotometer to a wavelength of 595 nm, and blank using the tube from step 3 which contains 0 BSA.
Wait 5 minutes and read each of the standards and each of the samples at 595 nm wavelength.
Plot the absorbance of the standards vs their concentration. Compute the extinction coefficient and calculate the concentrations of the unknown samples.
PROCEDURE (MICRO ASSAY, 1-10 µ g protein;
Prepare standard concentrations of BSA of 1, 5, 7.5 and 10 µ g/ml. Prepare a blank of NaCl only. Prepare a series of sample dilutions.
Add 100 µ l of each of the above to separate tubes (use microcentrifuge tubes) and add 1.0 ml of Coomasie Blue to each tube.
Turn on and adjust a spectrophotometer to a wavelength of 595 nm, and blank the spectrophotometer using 1.5 ml cuvettes.
Wait 2 minutes and read the absorbance of each standard and sample at 595 nm.
Plot the absorbance of the standards vs their concentration. Compute the extinction coefficient and calculate the concentrations of the unknown samples.
