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Spectrophotometers in Research

Spectrophotometers are used extensively in research. A spectrophotometer measures the amount of light at specific wavelengths that are either absorbed or transmitted by a sample placed in a container called a cuvette. The cuvettes used in the LabLearner program are made of glass and look like small test tubes. Research-grade cuvettes may be made of other, more expensive materials .

One of the most common uses of spectrophotometers is for determining the concentration of solutions. Basically, spectrometers work by shining a beam of light through a liquid sample contained in a cuvette. The light that passes through the sample is electronically detected by a photo-detector (see below). Light that is not absorbed by the sample is transmitted through it.

To make spectrophotometer readings,  the instrument must first be “blanked”. A blank is simply a sample of the pure liquid (solvent) in which the sample to be measured is dissolved in. The solvent is often water. When a spectrophotometer is blanked, a cuvette containing pure water is placed in the instrument and a blank button is pressed (0A/100%T). This sets the spectrophotometer so that a sample with just water in it has a transmittance of 100% and an absorbance of zero.

After blanking the spectrophotometer, the cuvette containing the blank solution is replaced with one containing a solution of a compound we wish to determine the concentration of. When this is done, the amount of light absorbed and transmitted by the solution of water (solvent) plus the compound to be measured (solute) is determined.

By reading transmittance and/or absorbance of various known concentrations of a given compound, a concentration curve can be drawn. Next, the absorbance and/or transmittance of samples of unknown solute concentrations can be determined and compared to the concentration curve. The exact wavelength of light that is set on the spectrophotometer is based on the properties of the molecules of the sample dissolved in the solvent.

Can you estimate the approximate concentration of the three samples shown in the incomplete data table in the concentration curve experiment above? The answers are shown at the bottom of this webpage.

The Oil Immersion Microscope

Students may find it interesting to know how and why an oil-immersion objective lens provides better high magnification images. It has to do with the refraction of light as it passes from the sample and toward the lens surface.

Light shines from below the stage and through the sample on the microscope slide. As the light leaves the glass of the cover slip of the slide, it enters the air. Air and glass have significantly different refractive indices, that is the degree to which a ray of light is bent as it moves from one medium to another. Therefore, much of the light coming up from the specimine is refracted upon reaching the air and doesn’t enter the objective lens. If light doesn’t enter the objective lens, we can’t see it and the specimine appears dark and fuzzy when we view it through the eyepiece.

On the other hand, immersion oil, has a refractive index much closer to that of the glass lenses and so light coming up from the specimeine is not refracted as much and more of the light is directed into the objective lens and subsequently to the eyepiece and to our eye. Oil immersion lenses give us the best look at specimines in light microscopy. Electron microscopes do not use light waves but rather a beam of electrons and produce much, much greater magnification and resolution. The resolution of transmission electron microscopes are about 2,000 times that of the best light microscopes!

LabLearner Tabs: Middle School Tools

Learn more about key LabLearner middle school scientific tools by clicking on one of the tabs below. It does not matter what order you learn about these instruments. You may want to review this section just prior to introducing each piece of equipment to your students.

Preparation of the Spectrophotometer

1. Turn on the spectrophotometer by the power switch (IO), located on the backside of the instrument. You should allow approximately 15 minutes for the instrument to warm up.

2. Once the instrument is warmed up, determine the wavelength at which you will begin your investigation. Set the wavelength with the wavelength control knob.

3. Check the sample compartment. Make sure that it is empty and close the cover.

4. Use the mode control key to set the display mode. Select either the T (transmittance mode) or A (absorbance mode) by pressing the mode button until the red light for T or A is lit.

Use of a Blank

1. Obtain a clean cuvette. Using a lab marker, place a line from the top lip of the cuvette down the side of the cuvette about 1 cm in length. Next, fill the cuvette with the solvent in which your solution has been prepared. This solution is referred to as the “blank.” This is used to zero the spectrophotometer. Wipe the cuvette to remove any excess solvent and fingerprints from the outside.

2. Place the cuvette in the sample compartment so that the line is lined up with the raised line at the top of the sample compartment. Push it into the compartment until you hear it click and then close the lid.

3. Set .000 A or 100% T with the 0A/100%T button. Once the spectrophotometer is properly “blanked” the letters BLA will flash on the digital readout.

4. Remove the cuvette from the sample compartment.

5. Empty the blank solution from the cuvette back into its original beaker or container.

Collection of Data

1. Fill the same cuvette with the solution to be tested. Wipe the test tube to remove any excess solution and fingerprints. Place the cuvette in the sample compartment so that the line is lined up with the raised line at the top of the sample compartment.

2. If measuring the transmittance, use the mode selection button to select the Transmittance mode. Record the percent transmittance from the display.

3. If measuring the absorbance, use the mode selection button to select the absorbance mode. Record the absorbance value from the display.

4. Remove the cuvette from the sample compartment and empty the solution back into its beaker or container.

5. Using a plastic dropper, rinse the cuvette with the blank. Pour the blank into a beaker labeled waste.

Repetition of Procedure

1. If obtaining a transmittance or absorption reading for more than one wavelength, select the next wavelength using the wavelength control knob.

2. Use the same “blank” as above to zero the spectrophotometer at this wavelength. Follow steps under Use of a Blank.

3. Repeat the steps under Collection of Data.

4. Continue with this process until you have gathered all of the required data.

5. Remember to rinse the cuvette with the blank solvent between each new solution tested and after the last solution you test.

Oxygen Meter Calibration

1. Disconnect the oxygen probe from the oxygen meter.

2. Turn on the oxygen meter.

3. Select the O2 mode by sliding the button so that it lines up with O2 rather than mg/L.

4. Press the zero button on the meter so that the meter’s display shows 0.

5. Reconnect the oxygen probe to the meter by inserting the plug into the input socket on top of the meter.

6. Wait five minutes until the temperature and oxygen values stabilize.

7. To calibrate the meter, press the button labeled O2 cal. The display will show either 20.8 or 20.9. This is the typical percentage of oxygen in the air.

8. If use of the oxygen meter is not occurring at sea level, the Height compensation must be adjusted. Press the button labeled Mt. so that the display shows an H for height. To increase the altitude, press the button labeled Factor Adj. until the desired value is reached. If the value displayed is greater than the desired value, press the factor adjustment button until the values cycle back to zero. To exit this mode, press the Mt. button once.

9. When calibration is complete, move the button from the O2 setting to the mg/L setting so that the meter is ready to measure dissolved oxygen content.

 

Oxygen Meter Resetting of the CPU System

* Note: If the value displayed during calibration or during use seems unreasonable or inaccurate, the CPU system may need to be reset. By following these procedures from the Traceable Digital Oxygen Meter Instructions, the system can be reset and inaccurate data avoided.

1. Select the O2 mode by sliding the button so that it lines up with O2 rather than mg/L.

2. Press the power button and turn off the oxygen meter.

3. Disconnect the oxygen probe from the oxygen meter.

4. Press the button labeled O2 cal continuously. While holding this button, push the power button again.

5. Press the zero button.

6. Connect the probe to the meter once again and wait until the reading has stabilized.

7. Press the O2 cal. button.

8. Turn off the meter by pressing the power button.

9. Recalibrate the oxygen meter and begin normal use.

 

Oxygen Meter Use and Operation in Liquid

1. Turn the switch on the oxygen probe to the dissolved oxygen (DO) setting.

2. If necessary, adjust the percent salt composition of the liquid in which the probe will be placed. Press the % salt button to display a screen which shows a letter ‘S’ for salinity. Press the factor adjustment button until the desired salinity is reached. Press the % salt button to return to the main screen.

3. If necessary, adjust the meter to your altitude. Press the Mt. button to display a screen that shows a letter ‘H’ for height. Each time you press the factor adjustment button, you will add 100 meters to your altitude. When you have reached the correct altitude, press the Mt. button to return to the main screen.

4. Place the oxygen probe in the liquid. When performing this part of the procedure, you may encounter a large air bubble on the tip of the probe. Such an air bubble causes irregularities in measurements. To avoid this, tilt the beaker slightly. Hold the oxygen probe so that it enters the liquid at an angle. Look at the tip of the probe to observe whether or not a large air bubble was formed. If it was, remove the probe and then reinsert it into the liquid until the tip of the probe is free of large bubbles. Small air bubbles are acceptable and will not affect the measurements.

5. To obtain accurate measurements of dissolved oxygen, the oxygen meter probe must be immersed to a depth so that the hole of the temperature sensor is immersed. Before obtaining any measurements, make sure the temperature sensor is covered by the liquid.

6. After the probe is in the liquid, gently stir the liquid. The liquid must be stirred in order to obtain accurate measurements with the oxygen probe. You may stir the liquid by moving the probe in the liquid or by keeping the probe stationary and gently moving the beaker.

7. Allow time for the temperature and oxygen readings to adjust and remain constant, approximately one or two minutes.

8. When the measurements become constant, put the meter into a record mode by pressing the record button. The letters “rec” will appear on the meter’s display.

9. Record the oxygen measurements for 30 seconds. Press the recall (CALL) button on the meter once to obtain the maximum recorded measurement, twice for the minimum, and three times for the average measurement.

 

Oxygen Meter Electrolyte Replacement

At the beginning of each school year, or if calibration and resetting the CPU system do not yield the desired results, check and refill the electrolyte using the following steps.

1. Unscrew the lower section of the probe by turning the ridged section. This is sometimes difficult to do by hand. Carefully use a pair of pliers if necessary, but avoid cracking the probe.

2. Pour any remaining old electrolyte solution onto a paper towel. Dispose of this in the trash. If dried old electrolyte solution is crusted inside the probe, rinse away with water.

3. Add electrolyte solution to the chamber using a disposable pipette. Fill the chamber approximately halfway.

4. Re-attach the separated chambers to the rest of the oxygen probe. Excess electrolyte solution will be expelled as you screw the probe together. Rinse probe with water to remove.

5. Re-calibrate the oxygen meter using the steps in Part A above.

 

Oxygen Meter Battery Replacement

Replace the battery if the letters “LBT” appear on the left corner of the display or if the steps in Part A or Part B do not yield the desired results.

1. Slide the battery cover of the back of the oxygen meter.

2. Replace the battery with a new 9-Volt alkaline battery. The battery must be an alkaline battery, not a regular or heavy-duty battery.

3. Replace the battery cover.

4. Re-calibrate the oxygen meter.

Microscope Use and Operation

1. Turn on the microscope.

2. Turn the diaphragm so that it is completely open and the maximum amount of light is visible through the eyepiece.

3. Turn the nosepiece so that the 4X objective is in place.

4. Make sure that the stage is raised completely. Place the slide on the stage. To do this, pull back the lever on the right side of the stage. Place the slide on the stage so that the coverslip is facing up toward the objective. Gently release the lever so that the holder falls into place against the slide.

5. Using the knobs located below the stage on the left side of the microscope, move your specimen so that it is directly below the objective.

6. Rotate the light intensity control knob until the appropriate amount of light shines through the slide.

7. Focus on the slide by using the large coarse adjustment knob. Since the stage is as high as it can be, you must focus on the slide by turning the coarse adjustment knob away from you.

8. After you have focused using the coarse adjustment knob, use the smaller fine focus knob to focus more sharply.

9. Rotate the nosepiece so that you view the slide through the 10X objective, then the 40X objective. Use only the fine focus knob to make adjustments. Do not rotate the coarse adjustment knob.

10. From the 40X objective, rotate the nosepiece clockwise slightly so that neither the 40X nor the 100X objectives are in place. Place one small drop of immersion oil on the slide. Rotate the nosepiece so that the 100X objective is in place.

 

* Note: When using the 100X objective, the slides you are viewing must be prepared slides, not wet mount slides. Placing immersion oil on the wet mount slide (as needed when using the 100X objective) could compromise the specimen. Prepared slides are sealed so the oil cannot go under the coverslip, and the oil can be wiped off the slide without difficulty.

In addition, once the 100X objective is in place and oil is on the slide, you may not view the slide with other objectives until the slide and the 100X object has been cleaned. For cleaning instructions, see step 12.

11. Focus on the slide, using only the fine focus knob.

12. Once you view a slide with the 100X objective, rotate the nosepiece clockwise until the 4X objective is in place. In this position, clean the bottom of the 100X objective using lens paper. Do not use a tissue, paper towel, or any other type of paper, as this may damage the objective. Wipe the objective until no more oil residue is removed.

13. Remove the slide and wipe it clean using a paper towel or tissue.

14. Turn off and unplug the microscope when finished.

Multimeters are instruments used for measuring voltage, current, and resistance.

Measuring Voltage

1. Turn the selector dial to the appropriate Direct Voltage range.

2. When using batteries as a power source or more batteries, use the 200V range.

3. Connect the red lead to the VΩMA jack.

4. Connect the black lead to the COM jack.

5. Hold the probe of the black lead against the negative terminal of the battery.

6. Hold the probe of the red lead against the positive terminal of the battery.

7. Record the value shown on the display. This is the number of volts being produced by the battery or batteries.

 

6. If using multiple batteries, hold the probes to the terminals of the two end batteries.

 

Measuring Current

1. Turn the selector dial to the 10A setting in the direct current range. The settings in the Direct Current range include 200μ, 2000 μ, 20m, 200m, and 10A. The 10A setting is located below the 200m setting.

2. Begin by assuming that the circuit holds a current greater than 200 mA and connect the red lead to the 10A jack. Connect the black lead to the COM jack as it was when reading voltage.

3. Current is measured by placing the multimeter into the circuit being constructed. For example, the multimeter may connect to one end of a device such as a light bulb, a resistor, or an electromagnet. The other end will connect to the wire or clip that is connected to the negative terminal of the battery.

4. Attach a clip or test lead from the positive terminal to the device in the circuit and attach another clip or test lead into the negative terminal of the battery.

5. Place the probe of the red lead into the device in the circuit, such a resistor.

6. Place the probe of the black lead in the clip leading to the negative terminal.

7. Record the value shown on the display.

8. If the current is less than 0.2 A, adjust the multimeter to the 200 mA (milliamp) range.

  • Remove the black probe from the clip (test lead) and the red probe from the device.
  • Turn the selection dial to the “Off position.

9. Remove the connector of the red probe from the 10A outlet and into the VΩmA outlet.

  • Turn the selection dial to the 200 mA mark in the Direct Current range.
  • Re-attach the red and black probes to create a closed circuit.
  • Record the value shown on the display in milliamps (mA, or 200m on dial).

19. If the current is less than 20 mA, open the circuit by removing the red probe from the device. Turn the selector dial to 20 mA (or 20m on the dial). Re-attach the red probe to the device and record the value on the display in milliamps (mA).

Overrange indicator

1. The multimeter will display a “1” on the left of the display screen when current is too great for the range setting.

2. Adjust the sensitivity of the multimeter by turning the selector dial to the next highest range. For example, if the setting was on 20m and a “1” appeared, turn the dial to 200m. If the over-range indicator is displayed when measuring current at the 200 mA range, move the red lead to the 10A jack and turn the dial to “10 A”.

ANSWERS TO CONCENTRATION CURVE EXPERIMENT ABOVE:

First sample: A = 0.30, approximate concentration = 0.30 g/ml

Second sample: A = 0.45, approximate concentration = 0.45 g/ml

Third sample: A = 0.75, approximate concentration is = 0.75 g/ml