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Abstract

Objective

Build a simple electronic device to measure light scattering in liquids, and use it to measure the effect of protease activity.

Introduction

Water in a mountain stream is usually murkier, or more turbid, after a rainfall because the rain washes soil and other small particles into the stream. Environmental engineers and others who track the quality of drinking water measure the turbidity of the water supply using a turbidity meter, called a turbidimeter. The turbidimeter measures light scattering. Whereas in light absorption, colored molecules in the liquid absorb the incoming light, in light scattering, the incoming light bounces off of small, suspended particles, and is thus redirected away from its initial path.

Instruments that measure light absorption or light scattering typically have different setups. For detecting light absorption, the instrument measures how much light passes directly through the liquid. For light scattering, the instrument measures how much light is redirected, or scattered, at a 90-degree angle from the direction of the incoming light. See Figure 1.

Figure 1. Some of the incoming light is scattered by suspended particles, and is then detected by the light meter at an angle of 90 degrees from the initial path of the light beam. Some light is also absorbed. The remaining light is transmitted through the liquid and emerges on the opposite side.

A number of factors affect the scattering of light by suspended particles. The concentration and the size of the particles are two such factors. The physical characteristics of the particles are clearly important, too; for example, fat globules in milk will behave differently than dust in the atmosphere.

Another factor is the wavelength of the light. Blue light is scattered by the atmosphere more than red light is, which is why the sky looks blue. As sunlight passes through the atmosphere, the blue light is redirected, and we "see" it here on the ground. Sunsets are red/orange for the same reason—the sunlight has been depleted of blue colors by passing through the atmosphere.

The goal of this science fair project is to measure the amount of light scattered by the suspended particles in milk, and to determine how the amount of scattered light is related to the concentration of the particles. (Can you predict what will happen at low, medium, and high concentrations?) The Experimental Procedure describes how to make a simple light detector so that the amount of light scattered can be measured. You will also use the light detector to track the enzymatic activity of proteases found in pineapple juice, measured by the reduction in turbidity of diluted milk. Enzymes speed up the rate of chemical reactions. Proteases are a class of enzymes that chop up proteins. The action of proteases causes the proteins in milk to coagulate, which makes the milk less turbid.

Terms, Concepts, and Questions to Start Background Research

• Turbidity
• Suspended particles
• Light scattering
• Light absorption
• Wavelength (of light)
• Enzyme
• Protease
• Coagulate
• Light transmission
• Light intensity
• Voltage drop
• Photoresistor
• Potentiometer
• Multimeter
• Water bath

Questions

• Based on your research, how does particle size affect light scattering?
• What is the scientific definition of the word turbidity?
• What is "Rayleigh scattering"?
• How does the wavelength of light affect light scattering?
• Is there a linear relationship between particle concentration and intensity of scattered light?
• What causes natural water sources to be turbid?
• What effect does water turbidity have on its oxygen content? (Hint: Turbid water absorbs more sunlight and thus, can be warmer than clear water).

Bibliography

• Omega Engineering. (2008). Turbidity Measurement. Retrieved November 10, 2008, from http://www.omega.com/techref/ph-6.html
• Water on the Web. (2008, January 17). Turbidity. Retrieved November 10, 2008, from http://waterontheweb.org/under/waterquality/turbidity.html

Materials and Equipment

The Experimental Procedure describes how to make the light-sensing circuit on a breadboard. Using the breadboard gives you a lot of control over how the circuit is set up. If you are not familiar with how to use a breadboard, read the Science Buddies page Use a Breadboard to Build and Test a Simple Circuit. If you do not feel comfortable working with a breadboard to build the circuit, you can use a RadioShack kit, called The Electronic Sensorslab, part # 280-278, which has numbered springs to connect the parts. Follow the directions in the workbook for the project entitled "Build a simple photoresistor light meter," page 85.

• Breadboard, 2 x 3 inches, RadioShack part #276-003
  1. As an alternative to a breadboard, you could use the kit from RadioShack, described above. The Electronic Sensorslab kit is part #280-278.
• Solderless Breadboard Wire Kit, RadioShack part #276-173
• 9-V battery
• 9-V snap connectors, RadioShack part #270-324
• 24-inch Insulated test/jumper leads, part #278-1157
• Potentiometer, 1 mega-ohm, RadioShack part #271-092
• Digital autoscaling multimeter; available at most hardware and auto supply stores or at Amazon.com
  1. Some multimeters require that you turn a knob to change the range of voltages the meter is able to read. Autoscaling multimeters do this automatically.
• Photoresistor, RadioShack part #276-1657, package of 5
• Jar lid (1)
• Black electrical tape
• Drill with 1/4-inch drill bit to make a hole in the jar lid. A stiff knife will work, too.
• Safety goggles
• 2- x 4-inch board (about 8 inches long)
• Laser pointer
• Clear glass jars or clear plastic cups, 1-cup or greater capacity (6)
• Permanent marker
• Masking tape
• Milk, fat-free
• Tap water
• Graduated cylinder; available at science supply stores, such as Carolina Biological: www.carolina.com
• Spoon
• Syringe that can measure 25 milliliters (mL)
• Dim light, such as a nightlight or LED flashlight
• Fresh or frozen pineapple; do not substitute canned pineapple as it will not work well
• Cheesecloth; any piece of cotton fabric (even an old cotton T-shirt) can be substituted
• Food grater
• Tablespoon
• Microwave-safe container
• Stopwatch
• Graph paper
• Lab notebook

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Experimental Procedure

Assembling the Light Meter

1. To begin this science fair project, assemble the parts needed to make the digital light-sensing meter. The circuit for the meter is shown in Figure 2, below. To learn how to use a breadboard, visit the Science Buddies page Use a Breadboard to Build and Test a Simple Circuit.
2. The circuit has a 9-V battery, a photoresistor, a potentiometer (the "R" with the crooked line) and a "Readout" meter, which will be the digital multimeter. The resistance of the photoresistor is decreased in the presence of light. The potentiometer, which has a knob that changes its resistance, is used to vary the sensitivity of the meter. In bright light, the voltage drop across the photoresistor is reduced, leading to an increase in the voltage drop across the potentiometer, which is the "readout" detected by the multimeter.

Figure 2. Circuit diagram for the light-sensing meter. It converts light energy (input) into voltage (output).

Making Your Circuit

1. Connect the battery terminals to the breadboard. The positive lead should be connected to the power bus and the negative lead should be connected to the ground bus.
   1. Insert the wire from the positive terminal into one of the holes in the power bus (marked on most breadboards as the row of holes next to the red line).
   2. Insert the wire from the negative terminal into one of the holes in the ground bus (the row of holes next to the black or blue line).

Connect the photoresistor to the circuit, as follows. The photoresistor should be attached to the breadboard with 24-inch insulated jumper leads (jumper wires have alligator clips on both sides). This will allow the photoresistor to be moved and positioned as needed.

1. Connect one lead from the photoresistor to an alligator clip on one of the jumper wires (green wire in Figure 3).
2. Connect the other end of the jumper wire to a small wire lead that is inserted into the breadboard (position, or socket, A1 on the breadboard).
3. Connect the other lead from the photoresistor to an alligator clip on another jumper wire (yellow wire in Figure 3).
4. Connect the other end of the jumper wire to the positive bus (yellow jumper attached to small white wire in Figure 3).

Figure 3. Picture of the light-sensing circuit.

Connect the potentiometer to the circuit.

6. Add two wires to the potentiometer, one of them in the middle. Attach the other wire on either side of the middle connection. Solder them for a permanent connection, or just twist them on if you prefer not to solder. (The potentiometer has two red wires attached to it in Figure 3.)
7. Attach one of the wires from the potentiometer to the ground bus on the breadboard.
8. Attach the other wire from the potentiometer to the breadboard so that it is connected to the photoresistor (position C1 on the breadboard in Figure 3).

Connect the multimeter to the circuit.

9. Connect the red wire (positive) from the multimeter to position E1, in the row that contains the leads from the photoresistor (position A1) and the potentiometer (C1).
10. Connect the black wire (ground) from the multimeter to the ground bus.

Attach the photoresistor to a jar lid. This will allow you to block unwanted light.

11. Cut a small 1/4-inch hole in a jar lid.
12. Cover the wires from the photoresistor with electrical wire so they can't touch any other metal. Leave a small amount of wire showing at the end in order to attach the alligator clip.
13. Place the photoresistor inside the hole in the jar and tape it in place with the black electrical tape. Make sure no light can enter through the hole.

Testing the Light-sensing Circuit

1. Turn on the multimeter.
2. Set it to read DC voltage, sometimes labeled as DCV.
   1. The voltage should range from less than 100 millivolts (mV) in the dark to around 8 V in bright light.
   2. Watch the units on the multimeter since it may change scales from millivolts to volts, or vice versa.
   3. Keep in mind that the photoresistor will pick up light from behind if it is not shielded.
3. Place the photoresistor in bright light. Record the voltage.
4. Place the photoresistor in total darkness. Record the voltage.
5. If the circuit does not work, try the following:
   1. Check all of your connections carefully.
   2. Make sure the battery is charged.
   3. Adjust the potentiometer, if necessary.

Setting Up the Laser Pointer

1. Tape the laser pointer onto the 2- X 4-inch board so that it points over the short end of the board.
   1. This will give the laser a steady mount at a fixed height.
   2. Any solid base will do.
2. Tape the base to the work surface to keep it stable.

Setting Up Your Samples

In this step, you will set up samples with decreasing amounts of light-scattering particles.

1. Label six clear glass jars or clear plastic cups 1 to 6 with the masking tape and permanent marker.
2. You will make a series of 1:10 dilutions. Use 25 mL in 250 mL.
3. The jars should have the following contents (there are four 1:10 dilutions), further described in steps 4–9:
   1. Water
   2. 100 percent fat-free milk
   3. 1/10 dilution
   4. 1/100 dilution
   5. 1/1,000 dilution
   6. 1/10,000 dilution
4. Put 250 mL of water in jar # 1.
5. Put 250 mL of milk in jar #2.
6. Put 250 mL of water into jar #3. Add 25 mL of milk. Stir with a clean spoon.
7. Put 250 mL of water into jar #4. Add 25 mL from jar #3. Stir with a clean spoon.
8. Put 250 mL of water into jar #5. Add 25 mL from jar #4. Stir with a clean spoon.
9. Put 250 mL of water into jar #6. Add 25 mL from jar #5. Stir with a clean spoon.

Measuring Scattered Light

1. Now you are ready to measure scattered light. Move the circuit and the jars to a work area with dim light. There should be just enough light for you to work and to read the multimeter. You could use a nightlight or a red LED flashlight as a work light.

Figure 4. The laser light enters the turbid liquid and scatters in all directions. To measure the scattered light, the photoresistor (attached to a jar lid) is placed on top of the container, over the liquid.

2. Place jar #2 in front of the laser pointer.
   1. Use jar #2 first, since it will have a high level of scattered light.
   2. You will later measure the scattered light from jar #1 (water), and the other jars.
3. The laser should be pointing into the liquid. If it is not, then adjust your setup.
4. Place the photoresistor on top of the jar, so that it will collect light scattered upward by the milk.
5. Turn off the room light and the laser light.
6. Read the voltage on the multimeter and record it in your lab notebook. It should be less than 0.5 V. Adjust the potentiometer, as needed.
7. Shine the laser into the milk.
8. Read the voltage on the multimeter.
9. Repeat steps 2–8 with the remaining jars.
   1. Record the voltage prior to turning on the laser for each sample.
   2. Record the voltage after turning on the laser for each sample.
   3. Keep the placement of the jars, the laser, and the sensor the same for all measurements.
10. Graph the voltage (scattered light) vs. the dilution of the milk.
11. Graph the voltage (scattered light) vs. the log of the dilution.

Tracking Enzyme Activity

Now that you have a working turbidity meter, you will use it to track an enzyme-dependent process. The process involves the clearing of milk by proteases found in fresh pineapple juice. The proteases cause the light-scattering particles in the milk to coagulate, so the milk becomes more transparent.

1. Label three clean jars 1 to 3 with your masking tape and permanent marker.
2. Make 500 mL of 10 percent milk by adding 50 mL of milk to 450 mL of water.
3. Add 150 mL of the 10 percent milk to each jar.
   1. The three jars should have the following contents when you've completed the rest of the steps:
      • 10 percent milk, with no additional ingredients
      • 10 percent milk, with inactivated proteases
      • 10 percent milk, with active proteases
4. Squeeze 100 mL of fresh pineapple juice into a clean container.
1. To get the pineapple juice, take a fresh pineapple, cut off the rind, and grate the flesh. Place the grated fruit in a piece of cheesecloth and squeeze over a clean container.
2. Any fresh pineapple fruit will work as will frozen pineapple that has been thawed at room temperature. Canned pineapple or refrigerated pineapple juice does not work as they are heat treated and heat destroys enzymes.
5. Transfer 50 mL of the juice into a small, microwave-safe container.
6. Microwave the pineapple juice for about 45 seconds, or as long as necessary to get the juice to boil for 3–5 seconds. Heat will destroy enzymes, but won't affect most chemicals. The point is to show that enzymatic activity, which is heat-sensitive, is what causes the milk to clear.
7. Start the stopwatch. Record the times at which the pineapple juice is added.
   1. Add 2 mL of heated pineapple juice to jar #2.
   2. Add 2 mL of fresh, unheated pineapple juice to jar #3.
   3. You may need to change the amount of juice you add, depending on how fast the reaction occurs under your experimental conditions. Add less pineapple juice to get a slower reaction, and more pineapple juice for a faster one.
8. Measure the amount of scattered light from each jar every 2 minutes for 20 minutes.
9. Graph the voltage (scattered light) vs. time for all three jars.
10. Repeat steps 1–8 of this section at least two more times.

Variations

• Measure the amount of scattered light at different angles. Set the photoresistor at the same level as the laser and measure the amount of light as you move in a circle around a jar. Use a concentration of milk that provides good scatter. Graph scattered light vs. angle.
• Use a green laser, RadioShack Catalog # 63-132, instead of the red laser. Graph Scattered light vs. Angle for red and green lasers.
• Determine how temperature affects the rate of milk clearing by proteases. Use 10 percent milk with active proteases, but place the milk and proteases in a water bath (such as warm or cold water in a cooler) at different temperatures.
• Change the amount of proteases added to the milk in the last section.
• Measure scattered light in other liquids with suspended particles, such as stomach antacid in water, or water from a local pond.
• When phosphate (PO₄^3-) and calcium (Ca²⁺) are mixed in a solution, the insoluble mineral calcium phosphate forms as a white substance that scatters light. Make solutions from antacid tablets (Ca²⁺) and phosphate-containing fertilizer, and use the sensor to follow the formation of calcium phosphate.

• For more science project ideas in this area of science, see Biotechnology Techniques Project Ideas.

Credits

David Whyte, PhD, Science Buddies

Last edit date: 2012-02-08 09:00:00