Lesson 3 – Spectrophotometer and Micropipette use

Description: The students will be introduced to two different pieces of equipment, the micropipette and the spectrophotometer. Both of these instruments will be used in a future investigation.

 

Objectives

What Students Learn:

  • The purpose of the micropipette is to measure and dispense small volumes of liquids.
  • A microliter (μL) is a unit of measure for small volumes of liquids in which 1 μL equals .000001L.
  • The proper use of a spectrophotometer including how to set the wavelength, measure the absorbance, and use a blank to zero the spectrophotometer between two readings.
  • A reference (blank) is needed to zero (calibrate) the spectrophotometer.
  • Optical density (O.D) is the unit used to measure absorbance.
  • When the number of particles (food coloring) present in a given volume of the sample increases, the light absorbance increases too.

What Students Do:

  • Students use three different sizes of micropipettes to practice measuring given volumes of liquid using the proper technique (reading and setting the correct volume, measuring and dispensing intended volumes of a sample).
  • Students use a spectrophotometer to measure the absorbance of various dilutions of food coloring and determine the relationship between absorbance and the number of particles in a sample.

Instructions

Overview

The students will be introduced to two different pieces of equipment, the micropipette and the spectrophotometer. Both of these instruments will be used in a future investigation. First the teacher will introduce how to use both the micropipette and spectrophotometer through a short video clip and through modeling the use of the equipment. The class will then be divided into groups. Half of the class will start by completing the spectrophotometry activity and half of the class will start with the micropipetting activity. After about 20 minutes the groups will switch. Upon completion of the two activities, students will know how to use the equipment properly and efficiently.

Teacher Background Information: This activity is designed to introduce the students to micropipettes and spectrophotometers. Micropipettes are one of the primary tools of the laboratory biologist. These instruments allow you to measure and dispense small and accurate volumes of liquid solutions. Using correct pipeting technique will greatly increase the chance that your student’s experiment will return meaningful data so that their laboratory experience can be both fun and academically enriching. These micropipettes will allow you to accurately measure volumes as small as 2µl and as large as 1000µl. A microliter is 1 millionth of a liter or 10-6 L.

A spectrophotometer is a device used to measure light intensity. A spectrophotometer can measure either absorbance or transmittance of light. A small beam of light with a specific wavelength is emitted from the spectrophotometer which goes through the sample in a small glass container called a cuvette. The spectrophotometer measures how much light is absorbed by the sample or how much of the light passes through the sample which is transmittance. In this activity students will be using the spectrophotometer to measure the amount of light that is absorbed when various dilutions of food coloring are used. The more particles (food coloring) that are present in a given volume of a sample, the more of the light is absorbed and the less light that is transmitted. In the spectrophotometer activity, it is important to use yellow food coloring with the specified wavelength of 540 nm. The reason 540 nm is used with the yellow food coloring is because the wavelength of yellow is ~600 nm. In order to get a reading with the spectrophotometer, you need to set the wavelength at the complementary wavelength to yellow which is blue ranging ~500 nm. Given the specified food coloring concentrations, a wavelength of 540 nm has the best results. S63166A
The light goes through an instrument that sets the wavelength. The small beam of light with a specific wavelength passes through the sample in a cuvette. Some of the light is absorbed and some of the light passes through. The amount of light that passes through is detected and a value is given by the spectrophotometer.

Before completing this lab, the teacher will need to check that they have all materials (see MATERIALS.doc (Google Doc | Word Doc)) There is also some advanced preparation required (see ADVANCE PREPARATION.doc (Google Doc | Word Doc)) in setting up the lab stations. The lesson is designed for 4 groups of 3-4 students to be working with the spectrophotometer and 6 groups of 3 students working with the micropipettes. The number of groups may vary depending on the number of students and the number of spectrophotometers available.

INTRODUCTION (As a whole Group)
    1. The teacher should remind students that in order to carry out the optimized procedure to test the affect of salinity on Halobacterium growth they will have to learn how to properly use two pieces of lab equipment: the micropipette and the spectrophotometer. In order to use these instruments, proper technique needs to be learned and practiced.

      Explain that half of the class will begin working with the spectrophotometer and half the class will be working with the micropipettes. After about 20 minutes, the students will switch. Tell the students before they can complete the two activities you will be giving an overview of proper technique and procedure for both the micropipetting and spectrophotometer activity.

    2. Introduce the micropipetting activity to the whole class. The teacher should give a brief introduction to what the micropipette is used for.

      A short inspirational video clip can be shown of a scientist using a micropipette in the lab (CSI or forensic science show). The teacher should emphasize that micropipettes are very expensive and need to be used properly to maintain the instrument and for accuracy in the lab.

    3. The teacher should next show a short instructional video (~7 minutes) on using a micropipette from ISB (Full_pipet.mov). This short video (~ 1 minute) can also be used if needed to review the use of micropipettes (Review_pipet.mov) right before using the micropipette.
    4. Distribute the student sheet for the micropipetting activity (Google Doc | Word Doc) and give an overview of the activity. As a class, read the Objectives, the Introduction and begin reading through the procedures. Have the students answer the questions on choosing the correct micropipette and setting the volume as you go through the procedures. When you get to Step 2 of Using the Micropipette, demonstrate each step at the front of the class. You may want to project the picture of the micropipette on the projector as well (micropipette.bmp). Be sure to emphasize the following as you demonstrate how to use the micropipette:
      • Once the pipet is set to the desired volume, make sure to lock the volume.
      • The plunger has two stops. The first stop is for drawing up the desired volume of liquid and the second stop is for completely dispensing the sample.
      • After dispensing the sample into its container, keep the plunger depressed until the tip is completely out of the container.

      Briefly go over the steps of the micropipetting activity. Be sure to emphasize the following when going over the micropipetting activity:

      • Do not leave the micropipette standing in a solution to avoid spilling the solution.
      • Do not allow the top of the micropipette tip to be completely submerged into any of the solutions.

      Explain that the students will be working in groups of three. Each student will be using all three of the micropipettes. Project on the classroom screen, Micropipette Reminders Slide (Google Slides | PowerPoint) during the activity so students can be further reminded of the important steps.

      Distribute the student sheet for the spectrophotometer lab (Google Doc | Word Doc) and give an overview of the activity. Explain that you are now going to go over how to use the spectrophotometer as a class. Read through the introduction to the spectrophotometer activity. Emphasize the picture showing how a spectrophotometer works. Explain that the light goes through a slit that sets the wavelength. The small beam of light with a specific wavelength passes through the sample in a cuvette. Some of the light is absorbed and some of the light passes through. The amount of light that passes through or the amount of light that is absorbed (this depends on the mode the spectrophotometer is set for, either absorbance or transmittance) is detected and a value is given by the spectrophotometer. We have found that drawing how a spec works on the board, while explaining it, helps students understand it. You can also add a short video, such as this one, to give an overview of how the spec works. Have the students read through the entire procedure. Be sure to emphasize that the spectrophotometer should be set to measure absorbance (not transmittance). Also, emphasize that the graduations on the Beral pipets go up to 1 mL. Students will need to measure 1 mL four times in order to put the desired amount of liquid into the cuvette.

      Background Note for the Teacher: Students should observe that as the number of particles (food coloring) present in a given volume of the sample increases, the light absorbance increases too.

    5. Discuss the importance of using a cuvette with water (blank) to zero the spectrophotometer using Spec.ppt. (Google Slides | PowerPoint)

      Ask students why the cuvette with water needs to be inserted into the spectrophotometer between each reading. Emphasize the importance of a blank. Show the PowerPoint of what a blank is. First, ask students to explain how they would find the mass of a substance like salt using an electronic balance, a beaker, and salt. Students may say that you can put the beaker on the balance and then zero the balance or they may say that you can find the mass of the beaker alone and the mass of the beaker with salt. You can then find the mass of the salt by subtracting the mass of the beaker from the mass of the beaker with salt. Explain that determining absorbance of a sample is similar to finding the mass of a substance in a beaker. The substance that serves as the blank may absorb some of the light (not all the light may be transmitted). For example, in the activity the cuvette with water may absorb some light. In order to find the absorbance just the food coloring, you need to subtract the initial absorbance of the water from the new absorbance of the water and food coloring. Just like with an electronic balance, the spectrophotometer can be zeroed after the blank (cuvette with water) is placed in the spectrophotometer. Now when the cuvette with the water and food coloring is added, any change in absorbance is due to the addition of the food coloring not the water.

      ACTIVITY (Working in Groups).

    6. Divide the class into two groups. Half of the class will be working with the spectrophotometer and half the class will be working with the micropipettes.

      Assign students to work in groups of three. However, during the spectrophotometer activity, students may need to work in larger groups depending on the class size and the number of spectrophotometers available. After 20 minutes, the teacher should have the groups switch.

    7. Collecting work. Use the Answer Key and Example Data.doc (Google Doc | Word Doc) to check. The teacher may either want to collect all student work including the paper towel from micropipetting lab or have students attach filter paper to their lab journal. If you are not going to look at the paper towel right away, you may want students to draw a circle around each of their dried samples from the micropipetting activity. The smaller volumes become hard to see.
    8. As an extension, students can practice converting metric units.

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Assessment

How will I know they know…

  1. Check student’s learning by grading the two worksheets specifically looking at metric conversions of the units, setting and reading micropipette volumes and the analysis questions.
  2. Check student’s performance using the micropipette by checking the diameter of the dots made on the filter paper. Partners should have similar diameters.
  3. Check the student’s performance using the spectrophotometer by comparing the student’s absorbance values with the values provided in the table below.
  4. Check the student’s performance by providing pre-made samples of food coloring to the student and have them use the spectrophotometer to check their absorbance. The teacher should have students use the same spectrophotometer and sample to do their readings for the assessment.

Resources

Accommodations

Help students understand difficult vocabulary by breaking apart and explaining key root words. For example – spectrophotometer: spectro – photo – meter. “Spectro” comes from the Latin, specere, meaning appearance or to look at – it indicates a range or distribution to look at (e.g. What is the spectrum of trees around your school?). Even more simply, spect = look (e.g. spectacles, inspect). “Photo” means “light” and “meter” indicates something used to measure, often with numbers or quantities (quantitatively). When stringing root words back together, do so one step at a time. A photometer describes something that measures light. A spectrophotometer describes something that measures the range of light. Going even more in depth, this is a machine that measures the many wavelengths of light as they move through a liquid sample. You can go as deep as needed by your students. For example, to extend this further, explain that light is made of wavelengths and these wavelengths result in various colors. Since a spectrophotometer measures the amount of light and the range of light, or the wavelengths, it also measures color and intensity.

Extension

As an extension, students can practice converting metric units. Examples are at the end of the student sheet for the micropipetting activity.

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