Lesson 3 – Spectrophotometer and Micropipette use
Course: Life Science, Integrated Science, STEM
Unit: Ecological Networks Part 1- Network interactions
- 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 .000001 L.
- 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.
- 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.
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.
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.