BIOL 207: Lab 3
BIOLOGY 207 LABORATORY 3
TECHNIQUES USED TO MEASURE GLUCOSE AND CHOLESTEROL
STUDENT LEARNING OBJECTIVES
1. Describe the function of the colorimeter.
2. Describe and understand Beer’s Law.
3. Understand the relationship between concentration and absorbance.
4. Calculate concentration from absorbance values.
5. Discuss hydrolysis reactions and condensation reactions.
6. Prepare and understand a standard curve.
GENERAL STATEMENT REGARDING SUBJECT MATTER
It is important that we become comfortable with basic techniques used to measure plasma glucose, protein
and lipid concentrations. Obviously, there are many situations in which you may need to know the
concentrations of these materials. After all, if you want to discuss the control of homeostasis, you have to
know if materials in the blood are within their normal range and that means you determine the
concentration.
There are many fundamental concepts needed in what seems to be the simple task of measuring the amount
of something in a liquid. We are going to use light to measure the concentration of glucose and cholesterol
in some samples. As long as the light you are using is monochromatic, or consists of one wavelength only,
and as long as the substance you are measuring absorbs that color of light, you might realize that the
amount of light absorbed by the material will be proportional to the amount of material in solution. The
more stuff in the solution, the more of a particular light is absorbed. What we need is a machine capable of
producing a single color of light and of measuring the amount of light absorbed. This instrument is referred
to as a colorimeter or spectrophotometer. The basic properties of light absorption and the relationship to
concentration are contained in Beer’s Law. We will discuss this below.
EXERCISE 3.1: Introduction
Beer’s Law is a fundamental law that describes the relationship between the amount of a specific material in
a solution and the amount of a specific wavelength or frequency of light that is absorbed by that material.
This law has been used for a very long time as the basis of measuring the concentrations of material in your
blood plasma. Simply stated, Beer’s Law states that the amount or concentration of a specific material, like
glucose, in solution is proportional to the amount of light absorbed by the solution. In other words, if you
have a solution of glucose, and there is a wavelength of light that is absorbed by glucose, you should be able
to determine the concentration of glucose in solution by measuring the amount of that specific wavelength
of light absorbed by the solution. There are, of course, other conditions that have to be true and we will
discuss those as we go through the exercises.
Beer’s Law assumes that the light being used is monochromatic or of a single wavelength. It’s possible to
separate white light into the various monochromatic light constituents by using a prism or other devices.
Many of you have seen this effect. You put a prism in a window, the sun shines on it, and you get a pattern
of different colors of light on your wall. That pattern is always the same in terms of sequence of colors. If
you block out all colors except the brightest red, you will then have nearly monochromatic red light. A
colorimeter (spectrophotometer) is an instrument that can produce white light and it also contains a
prism or grating capable of separating the white light into the various wavelengths, or into monochromatic
light. The colorimeter (spectrophotometer) can also measure the amount of light that enters a chamber,
3.1
, BIOL 207: Lab 3
and the amount of light that comes out. If you stick a test tube, or in this case a cuvette, into the chamber
and the material in the test tube absorbs some of the light, you can then measure the amount of light
absorbed by the solution in the colorimeter. According to Beer’s Law, the relationship between the
amount of light absorbed by a material in solution and its concentration is shown by
Concentration of solution 1 Absorbance of solution 1
=
Concentration of solution 2 Absorbance of solution 2
Again, this is absorbance at a specific wavelength by a specific component, say glucose, in the solution.
Using the above relationship, you can see that if you know the concentration of a material (glucose) in a
solution (this solution would be your standard), and if you can measure how much of a monochromatic light
the standard solution absorbs, and if you can measure the absorbance of a solution of unknown
concentration, then you should be able to use the above relationship to calculate the amount of material in
the solution of unknown concentration.
Example;
You want to know the amount of glucose in a solution or sample of plasma. You have a solution of glucose
that you know is 0.2 M in concentration. You measure the absorbance of the 0.2 M concentration material at
a specific wavelength and find the absorbance is 0.4 absorbance units. Now, to find out the concentration of
your unknown solution, you measure it’s absorbance at the same wavelength and find it is 0.3 absorbance
units. Just use the above relationship to calculate the concentration.
Concentration of known is 0.2 M = Absorbance of known is 0.4
Concentration of unknown is ? Absorbance of unknown is 0.3
Do the math: 0.2 M = 0.4
? 0.3
Concentration of the unknown = ? = 0.15 M
All of this assumes you set up the colorimeter in the proper way. This includes choosing the right
wavelength for your measurements, and setting the absorbance of the colorimeter so that a solution with a
concentration of 0 will have an absorbance of 0. We will discuss how to do this below.
There is another way of determining the concentration of the material in solution, and that is by using a
STANDARD CURVE. You will also use this method to determine the concentration of a material in
solution. Basically, you set up your colorimeter so that a solution with a 0 concentration of glucose has 0
absorbance, then you measure the absorbance of a series of solutions of known concentration and plot this
information on a standard curve.
For instance, let’s say you have the following solutions; solution 1; concentration of glucose 0,
absorbance in the colorimeter 0 solution 2; concentration of glucose 0.1 M, absorbance in the
3.2
BIOLOGY 207 LABORATORY 3
TECHNIQUES USED TO MEASURE GLUCOSE AND CHOLESTEROL
STUDENT LEARNING OBJECTIVES
1. Describe the function of the colorimeter.
2. Describe and understand Beer’s Law.
3. Understand the relationship between concentration and absorbance.
4. Calculate concentration from absorbance values.
5. Discuss hydrolysis reactions and condensation reactions.
6. Prepare and understand a standard curve.
GENERAL STATEMENT REGARDING SUBJECT MATTER
It is important that we become comfortable with basic techniques used to measure plasma glucose, protein
and lipid concentrations. Obviously, there are many situations in which you may need to know the
concentrations of these materials. After all, if you want to discuss the control of homeostasis, you have to
know if materials in the blood are within their normal range and that means you determine the
concentration.
There are many fundamental concepts needed in what seems to be the simple task of measuring the amount
of something in a liquid. We are going to use light to measure the concentration of glucose and cholesterol
in some samples. As long as the light you are using is monochromatic, or consists of one wavelength only,
and as long as the substance you are measuring absorbs that color of light, you might realize that the
amount of light absorbed by the material will be proportional to the amount of material in solution. The
more stuff in the solution, the more of a particular light is absorbed. What we need is a machine capable of
producing a single color of light and of measuring the amount of light absorbed. This instrument is referred
to as a colorimeter or spectrophotometer. The basic properties of light absorption and the relationship to
concentration are contained in Beer’s Law. We will discuss this below.
EXERCISE 3.1: Introduction
Beer’s Law is a fundamental law that describes the relationship between the amount of a specific material in
a solution and the amount of a specific wavelength or frequency of light that is absorbed by that material.
This law has been used for a very long time as the basis of measuring the concentrations of material in your
blood plasma. Simply stated, Beer’s Law states that the amount or concentration of a specific material, like
glucose, in solution is proportional to the amount of light absorbed by the solution. In other words, if you
have a solution of glucose, and there is a wavelength of light that is absorbed by glucose, you should be able
to determine the concentration of glucose in solution by measuring the amount of that specific wavelength
of light absorbed by the solution. There are, of course, other conditions that have to be true and we will
discuss those as we go through the exercises.
Beer’s Law assumes that the light being used is monochromatic or of a single wavelength. It’s possible to
separate white light into the various monochromatic light constituents by using a prism or other devices.
Many of you have seen this effect. You put a prism in a window, the sun shines on it, and you get a pattern
of different colors of light on your wall. That pattern is always the same in terms of sequence of colors. If
you block out all colors except the brightest red, you will then have nearly monochromatic red light. A
colorimeter (spectrophotometer) is an instrument that can produce white light and it also contains a
prism or grating capable of separating the white light into the various wavelengths, or into monochromatic
light. The colorimeter (spectrophotometer) can also measure the amount of light that enters a chamber,
3.1
, BIOL 207: Lab 3
and the amount of light that comes out. If you stick a test tube, or in this case a cuvette, into the chamber
and the material in the test tube absorbs some of the light, you can then measure the amount of light
absorbed by the solution in the colorimeter. According to Beer’s Law, the relationship between the
amount of light absorbed by a material in solution and its concentration is shown by
Concentration of solution 1 Absorbance of solution 1
=
Concentration of solution 2 Absorbance of solution 2
Again, this is absorbance at a specific wavelength by a specific component, say glucose, in the solution.
Using the above relationship, you can see that if you know the concentration of a material (glucose) in a
solution (this solution would be your standard), and if you can measure how much of a monochromatic light
the standard solution absorbs, and if you can measure the absorbance of a solution of unknown
concentration, then you should be able to use the above relationship to calculate the amount of material in
the solution of unknown concentration.
Example;
You want to know the amount of glucose in a solution or sample of plasma. You have a solution of glucose
that you know is 0.2 M in concentration. You measure the absorbance of the 0.2 M concentration material at
a specific wavelength and find the absorbance is 0.4 absorbance units. Now, to find out the concentration of
your unknown solution, you measure it’s absorbance at the same wavelength and find it is 0.3 absorbance
units. Just use the above relationship to calculate the concentration.
Concentration of known is 0.2 M = Absorbance of known is 0.4
Concentration of unknown is ? Absorbance of unknown is 0.3
Do the math: 0.2 M = 0.4
? 0.3
Concentration of the unknown = ? = 0.15 M
All of this assumes you set up the colorimeter in the proper way. This includes choosing the right
wavelength for your measurements, and setting the absorbance of the colorimeter so that a solution with a
concentration of 0 will have an absorbance of 0. We will discuss how to do this below.
There is another way of determining the concentration of the material in solution, and that is by using a
STANDARD CURVE. You will also use this method to determine the concentration of a material in
solution. Basically, you set up your colorimeter so that a solution with a 0 concentration of glucose has 0
absorbance, then you measure the absorbance of a series of solutions of known concentration and plot this
information on a standard curve.
For instance, let’s say you have the following solutions; solution 1; concentration of glucose 0,
absorbance in the colorimeter 0 solution 2; concentration of glucose 0.1 M, absorbance in the
3.2
