Biology DCN Exercise 1. Orientation lab.

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Biology DCN Exercise 1. Orientation lab.                                                                                          [pic 1]

GENERAL BIOLOGY II (101-DCN)

LAB EXERCISE # 1

ORIENTATION LAB

OBJECTIVES

1. To understand how a spectrophotometer measures absorbance and how it is used.

1. To produce a standard curve using computer software, and accurately determine the concentration of unknown solutions using this curve.

1. To be able to calculate the concentration of a substance after diluting it.

INTRODUCTION

In biochemistry, colorimetry is often used to determine the presence or measure the concentration of a substance. Colorimetry is a technique that uses the absorbance of coloured compounds to determine their concentration. It is based on the Beer-Lambert law, which states that the concentration of a solute is proportional to its absorbance. Certain substances are coloured (e.g., CuSO4 used in this lab activity) and their absorbance can be determined directly. Other substances (e.g., dissolved sugars or proteins used in lab exercise 2) do not have a significant absorbance; to colorimetrically determine their concentration, they will first be mixed with a reagent that produces a coloured compound. As an example, in lab exercise 2 we will see that when proteins react with the biuret reagent, a coloured compound is produced. When mixing protein solutions with the biuret reagent, the absorbance of the product will be proportional to the concentration of the proteins.

In this lab, you will become familiar with how a spectrophotometer can be used in colorimetry and you will calculate the concentration of an unknown CuSO4 solution using data provided by your instructor.

MEASURING CONCENTRATIONS OF SOLUTIONS USING A SPECTROPHOMETER

Coloured substances in a solution absorb light. The amount of light that is absorbed is proportional to the concentration of the coloured material in the solution (this is what is called the Beer-Lambert law). A spectrophotometer makes use of this fact to relate light absorption to concentration in order to determine the unknown concentration of substances in solutions.

In a spectrophotometer, light of a particular wavelength is passed through a clear tube containing the solution, and from there passes to a photo-electric cell which produces an electric current proportional to the amount of light striking it (Figure 1).

[pic 2]

Figure 1. Internal components of a spectrophotometer.

Modified from microbenotes.com/spectrophotometer-principle-instrumentation-applications/

By measuring the light absorption of known concentrations of a substance, a standard curve can be made.  Unknown concentrations of that substance can then be determined by measuring the light absorption of the unknown solution and referring to the standard curve.

PREPARING A STANDARD CURVE

In this course, we will generate standard curves (also known as calibration curves in analytical chemistry) to determine the relationships between the concentration of a substance and its absorbance of light at a particular wavelength. To generate a standard curve, you will create solutions of different concentrations and measure their absorbance using a spectrophotometer. Once you have this data, you can represent the relationship in a scatterplot and perform a linear regression to determine the function that best represents the relationship between the independent variable (concentration) and the dependent variable (absorbance). You can then use this function to determine the concentration of a substance with a known absorbance. Let’s look at the details of how to generate a good standard curve:

1. Choose the correct substance to generate your standard curve

Choose a substance that is chemically similar to the solution of unknown concentration whose concentration you are trying to determine. For example, in laboratory 2, your task is to determine the concentration of purified protein in a solution. To do this, you will use bovine serum albumin (BSA), an inexpensive protein, to generate a standard curve. You are making an assumption: when using the biuret protein assay, different proteins will have the same relationship between concentration by weight and absorbance of light.

1. Create solutions of known concentrations by diluting a stock concentration

When preparing the solutions of known concentration for your standard curve, be careful to make accurate measurements of your stock solution and solvent and to properly account for the addition of any reagent (see * below). Make sure that your solution is a homogeneous mixture by using the vortex on your bench or pipetting up and down with your pipette.

1. Measure the absorbance of your solutions

Make sure that you are measuring the absorbance at a wavelength that is optimal for your particular substance. This optimal wavelength will be indicated in the lab manual. When possible, set the absorbance of your spectrophotometer to 0 using a blank solution prior to measuring the absorbance of your test solutions. Make sure that your cuvette is free of scratches and clean.

1. Create a scatterplot

In Excel or other software, create a scatterplot using the known concentrations of your standards as your independent variable and the absorbance at a particular wavelength as your dependent variable. The scatter plot represents the individual data points and gives a good visual representation of the relationship between two variables. See Figure 3 below for an example of a standard curve.

Take a moment to look at your scatterplot and see if it is linear over the entire range of concentrations. In some cases, the absorbance of solutions at higher concentrations does not increase at the rate you would expect either because there is insufficient reagent to bind to the target substance, or because the absorbance of the substance is beyond the detection limit of the spectrophotometer.

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