Chapter 2. Laboratory Techniques: Spectrophotometer

Learning Objectives

Pre-Lab
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General Purpose

Conceptual

  • Understand the relationship between light absorption and the concentration of a substance
  • Understand how to use a standard curve

Procedural

  • Gain proficiency in the use of a spectrophotometer.

General Purpose

This lab will help familiarize you with some of the techniques you will be using in the exercises throughout this manual. It will also introduce you to some of the basic equipment needed to conduct the exercises in the manual.

General Background Information

Conducting experiments in biology requires the use of certain specialized equipment and techniques. To perform these techniques and use this equipment accurately and efficiently it is important to understand how the equipment works and to become practiced in the techniques. The techniques include pipetting and spectrophotometry. The instruments you are going to use during this exercise include pipets and spectrophotometers. You will be using these instruments and techniques during many of the other laboratory exercises this semester and in many other courses in the future. These instruments and techniques are also used in a wide variety of fields from industry to health care.

Spectrophotometry

Spectrophotometers are one of the most widely used laboratory instruments. Before continuing on, please read Appendix A, which describes the basic operation and use of a spectrophotometer in more detail. Cobalt chloride (CoCl2) solution is colored and follows Beer’s Law: When monochromatic radiation passes through a medium (e.g., solution) containing an absorbing substance, the decrease in the intensity of the light which is transmitted (or the increase in the intensity of the light absorbed) by the substance is directly proportional to the concentration of absorbing species. In other words as the concentration of a substance in solution increases, the amount of light absorbed by the solution increases. We can use a spectrophotometer to measure the concentration of CoCl2 in a solution as a function of the absorbance.

How a spectrophotometer works:

How to use a spectrophotometer:

Pre-Lab Quiz

Proceed to the Spectrophotometer Pre-Lab Quiz in the Spectrophotometer folder.