Using the Spectrophotometer

Using the Spectrophotometer Introduction In this exercise, you will learn the basic principals of spectrophotometry and and serial dilution and their practical application. You will need these skills to complete other exercises throughout the semester. It must be made clear that all this machine does is shine a beam of light filtered to a specific wavelength through a chamber, designed to hold a sample cuvette, and onto a light meter. The rest is inference and calculation from which you will examine basic experimental design and method. The instruments we will be using (Spectronic 401) will help you with some of this, and research-grade spectrophotometers can practically do it all for you. However, you must have a basic understanding of concepts and principals of spectrophotometry to be able to compensate for any errors in operation or experimental design. In short the machine will not think for you. You will have to learn to use these machines and to know what to expect from them so that you can detect problems and use these instruments with confidence later in the semester. Learning Objectives Conceptual You should understand: • the use of light transition and absorption to measure the concentration of chemicals in solution and the basic principles spectrophotometry. • how to determination of the A max for a compound. • the application of Beer’s law. Practical • basic operation of the spectrophotometer. • making dilutions. Underlying Science Basic principles of spectrophotometry An absorbance spectrophotometer is an instrument that measures the fraction of the incident light transmitted through a solution. In other words it they are used to measure the amount of light that passes through a sample material, and by comparison to the initial intensity of light they indirectly measure the amount of light absorbed by a sample. They are also designed to transmit light of narrow wavelength ranges (see Figure 1 the electromagnetic spectrum). It happens that a given material will always absorb light in the same way and not equally at all wavelengths of light-- that’s why things are different colors. Some compounds absorb light outside of the visible light spectrum, and that’s why there are colorless solutions like water. Because different compounds absorb light at different wavelengths a spectrophotometer can be used to distinguish compounds. Additionally, the amount of light absorption is directly proportional to the distance that the light traveled through a sample and the concentration of absorbing compounds in that sample. We will be using a spectrophotometer (the Spectronic 401) several times this semester to quantify the concentration of chemicals present in a solution. Spectrophotometers may also be used to estimate cell numbers and even identify what chemical are in solution.

400

500 Violet

600

700 nm

Blue Green Yellow Orange Red

10-5 nm Gamma rays

1013 nm X-rays

Ultraviolet

Visible Infrared

Microwave

Radio

Figure 1. The electromagnetic spectrum. Visible light (400-700 nm) constitutes only a small portion of the spectrum that ranges from gamma rays (less than 1 pm long) to radio waves that are thousands of meters long

When studying a compound in solution by spectrophotometry, you put it in a sample holder called a cuvette and place it in the spectrophotometer. Light of a particular wavelength passes through the solution inside of the cuvette and the amount of light transmitted (passed through the solution) or absorbed by the solution is recorded. You are interested only in the absorbance of the compound you are studying. To determine how much light is absorbed by the solvent and other components in the solution, we compare the absorbance of our test solution to a reference blank. For instance, in today’s lab exercise you will be measuring the absorbance of a dye, bromphenol blue that was dissolved in water. The reference blank in this case would be water alone. The amount of light transmitted through a solution is referred to as transmittance (T). The transmittance is defined as the ratio of the light energy transmitted by the sample (I) to the energy transmitted by the reference blank (I 0 ). T=I/I 0 This number is multiplied by 100 to determine the percent transmittance (%T), or the percentage of light transmitted by the substance. %T = I/I 0 * 100 Since the compound being tested is not present in the reference blank, the transmittance of the reference blank is defined as 100%T. A certain portion of the light will be absorbed by the compound in the test cuvette, therefore the %T will be lower. For most biological applications, we measure absorbance (A, also referred to as Optical Density or OD), the amount of light that is absorbed by a solution. Absorbance is related logarithmically to transmission. A=-log T

A=0 for the reference blank since there is no test compound present to absorb any light. Visible light (see Figure 2) is composed of wavelengths from 400 to 700 nm (nanometers). When visible light passes through a colored solution, some wavelengths are transmitted and some are absorbed. You see the color of the transmitted wavelengths. For instance, a red color results when a solution absorbs short wavelengths of light (green and blue) and transmits longer wavelengths (red). An absorbance spectrum (a plot of absorbance as a function of the wavelength of the incident light) is normally measured to determine the optimal wavelength (Amax) for measuring the absorbance of a given solution. The optimal wavelength (Amax) for measuring absorbance is that wavelength that is most absorbed by the compound in solution. A hypothetical absorbance spectrum is shown in Figure 2.

1.2

1.0 Absorbance 0.6

0.2 450

500

550 600 650 Wavelength (nm)

700

Figure 2. Absorption spectrum. A graph of absorbance vs. wavelength for a hypothetical compound. The Amax for this compound is about 500 nm.

The light from the spectrophotometer’s light source does not consist of a single wavelength, but a continuous portion of the electromagnetic spectrum. This light is separated into specific portions of the spectrum through the use of prisms or a diffraction grating. A small portion of the separated spectrum passes through a slit. When you adjust the wavelength on a spectrophotometer, you are changing the position of the prism or diffraction grating and different wavelengths of light are directed at the slit. The smaller the slit width, the better the ability of the instrument to resolve various compounds. The slit width in the Spectronic 401 is