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Spectrophotometry Page 1 of 10

Lab #4

SPECTROPHOTOMETRY

Adapted from: C. Greene MJC

INTRODUCTION:

In this exercise, you will learn the basic principles of spectrophotometry and serial dilution and

their practical applications. You will need these skills to complete other exercises throughout the

semester. A spectrophotometer is a very powerful tool used in both the biological and chemical

sciences yet operates by simply shining a beam of light, filtered to a specific wavelength (or very

narrow range of wavelengths), through a sample and onto a light meter. Some basic properties

of the sample can be determined by the wavelengths and amount of light absorbed by the

sample.

Objectives:

• State the basic mechanics of the spectrophotometer

• Describe the basic principles of spectrophotometry, including transmittance and

absorbance.

• Determine the Amax for a compound.

• Generate and use a standard curve to analyze data

• Perform serial dilutions

Background Information:

An absorbance spectrophotometer is an instrument that measures the fraction of the incident

light transmitted through a solution. In other words, it is used to measure the amount of light

that passes through a sample material and, by comparison to the initial intensity of light reaching

the sample, they indirectly measure the amount of light absorbed by that sample.

Spectrophotometers are designed to transmit light of narrow wavelength ranges. A given

compound will not absorb all wavelengths equally-that's why things are different colors (some

compounds absorb only wavelengths 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 by analyzing the

pattern of wavelengths absorbed by a given sample. Additionally, the amount of light absorbed is

directly proportional to the concentration of absorbing compounds in that sample, so a

spectrophotometer can also be used to determine concentrations of compounds in solution.

Finally, because particles in suspension will scatter light, thus preventing it from reaching the

light detector, spectrophotometers may also be used to estimate the number of cells in

suspension.

We will be using a spectrophotometer several times this semester to quantify the concentration

of chemicals present in a solution.

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 the cuvette and the amount of light transmitted passed through the

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Lab # 4

SPECTROPHOTOMETRY

Adapted from: C. Greene MJC INTRODUCTION: In this exercise, you will learn the basic principles of spectrophotometry and serial dilution and their practical applications. You will need these skills to complete other exercises throughout the semester. A spectrophotometer is a very powerful tool used in both the biological and chemical sciences yet operates by simply shining a beam of light, filtered to a specific wavelength (or very narrow range of wavelengths), through a sample and onto a light meter. Some basic properties of the sample can be determined by the wavelengths and amount of light absorbed by the sample. Objectives :

State the basic mechanics of the spectrophotometer
Describe the basic principles of spectrophotometry, including transmittance and absorbance.
Determine the A max for a compound.
Generate and use a standard curve to analyze data
Perform serial dilutions Background Information: An absorbance spectrophotometer is an instrument that measures the fraction of the incident light transmitted through a solution. In other words, it is used to measure the amount of light that passes through a sample material and, by comparison to the initial intensity of light reaching the sample, they indirectly measure the amount of light absorbed by that sample. Spectrophotometers are designed to transmit light of narrow wavelength ranges. A given compound will not absorb all wavelengths equally-that's why things are different colors (some compounds absorb only wavelengths 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 by analyzing the pattern of wavelengths absorbed by a given sample. Additionally, the amount of light absorbed is directly proportional to the concentration of absorbing compounds in that sample, so a spectrophotometer can also be used to determine concentrations of compounds in solution. Finally, because particles in suspension will scatter light, thus preventing it from reaching the light detector, spectrophotometers may also be used to estimate the number of cells in suspension. We will be using a spectrophotometer several times this semester to quantify the concentration of chemicals present in a solution. 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 the cuvette and the amount of light transmitted passed through the

solution – { Transmittance } or absorbed { Absorbance } by the solution is measured by a light meter. Note : 100% Absorbance = 0% Transmittance While a spectrophotometer can display measurements as either transmittance or absorbance, in biological applications we are usually interested in the absorbance of a given sample. Because other compounds in a solution (or the solvent itself) may absorb the same wavelengths as the compound being analyzed, we compare the absorbance of our test solution to a reference blank. Ideally, the reference blank should contain everything found in the sample solution except the substance you ate trying to analyze or measure. For instance, in today's lab exercise you will be measuring the absorbance of a dye, bromophenol 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 through the sample to the energy transmitted through the reference blank. Since the compound being tested is not present in the reference blank, the transmittance of the reference blank is defined as 100%T. (0% Absorbance) For most biological applications however, we measure absorbance ( A , also referred to as Optical Density or OD, where λ is the wavelength used for the measurements), the amount of light absorbed by a solution. Again, a reference blank is used. In this case, to 'zero out' any light absorbed by anything in the solution other than the compound of interest. By definition, the absorbance of the reference blank is set at zero (A = 0) Visible light (see your text) is composed of wavelengths from 400 to 700 nm (nanometers). When visible light passes through a colored solution, some wavelengths are transmitted and others are absorbed. You see the color of the transmitted wavelengths. For instance, a red color results when a solution absorbs short wavelengths (green and blue) and transmits longer wavelengths (red). An absorbance spectrum (a plot of absorbance as a function of wavelength) is determined to select the optimal wavelength for analyzing a given compound. The optimal wavelength (Amax} for measuring absorbance is that wavelength that is most absorbed by the compound in question. This provides maximum sensitivity for your measurements.

SPECTRONIC 20

Power
LCD Display
Sample Door
Keyboard
Optional Printer Connect
Lamp Compartment Door

1. DETERMINATION OF THE A max OF BROMOPHENOL BLUE:

MATERIALS:

Beaker of dH Tube of Bromophenol blue (BPB) (18.6μM} Cuvettes P-1 000 Micropipettor and blue tips Spectrophotometer PROCEDURE: Watch the demonstration on how to use the Spec in class and follow the instructions below. DETERMINATION OF THE A max of BROMOPHENOL BLUE

Look at the bromophenol blue (BPB) dye. What color light is being transmitted? What color light is being absorbed? Using your knowledge of color and wavelengths, estimate what the wavelength might be the A max. Record this on your worksheet
To determine the Amax of the compound, each team will be assigned a range of 100 nm within the visible range of the spectrum [400nm to 700 nm). Read the absorbance of the sample every 10 nm within your team's assigned range. You will then write your data on the board and compile and graph using the data for the whole class to determine the A max for BPB.
Follow the Spec instructions above to set the wavelength of the spectrophotometer to the lowest wavelength in your range.
Place the reference blank cuvette (usually referred to as a "blank") into the spectrophotometer ("spec") and follow the above directions to zero the absorbance.
Remove your blank and place the cuvette containing the bromophenol blue into the spec. Read the absorbance at the first wavelength in your range and record it in a table in your lab book.
Repeat steps 6 - 8 to read the absorbance of the bromophenol blue every 10 nm for the rest of your range.
You must "re-zero" the spec at each wavelength using the blank. Don't worry if the display of the spec starts flashing "UNFL." This will go away when you hit the AUTO ZERO button.
Rinse out your two cuvettes with dH20 and place upside down on a Kim Wipe to drain, you will use them again in Part B.
- Record your numbers in the results section on your worksheet.
- Once all the data is obtained and present on the board, draw a quick graph in your lab book and determine the Amax for bromophenol blue. This is the wavelength you will use in Part 2.

Single Dilutions (Background) Stock solutions are constantly diluted in biological lab settings. It is critical that you master both serial and simple (single) dilutions. The following formula can easily be used to carry out a simple dilution. Calculation of Concentration Using C1V1 = C2V To make a fixed amount of a dilute solution from a stock solution, you can use the formula: C1V1 = C2V2 where:

V1 = Volume of stock solution needed to make the new solution
C1 = Concentration of stock solution
V2 = Final volume of new solution
C2 = Final concentration of new solution Example: Make 5 mL of a 0.25 M solution from a 1 M solution.
Formula: C1V1 = C2V
Plug values in: (V1)(1 M) = (0.25 M) (5 ml)

• Solve for V1 : V1 = [ (0.25 M) (5 ml)] / (1 M)

V1 = 1.25 ml
Answer: Place 1.25 mL of the 1 M solution into V 2 - V 1 (5ml – 1.25 ml) = 3.75 ml of diluent Practice this technique – if you are going to be a successful biology student you will need to be very comfortable carrying out this type of activity.

EXAMPLES

2A: THE EFFECT OF CONCENTRATION ON ABSORBANCE: ( use of serial dilutions ) MATERIALS: Bromophenol blue (BPB) (18.6μM} Cuvettes P- 1000 Micropipettor and blue tips Spectrophotometer Test tubes Unknown solution of bromophenol blue PROCEDURE:

Obtain 4 test tubes. Number them 1 to 4.
Use a P1000 to pipet 2000 μI of dH 2 0 into each tube.
Add 2000 μI of 18.6 μM bromophenol blue to tube 1. This tube represents a 50% (or a 1: 2 dilution ). The concentration of bromophenol blue in this tube is 9.3 μM (18.6 μM * 0.5). To mix the contents of tube 1, use a vortex mixer if available. Place your tube in the black rubber cup of the vortex and press down to mix your sample.
Determine the Absorbance of your solution in tube 1 and record it on your worksheet
Pipet 2000 μI of the Tube 1 contents into Tube 2. Mix using the vortex. Tube 2 is a 25% (or 1:4 dilution ). (You made a 50% dilution of a 50% dilution. 0.5 * 0.5 = 0.25) What is the concentration of bromophenol blue in this tube?
Determine the Absorbance of your solution in tube 2 and record it on your worksheet
Transfer 2000 μI of Tube 2 into Tube 3. What dilution is this? You made a 50% dilution of a 1:4 dilution, making it a 1:8 dilution. What is the concentration of bromophenol blue in this tube?
Determine the Absorbance of your solution in tube 3 and record it on your worksheet
Transfer 2000 μI of Tube 3 into Tube 4. ( 1:16 dilution )
Set the spec. to the A max wavelength that you determined in Part 1 of this exercise.
Zero the spec using your blank cuvette.
Determine the Absorbance of your solution in tube 4 and record it on your worksheet

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Lab # 4

SPECTROPHOTOMETRY

SPECTRONIC 20

1. DETERMINATION OF THE A max OF BROMOPHENOL BLUE:

MATERIALS:

• Solve for V1 : V1 = [ (0.25 M) (5 ml)] / (1 M)

EXAMPLES

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