Pre-Lab E9: Spectrophotometry ( 10 pts) · PDF filePre-Lab E9: Spectrophotometry ( 10 pts) ......

Pre-Lab E9: Spectrophotometry ( 10 pts) Name: _______________________________; Lab Section: _______________; Grade: _______

1. A spectrophotometer measures: a. the wavelength b. the absorbance of light

2. If the absorbance value is 2, it means a. 99% of light is absorbed b. 90% of light is absorbed c. 10% of light is absorbed

3. The molar absorptivity of a chemical (ε) is always constant at any wavelength (ie – it would

be the same at 300nm and 400nm and 500nm). a. True b. False

4. Which of the following wavelength range describes visible light? a. 400-700nm b. 200-400 nm c. 700-1000 nm

5. Arrange the following in the proper order for today’s experiment: ___determine the absorbance of the KMnO4 solution of an unknown concentration.at λmax ___measure the absorbance of a KMnO4 solution at different wavelength to determine the λmax ___construct the standard curve of various KMnO4 solutions at λmax ___determine the concentration of the unknown solution using the standard curve ___ Make various KMnO4 solutions using parallel dilution method.

6. What are the two reasons for measuring the absorbance of a sample at its λmax? 7. What is the purpose of using a blank specimen in calibrating the spectrophotometer (to

autozero the machine)?

8. Complete the dilution table using parallel dilution method (example in procedure). Each of the following solutions should have a final volume of 5 ml. ( show calculation of Tube D to get full credit, 3 pts)

Table 1: Preparation of Seven Known Dilutions of KMnO4 Final Concentration of KMnO4 1 mM of KMnO4 Stock

(ml) Distilled water (ml)

Tube A: 0 mM 0 ml 5 ml Tube B: 0.05 mM Tube C: 0.10 mM Tube D: 0.15 mM Tube E: 0.20 mM Tube F: 0.30 mM Tube G: 0.40 mM 2 ml 3 ml Calculation of Tube D:

For more graph paper see: w

ww

.vvi.com/graphpaper

© Copyright 2004; VVimaging, Inc. All Rights Reserved.

Biol 2281, Spring 2016 E9: Spectrophotometry Procedure/Report

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Experiment 9: Spectrophotometry

Objectives: At the end of the exercise, you should be able to

1. demonstrate the techniques of using a spectrophotometer to determine the absorbances of solutions;

2. understand the concept of standard curves and be able to apply this concept in solving other similar problems;

3. understand the concepts and principles of Beer’s law; 4. learn how to manually draw graphs of experimental data and how to use Excel program

to graph and perform linear regression analysis. A. Materials Provided for Each student

1. 1 mM KMnO4 and a bottle of water 2. One of P1000 Micropipet and blue tips box 3. One of 5 ml Pipet for water 4. Eight of cuvettes (pathlength= 1 cm) and a cuvette holder 5. marker, ruler, masking tape and kim wipe. 6. One orange testube rack. Small Test tubes (7): DO NOT WRITE DIRECTLY ON THE

TUBES. LABEL THEM WITH MASKING TAPE; At the end of the lab, you need to remove the label and wash the tubes.

7. Numbered unknown KMnO4 solutions (provided to you by TA) B. Procedure (include a maximum of 8 points)

I. Making KMnO4 solutions Prepare seven solutions in small test tubes with the mixture of distilled water and stock solution of KMnO4 (1 mM) as listed in Table 1. The final volume of each solution should be 5 ml. Fill in the table completely and have your instructor check your numbers before you begin. Only tube A and tube G are needed to determine the absorption spectrum. You will use the others later in the lab period.

Table 1: Preparation of Seven Known Dilutions of KMnO4 Final Concentration of KMnO4 1 mM of KMnO4 Stock

(ml) Distilled water (ml)

Tube A: 0 mM 0 ml 5 ml Tube B: 0.05 mM Tube C: 0.10 mM Tube D: 0.15 mM Tube E: 0.20 mM Tube F: 0.30 mM Tube G: 0.40 mM 2 ml 3 ml Note: To complete the above table, you will need to learn how to perform “parallel dilutions”: dilute a concentrated stock solution to make solutions of lower concentrations. The formula for parallel dilution calculation is as follows: C1V1=C2V2 Where C1 and V1 are the concentration and volume of your stock solutions, and C2 and V2 are the concentration and volume of your dilute solution.


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For example, C1 = 1 mM for the stock solution, C2 = 0.4 mM (final concentration) and V2 = 5 ml (final volume) for the diluted solution. Therefore, the volume of stock solution V1 required can be determined as: (1 mM) V1 = (0.4 mM) ( 5 ml)

V1 = 2 ml The volume of water to make the dilution should be ____________.

II. Determine the absorption spectrum of KMnO4 in order to discover lambda max (λmax ) of the solution

1. Prepare the spectrophotometer: a. Turn on the spectrophotometer via the switch in the back. b. Make sure the spectrophotometer is on Basic ATC mode. If unsure, pressing ESC

will take you back to the menu, and Basic ATC will be the option on the bottom right.

c. After on Basic ATC mode, change mode (top left corner) to "Absorbance" via the Change mode button at the bottom of the screen.

d. Set the wavelength to 400 nm by pressing Set nm and entering 400.

2. Autozero

a. Prepare blank by using transfer pipet to fill a clean cuvette with about 1 ml of H2O (from tube A).

b. Open sample compartment, place the blank cuvette inside the B slot of the 6-positon cuvette holder. Make sure the arrow on the side of the cuvette is in-line with the circle of cuvette holder, not facing into or out of the circle. Close the lid.

c. Change the wavelength value by pressing Set nm and entering the new wavelength. Press “B” on cuvette position button to measure the blank under new wavelength. Press Measure Blank.

3. Measure the absorbance of 0.4mM KMnO4 at various wavelengths.

a. Place a cuvette with around 1 ml of 0.4mM KMnO4 (from tube G) in sample slot 1, then close the lid.

b. Press “1” on cuvette position button to measure sample. c. Record the absorbance value, then change the wavelength value by pressing Set

nm and entering the new wavelength. d. Press “B” on cuvette position button to measure the blank under new

wavelength. Press Measure Blank. e. Press “1” on cuvette position button to measure sample. f. Record the new absorbance, then repeat steps c, d,e to complete all

measurements-record results in Table 2.


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Table 2: Measurement of Absorption Spectrum of KMnO4 Wavelength Absorbance Wavelength Absorbance

400 nm 520 nm 420 nm 540 nm 440 nm 560 nm 460 nm 580 nm 480 nm 600 nm 500 nm 620 nm

4. Plot the data from Table 2 on graph paper with proper labeling. What is the wavelength maximum for KMnO4? λmax = ________ (3 pts for graph: title, linear scales, label of each axis and unit of measurements, correct plotting of curve and any other relevant information).

III. Construct the standard curve for solutions of KMnO4

A graph showing a chemical’s absorbance at its wavelength maximum versus its concentration is called a standard curve, and the relationship should be a linear. 1. Set the wavelength of the spectrophotometer at the wavelength maximum for KMnO4. 2. Autozero (step 2 at previous page) the spectrophotometer using the 0mM solution (tube A). 3. For each solution in Table 1, using a separate cuvette for each one, measure the

absorbance of each solution at the wavelength maximum and record results in Table 3. Do you need to autozero between each measurement? _____

a. To measure absorbance of multiple solutions-put 1ml of each solution in a cuvette, then place them in slots 1-5 of the spectrophotometer.

b. After autozeroing the spectrophotometer, press the corresponding button of the 6-position cell changer to measure the absorbance of each solution.

c. 1-5 will allow for measurement of 0.05mM—0.30mM, so you will need to replace the cuvette in slot 5 with another one containing the 0.40mM solution to measure its absorbance. Do not remove samples from cuvettes until your instructor check your data.

4. Plot the data from Table 3 onto the 2nd sheet of graph paper, and show your instructor. The relationship between concentration and absorbance is linear, so you should draw a straight line that lies as close as possible to each data point (best-fit). Do not simply connect the dots. The line should start from “0” (intercept at zero). (3 pts for the graph; grading criteria same as above)

Table 3: Determination of Standard Curve of Absorption vs. Concentration of KMnO4 . Determined at Wavelength maximum (λmax ) = ________nm

Concentration of KMnO4 Standards Absorbance Tube A: 0 mM 0 Tube B: 0.05 mM Tube C: 0.10 mM Tube D: 0.15 mM Tube E: 0.20 mM Tube F: 0.30 mM Tube G: 0.40 mM


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IV. Using the standard curve to measure the unknown concentration of a solution

1. Each student: obtain a tube with an unknown KMnO4 solution and record the tube number in table 4.

2. Use the blank cuvette to zero the spec at the wavelength maximum for KMnO4 3. Measure the absorbance of the unknown solution and record this value in Table 4. 4. Find this absorbance value on the vertical axis of your standard curve and then figure out the

corresponding concentration on the horizontal axis. Record the concentration in Table 4 and also on your standard curve graph. Ask your instructor to check your result. (2 pts)

Table 4: Analysis of Unknown Concentration of KMnO4 Your Unknown Tube # __________ Absorbance Concentration Determined From the

Standard Curve

V. Cleaning Up

a. Remove all cuvettes from spectrophotometer. Rinse all cuvettes thoroughly with tap water three times then with de-ionized water (clear-top spray bottle) twice, place them upside down on paper towel to remove residual water.

b. Turn off the spectrophotometer. c. Take off the masking tape label from all of your test tubes. Thoroughly rinse the test tubes

with tap water at least 3 times for each tube. Replace them upside down in a metal basket. d. Refill blue tip box and diH2O squirt bottle. e. Empty trash container into regular trash. Discard the long 5ml pipette in a tall jar next to back

sink.

VI. Post Lab Report (20 pts total): Include a title page and questions (1pt)

1. Copy your completed Table 3 into your report. Use Excel to generate a standard curve of absorption vs mM of KMnO4. Make sure you set the intercept at zero and include the equation and R2 value on your graph. (2 pts)

2. Explain the purpose of the standard curve you made in question 1. (1 pt)

3. Based on the equation generated by the standard curve and the absorption values recorded in Table 4, calculate the concentration of your unknown. ( 2 pt)

4. Write out Beer’s law and define each of the terms it contains. Then calculate the molar absorptivity at the λmax of KMnO4 using Molar ( M: mole/Liter) as the unit of concentration. (2pts) Note: The unit of concentration for KMnO4 in question 1 is mM (mmole/Liter). You need to convert the unit to M (mole/Liter) first.

5. The concept of a standard curve is very important. It is routinely used in biology and chemistry laboratories to determine unknown parameters. The following exercise demonstrates the use of a standard curve in determining the concentration of a protein


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solution. The standard curve is based on data showing how absorbance of UV light at 280 nm correlates with protein concentration:

Protein concentrations (ug/ml) Absorbance at 280 nm 0 0 10 0.033 20 0.052 40 0.095 80 0.195

150 0.312 200 0.400 400 0.912 600 1.290

a. Use Excel to make a scatter diagram using data in the table above; include the table in your report;(1 pt)

b. then calculate the best fitting straight line by using linear regression. Present the graph with the equations on your graph. ( 1 pt)

c. What makes you conclude that the protein concentration and UV absorbance are linearly related over this concentration range of protein? ( 1 pt)

d. If the absorbance of a protein solution whose concentration is unknown is 0.45, what is the protein concentration of the solution? Show your calculation. ( 1 pt)

6. Staple your two hand-drawn graphs (Absorption spectrum graph and the standard curve

graph) in the lab to your report. (8 pts, completed in the lab) Graphing Data and Linear Regression Analysis A. SETTING UP A GRAPH You will often make scatter diagrams and line graphs to illustrate the data that you collect. Scatter diagrams are often used to show the relationship between two variables. For example, in an absorbance spectrum, the variables would be the wavelength of light and the amount of light absorbed. Although this data is recorded in a table, a scatter diagram can illustrate in a more visual way the relationship between the two data sets: absorbance and wavelength. Perhaps the number one rule for graphic display has to do with the axes. Given a two-dimensional graph, with two values (x and y), which value is x and which is y? The answer is always the same - the KNOWN value (the “independent variable”) is always the ordinate (x) value. The value that is MEASURED (the “dependent variable”) is the abscissa (y) value. The horizontal line on the graph paper is identified as the x-axis and the vertical line is the y-axis. Each axis is labeled with an appropriate unit of measurement. Each increment of these lines represents the same amount of the measurement. For example, if you are drawing a scatter diagram of an absorbance spectrum and if one square of the x-axis represents 10 nm of wavelength of light, each other square also represents 10 nm of the wavelength of light along the x-axis. (This is called a “linear scale”.)


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If the data collected involves two or more sets of data having a common x axis, but varying y axes (or values), then a multiple graph may be used. The rules for graphing apply to each set of data, with the following provision: make sure that you have a figure legend that identifies which data points belong to each line. B. APPROXIMATING A “BEST FIT” LINE FOR A SCATTER DIAGRAM If you look at your scatter plot and several points on the graph are close to forming a straight line, it is reasonable to conclude that the relationship between the independent and dependent variables is linear. The straight line defines this linear relationship. If, for example, you have studied the effects of a particular fertilizer on fruit production in apple trees, your independent variable is the amount of fertilizer applied, while your dependent variable is the weight of apples harvested at each level of fertilizer application. If you were to plot these results, you would have a line that tells you exactly how much fruit you could expect from apple trees at a given level of fertilizer application. It is unlikely that your data points in the apple experiment described above would all lie on a perfect line, due to random variation in conditions and trees. To be able to get the best approximation of fruit yield per fertilizer application, you would want to know the average result. The best way to do this is to draw the “best-fit straight line” through your scatter plot. The most valid best fit straight lines that illustrate a linear relationship are determined by using a type of statistical analysis called linear regression analysis, which you will learn to use in this lab exercise. Often, you will be graphing data that illustrates a relationship that is generally linear but then the linearity breaks down at the extremes of changing conditions. In these cases, some of the data points on your graph will form a nearly straight line. This is an indication that at a certain point, the data that you collected no longer have a linear relationship. However, the information in the linear part of the graph may still be valuable. In that case, place your straight line so that it is as close as possible to as many points that lie along the linear part of your graph as possible. C. GRAPHING WITH MICROSOFT EXCEL 2000 1. Enter data in a table. 2. Select the data by highlighting them; Go to Menu “Insert”, and then “Chart”. 3. Select the XY(SCATTER) “Chart Type”. Select the scatter “Chart Subtype” described as

“Compare pairs of values”. Click on NEXT. 4. Make sure that the correct data appear in the “Data Range” box. Under “Series”, make sure

that the X and Y value(s) correspond to the correct columns. Click on NEXT. 5. Under “Chart Options”, add a title for the chart and titles for the X and Y axes. Be sure to add

the correct units of measurement. Click on NEXT. 6. Under “Chart Location”, you can select AS NEW SHEET if you want the graph to appear

separately, and AS OBJECT IN if you want the graph to appear with you data table (“Sheet 1”). Click on FINISH.

7. If you want to modify your chart, such as changing chart titles and units of measurements for the X and Y axes, you may always select “Chart Options” under “Chart” menu.

D. LINEAR REGRESSION WITH MICROSOFT EXCEL 2000 You will often be drawing graphs of standard curves which will be used later for extrapolation of raw data of measurements. You can do your extrapolation manually from your graph, or you may derive an equation for your best-fit straight line and use the equation for your determinations. The equation Y = aX + b, where a stands for the slope and b stands for the Y-intercept, can


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be derived by a spreadsheet program automatically. The following instructions will show you how to use Microsoft Excel to derive an equation from your data. 1. Make a graph, following the instructions listed above. 2. Selecting the graph, go to “Chart” menu and select “add trendline” 3. Under “Type” tab, select “Linear”; Click “Options” tab, and put check marks on all of the

following choices: “Set intercept =0”, “Display equation on chart”, “Display R-squared value on chart”. Click OK.

4. Your equation will be displayed somewhere on your graph. The Correlation Coefficient or “R” value is a measure of how well your data agrees with a linear relationship. The value of this coefficient will be between –1 and +1. The closer the number is to –1 or +1, the better your data fit a straight line. In order to conclude that 2 sets of variables are linearly related, the R value greater than 0.95 (or less than –0.95) would be sought for most types of experiments in cell or molecular biology.

E. Graphing/Linear Regression Analysis WITH MICROSOFT EXCEL 2007 1. Enter data in a table. 2. Select the data by highlighting them. 3. Go to Menu “Insert”. One of the options under “Insert” is “Chart”, and some default chart types

such as “Column” and “Line” may already be shown. Look for a “scatter” chart type; If you don’t have “scatter” as one of the default selections, you can click on “other charts” and select “All Chart Types” to locate the scatter chart type.

4. Once the “scatter” chart is selected, a chart with scattered data points will be plotted and shown on the same worksheet where you have your data. Now you need to label the chart and the X, Y axes. You can do that by first selecting the chart, and then click on the “Layout” tab on the file menu. Among many options under “Layout” are “Chart title” and “Axis title”.

5. Also under the “Layout” menu, there is a tab for performing linear regression analysis. It is called “Trendline”. Select “More trendline options” once you click on “Trendline” tab. Select “Linear” under “Trend/Regression Type”, and then check boxes in front of the following: • “Set intercept =0”, • “Display equation on chart”, • “Display R-squared value on chart”.

Biol 2281, Expt. 9

Dr. Wenju Lin, Spring 2016 1

Experiment 9:Spectrophotometry:An Analytical Tool

1

lights Photo-

Sample: I < Io

The process of light being absorbed by a solution

2

Io I

Cuvette withPathlength, b,

containing solution

source detector

bAs concentration increased, less light was transmitted (more light absorbed).

Filter

Some terminology1. I – intensity Io = initial intensity I = the intensity of light that pass through the sample

2. T – transmittance: fraction of light that pass through the

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sample (T=0 to 1)T = I/ Io

3. A – absorbance A = - log T = -log I/ Io , log10(100) = ?, log10(1/100) = ? usually ranges from 0 (no absorption) to 2 (99% absorption) A= 0, what does it mean? A= 1, what does it mean? A=2, what does it mean?

Beer’s Law• A(absorbance) = bc, where

– : molar absorptivity or molar extinction coefficient• a constant dependent on both wavelength and substance (molar

absorptivity); L mol-1 cm-1

• At a given wavelength, it describes relative strength of light absorbing function of the absorbing molecules

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– b: pathlength; cm– c: molar concentration; mol/L– A: no unit

• A linear relationship exists between absorbance and concentration at each wavelength for any given chemical.

• A linear relationship between concentration of a colored compound in solution and the light absorption of the solution.

Electromagnetic Spectrum

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• UV-visible spectrometers work with visible light (400-800nm) and UV(200-400nm) as light sources.

<>

Biol 2281, Expt. 9


Analyze at what wavelength?characteristic max

Crystal Violet Absorption Spectrum

0.6

0.8

1

1.2

1.4

sorb

ance

7

0

0.2

0.4

200 250 300 350 400 450 500 550 600 650 700 750wavelength, nm

Abs

max

max - wavelength where maximum absorbance occurs, 1. Why measure at max? For ___________and _____________;2. A chemical solution is colorless, does it have max?

Some Common Uses of Spectrophotometry in Biology

Determination of analyte concentrationusing Beer’s LawKMnO or other chemicals (Experiment 9 plastic

8

KMnO4 or other chemicals (Experiment 9, plastic cuvettes)

Proteins (quartz cuvettes)Nucleic acids (DNA and RNA)

Determination of the rate of enzyme catalyzed reactions (Experiment 10)

Experiment 9 Content/Goal• Know how to properly autozero the spectrophotometer

using solvent blank.• Determine the absorption spectrum and max of KMnO4

• Construct a standard concentration curve for KMnO4

9

4

• Determine the concentration of a KMnO4 solution based on ____________ and _____________;

• Graphing and data processing:– manually plot the data onto graphing paper and extrapolate

information from the graph (in the lab on graphing paper)– use Excel to graph data and perform linear regression analysis

(at home)

The BLANKThe blank contains all substances (solvents)

except the analyte. Is used to set the absorbance to zero:

A 0

10

Ablank = 0This removes any absorption of light due to

these solvents and the cuvette.After autozeroing, all measured absorbance is

due to the analyte.Need to autozero every time a new wavelength is

set.

Making KMnO4 solutions using parallel dilution method

Final Concentration of KMnO4 1 mM of KMnO4 Stock (ml) Distilled water (ml)

Tube A: 0 mM 0 ml 5 ml

Tube B: 0.05 mM

Tube C: 0.10 mM

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Tube D: 0.15 mM

Tube E: 0.20 mM

Tube F: 0.30 mM

Tube G: 0.40 mM 2 ml 3 ml

C1V1=C2V2(1 mM) V1 = (0.4 mM) ( 5 ml)V1 = 2 ml , volume of water = 5 ml-2 ml

Measure the Absorption Spectrum to determine max

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• Performed with 0.4 mM of KMnO4

• Autozero the spec everytime the wavelength is changed• When plotting the data: (3 pts)

– Using linear scale on axes– A smooth curve fitted to data points– Title, unit and label for each axis – (max )

Biol 2281, Expt. 9


Construct a standard concentration curve for KMnO4

Table 3: Determination of Standard Curve of Absorption vs. Concentration of KMnO4 . Determined at Wavelength maximum (max ) = ________nm

Concentration of KMnO4 Standards Absorbance

1. mM 0

0.05 mM

0.1 mM

0.15 mM

0 2 mM

13

• Prepare each of the solutions from a stock solution (1 mM of KMnO4 )• Measured at one wavelength: max• Autozero the spec once • When plotting data: ( 3 pts)

– Title, label, units of measurements– A straight line

0.2 mM

0.3 mM

4. mM

Use the standard curve to analyze the unknown concentration

• Standard curve:– A graph that relates

absorbances to concentrations of a

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chemical solution.

– Based on Beer’s law:

• A= constant * [concentration]

At home: Graphing Data Using ExcelInstructions for Excel 2000 and Excel 2007 Provided with

the procedure handout

Standard Concentration Curve for KMnO4

0.6

0.8

1

ce a

t __

_ n

m

15

• Step 1: Enter data in Excel• Step 2: Insert “Chart”: make a scatter

diagram of data with proper labels

0

0.2

0.4

0 0.1 0.2 0.3 0.4 0.5

mM of KMnO4

Ab

sorb

anc

At home: Graphing Data Using ExcelStandard Concentration Curve for KMnO4

y = 2.2177xR2 = 0.9994

0 2

0.4

0.6

0.8

1rb

ance

at

___

nm

16

• Step 3: Perform Linear Regression Analysis– “chart”, “Add Trendline”, make sure to select “display

equation”, “display R2”, “Set intercept = 0”

• Step 4: Calculate the concentration of unknown using the equation

0

0.2

0 0.1 0.2 0.3 0.4 0.5

mM of KMnO4

Ab

sor

Pre-Lab E9: Spectrophotometry ( 10 pts) · PDF filePre-Lab E9: Spectrophotometry ( 10 pts) ......

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