AP® InvestIgAtIon #13 - hintonhinton.harvardmedk12.wikispaces.net/.../APBio_no13-Teacher_manual.pdfPrE-lAb PrEPArAtiOn 9 studEnt GuidE cOntEnts bAckGrOund 12 ... There is a shift

AP® InvestIgAtIon #13 EnzymE Activity – tEAchEr’s GuidE

©2012, Ward’s Natural Science

All Rights Reserved, Printed in the U.S.A.

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250-7462 v.5/12

Kit # 36-7413

AbstrAct 1

GEnErAl OvErviEw 2

rEcOrdinG dAtA 3

mAtEriAls chEcklist 5

curriculum AliGnmEnt 6

lEArninG ObjEctivEs 7

timE rEquirEmEnts 7

sAfEty PrEcAutiOns 8

PrE-lAb PrEPArAtiOn 9

studEnt GuidE cOntEnts

bAckGrOund 12

PArt 1: structurEd inquiry – EstAblishinG A bAsEllinE 19

PArt 2: GuidEd inquiry – vAriAblEs thAt AffEct thE rAtE Of EnzymE rEActiOn 22

PArt 3: OPEn inquiry – dEsiGn An ExPErimEnt 25

mAtEriAl sAfEty dAtA shEEts 27

table of Contents

**AP® and the Advanced Placement Program are registered trademarks of the College Entrance Examination Board. The activity and materials in this kit were developed and prepared by Ward’s Natural Science Establishment, which bears sole responsibility for their contents.



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EnzymE Activity: tEAchEr’s GuidE Kit # 36-7413

AbstrACtChemical reactions underlie metabolism. Organisms have evolved catalytic proteins, called enzymes, that can make the reactions more efficient by lowering the activation energy of a chemical reaction. Catalytic efficiency of enzymes is dependent upon the precise shape of the active site in the protein that interacts with substrates and products. The abiotic conditions of the reaction affect the rate of enzyme-mediated conversion of substrate to product by affecting the conformation of this active site. In this set of investigations, students will use an extract of turnips containing the enzyme peroxidase to react with its substrate (hydrogen peroxide) and a color indicator (guaiacol) for formation of product (O2), to estimate the rate of peroxidase activity under a variety of experimental conditions.



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generAl overvIewThe College Board has revised the AP Biology curriculum to begin implementation in the fall of 2012. Advanced Placement (AP) is a registered trademark of the College Entrance Examination Board. The revisions were designed to reduce the range of topics covered, to allow more depth of study and increased conceptual understanding for students. There is a shift in laboratory emphasis from instructor-designed demonstrations to student-designed investigations. While students may be introduced to concepts and methods as before, it is expected that they will develop more independent inquiry skills. Lab investigations now incorporate more student-questioning and experimental design. To accomplish this, the College Board has decreased the minimum number of required labs from 12 to 8 while keeping the same time requirement (25% of instruction time devoted to laboratory study). The College Board has defined seven science practices that students must learn to apply over the course of laboratory study. In brief, students must:

1. Use models

2. Use mathematics (quantitative skills)

3. Formulate questions

4. Plan and execute data collection strategies

5. Analyze and evaluate data

6. Explain results

7. Generalize data across domains

The College Board published 13 recommended laboratories in the spring of 2011. They can be found at: http://advancesinap.collegeboard.org/science/biology/lab

Many of these laboratories are extensions of those described in the 12 classic labs that the College Board has used in the past. The materials provided in this lab activity have been prepared by Ward’s to adapt to the specifications outlined in AP Biology Investigative Labs: An Inquiry-Based Approach (2012, The College Board). Ward’s has provided instructions and materials in the AP Biology Investigation series that complement the descriptions in this College Board publication. We recommend that all teachers review the College Board material as well as the instructions here to get the best understanding of what the learning goals are. Ward’s has structured each new AP investigation to have at least three parts: Structured, Guided, and Open Inquiry. Depending on a teacher’s syllabus, they may choose to do all or only parts of the investigations in scheduled lab periods.

The College Board requires that a syllabus describe how students communicate their experimental designs and results. It is up to the teacher to define how this requirement will be met. Having students keep a laboratory notebook is one straightforward way to do this.



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reCordIng dAtA In A lAborAtory notebook

All of the Ward’s Investigations assume that students will keep a laboratory notebook for student-directed investigations. A brief outline of recommended practices to set up a notebook, and one possible format, are provided below.

1. A composition book with bound pages is highly recommended. These can be found in most stationary stores. Ward’s offers several options with pre-numbered pages (for instance, item numbers 32-8040 and 15-8332). This prevents pages from being lost or mixed up over the course of an experiment.

2. The title page should contain, at the minimum, the student’s name. Pages should be numbered in succession.

3. After the title page, two to six pages should be reserved for a table of contents to be updated as experiments are done so they are easily found.

4. All entries should be made in permanent ink. Mistakes should be crossed out with a single line and should be initialed and dated. This clearly documents the actual sequence of events and methods of calculation. When in doubt, over-explain. In research labs, clear documentation may be required to audit and repeat results or obtain a patent.

5. It is good practice to permanently adhere a laboratory safety contract to the front cover of the notebook as a constant reminder to be safe.

6. It is professional lab practice to sign and date the bottom of every page. The instructor or lab partner can also sign and date as a witness to the veracity of the recording.

7. Any photos, data print-outs, or other type of documentation should be firmly adhered in the notebook. It is professional practice to draw a line from the notebook page over the inserted material to indicate that there has been no tampering with the records.

For student-directed investigations, it is expected that the student will provide an experimental plan for the teacher to approve before beginning any experiment. The general plan format follows that of writing a grant to fund a research project.

1. Define the question or testable hypothesis.

2. Describe the background information. Include previous experiments.

3. Describe the experimental design with controls, variables, and observations.

4. Describe the possible results and how they would be interpreted.

5. List the materials and methods to be used.

6. Note potential safety issues.

(continued on next page)



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reCordIng dAtA In A lAborAtory notebook (continued)

After the plan is approved: 7. The step-by-step procedure should be documented in the lab notebook. This includes recording

the calculations of concentrations, etc., as well as the weights and volumes used.

8. The results should be recorded (including drawings, photos, data print outs, etc.).

9. The analysis of results should be recorded.

10. Draw conclusions based on how the results compared to the predictions.

11. Limitations of the conclusions should be discussed, including thoughts about improving the experimental design, statistical significance, and uncontrolled variables.

12. Further study direction should be considered.

The College Board encourages peer review of student investigations through both formal and informal presentation with feedback and discussion. Assessment questions similar to those on the AP exam might resemble the following questions, which also might arise in peer review:

• Explain the purpose of a procedural step.

• Identify the independent variables and the dependent variables in an experiment.

• What results would you expect to see in the control group? The experimental group?

• How does XXXX concept account for YYYY findings?

• Describe a method to determine XXXX.



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MAterIAls ProvIded In kIt MAterIAls needed bUt not ProvIded

Units per kit/

group????Description Test tube racks\

1 pH paper, range 1-14, Vial/100 Ice

1 pkg./12 Glass stirring rod, 6 mm x 150 mm Beakers, 1000 mL, 150 mL, 50 mL

48?? Tubes, 15 x 125 mm Parafilm

1 Guaiacol, 250 mL Distilled water

24 Syringe, 2.5 mL Timer

16 Syringe, 10 mL Brown bottle, 500 mL or bottle wrapped in foil

1 Hydrogen peroxide, 3%, lab grade Beakers or flasks, 500 mL

1 pkg./15 Pipet, 6” Graduated cylinders, 20-500 mL and 1L

1 Scholar Chemistry MSDS CD-Rom Refrigerator

3 Cheesecloth, 9 x 12 piece Blender

1 pkg./20 Disposable pipet. 9”Other materials as determined by students’

experimental design

1

Buffer set:

Includes envelopes of pH 2-11

(one each, for a total of 10 envelopes),

500 mL buffer

Personal protective equipment (gloves, apron,

safety glasses)

1 Instructions (this booklet) oPtIonAl MAterIAls (not ProvIded)

Light probe or spectrophotometer

MAterIAls CheCklIst

Call “Us” at 1.800.962.2660 for

Technical Assistance Visit “Us” on-line at

www.wardsci.com

for U.S. Customers

www.wardsci.ca

for Canadian Customers

or



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CUrrICUlUM AlIgnMentBig Ideas

Big Idea 4: Biological systems interact, and these interactions possess complex properties

Also connects to:

Big Idea 2: Biological systems utilize energy and molecular building blocks to grow, to reproduce, and to maintain homeostasis.

Enduring Understandings

2.D.1: All biological systems from cells and organisms to populations, communities and ecosystems are affected by complex biotic and abiotic interactions involving exchange of matter and free energy.

4A1: The subcomponents of biological molecules and their sequence determine the properties of that molecule.

4B1: Interactions between molecules affect their structure and function.

Science Practices

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Kit # 36-7413

This lab activity is aligned with the 2012 AP Biology Curriculum (registered trademark of the College Board). Listed below are the aligned Content Areas (Big Ideas and Enduring Understandings), the Science Practices, and the Learning Objectives of the lab as described in AP Biology Investigative Labs: An Inquiry Approach (2012). This is a publication of the College Board that can be found at http://advancesinap.collegeboard.org/science/biology/lab.

don’t see any science practices listed in word doc.



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leArnIng objeCtIvesThe student is able to design a plan for collecting data to show that all biological systems are affected by complex biotic and abiotic interactions (2D1 & SP 4.2, SP 7.2).

The student is able to use models to predict and justify that changes in the subcomponents of a biological polymer affect the functionality of the molecule (4A1 & SP 6.1, SP 6.4).

The student is able to analyze data to identify how molecular interactions affect structure and function (4B1 & SP 5.1).

tIMe reqUIreMents

Structured Inquiry 10 minutesGuided Inquiry 45 minutesOpen Inquiry 45 minutes

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sAfety PreCAUtIons Lab-Specific Safety

Guaiacol is a poison and flammable. Review the Material Safety Data Sheet (MSDS) for this chemical.

Remind students to use proper pipeting techniques. Never mouth pipet

General Safety

The teacher should be familiar with safety practices and regulations in their school (district and state). The teacher should know what needs to be treated as hazardous waste and how to properly dispose of non-hazardous chemicals or biological material.

Consider establishing a safety contract that students and their parents must read and sign off on. This is a good way to identify students with allergies to things like latex so that you (and they) will be reminded of what particular things may be risks to individuals. A good practice is to include a copy of this contract in the student lab book (glued to the inside cover).

Students should know where all emergency equipment (safety shower, eyewash station, fire extinguisher, fire blanket, first aid kit etc.) is located.

Make sure students remove all dangling jewelry and tie back long hair before they begin.

Remind students to read all instructions, Material Safety Data Sheets (MSDSs) and live care sheets before starting the lab activities and to ask questions about safety and safe laboratory procedures. Appropriate MSDSs and live care sheets can be found on the last pages of this booklet. Additionally, the most updated versions of these resources can be found at www.wardsci.com, under Living Materials http://wardsci.com/article.asp?ai=1346. (Note that in this particular lab, there are no live materials that require a live care sheet.

In student directed investigations, make sure that collecting safety information (like MSDSs) is part of the experimental proposal.

As general laboratory practice, it is recommended that students wear proper protective equipment, such as gloves, safety goggles, and a lab apron.

At end of lab:

All laboratory bench tops should be wiped down with a 20% bleach solution or disinfectant to ensure cleanliness.

Remind students to wash their hands thoroughly with soap and water before leaving the laboratory.

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Pre-lAborAtory PrePArAtIon1. Make copies of Student Guide.

Copy pages __ to __ of the student copymaster prior to starting class.

2. Dilute hydrogen peroxide solution

NOTE: This can be made a day ahead and stored overnight in the refrigerator protected from light in either a brown bottle or foil wrapped bottle.

Prepare 2 liters of the dilute solution by adding 66 mL of the stock 3% hydrogen peroxide solution to 2 liters of distilled water. Dispense into 8 bottles and label this solution 0.1% H

2O

2.

NOTE: Be extremely precise in your measurements of all reagents to avoid erroneous results.

3. Prepare pH buffer solutions.

NOTE: This can be prepared a day ahead and stored covered overnight at room temperature.

a) Label each of 6 large beakers so that there is one beaker per pH buffer envelope.

b) Measure 500 mL of distilled water and pour 500 mL of water into each beaker.

c) Empty one entire powder-filled envelope over into the filled beaker, and label the beaker that matches the pH indicated on the envelope. Mix by stirring the powder into solution.

d) Measure the pH of the buffer solution to confirm that the desired pH is achieved.

OPTIONAL: Dispense buffer into 8 bottles, one for each lab group.

4. Prepare stock indicator solution.

NOTE: This can be prepared the day before the experiment and stored at 4 °C, protected from light.

NOTE: Keep the guaiacol solution on ice in a designated area throughout the course of the lab to maintain stability.

Dilute 1.5 mL guaiacol in 500 mL water in either a brown bottle or a bottle covered with aluminum foil.

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5. Prepare stock enzyme solution.

NOTE: The turnip peroxidase solution can be prepared the day before the experiment. The solution is stable for up to one week if stored at 4 °C and protected from light.

The enzyme solution is an extract made from a fresh turnip (purple topped or other available at the grocery store). Weigh out 25 g of a peeled turnip. Place into a blender and cover with 400 mL of cold distilled water. Blend on high speed for 1 minute. Filter the puree through three layers of cheesecloth. Discard the cheesecloth with the solids. Pour the solution into either in a brown bottle or a bottle covered with aluminum foil, and store in refrigerator, protected from light. Let any starch precipitate settle to bottom of bottle.

TIP: It is always best to use distilled water, but tap water may be used as well.

NOTE: Keep the turnip peroxidase solution on ice in a designated area throughout the course of the lab to keep the enzyme activity stable.

6. Prepare the color palette.

NOTE: Prepare the standardized color palette the day before the lab activity to ensure the reactions have maximized the production of the enzyme-substrate reaction product. This will represent your “brownest” sample (10). This color palette will be used by the students to approximately quantify the reaction rate of the enzyme-substrate reaction. Store in refrigerator overnight.

a. Prepare Enzyme-substrate mixture (enough to make one color palette)

In a 150 mL beaker add each of the following:

15 mL stock turnip peroxidase solution

30 mL 0.1% H2O

2 stock

and

15 mL stock indicator – guaiacol solution

NOTE: Be sure to change the disposable pipets in between solutions so there is no carry over.

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Pre-lAborAtory PrePArAtIon (ContInUed)




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Pre-lAborAtory PrePArAtIon (ContInUed)


notes6. Prepare the color palette (continued)

b. Label 11 test tubes with the numbers 0-10 respectively. Using the chart below, fill the respective tube with the specific amounts of each reagent:

Test

Tube

Number

Final Solution

Quantity Desired

(mL)

Concentration

of

Final Solution

(%)

mL of

Enzyme-

Substrate

Mixture

mL of

Distilled Water

10 7.5 100 7.50 0.009 7.5 90 6.75 0.758 7.5 80 6.00 1.50.7 7.5 70 5.25 2.256 7.5 60 4.50 3.005 7.5 50 3.75 3.754 7.5 40 3.00 4.503 7.5 30 2.25 5.252 7.5 20 1.50 1.501 7.5 10 0.75 6.750 7.5 0 0.00 7.50

OPTIONAL: The color palette can be quantified using a spectrophotometer to measure absorbance at a wavelength of 436 nm.

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bACkgroUndEnzymes, Substrates and Products

Chemical reactions underlie metabolism. Organisms have evolved catalytic proteins, called enzymes, that can make the reactions more efficient by lowering the activation energy of a chemical reaction. Catalytic efficiency of enzymes is dependent upon the precise shape (conformation) of the active site in the protein that interacts with substrates and products.

When the enzyme is reacting with the substrate, a complex is formed. This interaction can be expressed as:

Figure 1

In the induced fit model of enzymatic activity, enzymes change shape after binding to a substrate, improving the “fit” between the enzyme and the substrate.

That fit between the enzyme and the substrate is responsible for lowering the activation energy required to transform substrates into products. The graph below illustrates the differences between the amounts of activation energy (Ea) of an uncatalyzed reaction and the lower activation of an enzyme-catalyzed reaction. Note that there is no difference in the final amount of free energy (Δ G).

Figure 2: Course of Reaction

objeCtIves

Design a plan for collecting data to show that all biological systems are affected by complex biotic and abiotic interactions.

Use models to predict and justify that changes in the subcomponents of a biological polymer affect the functionality of the molecule.

Analyze data to identify how molecular interactions affect structure and function.

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bACkgroUnd (ContInUed)

The efficiency of an enzyme in facilitating the chemical reaction that transforms substrates to products is changed by conditions that affect the shape of the active site. Optimal enzyme efficiency requires optimal environmental conditions, such as pH and temperature. Raising the temperature of any substance will cause a rise in its average kinetic energy, because the heat in any given system is expressed as molecular motion. The more heat that is added, the more molecular motion will occur. As a general rule, an increase of 10 °C doubles the rate of most chemical reactions. Enzymes are proteins, however, and so are subject to heat-induced alteration of their shape or tertiary structure. The protein has become denatured when the tertiary structure is altered so much that it becomes permanently inactive.

The relative acidity or alkalinity of an enzyme’s surroundings also influences its activity through a similar mechanism as temperature – by altering the enzyme’s interaction with substrates through conformational changes in the active site. The pH range that supports an enzyme’s optimal activity is associated with the natural environment in which it evolved. The ionization state of the R groups of the amino acids that make up the protein are affected by the pH of the environment. Therefore, the charge associated with an R group in an active site will affect how efficiently the enzyme interacts with substrates and products. Some enzymes will not be optimally active until negative charges are neutralized or, conversely, until R groups become charged. Two examples are illustrated in the graph below. Pepsin is a hydrolytic enzyme that is required to be active in the acidic environment of the stomach. Trypsin is hydrolytic intestinal enzyme with an optimal enzymatic activity in the slightly basic range that is produced in the pancreas.

Figure 3


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Enzyme-facilitated reactions follow the Law of Mass Action: the direction taken by an enzyme-catalyzed reaction is directly dependent on the relative concentration of enzyme, substrate, and product. When there is a great deal of substrate and little product, the reaction will form more products. Conversely, when there is a great deal of product, the reaction may form more substrate. However, here are two situations where the Law of Mass Action is circumvented. When the product is immediately metabolized or transported away from the enzyme, the product concentration does not rise. In highly exergonic reactions, the product has little free energy and the opposite reaction requires a large, if not unachievable, amount of energy to reverse the process.

Figure 4

Note that in the above graph, the initial reaction is very steep. As more product forms, the rate of formation levels off as an equilibrated ratio of substrate and product is produced.

Enzymes are often tightly bound to a prosthetic group (cofactor), which is either a metal ion, an organic molecule/metal ion complex, or a small organic molecule (coenzyme). Metal ions such as Fe+3 and Zn+2 are generally involved in reactions which require electron removal from a substrate or which can electrically bond an enzyme to a substrate. Coenzymes have a much more varied role. Some are not tightly bound and can move from enzyme to enzyme, transferring electrons or protons. Some alter substrates to better fit with the enzyme. Still others, bound into membranes, are essential to the energy conversion reactions of photosynthesis and respiration. Many coenzymes must be taken in by animals and are not synthesized; these are collectively referred to as vitamins.



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Competitive Inhibitors

Molecules that bind to the active site and compete with the substrate are called competitive inhibitors. Note that the inhibitor is similar enough to the enzyme that it can fit in the active site, but it is not the same as the substrate. The inhibitor binds the enzyme, therefore blocking substrates from binding, and no product is produced. Competitive inhibition is reversible and behaves the same as an enzyme–substrate complex, with constant binding and unbinding of the inhibitor due to the Law of Mass Action. If the concentration of the inhibitor is large enough, the reaction with substrate will slow down; otherwise, the inhibitor has little effect. Increasing the concentration of the substrate can overcome the effect of a competitive inhibitor. The figure below illustrates the effect of a competitive inhibitor on an enzyme. The reaction without an inhibitor proceeds very rapidly, whereas the reaction that is competitively inhibited proceeds more slowly.

Figure 5: Substrate Concentration

Unlike competitive inhibitors, noncompetitive inhibitors bind to a region of the enzyme other than the active site, causing a shape change in the enzyme that will impair the function of the active site. Since there is no competition for the active site, the Law of Mass Action will not come into play in this situation, and a buildup of substrate will not make a difference in accelerating the reaction.



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notes

Figure 6

More complex enzymes that have quaternary structures contain sites in addition to the active sites which are called effector bonds. The effector renders the enzyme inactive only while it is on the enzyme. This is a common mechanism of control in metabolic pathways. In the pathways, enzymes exist in two forms: active and passive. The active form is rendered inactive by an effector, often a product of a later enzyme reaction, as illustrated in the diagram below. When the products of a metabolic pathway inhibit an earlier step in the pathway, it is referred to as feedback inhibition, or negative feedback.

Figure 7???

Not all effectors inhibit an enzyme. In allosteric promotion, the effector activates an inactive enzyme. Both of these regulatory mechanisms are extremely effective and can work together, allowing the cell to store enzymes in both their active and inactive forms. In allosteric inhibition, if a product down the metabolic line begins to build up, it is not to the cell’s advantage to continue to make it. The product itself will “turn down” the reaction until most of the product has been metabolized. In allosteric promotion, that same product (or a different one) will activate an enzyme to begin reaction with a substrate.



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notes The Role of Turnip Peroxidase

Hydrogen peroxide is a highly reactive oxidizing agent that is produced in most cells, formed spontaneously as metabolic waste. The enzyme turnip peroxidase is found in a cell organelle called the peroxisome that serves to sequester and break down hydrogen peroxide into relatively inert water and oxygen gas, preventing chemical damage that would be caused by free, reactive peroxide.

Turnip cells use turnip peroxidase to break down hydrogen peroxide into water and oxygen (Figure 6). How efficiently the enzymes are able perform this reaction is influenced by abiotic and biotic factors. The rate of enzymatic activity is determined by how fast 1 unit of enzyme can convert 1µM of hydrogen peroxide to oxygen. In this lab activity, you will perform an enzymatic activity assay in which you will assess the amount of oxygen produced by comparing the color development of the reaction to a color palette.

2 H2O

2 2 H

2O + O

2

Substrate Enzyme Products

turnip peroxidase

Figure 7?? 8??

Is this number reference correct?



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notessAfety PreCAUtIons

As general safe laboratory practice, it is recommended that you wear proper protective equipment, such as gloves, safety goggles, and a lab apron.

As general lab practice, read the lab through completely before starting, including any Material Safety Data Sheets (MSDSs) and live materials care sheets at the end of this booklet as well as any appropriate MSDSs for any additional substances you would like to test. One of the best sources is the vendor for the material. For example, when purchased at Wards, searching for the chemical on the Ward’s website will direct you to a link for the MSDS. (Note: There are no live materials care sheets included in this particular lab.)

At the end of the lab:

All laboratory bench tops should be wiped down with a 20% bleach solution or disinfectant to ensure cleanliness.

Wash your hands thoroughly with soap and water before leaving the laboratory.

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ProCedUre tIPs

When performing this lab activity, all data should be recorded in a lab notebook. You will need to construct your own data tables, where appropriate, in order to accurately capture the data from the investigation.

If directed to do so by your teacher, this part of the lab may be done at the same time as Part 2 of the lab.

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PArt 1 – strUCtUred InqUIry: estAblIshIng A bAselIne

MAterIAls needed Per lAb groUP2 1 mL syringes1 2.5 mL syringe1 10 mL syringe5 15 x 125 mm tubes1 Disposable 9” transfer pipet1 mL Guaiacol1 mL Turnip peroxidase solution2 mL Dilute hydrogen peroxide4 mL pH 7 buffer solution Timer

Shared Materials

1 box of Parafilm

PArt 1 – ProCedUre: strUCtUred InqUIry1. Label the test tubes and syringes, as follows:

2.5 mL syringe labeled ‘E’ for enzyme – turnip peroxidase solution.

2.5 mL syringe labeled ‘P’ for product as represented by indicator . Guaiacol reacts with free O

2 (product) to form brown

color.

10 mL syringe labeled ‘NB’ for neutral buffer – pH 7.

2.5 mL syringe labeled ‘S’ for substrate – 0.1% H2O

2

Test tube labeled ‘SPNB’ for mixture A

Test tube labeled ‘ENB’ for mixture B

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2. Fill and prepare the labeled syringes and tubes with the appropriate solutions (provided).

a. Dispense the following reagents in “Tube SPNB”:

2 mL of ‘S’ substrate

1 mL of ‘P’ indicator for product

1 mL of ‘NB’ neutral buffer

b. Cap tube ‘SPNB’ with parafilm and gently invert two times to mix.

c. Dispense the following reagents in “Tube ENB”:

1 mL ‘E’ enzyme (turnip peroxidase)

3 mL‘NB’ neutral buffer

d. Cap tube ‘ENB’ with parafilm and gently invert two times to mix.

e. Using a disposable transfer pipet, transfer the mixture from Tube ‘SPNB’ into Tube ‘ENB’.

f. Cap with parafilm and invert two times to mix.

3. Make observations and record data.

a. Using the color palette provided by your instructor, immediately observe and compare the color of your reaction to the color palette and record the tube # (color) of the mixture over time. This data should be recorded in 1-minute intervals for a total incubation period of five minutes.

OPTIONAL: If the class has access to a probe to measure the transmittance of light or to a spectrophotometer, more quantitative results with better resolving power can be generated by measuring the changes in the transmittance or absorbance of light.

b. Plot the increase in color intensity (product formation) relative to your color palette over the 5-minute interval and calculate the rate of enzymatic reaction under the baseline conditions of this experiment.

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ProCedUre – PArt 1: strUCtUred InqUIry (ContInUed)

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ProCedUre – PArt 1: strUCtUred InqUIry (ContInUed)

notes 3. Make observations and record data (continued)

Note: The color palette represents a range of indicator color that increases 10% between tubes 1-10. Tube 0 represents no indicator, no color.

NOTE: The color intensity is used as way to quantify the amount of oxygen that is produced in the enzymatic reaction. The brown color is produced when the guaiacol reacts with oxygen (product of the enzyme substrate reaction). Therefore, the more intense the color, the more oxygen is produced in the reaction.

4. Set your labeled syringes aside for use in the following parts of this investigation.

NOTE: Do not throw away your labeled syringes. You will need them for the remaining parts of this investigation.

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PArt 2 – gUIded InqUIry: vArIAbles thAt AffeCt the rAte of enzyMe reACtIon

MAterIAls needed Per lAb groUP12 15 x 125 mm tubes3 2.5 mL syringes (from Part 1)1 10 mL syringe (from Part 1)6 mL ‘P’ product guaiacol6 mL ‘E’ enzyme turnip peroxidase solution12 mL ‘S’ substrate dilute hydrogen peroxide (0.1% H

2O

2)

Shared Materials

1 ‘NB’ 10 mL Syringe to dispense all buffer solutions500 mL pH 3 Buffer Solution500 mL pH 5 Buffer Solution500 mL pH 6 Buffer Solution500 mL pH 7 Buffer Solution500 mL pH 8 Buffer Solution500 mL pH 10 Buffer Solution

PArt 2 – ProCedUre: gUIded InqUIry1. In your laboratory notebook or sheet, record the baseline rate

(from Part 1???).

2. Label twelve 13 x 100 mm tubes 1 through 12, respectively.

To simplify the process, pair the tubes according to the chart below:

Tube 1 2 4 9 11 12Tube/pH 3 5 6 7 8 10

NOTE: All of the pH tubes will contain the respective pH buffer solution, in case the tubes get mixed up.

qqqqqq

qqqqqqq

‹

ProCedUre tIPs

When performing this lab activity, all data should be recorded in a lab notebook. You will need to construct your own data tables, where appropriate, in order to accurately capture the data from the investigation.

If directed to do so by your teacher, this part of the lab may be done at the same time as Part 1 of the lab.

‹

‹


Jana, please note...add p, S, etc.



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3. In each of tubes 1, 2, 4, 9, 11, and 12, dispense:

2 mL ‘S’ substrate dilute hydrogen peroxide using ‘S’ syringe

1 mL‘P’ product indicator guaiacol using ‘P’ syringe

1 mL ‘NB’ neutral buffer pH 7

NOTE: All of these tubes contain the substrate.

4. In tube 3 dispense the following volumes of reagents:

1 mL of turnip peroxidase solution, using the syringe labeled ‘E’

3 mL of pH 3 solution, using the rinsed ‘NB’ syringe

5. Repeat step 4 for tubes 5, 6, 7, 8, and 10 with their respective pH buffers.

6. Using a disposable 9” transfer pipet, combine the reagents from tube 1 with the reagents in tube/pH 3.

7. Observe the enzyme reaction mixtures every minute for 5 minutes by comparing to the color palette (or optional measurement of absorbance/transmittance). Record your observations in your laboratory notebook.

8. Refer back to the tube pairing chart and mix the remaining pairs of tubes. Repeat Steps 6 and 7 for the remaining pairs of tubes.

9. Calculate the rate of reaction for each tube as described in Part 1. In your laboratory notebook, graph your rate results relative to pH.

‹

ProCedUre – PArt 2: gUIded InqUIry (ContInUed)

notes



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PArt 2 AssessMent qUestIons

1. Based on the graph and the overall slope of the line, what can you conclude about the effect of pH on reaction rate? Why do you think that occurs?

The turnip peroxidase enzyme is most effective at the pH of 7. As the pH moves away from neutral, the conformation of the protein impairs its enzymatic activity.

2. What happens to turnip peroxidase during and after the reaction?

Turnip peroxidase remains the same throughout the whole reaction and is present once the reaction stops. It is not considered a product of the reaction as it never changes composition, although it may lose activity due to conformational changes.

3. What would the reaction look like if you left parts of the reaction out of the mix?

If the enzyme was left out, the reaction would be very slow, perhaps speeded with heating.

If the substrate was left out, you might expect no reaction, however, there might be some substrate present in the turnip extract, but at a much lower concentration (would slow the reaction).

If the indicator was left out, the reaction would occur but we would not be able to detect the formation of product. Since the product reacts with the indicator, the product may build up faster without the indicator, possibly reaching a lower equilibrium rate.

4. What other factors may influence enzyme activity (rate of reaction)?

Temperature, starting substrate concentration, cofactors in other enzymes, inhibitor concentrations, etc.

5. Peroxidase breaks down hydrogen peroxide. What other types of enzymes might be needed in an organism?

Enzymes that build or break chemical bonds of biological molecules like proteins, lipids, carbohydrates, and nucleic acids. Proteases, lipases, glycolytic enzymes, DNA polymerase, fatty acid synthase, etc.

6. You investigated peroxidase from a turnip. How might the activity of peroxidase from a mammal be different?

Optimal temperature would be closer to body temperature, pH optima may be different. The sequence of the protein would also be likely to be different – but the peptides forming the active site might be conserved during evolution.



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PArt 3: enzyMe ACtIvIty

oPen InqUIry: desIgn An exPerIMentWhat questions occurred to you as you completed your studies of peroxidase activity? Now that you are familiar with enzymatic activity and ways to assess that activity, design an experiment to investigate one of your questions. Questions may involve assessing a range of abiotic factor effects on peroxidase activity, modeling the kinetics of enzyme activity, comparing protein sequence or optimal conditions for peroxidases from different sources, testing potential chemical inhibitors of enzyme activity, comparing sensitivities of different kinds of enzymes to the same abiotic factors, or identifying indicators for different kinds of enzyme activity.

Before starting your experiment, plan your investigation in your lab notebook. Have your teacher check over and initial your experiment design. Once your design is approved, investigate your hypothesis. Be sure to record all observations and data in your laboratory sheet or notebook.

Use the following steps when designing your experiment.

1. Define the question or testable hypothesis.

2. Describe the background information. Include previous experiments.

3. Describe the experimental design with controls, variables, and observations.

4. Describe the possible results and how they would be interpreted.

5. List the materials and methods to be used.

6. Note potential safety issues.

After the plan is approved by your teacher:

7. The step by step procedure should be documented in the lab notebook. This includes recording the calculations of concentrations, etc. as well as the actual weights and volumes used.

8. The results should be recorded (including drawings, photos, data print outs).

exPerIMent desIgn tIPs

The College Board encourages peer review of student investigations through both formal and informal presentation with feedback and discussion. Assessment questions similar to those on the AP exam might resemble the following ques-tions, which also might arise in peer review:

Explain the purpose of a procedural step.

Identify the independent variables and the dependent variables in an experiment.

What results would you expect to see in the control group? The experimental group?

How does XXXX concept account for YYYY findings?

Describe a method to determine XXXX.

‹

‹

‹

‹

‹




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9. The analysis of results should be recorded.

10. Draw conclusions based on how the results compared to the predictions.

11. Limitations of the conclusions should be discussed, including thoughts about improving the experimental design, statistical significance and uncontrolled variables.

12. Further study direction should be considered.

notes

PArt 3: oPen InqUIry (ContInUed)



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MAterIAl sAfety dAtA sheets


Material Safety Data Sheet Page 1 of 2

© 2009, Scholar Chemistry. All Rights Reserved. 1/20/2012

Buffer Capsule, pH 2.00 MSDS # 126.30

Section 1: Product and Company Identification Buffer Capsule, pH 2.00

Synonyms/General Names: pH 2 Buffer Capsule Product Use: For educational use onlyManufacturer: Micro Essential Laboratory Inc., Brooklyn, NY 11210

24 Hour Emergency Information Telephone Numbers CHEMTREC (USA): 800-424-9300 CANUTEC (Canada): 613-424-6666

ScholAR Chemistry; 5100 W. Henrietta Rd, Rochester, NY 14586; (866) 260-0501; www.Scholarchemistry.com

Section 2: Hazards Identification Capsule containing white powder; no odor. HMIS (0 to 4)

CAUTION! Powder is a body tissue irritant. Target organs: None known

This material is not considered hazardous by the OSHA Hazard Communication Standard (29 CFR 1910.1200) if used properly.

Section 3: Composition / Information on Ingredients Sulphamic Acid (5329-14-6) 30 to 40% Potassium Biphthalate ( 877-24-7) 60 to 70%

Section 4: First Aid Measures Always seek professional medical attention after first aid measures are provided.

Eyes: Immediately flush eyes with excess water for 15 minutes, lifting lower and upper eyelids occasionally.Skin: Immediately flush skin with excess water for 15 minutes while removing contaminated clothing. Ingestion: Call Poison Control immediately. Rinse mouth with cold water. Give victim 1-2 cups of water or milk to drink.

Induce vomiting immediately. Inhalation: Remove to fresh air. If not breathing, give artificial respiration.

Section 5: Fire Fighting Measures Noncombustible solution. When heated to decomposition, emits acrid fumes. 0 Protective equipment and precautions for firefighters: Use foam or dry chemical to extinguish fire. 0 0

Firefighters should wear full fire fighting turn-out gear and respiratory protection (SCBA). Cool container with water spray. Material is not sensitive to mechanical impact or static discharge.

Section 6: Accidental Release Measures Use personal protection recommended in Section 8. Isolate the hazard area and deny entry to unnecessary and unprotected personnel. Sweep up spill and place in sealed bag or container for disposal. Wash spill area after pickup is complete. See Section 13 for disposal information.

Section 7: Handling and Storage Green Handling: Use with adequate ventilation and do not breathe dust or vapor. Avoid contact with skin, eyes, or clothing. Wash

hands thoroughly after handling. Storage: Store in General Storage Area [Green Storage] with other items with no specific storage hazards. Store in a cool, dry,

well-ventilated, locked store room away from incompatible materials.

Section 8: Exposure Controls / Personal ProtectionUse ventilation to keep airborne concentrations below exposure limits. Have approved eyewash facility, safety shower, and fire extinguishers readily available. Wear chemical splash goggles and chemical resistant clothing such as gloves and aprons. Wash hands thoroughly after handling material and before eating or drinking. Exposure guidelines: Buffer pH 2 capsule: OSHA PEL: N/A, ACGIH: TLV: N/A, STEL: N/A.

Health 1 Fire Hazard 0 Reactivity 0



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MAterIAl sAfety dAtA sheetsMaterial Safety Data Sheet Page 2 of 2


MSDS # 126.30 Buffer Capsule, pH 2.00 Scholar Chemistry Section 9: Physical and Chemical Properties Molecular formula N/A. Appearance Capsule with white solid.Molecular weight N/A. Odor No odor.Specific Gravity N/A Odor Threshold N/A. Vapor Density (air=1) N/A Solubility Complete. Melting Point N/A Evaporation rate N/A (Butyl acetate = 1). Boiling Point/Range N/A. Partition Coefficient N/A (log POW). Vapor Pressure (20°C) N/A. pH 2.0, acidic. Flash Point: N/A. LEL N/A. Autoignition Temp.: N/A. UEL N/A.

N/A = Not available or applicable

Section 10: Stability and Reactivity Avoid heat and moisture.Stability: Stable under normal conditions of use and storage. Incompatibility: Acids, alkalis, Shelf life: Indefinite if stored properly.

Section 11: Toxicology Information Acute Symptoms/Signs of exposure: Eyes: Redness, tearing, itching, burning, conjunctivitis. Skin: Redness, itching. Ingestion: Irritation and burning sensations of mouth and throat, nausea, vomiting and abdominal pain. Inhalation: Irritation of mucous membranes, coughing, wheezing, shortness of breath, Chronic Effects: No information found. Sensitization: none expected pH 2 Buffer: LD50 [oral, rabbit]; N/A; LC50 [rat]; N/A; LD50 Dermal [rabbit]; N/A Material has not been found to be a carcinogen nor produce genetic, reproductive, or developmental effects.

Section 12: Ecological Information Ecotoxicity (aquatic and terrestrial): Ecological impact has not been determined.

Section 13: Disposal Considerations Check with all applicable local, regional, and national laws and regulations. Local regulations may be more stringent than regional or national regulations. Small amounts of this material may be suitable for sanitary sewer or trash disposal.

Section 14: Transport Information DOT Shipping Name: Not regulated by DOT. Canada TDG: Not regulated by TDG. DOT Hazard Class: Hazard Class: Identification Number: UN Number:

Section 15: Regulatory Information EINECS: Not listed . WHMIS Canada: Not WHMIS Controlled. TSCA: All components are listed or are exempt. California Proposition 65: Not listed.

The product has been classified in accordance with the hazard criteria of the Controlled Products Regulations and the MSDS contains all the information required by the Controlled Products Regulations.

Section 16: Other Information Current Issue Date: January 20, 2012 Disclaimer: Scholar Chemistry and Columbus Chemical Industries, Inc., (“S&C”) believes that the information herein is factual but is not intended to be all inclusive. The information relates only to the specific material designated and does not relate to its use in combination with other materials or its use as to any particular process. Because safety standards and regulations are subject to change and because S&C has no continuing control over the material, those handling, storing or using the material should satisfy themselves that they have current information regarding the particular way the material is handled, stored or used and that the same is done in accordance with federal, state and local law. S&C makes no warranty, expressed or implied, including (without limitation) warranties with respect to the completeness or continuing accuracy of the information contained herein or with respect to fitness for any particular use.



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CAUTION! Powder is a body tissue irritant. Target organs: None known


Section 3: Composition / Information on Ingredients Sulphamic Acid (5329-14-6) 20 to 30% Potassium Biphthalate ( 877-24-7) 70 to 80%










Section 8: Exposure Controls / Personal ProtectionUse ventilation to keep airborne concentrations below exposure limits. Have approved eyewash facility, safety shower, and fire extinguishers readily available. Wear chemical splash goggles and chemical resistant clothing such as gloves and aprons. Wash hands thoroughly after handling material and before eating or drinking. Exposure guidelines: Buffer pH 3 capsule: OSHA PEL: N/A, ACGIH: TLV: N/A, STEL: N/A.




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Section 11: Toxicology Information Acute Symptoms/Signs of exposure: Eyes: Redness, tearing, itching, burning, conjunctivitis. Skin: Redness, itching. Ingestion: Irritation and burning sensations of mouth and throat, nausea, vomiting and abdominal pain. Inhalation: Irritation of mucous membranes, coughing, wheezing, shortness of breath, Chronic Effects: No information found. Sensitization: none expected pH 3 Buffer: LD50 [oral, rabbit]; N/A; LC50 [rat]; N/A; LD50 Dermal [rabbit]; N/A Material has not been found to be a carcinogen nor produce genetic, reproductive, or developmental effects.









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CAUTION! Powder may be a slight body tissue irritant. Target organs: None known


Section 3: Composition / Information on Ingredients Potassium Biphthalate ( 877-24-7) 100%










Section 8: Exposure Controls / Personal ProtectionUse ventilation to keep airborne concentrations below exposure limits. Have approved eyewash facility, safety shower, and fire extinguishers readily available. Wear chemical splash goggles and chemical resistant clothing such as gloves and aprons. Wash hands thoroughly after handling material and before eating or drinking. Exposure guidelines: Potassium Biphthalate: OSHA PEL: N/A, ACGIH: TLV: N/A, STEL: N/A.





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Section 11: Toxicology Information Acute Symptoms/Signs of exposure: Eyes: Redness, tearing, itching, burning, conjunctivitis. Skin: Redness, itching. Ingestion: Irritation and burning sensations of mouth and throat, nausea, vomiting and abdominal pain. Inhalation: Irritation of mucous membranes, coughing, wheezing, shortness of breath, Chronic Effects: No information found. Sensitization: none expected Potassium Biphthalate : LD50 [oral, rabbit];3200 mg/kg; LC50 [rat]; N/A; LD50 Dermal [rabbit]; N/A Material has not been found to be a carcinogen nor produce genetic, reproductive, or developmental effects.









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Section 3: Composition / Information on Ingredients Potassium Biphthalate ( 877-24-7), 75 to 85% Sodium Phosphate Dibasic, (7558-79-4), 15 to 25%










Section 8: Exposure Controls / Personal ProtectionUse ventilation to keep airborne concentrations below exposure limits. Have approved eyewash facility, safety shower, and fire extinguishers readily available. Wear chemical splash goggles and chemical resistant clothing such as gloves and aprons. Wash hands thoroughly after handling material and before eating or drinking. Exposure guidelines: Potassium Biphthalate: OSHA PEL: N/A, ACGIH: TLV: N/A, STEL: N/A.





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Section 11: Toxicology Information Acute Symptoms/Signs of exposure: Eyes: Redness, tearing, itching, burning, conjunctivitis. Skin: Redness, itching. Ingestion: Irritation and burning sensations of mouth and throat, nausea, vomiting and abdominal pain. Inhalation: Irritation of mucous membranes, coughing, wheezing, shortness of breath, Chronic Effects: No information found. Sensitization: none expected pH 6 buffer capsule : LD50 [oral, rabbit]; N/A; LC50 [rat]; N/A; LD50 Dermal [rabbit]; N/A Material has not been found to be a carcinogen nor produce genetic, reproductive, or developmental effects.









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Section 3: Composition / Information on Ingredients Sodium Phosphate Dibasic, (7558-79-4), 60 to 70% Potassium Phosphate monobasic (7778-77-0), 30 to 40%










Section 8: Exposure Controls / Personal ProtectionUse ventilation to keep airborne concentrations below exposure limits. Have approved eyewash facility, safety shower, and fire extinguishers readily available. Wear chemical splash goggles and chemical resistant clothing such as gloves and aprons. Wash hands thoroughly after handling material and before eating or drinking. Exposure guidelines: pH 7 Buffer: OSHA PEL: N/A, ACGIH: TLV: N/A, STEL: N/A.





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MSDS # 132.30 Buffer Capsule, pH 7.00 Scholar Chemistry Section 9: Physical and Chemical Properties Molecular formula N/A. Appearance Capsule with white solid.Molecular weight N/A. Odor No odor.Specific Gravity N/A Odor Threshold N/A. Vapor Density (air=1) N/A Solubility Complete. Melting Point N/A Evaporation rate N/A (Butyl acetate = 1). Boiling Point/Range N/A. Partition Coefficient N/A (log POW). Vapor Pressure (20°C) N/A. pH 7.0, neutral. Flash Point: N/A. LEL N/A. Autoignition Temp.: N/A. UEL N/A.












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Section 3: Composition / Information on Ingredients Sodium Phosphate Dibasic, (7558-79-4), 80 to 90% Potassium Phosphate monobasic (7778-77-0), 10 to 20%















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Section 3: Composition / Information on Ingredients Sodium Carbonate, (497-19-8), 20 to 30% Sodium Bicarbonate (144-55-8), 70 to 80%















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Section 3: Composition / Information on Ingredients Sodium Carbonate, (497-19-8), 50 to 70% Sodium Bicarbonate (144-55-8), 30 to 50%















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Section 3: Composition / Information on Ingredients Sodium Phosphate, tribasic, (7601-54-9), 20 to 30% Sodium Bicarbonate (144-55-8), 70 to 80%















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Guaiacol MSDS # 320.00 Section 1: Product and Company Identification

Guaiacol Synonyms/General Names: 2-Methoxyphenol Product Use: For educational use only Manufacturer: Columbus Chemical Industries, Inc., Columbus, WI 53925.



Section 2: Hazards Identification Pale, yellow liquid or solid [melting point is RT], characteristic odor. HMIS (0 to 4) CAUTION! Combustible liquid, keep away from all ignition sources. Moderately toxic by ingestion and skin absorption. Body tissue irritant. Target organs: Skin This material is considered hazardous by the OSHA Hazard Communication Standard (29 CFR 1910.1200).

Section 3: Composition / Information on Ingredients Guaiacol (90-05-1), 98%


Eyes: Immediately flush eyes with excess water for 15 minutes, lifting lower and upper eyelids occasionally. Skin: Immediately flush skin with excess water for 15 minutes while removing contaminated clothing. Ingestion: Call Poison Control immediately. Aspiration hazard. Rinse mouth with cold water. Give victim 1-2 tbsp of

activated charcoal mixed with 8 oz water. Inhalation: Remove to fresh air. If not breathing, give artificial respiration.

Section 5: Fire Fighting Measures Combustible Liquid. When heated to decomposition, emits acrid fumes 2 Protective equipment and precautions for firefighters: Use foam or dry chemical to extinguish fire. 2 1 Firefighters should wear full fire fighting turn-out gear and respiratory protection (SCBA). Cool container with water spray. Material is not sensitive to mechanical impact. Material is sensitive to static discharge.

Section 6: Accidental Release Measures Use personal protection recommended in Section 8. Isolate the hazard area and deny entry to unnecessary and unprotected personnel. Remove all ignition sources and ventilate area. Contain spill with sand or absorbent material and place material in a sealed bag or container for disposal. Wash spill area after pickup is complete. See Section 13 for disposal information.


hands thoroughly after handling. Storage: Store in Flammable Area [Red Storage] with other flammable materials and away from any strong oxidizers. Store in a

dedicated flammables cabinet. Store in a cool, dry, well-ventilated, locked store room away from incompatible materials.

Section 8: Exposure Controls / Personal Protection Use ventilation to keep airborne concentrations below exposure limits. Have approved eyewash facility, safety shower, and fire extinguishers readily available. Wear chemical splash goggles and chemical resistant clothing such as gloves and aprons. Wash hands thoroughly after handling material and before eating or drinking. Use NIOSH-approved respirator with an acid/organic cartridge. Exposure guidelines: Guaiacol: OSHA PEL: N/A, ACGIH TLV: N/A, STEL: N/A.





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MSDS # 320.00 Guaiacol Scholar Chemistry Section 9: Physical and Chemical Properties Molecular formula CH3OC6H4OH. Appearance Pale, yellow liquid. Molecular weight 124.14. Odor Characteristic odor. Specific Gravity 1.129 g/mL @ 20°C. Odor Threshold N/A. Vapor Density (air=1) 4.27. Solubility Alcohol, ether, acetic acid. Melting Point 28°C. Evaporation rate N/A . (Butyl acetate = 1). Boiling Point/Range 205°C. Partition Coefficient N/A. (log POW). Vapor Pressure (20°C) N/A. pH N/A. Flash Point: 82°C (179°F) CC. LEL 3.3%. Autoignition Temp.: 385°C (725°F). UEL 19 %.


Section 10: Stability and Reactivity Avoid heat and ignition sources. Stability: Stable under normal conditions of use. Incompatibility: Oxidizers Shelf life: Indefinite if stored properly.

Section 11: Toxicology Information Acute Symptoms/Signs of exposure: Eyes: Stinging pain, watering of eyes, inflammation of eyelids and conjunctivitis. Skin: Insensitivity to pain, feel of coolness or cold, skin looks white and feels hard and cold. Ingestion: Breath has sweet, organic odor, mental confusion, drowsiness, nausea, vomiting and headache. Inhalation: Rapid irregular breathing, headache, fatigue, mental confusion, nausea and vomiting, giddiness and poor judgment, convulsions and death. Chronic Effects: Repeated/prolonged skin contact may cause dryness or rashes. Sensitization: none expected Guaiacol: LD50 [oral, rat]; 520 mg/kg; LC50 [rat]; N/A; LD50 Dermal [rabbit]; 5020 mg/24H Severe Material has not been found to be a carcinogen nor produce genetic, reproductive, or developmental effects.

Section 12: Ecological Information Ecotoxicity (aquatic and terrestrial): EC50 – 25.9mg/l – 48h – Daphnia magna.


Section 14: Transport Information DOT Shipping Name: Not regulated by DOT. Canada TDG: Not reulated by TDG. DOT Hazard Class: Hazard Class: Identification Number: UN Number:

Section 15: Regulatory Information EINECS: Listed (201-964-7). WHMIS Canada: Not WHMIS controlled. TSCA: All components are listed or are exempt. California Proposition 65: Not listed. The product has been classified in accordance with the hazard criteria of the Controlled Products Regulations and the MSDS contains all the information required by the Controlled Products Regulations.

Section 16: Other Information Current Issue Date: December 21, 2011 Disclaimer: Scholar Chemistry and Columbus Chemical Industries, Inc., (“S&C”) believes that the information herein is factual but is not intended to be all inclusive. The information relates only to the specific material designated and does not relate to its use in combination with other materials or its use as to any particular process. Because safety standards and regulations are subject to change and because S&C has no continuing control over the material, those handling, storing or using the material should satisfy themselves that they have current information regarding the particular way the material is handled, stored or used and that the same is done in accordance with federal, state and local law. S&C makes no warranty, expressed or implied, including (without limitation) warranties with respect to the completeness or continuing accuracy of the information contained herein or with respect to fitness for any particular use.



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Hydrogen Peroxide, 3% MSDS # 345.00 Section 1: Product and Company Identification

Hydrogen Peroxide, 3% Synonyms/General Names: N/A Product Use: For educational use only Manufacturer: Columbus Chemical Industries, Inc., Columbus, WI 53925.



Section 2: Hazards Identification Clear, colorless liquid, slight odor HMIS (0 to4) WARNING! Oxidizing agent and body tissue irritant. Target organs: None known. This material is considered hazardous by the OSHA Hazard Communication Standard (29 CFR 1910.1200).

Section 3: Composition / Information on Ingredients Hydrogen Peroxide, 35% (7722-84-1), 3%. Water (7732-18-5), 97%. Section 4: First Aid Measures

Always seek professional medical attention after first aid measures are provided. Eyes: Immediately flush eyes with excess water for 15 minutes, lifting lower and upper eyelids occasionally. Skin: Immediately flush skin with excess water for 15 minutes while removing contaminated clothing. Ingestion: Call Poison Control immediately. Do not induce vomiting. Rinse mouth with cold water. Give victim 1-2 cups of

water or milk to drink. Inhalation: Remove to fresh air. If not breathing, give artificial respiration.

Section 5: Fire Fighting Measures Noncombustible solution. When heated to decomposition, produces oxygen gas. 0 Protective equipment and precautions for firefighters: Use foam or dry chemical to extinguish fire. 1 1


Section 6: Accidental Release Measures Use personal protection recommended in Section 8. Isolate the hazard area and deny entry to unnecessary and unprotected personnel. Remove all ignition sources and ventilate area. Contain spill with sand or absorbent material and place material in a sealed bag or container for disposal. Wash spill area after pickup is complete. See Section 13 for disposal information.




Section 8: Exposure Controls / Personal Protection Use ventilation to keep airborne concentrations below exposure limits. Have approved eyewash facility, safety shower, and fire extinguishers readily available. Wear chemical splash goggles and chemical resistant clothing such as gloves and aprons. Wash hands thoroughly after handling material and before eating or drinking. Use NIOSH-approved respirator with an acid/organic cartridge. Exposure guidelines Hydrogen Peroxide: OSHA PEL: 1.4 mg/m3; ACGIH TLV: 1.4 mg/m3; STEL:N/A.





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MSDS # 345.00 Hydrogen Peroxide, 3% Scholar Chemistry Section 9: Physical and Chemical Properties Molecular formula H2O2. Appearance Clear, colorless liquid. Molecular weight 34.01. Odor Slight odor. Specific Gravity 1.01 g/mL @ 20°C. Odor Threshold N/A. Vapor Density (air=1) 0.7. Solubility Completely soluble in water. Melting Point 0°C. Evaporation rate < 1 (Butyl acetate = 1). Boiling Point/Range 100°C. Partition Coefficient N/A. (log POW). Vapor Pressure (20°C) 14. pH N/A. Flash Point: N/A. LEL N/A. Autoignition Temp.: N/A. UEL N/A.

Section 10: Stability and Reactivity Avoid heat and ignition sources. Stability: Instable, many materials will catalyze the decomposition of hydrogen peroxide to produce oxygen, water, and heat. Incompatibility: Reducing agents, alkalis, organic materials, metals, acids, bases, metal salts, dust and dirt contaminants and flammable substances. Shelf life: Fair shelf life, store in a cool, dry environment.

Section 11: Toxicology Information Acute Symptoms/Signs of exposure: Eyes: Redness, tearing, itching, burning, conjunctivitis. Skin: Redness, itching. Ingestion: Irritation and burning sensations of mouth and throat, nausea, vomiting and abdominal pain. Inhalation: Irritation of mucous membranes, coughing, wheezing, shortness of breath. Chronic Effects: Repeated/prolonged skin contact may cause thickening, blackening or cracking. Repeated eye exposure may cause corneal erosion or loss of vision. Sensitization: none expected Hydrogen Peroxide: LD50 [oral, rat]; N/A; LC50 [rat]; N/A; LD50 Dermal [rabbit]; N/A Material has not been found to be a carcinogen nor produce genetic, reproductive, or developmental effects.

Section 12: Ecological Information Ecotoxicity (aquatic and terrestrial): Toxic to beneficial microorganisms (e.g. soil and sewage treatment microorganisms). Do not release to environment.


Section 14: Transport Information DOT Shipping Name: Not regulated by DOT. Canada TDG: Not regulated by TDG . DOT Hazard Class: Hazard Class: Identification Number: UN Number:

Section 15: Regulatory Information EINECS: Listed (231-765-0) . WHMIS Canada: Not WHMIS controlled. TSCA: All components are listed or are exempt. California Proposition 65: Not listed. The product has been classified in accordance with the hazard criteria of the Controlled Products Regulations and the MSDS contains all the information required by the Controlled Products Regulations.

Section 16: Other Information Current Issue Date: December 21, 2011 Disclaimer: Scholar Chemistry and Columbus Chemical Industries, Inc., (“S&C”) believes that the information herein is factual but is not intended to be all inclusive. The information relates only to the specific material designated and does not relate to its use in combination with other materials or its use as to any particular process. Because safety standards and regulations are subject to change and because S&C has no continuing control over the material, those handling, storing or using the material should satisfy themselves that they have current information regarding the particular way the material is handled, stored or used and that the same is done in accordance with federal, state and local law. S&C makes no warranty, expressed or implied, including (without limitation) warranties with respect to the completeness or continuing accuracy of the information contained herein or with respect to fitness for any particular use.

Documents

AP® InvestIgAtIon #13 - hintonhinton.harvardmedk12.wikispaces.net/.../APBio_no13-Teacher_manual.pdfPrE-lAb PrEPArAtiOn 9 studEnt GuidE cOntEnts bAckGrOund 12 ... There is a shift