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1/ 11• Procedure

Write a Pre-Lab assl notebook R . gnment based on the procedure below. This Information must be In your lab • emember to Include a title, objective and a summarized procedure In your own words.

Instead of using a b tt • 1· · f 'd b ure e to titrate your sample you will use a pipette to add SOµL a iquots O acr or

1 ase, as this method is much more accurate. w'hich pipettor should you use: P-10, P-SO, P-200 or P- OOO? 1ndicate this in your notebook or prelab. You will titrate 20ml of a solution containing an

u~known an,ino acid with 1M HCI until the pH re~ches Nl.5. You will then titrate a separate 20ml aliquot With lM NaOH u~til the pH reaches Nll.5. . ·.

Materials • Unknown amino acid solution (50ml; 0.1M) • lM NaOH (Nl0mL) • lM HCI (Nl0mL) • pH meter, stir plate, and stir bar • S0µL pipettor and tips • 30ml beaker • Set of three standard buffers: pH 4, 7, and 10 for pH meter calibration

An important note about beakers: Use the smallest possible beaker (30ml). This will ensure that the electrode is able to be submerged deep enough. If your pH reading is rapidly fluctuating, the electrode likely needs to be submerged more. It often needs to be at the bottom of the beaker.

pH Meter Calibration: Perform a three-point calibration of the HANNA Instruments Hl2202 Edge Blu pH Meter using pH 4.01, 7.01, and 10.01 buffers: ·

1. Your tech will have already turned on the pH meters and paired them to the correct Bluetooth electrode. Check the battery life of the meter. Ask for a charger if you are running low.

2. Remove the electrode from the storage solution by first turning the black collar on the storage cap counterclockwise (see illustration to the right). Rinse the electrode with RO water and gently blot dry with a Kimwipe. Do not.use paper towels.

*** NEVER LEAVE THE ELECTRODE OUT OF SOLUTION *** 3. Place the electrode directly in the tube of pH 7.01 buffer. Leave the tube in the

rack; don't hold it in your hand. Briefly and gently stir the electrode in the buffer, then let go and let it rest in the tube.

a. Press the "CAL/MODIFY" button to enter calibration mode.

b. "7.01" will be displayed in the bottom right corner. That means the electrode wants to be in the 7.01 ,buffer, which it is, so now you just have to wait for the reading to stabilize. "WAIT" will be displayed across the bottom of the screen.

c. Once the reading is stable, the pH meter will beep and "CFM" (meaning "CONFIRM") will flash near the left side of the screen. Press the "GLP/CFM" button to confirm the measurement.

••

.11 say pH 4 01. That means the . h b ·tt right corner wI . d. After confirming the first pomt, t e o om electrode wants to be in the pH 4.01 buffer.

. . . the H 4 01 buffer. Briefly and 4. Clean the electrode with RO water and a Kimwipe. Place it '" P · gently stir the electrode in the buffer, then let go. h

,, . d" I d across the bottom oft e screen. a. Wait for the reading to stabilize. "WAIT will be Isp aye . b d "CFM" will flash . Press the b. Once the reading is stable, the P!"i meter will eep an

nGLP/CFM" button to confirm the measurement. · . h ·11 y 10 01 That means the c. After confirming the second point, the bottom rig t corner WI sa · ·

electrode wants to be in the 10.01 buffer.

5. Clean the electrode with RO water and a Kimwipe.· Place it in the pH 10.01 buffer. Briefly and gently stir, then let go.

a. Wait for the reading to stabilize. "WAIT" will be displayed across the bottom of the screen.

b. Once the reading is stable, the pH meter will beep and "CFM" will flash. Press the "GLP/CFM" button to confirm the measurement.

c. ****IMPORTANT/ .... After confirming the 10.01 measurement, some meters will display 12.01 in the bottom right corner:

• If your meter DOES NOT ask for the pH 12.01 buffer: DO NOT press any more buttons. Your meter will automatically save the data, exit calibration mode, and is ready to use.

• If your meter DOES ask for the pH 12.01 buffer: Press the "CAL/MODIFY" button to save the calibration. Once saved, your meter will exit calibration mode and is ready to use. · '

--•oo not press "CAL/MODIFY" again, or you will have to perform a new calibration•••

The pH meter automatically reads and displays the pH as it changes. After saving the calibration data, you do not need to press any buttons qt all for any reason ever again.

Part A. Acid titration:

1. Obtain 50ml of a O.lM solution of unknown amino acid. Make sure you sign out your unknown. Be careful with your sample, as we usually do not have enough to provide a new sample in case of mishaps. Record your unknown number in your notebook and write your name on the unknown tube to prevent mix-ups.

2. Transfer 20ml of your unknown to a 30ml beaker. Record the volume to the nearest 0.1ml. Use a graduated cylinder; the markings on the beaker are not accurate.

3. Set up the pH meter, magnetic stir bar, and stirring plate so your sample can be titrated while under constant stirring. Make sure the stir bar does not hit the electrode. Also make sure the electrode is sufficiently submerged to get a $table reading; it usually needs to be at the very

y bottom of the be k initial readin of a a er. ~II unknowns have an Initial pH -u If your unknown does not have an

g pprox1mately 7, talk to your instructor or CL T. 4· Record the in·r 1 1 ,a PH of your unknown ~.efore adding any acid.

5. Begin tit f · · · ra mg your sample with lM Hq. A few things to keep in mind: • ~hen transferring the acid or b~s~, make sure there are no droplets on the outside of the

tip, as that will decrease the ac.curacy of your results and make it more difficult for you determine the identity of your unknown.

• When dispensing the acid or base into your unknown, submerge the tip only a few millimeters into the solution and carefully pipette up and down (only to the first stop) a few times to rinse the inside of the tip, then go to the second stop to expel all the liquid. Remember to change tips after each aliquot.

After adding SOµL of acid, wait for the reading to stabilize (the hourglass icon near the left side of the screen will disappear). Record the pH each time you add SOµL; you can use the worksheets on the next few pages. You might not fill the entire table or you may need to add more entries to theses tables. These tables are just a guide.

6. ntrate your sample with HCI until the p~ reaches°':;;1.5.

7. Once you are done with the acid titrati~n, wipe the end of the pipettor to remove any acid residue that can contaminate your n.ext:titration or damage the pipettor. Rinse and blot the electrode as before. Put it back in the si orage bottle, make sure it is fully submerged in the storage buffer, then turn the black collar on the cap to tighten.

*** DO NOT leave the electrode out of solution***

Part B. Base titration:

1. Transfer 20ml of your unknown to a clean 30ml beaker. Record the volume to the nearest 0.1ml.

2. Record the initial pH of your unknown before adding any base. Again, it should be close to 7. It is okay if this reading and the initial reading for your acid titration are not exactly the same. Sometimes differences in initial pH measurements are due to changes in temperature.

3. Begin titrating your sample in the same manner as before, but this time add SOµL aliquots of lM NaOH. Record the pH each time you add SOµL of base; you can use the worksheets on the next few pages. Again, you might not fill the entire table or you may need to add more entries to theses tables. These tables are just a guide. ·

I 4. Titrate your sample with NaOH until the pH reaches "'11.5

5. Once you have finished the base titration, wipe the end of the pipettorto remove any base residue that can damage the pipettor. Rinse and blot the electrode as before. Put it back in the storage bottle, make sure it is fully submerged in the storage buffer, then turn the black collar on the cap to tighten.

*** DO NOT leave the electrode out of solution***

Table 1. Acid Titration of Unknown A,i,._o~ -61 . Add 1M HCI until you reach pH "'1.5

VolumeoflM Total Volume of lM HO pH HO added(ml) added to unknown(ml)

0 0.00 =,-. (0 0.05 2.30 0.05 0.05 J•f"1" 0.05 2.35 0.05 0.10 3· IIS 0.05 2.40 0.05 0.15 3.522. 0.05 2.45 0.05 0.20 3.ot 0.05 2.50 0.05 0.25 J_, q5 0.05 2.55 0.05 0.30 l, &5 0.05 2.60 0.05 0.35 1..2-, :,1 0.05 2.65 0.05 0.40 ..2. 6'f 0.05 2.70 0.05 0.45 IJ-6.3 0.05 2.75 0.05 0.50 ij_. Sl, 0.05 2.80 0.05 0.55 J-5/ 0.05 2.85 0.05 0.60 :1, 4-6 0.05 2.90 0.05 0.65 j_. 41 0.05 2.95 0.05 0.70 .2-36 0.05 3.00 0.05 0.75 .2-52 0.05 3.05 0.05 0.80 :)..2,0 0.05 3.10 0.05 0.85 .J.. I b 0.05 3.15 0.05 0.90 2, l:l 0.05 3.20 0.05 0.95 ).. O'I 0.05 3.25 0.05 1.00 ..f). OS 0.05 3.30 0.05 1.05 ..J.. O.l 0.05 3.35 0.05 1.10 i, q'I 0.05 3.40 0.05 1.15 1·% 0.05 3.45

0.05 1.20 I · tr ..l 0.05 3.50

0.05 1.25 I , !"f 0.05 3.55

0.05 1.30 , • ..81!. 0.05 3.60

0.05 1.35 /·8'?> 0.05 3.65

0.05 1.40 I. to 0.05 3.70

0.05 1.45 I. -:,-=, 0.05 3.75

0.05 1.50 I, =,-4- 0.05 3.80

0.05 1.55 , . .:;~ 0.05 3.85

0.05 1.60 l·il 0.05 3.90

0.05 1.65 l_. 66 0.05 3.95

0.05 1.70 1-l,l/- 0.05 4.00

0.05 1.75 1 · "' 0.05 4.05

0.05 1.80 ,. 58 0.05 4.10

0.05 1.85 I, 56 0.05 4.15

0.05 1.90 l, .~ 1 0.05 4.20

0.05 1.95 /, SI 0.05 4.25

0.05 2.00 I• I.ft 0.05 4.30

0.05 2.05 0.05 4.35

0.05 2.10 0.05 4.40

0.05 2.15

0.05 2.20

0.05 2.25

0.05 4.45

0.05 4.50

0.05 4.55

Table 2. Base Titration of Unknown ~ o3-o I • Add 1M NaOH until you reach pH"'ll.5

VolumeoflM Total Volume of 1M NaOH pH NaOH added(ml ) added to unknown(ml)

0 0.00 1-0 0.05 0.05 1. -:,., 0.05 0.10 ~- 0 ~

0.05 0.15 f,25 0.05 0.20 ~ -?>8 0.05 0.25 lc--14 & 0.05 0.30 X' • C,l.j,

0.05 0.35 it;b~ 0.05 0.40 g~ 71 0.05 0.45 g. 1=1- 0.05 0.50 ·f< . &'3 0.05 0.55 Jc . 8"1 0.05 0.60 Ir· 'il.f- 0.05 0.65 ''1'f 0.05 . 0.70 ~.Ol{- 0.05 - 0.75 14 . 01 0.05 · 0.80 · q . I 5 0.05 0.85 a. 1·~ 0.05 0.90 ~-J-1 0.05 0.95 q •? 0.05 1.00 'i . ,l.'I 0.05 1.05 q . ~~ 0.05 1.10 tf .:,6'. .. , 0.05 1.15 er .40 0.05· 1.20 q. 4'1 0.05 1.25 Q , qg 0.05 1.30 Lt•~ I 0.05 1.35 {,\ . ~c,. 0.05 1.40 ~. ,;q 0.05 1.45 "!.-ht.I- 0.05 1.50 ct , (.,K 0.05 1.55 It . J.--1 0.05 1.60 '¾.

Volumeof l M Total Volume of 1M NaOH pH NaOH added (ml) added to unl<nown(ml)

0.05 2.30 10 •. l\'f 0.05 2.35 0 '•3l 0.05 2.40 \D, ~ I 0.05 2.45 lo . 4~ 0.05 2.50 l0 • 4 C, 0.05 2.55 iO., .. 'oJ. 0.05 2.60 \o· 56 0.05 2.65 10 -· b1 0.05 2.70 \0. (,2,

0.05 2.75 lo· (,:t 0.05 . 2.80 \O· ;JO

0.05 2.85 \0 ·~4 0.05 2.90 \O· ~ 0.05

) . 2.95 ,o -81 0.05 3.00 l0·&'1t 0.05 3.05 10 • '\ I 0.05 3.10 \O · ,4 0.05 . 3.15 \0 . f\C\ 0.05 3.20 .. tt • ·0\ 0.05 3.25 \l .os 0.05 3.30 ll • e><l 0.05 3.35 \l t , , •

0.05 ;..· 3.40 n· .. ,~ 0.05 3.45 H . l'1 0.05 3.50 ~I -~ 0.05 3.55 l \ • .a.:,- 0.05 3.60 ft. . ,-2.\ .· 0.05 3.65 \l. ,;a.c:;. 0.05 3.70 1 l - ~" 0.05 3.75 tl . 4-<f 0.05 3.80 I\ . 4t 0.05 3.85 \l- 0.05 3.90

0.05 1.65 £. g-i 0.05 3.95

0.05 1.70 l • i'C.. 0.05 4.00

0.05 1.75 (I . ~ 0.05 4.05

0.05 1.80 1:1 . '1~ 0.05 4.10

0.05• 1.85 '1-'\~ 0.05 4.15

0.05 1.90 ~O· ol 0.05 4.20

0.05 1.95 0 , OC., , 0.05 4.25

0.05 2.00 o .o 'I 0.05 4.30

0.05 2.05 . \D. \4,; . .._ 0.05 4.35

0.05 2.10 h'\. \I 0.05 4.40

0.05 2.15 \Q. j2. 0.05 4.45

0.05 2.20 \0•.2.b 0.05 4.50

0.05 2.25 l r'\ -.J.~ 0.05 4.55

111. Data Analysis Analyze your data and create a titration curve to determine the pK. and pl values of your amino acid. You will use those values to determine the Identity of your unknown amino acid.

1. Use the pH vs. volume data recorded during lab ..

2. A detailed tutorial on how to make the pH vs. molar equivalents graph (titration curve) is available in your Lab 3 folder on Blackboard.

3. Using the data from your tables, convert the totai volume of titrant (HCI or NaOH) that was added to molar equivalents (Meq) of acid or base needed to titrate the amino acid sample to any given pH. Excel can calculate this for you. Remember to convert the volume from ml to L first.

Me = moles oftitrant _ [titrant] x total volume oftitrant (in L) q moles of amino acid - [amino acid] volume of amino acid titrated (in L)

.•

(titrant) is the molar concentration of acid or base (amino acid] is the molar concentration of amin'o acid

4· When plotting your data, pH is on the y-axis and molar equivalents is on the x-axis. Do not use volume for the x-axis.

! 5. Combine the acid and base titration curves into a single curve that spans pH 1.5-11.5

The equivalents of titrant consumed in one :full inflection of a curve (in the zone of a single ionization) represent the quantity of acid or base that is required to titrate one ionizable group of your amino acid sample. All unknown amino acids are triprotic, therefor they all contain three ionizable groups. Based on this information, how many molar equivalents of acid or base were needed to titrate your amino acid? The end point of titration should be recognizable as the point at which the pH changes sharply upon addition of titrant. This point tends to be more accurately determined from the titration of the amino acid with base.

6. Determine the three pK. values from the titration curve; they are equal to the pH at the half equivalency points. If your acid titration was longer than the base, the pK1 values will be at -1.5, -0.5 and 0.5 Meq. If your base titration was longer, the pK1 values will be at -0.5, 0.5 and 1.5 Meq. You can interpolate on the graph to find the pH •at those half equivalency points or you can look at your pH vs Meq table. Remember that at, the half equivalency points, [A-] == [HA] (for that particular ionizable group), therefore pH == pK.. ·

7. Once you have determined the pK. values, compare them to the reference pK. values of triprotic amino acids from the literature (you can use the ones presented in this manual) and calculate the

I % error of each. According to the% errors, what is the identity of your unknown? Keep in mind that all amino acids have a pK. of ~2 and ~9-10 for the a-carboxyl and a-amino group, respectively. Which of your three pK. values will be the most helpful when deciding the identity of your unknown?

8. Look closely at the shape of your titration curve; it also gives you clues that can help you determine the identity of the unknown amino acid . Does the curve have a longer acid side or basic side? How many buffer zones and inflection points do you see? Based on the pK.s, where on the graph should the buffer zones be?

IV.

9. Once you hav d lit e etermlned the identi

erature value, and calculate th a., ty of your unknown, calculate the pl compare it to the e 7Q error. '

Assignment: Results and Questions For this assignment you will . _ . raw data and

1 ' write the Results section and include References and an Appendix with

samp e calculat' y -1 · B I is a g 'd h ions. ou w1 I also answer some questions regarding your results. e ow u, eon ow to writ th · · bl · the L b

3 f

I e ese sections. More details on how to write a lab report are availa e '"

. a .0 der on Blackboard. Your assignment must be typed using a word processing program that is compatible with T · · ' · urnitm.com. Check your syllabus for a list of compatible programs. Do not copy and paste the guide into your document.

1. Results (SO pts) ~rit~ this section using paragraphs. Pay close attention to the flow of the information presented m this section. The transitions from point to point should be smooth. Your descriptions should not read like you are answering short questions or bullet points.

You. will present your analyzed data, results, and how they were obtained. Do not include explanations or interpretations of your results or limitations and importance of the study; those are presented in the Discussion section.

A. Start the Results section with a brief introduction that states the purpose the experiment and what was done. Include the number of your unknown. (No more than 5 sentences).

B. Include a figure of the titration curve. The figure can be inserted within the text. When making your figure, keep in mind th~t you must include:

• A graph with a proper title and labeled axis (remember to include units). • pl<., and pl values on the graph. • A caption below the gra·ph. The caption must have a title and description of the figure.

Figures are standalone entitle~. All information presented in the caption must also be included in the main text of: the Results section.

• Your figure should look similar to the one below:

Title of Figure

pl(or • 11.07

I IIL,•7.05

0 o_.s · 1.5

Molar Equivalents

Figure 2. Title of Figure. Tliis Is the titration curve of... followed by a description of the graph.

C. Write a description of your results. You must include the following (in no particular order): • Describe the graph: what data was used to construct it, what information can it

provide and how did you-get that information. • Indicate the identity of your unknown amino acid. • State the pKa and pl values that you obtained from your data. Compare these values

to the literature (calculate% error). • Reference the raw data and calculations in the Appendix.

2. Questions (30pts; 10pts each) Answer the following questions regarding your un~~own amino acid. You do not need to copy/paste the questions in your assignment. However, you must answer each question separately and thoroughly. Number your .inswe_rs.· Do not combine all answers into one paragraph.

A. What is happening to your amino acid throughout the titration process in terms of structure and charges? If you add a figure of your amino acid to aid in your explanation, it must have a caption.

B. What is happening to your amino acid at each pKa and the pl?

C. Explain the shape of the titration curve and how it relates to the behavior of a buffer (refer to #8 in the Data Analysis).

3. References (lOpts) You will use references for your literature values and to answer the questions above. When referencing literature for this lab you must:

A. Include at least three references, two of which must be outside references • Inside references: Any material given to you by the lecture or lab instructor

(Biochemistry text book, lab manual, PPT, etc.) • Outside references: Material not given to you by the lecture or lab instructor (other

class textbooks, research papers, reviews, etc.) '

B. Use in-text citations (MLA or APA is fine)

4. Appendix (10pts) A. Include a sample calculation (one of each; choose any data point) for molar equivalents,%

error, and Pl. B. Include a table with raw data for pH, volumes of titrant, and molar equivalents.

s. Observations (-5 if not included) You must include a copy for your handwritten observations and data collected during the lab. Include a scan or a picture. Do not re-type your observations.