BIOCHEMISTRY 3723
EXPERIMENT 3
AMINO ACID TITRATION
Report Author:
Lab Partner:
Date Due:
Date Turned In:
Unknown #:
Unknown Identification:
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Procedural Changes: Indicate any changes you made from the procedure in the lab manual.
Explain why these changes were made and how they affect the results.
Results:
I. Raw Data
A. Standardization for acid titration:
Electrode # _______________
1. The pH reading with pH 7.00 buffer was:
2. The pH reading with pH 4.00 buffer was:
B. Acid Titrations: The exact HCl concentration was:
1. Enter acid titration data for the 40.0 mL water sample in the following table:
Buret
reading
2.
Buret
reading
total mL
HCl
0.00
pH
Buret
reading
total mL
HCl
pH
Buret
reading
total mL
HCl
Enter acid titration data for the 40.0 mL amino acid solution in the table below:
total mL
pH
Buret
total mL
pH
Buret
total mL
HCl
reading
HCl
reading
HCl
0.00
C. Standardization for base titrations:
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pH
pH
1. The pH reading with pH 7.00 buffer was:
2. The pH reading with pH 10.00 buffer was:
D. Base Titration: The exact NaOH concentration was:
1. Enter base titration data for the 40.0 mL water sample in the following table:
Buret
reading
2.
Buret
reading
total mL
NaOH
0.00
pH
Buret
reading
total mL
NaOH
pH
Buret
reading
total mL
NaOH
Enter base titration data for the 40.0 mL amino acid solution in the table below:
total mL
pH
Buret
total mL
pH
Buret
total mL
NaOH
reading
NaOH
reading
NaOH
0.00
II. Computer analysis--Include all computer printouts in lab report.
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pH
pH
A. Endpoint of titration and molecular weight determination:
1. From the titration data (inspect the uncorrected titration curve) determine the
endpoint volume and meq of NaOH added. It is best to use the point after pH 11 or
so where the slope on the Raw Data curve is the greatest. The computer has corrected
for the amount of NaOH used to titrate water to each pH. For the end point value you
use, show the calculation for the water correction. (This is the correction made by the
computer: subtract the volume of NaOH needed to titrate water to the endpoint pH
from the volume of NaOH needed to titrate the amino acid solution to that same pH).
Also show the calculation for the conversion of net volume NaOH to net meq NaOH
(mL x meq/mL = meq).
Endpoint pH used: ______________
Volume NaOH added to AA in water: __________
Volume NaOH added to H2O; _____________
Net endpoint volume of NaOH: __________
Net endpoint meq of NaOH: ____________
2. Using the endpoint meq of NaOH, calculate the concentration (in N) of the amino
acid you titrated. (meqNaOH = meqAA = VAA x CAA)
meqAA: ________________
CAA:
_______________N
3. By inspecting the graphs, determine how many functional groups on the amino acid
were titrated with the NaOH (equivalents/mole). Use this information to determine
the molar concentration of the amino acid. Show calculation.
(Equivalents/liter equivalents/mole = moles/liter)
# functional groups titrated by NaOH: _______________
CAA:
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______________M
4. Calculate the molecular weight of the amino acid using the formula:
CAA(g/liter) = CAA (moles/liter) x Molecular weightAA (g/mole)
(Remember that all amino acid unknowns were 20.00 mg/mL).
amino acid molecular weight: ____________________
B. Determination of pKas
1. List the pKas determined by computer analysis (with appropriate significant figures):
pKas = _____________________
2. The pKa is the pH achieved after half an equivalent of titrant has been added for each
equivalent of group being ionized. This gives us another way to determine pKas.
Using this method, determine each of the pKas of your amino acid and list them: To
do this, determine the base endpoint in meq and divide by two if one functional group
was titrated with the NaOH. If two functional groups were titrated with NaOH
(histidine unknown), divide by four. This gives the meq needed to titrate to the first
basic pKa. For histidine, the second basic pKa is reached after 3/4 the base needed to
reach the endpoint has been added. The pH value reached when this amount of
NaOH has been added is the pKa. Since the equivalents of -COO– equal the
equivalents of -NH3+, the endpoint determined for the base titration can also be used
to find the pKa for the acid functional groups.
meq NaOH at endpoint: ___________
meq (acid or base?) to titrate to pKa 1: ______________
meq (acid or base?) to titrate to pKa 2: ______________
meq (acid or base?) to titrate to pKa 3: ______________
pKa1: ______________
pKa2: ______________
pKa3: ______________
Discussion:
A. Identification:
1. Based on the molecular weight, amino acid # _____ is
MW of _________ and pKs of _______________.
, which has a
2. Are there any other amino acids on the unknown list that could be your unknown?
Which ones and how did you eliminate them?
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B. Error analysis:
1. How closely does your calculated molecular weight agree with the theoretical
molecular weight of your amino acid? (Calculate accuracy). What is the most likely
source of error in determining the molecular weight of the amino acid? What are
other likely sources of error in this experiment?
2. How well do the pKas determined by the computer program (from the second
derivitive) agree with the pKas of the amino acid you identified? What are the most
likely sources of error in determining the pKas?
3. How well do the pKas determined from the number of meqs used to titrate to the
endpoint agree with the pKas of the amino acid you identified? What are the most
likely sources of error in determining the pKas by this method?
4. How closely do the pKs calculated by computer and from the endpoint meqs agree
with each other? Which do you think are more reliable and why?
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