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A Titration Analysis to Determine the Identity of an Unknown Amino Acid

Author: Katherine Liberty Dyer

Laboratory Partner: Abraham Ray Miller

10/10/2017
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Introduction:

Titrations are one of the most commonly performed experimental techniques in secondary and

undergraduate chemistry laboratory courses due to their simplicity, relative cost-effectiveness, and ease of

obtaining precise results without significant skill.1 A titration, in which a known quantity of an acid or base is

gradually combined with serial additions of a small quantity of base or acid to allow the performing individual

to observe the effect on pH either through a device such as a pH meter or a color-changing indicator buffer, is

an effective method of determining the identity of an unknown acid or base. This relies on the idea that, as pH

changes, the substance will either protonate or deprotonate (depending on the direction of the titration), and

by comparing the pH where equivalence points are found, the identity of the substance can be determined by

comparing these to known values belonging to possible identities. Amino acids have a distinctive appearance

when creating titration curves; because they possess a carboxyl group and amino group, and possibly an

additional R group, that can be expected to ionize during a titration, the titration curve of an amino acid will

have 2-3 equivalence points.2 The reactions that occur when an amino acid (one possessing an acidic R group

that is neutrally charged when protonated) is titrated with sodium hydroxide, a strong base, can be described

as such:

H3A+2 + NaOH -> H2A+ + H2O + Na+

H2A+ + NaOH -> HA + H2O + Na+

HA + NaOH -> A- + H2O + Na+

The first equation describes the deprotonation of the carboxyl group, which has a pKa of <3. The pKa of

an acid (or a functional group) describes the pH at which 50% of the substance will be deprotonated. The

second equation in this case describes the deprotonation of the R group, which typically has a pKa between

pH 3-9. The final equation described the deprotonation of the amino group, with a pKa usually somewhere
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between 9-10. (Note, some amino acids, typically those whose R groups contain an amino group, would have

different charges for each equation, and the amino group attached to their chiral carbon would deprotonate

before the one on their R group.)3

Hypothesis:

Titration of the unknown amino acid by sodium hydroxide will reveal a polyprotic acid with an ionizable

R-group, thus generating 3 separate pKas.

Procedure:

The Hanna pH meter was calibrated in the following manner: 2 250-mL beakers were placed next to the

meter, one containing deionized (“DI”) water (“wash beaker”), and one empty (“rinse beaker”), together with

a squirt bottle containing DI water (“rinse bottle”). The protective cap was completely removed from the

probe and set aside and the pH meter was turned on. The pH meter probe was placed in the wash beaker and

shaken back and forth to remove residue from the probe. The probe was then removed from the wash beaker

and held inside the wash beaker, not touching any part of the beaker, and rinsed by squirting it with DI water

from the rinse bottle. The probe was dried with a Kim wipe and then placed into a plastic conical sample tube

containing pH 7 calibration solution. The temperature was obtained by consulting a thermometer inserted in

the solution and found to be 21C. The temperature button on the pH meter was pressed and the temperature

set to 21C. The offset was calibrated to pH 7. The probe was removed from solution, placed in the wash

beaker, shaken back and forth, held in the rinse beaker, rinsed with DI water, dried with a Kim wipe, and

placed in a plastic conical sample tube containing pH 4 calibration solution. The slope was then calibrated to

pH 4.

The titration was set up in the following manner: a 50-mL buret was obtained, cleaned with soap and tap

water, rinsed thoroughly with tap water, and rinsed thoroughly with DI water (“washed and rinsed”). The
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stopcock was tested to rule out leaks. A quantity of approximately 50-mL of sodium hydroxide was obtained

and placed in a washed and rinsed and dried 250-mL beaker. The buret was rinsed with 5-mL of sodium

hydroxide, which was discarded in a 400-mL beaker marked “toxic waste” (“waste beaker”). The buret was

then placed in a buret clamp on a stand and the waste beaker was placed underneath it. Using a funnel to

prevent spillage, approximately 41-mL of sodium hydroxide was poured into the buret. The stopcock was

turned to allow the sodium hydroxide to flow into the beaker until there were no air bubbles present in the tip

of the buret, and the amount of sodium hydroxide remaining in the beaker was noted to be 39.71-mL. The

waste beaker was set aside and a 50-mL beaker was washed and rinsed and placed inside a 1000-mL beaker

underneath the buret. (The 1000-mL beaker was used to ensure that the pH probe would not overbalance and

cause the 50-mL beaker to tip.) An approximately 35-mL quantity of the unknown amino acid to be titrated

was obtained and placed in a washed and rinsed and dried Erlenmeyer flask (“analyte”). A 25-mL volumetric

pipet was washed and rinsed and used to transfer 25.00-mL of unknown amino acid to the 50-mL beaker. The

pH probe was then removed from solution, washed in the wash beaker, rinsed in the rinse beaker, dried with a

Kim wipe, and placed into the analyte. (Initially this was performed with a 150-mL beaker; however, the

analyte was not sufficiently deep to cover the tip of the probe to provide an accurate pH reading, and the

analyte was transferred to the 150-mL beaker.) An initial pH of 1.42 was obtained.
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Figure 1: Titration setup.

The titration was begun in the following manner: sodium hydroxide was added to the analyte in increments of

0.50-mL. After each increment was added, the pH probe was swirled in the liquid for 3 seconds. When the

liquid ceased visible movement, pH was measured and recorded to 2 significant digits (e.g. 1.42). The titration

was continued in this manner until the pH began to change by greater than 0.1 per increment, at which point

the titration was continued in increments of 0.10-mL. This was continued until approximately 22.5-mL of

sodium hydroxide had been added to the analyte; at this point, the solution was transferred to a washed and

rinsed and dried 150-mL beaker to accommodate a larger volume of solution. The titration was continued until

a pH of 8.58 was reached. (Note, the pH meter should have been recalibrated once pH 7.00 was reached as

per the instructions provided; however, the meter was not recalibrated until pH 8.58).

The pH meter was recalibrated as follows: the probe was removed from solution, placed in the wash beaker,

shaken back and forth, held in the rinse beaker, rinsed with DI water, dried with a Kim wipe, and placed in a

plastic conical sample tube containing pH 7 calibration solution. The temperature was obtained by consulting

a thermometer inserted in the solution and found to be 21C, consistent with the previous setting. The offset

was calibrated to pH 7. The probe was removed from solution, placed in the wash beaker, shaken back and
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forth, held in the rinse beaker, rinsed with DI water, dried with a Kim wipe, and placed in a plastic conical

sample tube containing pH 10 calibration solution. The slope was then calibrated to pH 10.

The titration was continued in the following manner: the probe was removed from solution, placed in the

wash beaker, shaken back and forth, held in the rinse beaker, rinsed with DI water, dried with a Kim wipe, and

replaced in the analyte solution. The pH of the solution was rechecked with the recalibrated pH meter and

noted. Sodium hydroxide was added to the analyte in increments of 0.10-mL. After each increment was

added, the pH probe was swirled in the liquid for 3 seconds. When the liquid ceased visible movement, pH was

measured and recorded to 2 significant digits. The titration was continued in this manner until the level of the

buret reached 50.00-mL. At this point, the titration had not yet reached a pH of 12 (the desired endpoint), and

the buret was refilled with additional sodium hydroxide as described above. A volume slightly greater than 50-

mL was placed in the buret and the excess was drained into the waste beaker until the total volume was

50.00-mL. The titration was continued in increments of 0.10mL until pH of 11.37, at which point the pH was

changing very little in response to added sodium hydroxide. The titration was continued in increments of 0.50-

mL until a pH of 11.76 was reached, at which point it was continued in increments of 1.00-mL until a pH of 12

was reached.

The titration was repeated in the following manner: the analyte solution was neutralized with the addition of

baking soda (sodium bicarbonate) and disposed of in the lab sink. The 50-mL and 150-mL beakers were

washed and rinsed and dried, and the buret was again refilled until it contained 50.00-mL as described above.

The pH meter was recalibrated to pH 4 and the titration was repeated in the same manner as described as

above, with intervals of 0.10-mL, 0.50-mL, and 1.00-mL used to focus on points in the graph that appeared to

be equivalence points. The titration was repeated a 3rd time focusing on the intervals between 18.00-19.50-mL

added and 23.00-32.00-mL.

Results:
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Titration 1 - NaOH: 0.1006 M Unknown Amino Acid: 12.4138 g/2L Analyte vol: 25.00 mL

NaOH 18.21 2.97 23.41 5.97


Given 18.69 3.23 23.51 5.99
(mL) pH 18.82 3.34 23.58 6.00
0.00 1.42 18.90 3.42 23.71 6.02
0.69 1.42 19.01 3.56 23.81 6.04
1.18 1.45 19.11 3.70 24.11 6.09
1.70 1.47 19.21 3.90 24.21 6.11
2.21 1.49 19.29 4.09 24.31 6.13
2.69 1.51 19.40 4.31 24.41 6.14
3.21 1.55 19.49 4.52 24.51 6.15
3.69 1.57 19.61 4.68 24.61 6.16
4.21 1.59 19.70 4.78 24.71 6.19
4.68 1.62 19.81 4.88 24.81 6.22
5.21 1.63 19.90 4.95 24.91 6.24
5.70 1.66 20.01 5.02 25.01 6.25
6.21 1.69 20.11 5.08 25.11 6.27
6.71 1.71 20.21 5.13 25.21 6.30
7.21 1.75 20.69 5.34 25.31 6.30
7.71 1.77 21.20 5.50 25.41 6.32
8.21 1.79 21.30 5.53 25.51 6.33
8.69 1.82 21.41 5.56 25.61 6.34
9.21 1.86 21.50 5.57 25.71 6.38
9.71 1.89 21.61 5.60 25.81 6.40
10.20 1.91 21.71 5.62 25.91 6.41
10.72 1.95 21.81 5.65 26.01 6.43
11.20 1.99 21.91 5.67 26.11 6.46
11.71 2.03 22.01 5.69 26.21 6.48
12.21 2.06 22.19 5.74 26.31 6.50
12.68 2.10 22.31 5.76 26.41 6.52
13.21 2.15 22.41 5.78 26.51 6.53
13.69 2.19 22.51 5.80 26.61 6.55
14.21 2.24 22.61 5.82 26.71 6.57
14.69 2.28 22.71 5.84 26.81 6.59
15.20 2.35 22.83 5.86 26.91 6.62
15.69 2.41 22.91 5.87 27.01 6.67
16.20 2.47 23.01 5.90 27.11 6.69
16.67 2.57 23.11 5.92 27.21 6.71
17.19 2.67 23.21 5.93 27.41 6.77
17.68 2.80 1 drop of NaOH lost 23.31 5.95 27.53 6.81
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27.61 6.82 31.91 8.83 36.11 9.58


27.70 6.83 32.01 8.84 36.21 9.60
27.81 6.87 32.11 8.87 36.31 9.61
27.91 6.92 32.21 8.89 36.44 9.64
28.01 6.97 32.31 8.91 36.51 9.66
28.11 7.03 32.41 8.92 36.61 9.68
28.23 7.07 32.51 8.95 36.71 9.70
28.31 7.12 32.61 8.96 36.81 9.73
28.41 7.15 32.71 8.98 36.91 9.74
28.51 7.20 32.81 8.99 37.01 9.77
28.61 7.27 32.91 9.03 37.11 9.79
28.71 7.33 33.01 9.04 37.21 9.81
28.81 7.42 33.11 9.05 37.31 9.83
28.91 7.47 33.21 9.07 37.41 9.88
29.01 7.58 33.31 9.09 37.51 9.91
29.11 7.65 33.41 9.12 37.61 9.94
29.21 7.74 33.51 9.13 37.71 9.96
29.21 7.74 33.61 9.15 37.81 9.99
29.31 7.83 33.71 9.16 37.92 10.04
29.41 7.92 33.81 9.17 38.02 10.06
29.61 8.04 33.93 9.20 38.11 10.09
29.71 8.12 34.01 9.21 38.21 10.12
29.81 8.15 34.11 9.23 38.31 10.16
29.91 8.23 34.21 9.25 38.42 10.21
30.00 8.26 34.31 9.26 38.51 10.28
30.12 8.31 34.51 9.30 38.59 10.29
30.21 8.35 34.59 9.31 38.71 10.33
30.31 8.38 34.71 9.33 38.81 10.38
30.41 8.42 34.81 9.34 38.91 10.43
30.51 8.74 34.91 9.37 39.01 10.48
30.61 8.50 35.01 9.38 39.13 10.55
30.71 8.52 35.11 9.40 39.21 10.59
30.81 8.58 35.21 9.42 39.31 10.65
30.91 8.60 35.31 9.44 39.42 10.71
31.01 8.62 35.41 9.45 39.51 10.77
31.21 8.67 35.51 9.46 39.61 10.84
31.31 8.69 35.61 9.47 39.71 10.88 25 minutes elapsed
31.41 8.71 35.72 9.51 39.71 10.69 and pH changed
31.51 8.74 35.81 9.52 39.86 10.72
31.71 8.78 35.91 9.54 39.91 10.74
31.81 8.80 36.03 9.56 40.01 10.79
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40.11 10.84 41.41 11.23 44.71 11.64


40.24 10.91 41.51 11.26 45.21 11.67
40.31 10.92 41.61 11.27 45.71 11.71
40.41 10.95 41.71 11.29 46.21 11.74
40.51 11.00 41.81 11.31 46.79 11.76
40.62 11.05 41.91 11.33 47.71 11.80
40.71 11.07 42.01 11.35 48.71 11.85
40.84 11.10 42.11 11.36 49.71 11.89
40.91 11.12 42.21 11.37 50.71 11.92
41.01 11.13 42.72 11.43 51.71 11.96
41.11 11.17 43.21 11.51 52.71 11.98
41.21 11.19 43.74 11.57 53.71 12.00
41.31 11.22 44.21 11.60

Titration 1 of Unknown Amino Acid


14.00

12.00

10.00

8.00
pH

6.00

4.00

2.00

0.00
0.00 10.00 20.00 30.00 40.00 50.00 60.00
NaOH Given (mL)

Figure 2
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Standard Deviation 1 of Unknown Amino Acid


4.00
3.00
2.00
1.00
Slope

0.00
-1.00 0.00 10.00 20.00 30.00 40.00 50.00 60.00

-2.00
-3.00
NaOH Given (mL)

Figure 3

Titration 2 - NaOH: 0.1006 M Unknown Amino Acid: 12.4138 g/2L Analyte vol: 25.00 mL

NaOH 11.00 2.03 19.30 4.68


Given 11.50 2.07 19.40 4.77
(mL) pH 12.00 2.11 19.50 4.89
0.00 1.45 12.50 2.15 19.60 4.94
0.50 1.46 13.00 2.19 19.70 5.03
1.00 1.47 13.50 2.24 19.80 5.03
1.89 1.50 14.00 2.29 19.89 5.17
2.00 1.52 15.50 2.48 20.00 0.52
2.50 1.55 16.00 2.55 20.10 5.28
1.99 1.58 16.50 2.63 20.20 5.32
3.50 1.60 17.01 2.74 20.30 5.35
4.00 1.63 17.50 2.85 20.40 5.39
4.50 1.65 18.00 3.04 20.50 5.42
5.00 1.67 18.10 3.07 21.00 5.59
5.50 1.70 18.20 3.13 21.50 5.71
6.01 1.72 18.30 3.17 22.00 5.82
6.50 1.74 18.40 3.24 22.50 5.92
7.00 1.78 18.50 3.32 23.00 6.01
7.50 1.80 18.60 3.38 23.51 6.10
8.00 1.84 18.70 3.48 24.00 6.17
8.50 1.87 18.80 3.59 24.50 6.26
9.00 1.89 18.90 3.76 25.00 6.35
9.50 1.93 18.98 3.95 26.00 6.51
10.00 1.97 19.10 4.22 27.02 6.73
10.54 2.01 19.20 4.44 28.00 7.03
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29.00 7.33 31.50 8.65 41.50 10.69


29.10 7.38 32.01 8.80 42.02 10.87
29.21 7.46 32.50 8.95 42.50 11.06
29.30 7.52 33.00 9.01 43.00 11.25
29.40 7.55 34.00 9.18 43.50 11.32
29.50 7.64 35.00 9.35 44.00 11.39
29.60 7.69 36.00 9.48 45.03 11.54
29.70 7.81 37.00 9.58 46.00 11.66
29.80 7.88 38.03 9.73 47.02 11.71
29.90 7.94 39.00 9.91 48.00 11.79
30.00 8.01 40.00 10.15 48.98 11.85
30.10 8.08 40.10 10.17 50.00 11.89
30.20 8.11 40.20 10.21 51.00 11.92
30.30 8.19 40.30 10.24 52.00 11.95
30.40 8.26 40.40 10.26 54.04 12.00
30.50 8.29 40.50 10.29
31.03 8.51 41.00 10.46

Titration 2 of Unknown Amino Acid


14.00

12.00

10.00

8.00
pH

6.00

4.00

2.00

0.00
0.00 10.00 20.00 30.00 40.00 50.00 60.00
NaOH Given (mL)

Figure 4
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Standard Deviation 2 of Unknown Amino Acid


3.00
2.50
2.00
1.50
Slope

1.00
0.50
0.00
-0.50 0.00 10.00 20.00 30.00 40.00 50.00 60.00
NaOH Given (mL)

Figure 5

Titration 3 - NaOH: 0.1006 M Unknown Amino Acid: 12.4138 g/2L Analyte vol: 25.00 mL

NaOH 19.20 4.96 29.20 7.29


Given 19.30 5.03 29.30 7.32
(mL) pH 19.41 5.10 29.40 7.41
0.00 1.45 19.50 5.15 29.50 7.45
18.00 3.25 23.00 6.02 29.60 7.51
18.10 3.30 23.50 6.10 29.70 7.60
18.20 3.42 24.00 6.18 29.80 7.65
18.29 3.52 24.50 6.26 29.90 7.72
18.41 3.67 25.03 6.36 30.00 7.82
18.50 3.82 25.51 6.48 30.10 7.87
18.60 4.11 25.96 6.59 30.20 7.94
18.70 4.32 26.10 6.53 30.30 8.01
18.80 4.53 26.50 6.60 30.40 8.05
18.90 4.68 27.00 6.70 30.50 8.08
19.00 4.78 28.02 6.96 31.00 8.28
19.10 4.90 29.08 7.25 32.00 8.60
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Titration 3 of Unknown Amino Acid


10.00

8.00

6.00
pH
4.00

2.00

0.00
0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00
NaOH Given (mL)

Figure 6

Standard Deviation 3 of Unknown Amino Acid


4

2
Slope

0
0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00
-1
NaOH Given (mL)

Figure 7
Calculations:
Equivalence point - moles of titrant = moles of analyte

H3A+2 + NaOH -> H2A+ + H2O + Na+

H2A+ + NaOH -> HA + H2O + Na+

HA + NaOH -> A- + H2O + Na+

Because NaOH is a strong base, it will react fully.

Per standard deviation of titration #1, 1st equivalence point is between 19.49 and 19.61 mL of NaOH given.

1st equivalence point = (19.49 + 19.61)/2

= 19.55 mL NaOH

0.1006 M NaOH * 19.55 mL * (1 L/1000 mL) = 0.001967 mol NaOH.


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Analyte (UK): 12.4138 g/2L = 6.2069 g/L

6.2069 g/L * 25.00 mL * (1 L/1000mL) * 1 mol NaOH/1 mol UK * 1/(0.001967 mol NaOH) = 78.89 g/mol UK

At ½ equivalence point, pH = pKa.

½ 1st equivalence point = 19.55 mL NaOH/2 = 9.775 mL NaOH

Per data, at 9.71 mL NaOH, pH = 1.89 – 1st pKa

Per standard deviation of titration #2 (used due to suspected error in titration #1 at this point), 2nd
equivalence point is 29.70 mL of NaOH given.

0.1006 M NaOH * 29.70 mL * (1 L/1000 mL) = 0.002988 mol NaOH.

Analyte (UK): 12.4138 g/2L = 6.2069 g/L

6.2069 g/L * 25.00 mL * (1 L/1000mL) * 2 mol NaOH/1 mol UK * 1/(0.002988 mol NaOH) = 103.86 g/mol UK

At ½ equivalence point, pH = pKa.

½ 2nd equivalence point = (29.70 mL +19.55 mL) /2 = 24.625 mL NaOH

Per data, at 24.625 mL NaOH, pH = 6.26 – 2nd pKa

Per standard deviation of titration #, 3rd equivalence point is between 40.11 and 40.24 mL of NaOH given.

0.1006 M NaOH * (40.11 + 40.24)/2 * (1 L/1000 mL) = 0.004042 mol NaOH.

Analyte (UK): 12.4138 g/2L = 6.2069 g/L

6.2069 g/L * 25.00 mL * (1 L/1000mL) * 3 mol NaOH/1 mol UK * 1/(0.004042 mol NaOH) = 115.17 g/mol UK

At ½ equivalence point, pH = pKa.

½ 3rd equivalence point = (29.70 mL + 40.175 mL) /2 = 34.9375 mL NaOH

Per data, at 35.00 mL NaOH, pH = 9.35 – 3rd pKa

Discussion:
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Based on the three pKas calculated, the amino acid is histidine, which has pK1 of 1.82, pK2 of 9.17, and pKR of

6.00. This supports the hypothesis that the amino acid would be a polyprotic acid with an ionizable R group,

generating 3 pKas. Based on the known pKas of the amino acid histidine, the percent difference is as follows:

Known Value Experimental Value Percent Difference

1.82 1.89 (1.89-1.82)/1.89 = 3.7%

6.00 6.26 (6.00-6.26)/6.00 = -4.3%

9.35 9.17 (9.35-9.17)/9.35 = 1.9%

As shown above, the experimental values were similar to the known values for the pKa, with less than 5%

difference between each set of values. The molar mass, however, was very dissimilar from the known mass for

histidine, as shown below:

Known Value4 Experimental Value Percent Difference

155.2 g/mol 78.89 g/mol (155.2-78.89)/155.2 = 49.2%

155.2 g/mol 103.86 g/mol (155.2-103.86)/155.2 = 33.1%

155.2 g/mol 115.17 g/mol (155.2-115.17)/155.2 = 25.8%

These discrepancies suggest that either the amount of amino acid in the solution was inaccurate.

Sources of error were also found in the form of discrepancies in titration data. In the first titration, a drop of

sodium hydroxide was wasted and not added to the solution. As this represents less than 0.10-mL of sodium

hydroxide, this does not represent a significant source of error. Additionally, the pH meter was not

recalibrated at the proper point during the first titration, resulting in a pH correction of 0.02, also not a

significant difference. Finally, the analyte solution sat for approximately 25 minutes partway through the first
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titration and the pH decreased by 0.19, possibly a more significant source of error, although based on the

titration curve and the relationship of the experimental pKas to known pKas, it does not seem to have caused

a significant discrepancy, although when looking at the calculated molar weight, these sources of error seem

more significant.

Conclusion:

The experimental pKas of the unknown amino acid were 1.89, 6.26, and 9.17. Based on this information, the

identity of the unknown was found to be histidine. The molecular weight of the unknown was found to be

78.89 g/mol, 103.86 g/mol, and 115.17 g/mol based on the three equivalence points of the titration curve; this

is significantly dissimilar from the known molar mass of histidine at 155.2 g/mol. Based on this information,

the lab could possibly be improved through specifically measuring the mass per liter of amino acid rather than

using a stock solution.


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References:

1. Lim, K. A tabular approach to titration calculations. Teaching Science. 2012, 58(3), 33-39.

2. Yahya Nural, H. Ali Döndaş, Hayati Sarı, Hasan Atabey, Samet Belveren, & Müge Gemili. (2014).

Determination of Acid Dissociation Constants (pKa) of Bicyclic Thiohydantoin-Pyrrolidine Compounds in

20% Ethanol-Water Hydroorganic Solvent. International Journal of Analytical Chemistry, 01 January

2014, Vol.2014.

3. Cox, M.; Nelson, D. Lehninger Principles of Biochemistry, 6th ed.; W. H. Freeman and Company: New

York, 2013; pp 76-79.

4. ThermoFisher Reference Tools – Proteins and Amino Acids.

https://www.thermofisher.com/us/en/home/references/ambion-tech-support/rna-tools-and-

calculators/proteins-and-amino-acids.html (accessed 10/16/17).