PROJECT FINAL REPORT COVER PAGE
GROUP NUMBER_____W6_____
PROJEC TITLE Determining the Effect of Nonpolar Side Chain Length on H C of Amino Acids
DATE SUBMITTED__May 11, 2000__
ROLE ASSIGNMENTS
ROLE
GROUP MEMBER
FACILITATOR………………………..PRASHANTH JAYARAM
TIME & TASK KEEPER………………CHUNGPENG SHEN
SCRIBE………………………………..TONY YEUNG
PRESENTER………………………….GREG MILLER
SUMMARY OF PROJECT CONCLUSIONS
The relationship between nonpolar side chain length of amino acids and their heats of
combustion was determined by combusting glycine, valine, and leucine in the Parr1341 Oxygen
Bomb Calorimeter. The heats of combustion of the aforementioned amino acids were found to be
233.21kcal/mol  0.93%CL, 684.25 kcal/mol  1.59%CL, and 846.57 kcal/mol  1.56%CL,
respectively. Their percentage standard deviations were found to be 0.75%, 1.28%, and
1.36%, respectively. The heats of combustion of glycine, valine, and leucine only deviated from
their respective literature values by 0.17%, -1.64%, and -0.78%. The relationship between the
side chain and the heats of combustion of nonpolar amino acids was determined to be linear. It
was found that the HC of nonpolar amino acid with linear side chain composed of only CH,
methyl and methylene groups is equal to 231.83 kcal/mol  3.75% CL plus the product of 152.65
kcal/mol 1.98% CL and total number of CH, methyl and methylene group(s).
The
aforementioned relationship was very consistent with the slope and the intercept of the line from
literature values with % deviation of -0.262% and -1.47% respectively. Furthermore, the 95%
confidence limits were  3.75% and  1.98%; indicating a high level of confidence of the
experimental values of the slope and intercept. However, the above relationship should only be
applied to nonpolar amino acids with fairly linear side chains consisting of methyl and methylene
groups. Using the experimental linear relationship between HC and the length of side chain, HC
of nonpolar amino acids with nonlinear structure, double bonds, nitrogen, and sulfur in the side
chains could depart from their literature values by as much as 19.4%.
OBJECTIVE
The objective of this experiment was to determine the effect of non-polar side chain
length on the heats of combustion of amino acids using the non-adiabatic Parr-Bomb
Calorimeter.
To achieve this objective, the following tasks were completed: benzoic acid
standardization of the bomb calorimeter, selection of appropriate nonpolar amino acids,
determination of the heats of combustion of amino acids, the precision of the experimental
values and their deviations with respect to literature values, construction of a plot of heat of
combustion values versus side chain length of amino acids, and determination of the correlation
from this plot.
The specific aims of our project were as follows:
1. To determine 3 amino acids to be tested based on side chain polarity, spread of heat
combustion literature values, availability, and ease of pellet formation.
2. To determine heats of combustion of selected amino acids to a precision of 2%.
(Precision derived from experimental calibration data.)
3. To determine the correlation between the amino acid side chain length and the
empirically determined heats of combustion.
Our hypothesis was that the heat of combustion of a non-polar amino acid is directly
proportional to the length of its side chain.
BACKGROUND
NIST Web Book: Literature values of the heats of combustion of non-polar amino acids were
obtained from this source. The heats of combustion for glycine, valine, and leucine were found
to be 232.8 + 0.1 kcal/mol, 695.67+ 0.45 kcal/mol, and 853.258+ 0.459 kcal/mol respectively.
Parr Bomb Operating Instructions: The specific protocol to be followed when the non-adiabatic
bomb calorimetry was performed was obtained from this source. The preliminary calculations
with respect to benzoic acid bomb standardization were also obtained from this manual.
Biochemistry, Pg. 756-792, Voet and Voet: An understanding of amino acids, sufficient to
perform the experiment, was obtained from this source.
Primary, secondary, and tertiary
structures of the amino acids were studied in relation to the amino acids specific role(s).
Generally speaking, polar amino acids are located on the exterior of proteins whereas non-polar
amino acids are located in the interior. This positioning is employed so as to facilitate more
efficient packing of the amino acids within a protein.
THEORY
In order to determine the effect of nonpolar amino acid side chain length on the heat of
combustion, the side chain length had to be first properly defined and quantified. The side chain
of different amino acids contained various CH, methyl and methylene groups. Thus, problems
arose because each side chain group could be seen as an independent variable. On the other
hand, all the side chain groups were composed of C-C and C-H bonds. Thus, a method was
devised to quantify the side chain group by the number of C-C bond and C-H bond in it.
But the difference between the C-C and C-H bonds needed to be determined. The bond
energies of C-C and C-H were determined by calculation using the heat of combustion of
methane (CH4) and ethane (C2H6). It was decided that the heat of combustion of Methane could
be divided by four to obtain the bond energy for each C-H bond. This was based on the
assumption that each of the four C-H bonds in Methane was nearly identical with the other. The
value of C-H bond was found to be 53.22 kcal/mol. This value was also used as the C-H bond
energy in side chain of other amino acids. This assumption was based on the fact that the side
chains of the different non-polar amino acids in this experiment had similar structure, for instant,
linear or branched carbon chain. Then, the C-C bond energy was calculated by using this C-H
value and the heat of combustion of ethane. Since ethane has a total of six C-H bonds and one
C-C bond, its heat of combustion was first subtracted by the bond energy of six C-H bonds. The
remaining value in the heat of combustion was assumed to be the bond energy of the C-C bond.
The bond energy of C-C was found to be 53.69 kcal/mol.
From the two calculations, it was observed that the bond energy of C-H was very close to
the bond energy of C-C. Thus, C-C bonds and C-H bonds were not differentiated in this
experiment.
This similarity became very useful because the C-C bonds could be used to
compensate for the differences in C-H bonds between CH, methyl and methylene groups.
From the structures of the nonpolar amino acids, it was seen that each CH group was
always accompanied by two other C-C bonds in the side chain. It was proposed that these two
C-C bonds could be seen as two C-H bonds because it was found that the difference between a
C-C and a C-H bond is minimal. Thus, a CH group could be seen as a methyl group with the
additional C-C bonds. For methylene, it was also found that there was always another C-C bond
which could be used to compensate its difference of one C-H bond with the methyl group.
Therefore, the differences between CH, methylene and methyl group were compensated
by the C-C bonds in the side chain and the side chain length was then defined by the total
number of CH, methylene and methyl groups.
APPARATUS AND EQUIPMENT
Main equipment included the Parr 1341 Oxygen Bomb Calorimeter and the Parr 1108
Oxygen Combustion Bomb. The amino acid pellets were all made from research grade amino
acids. (please refer to Parr Operating Instructions for further details)
METHODS
The measurements of the heat of combustion were performed using the method of bomb
calorimetry. The amino acid pellets were burned inside a 25 atm oxygen environment to ensure
complete combustion. The bomb was first calibrated to obtain the bomb constant, W, before the
main experimentation. (please refer to Parr Operating Instructions for background information)
A total of five trials for each amino acid were performed. Further trials did not appear to
be necessary because all results sufficiently satisfied the required precision of two percent.
CALCULATION AND RESULTS
The Calorimeter was standardized using standard benzoic acid pellet samples in order to
determine the energy equivalent or effective heat capacity for the system. The energy equivalent
(W) is computed by substituting in to the following equation:
W
H m  e1  e3
t
(1)
where H is the heat of combustion of the standard benzoic acid sample in calories per gram (H =
6317.6 cal/g) 1, m is the mass of the standard benzoic acid sample in grams, e1 is the correction
for heat of formation of nitric acid in calories, and e3 is the correction for heat of combustion of
the firing wire in calories. The mean value of the energy equivalent of the calorimeter can be
found in the table below.
W (cal/C)
 Uncertainty
(cal/C)
Trial 1
Trial 2
Trial 3
2478.10
2493.97
2522.76
39.22
36.69
28.35
Mean
(cal/C)
2498.28
% STDEV
T
0.906
4.30
95% Confidence
Limit (%)
2.25
Table 1: Standardization of the Parr 1341 Oxygen Bomb Calorimeter
Temperature rise curves (temperature versus time) were plotted for all amino acid trials.
A sample temperature rise curve for Valine trial can be found in Figure 1 below. From the
temperature vs. time graphs the net corrected temperature rise, t, was calculated by substituting
into the following equation:
t  tc  ta  r1 (b  a )  r2 (c  b)
(2)
where tc is the temp at which rate is constant (after ignition), ta is the temp at ignition, r1 is the
rate temp rise in 5 min preignition period, r2 is the rate of temp rise during 5 min period after rate
has become constant, a is the time of ignition, b is the time when temp equals 60% of total
temperature rise, c is the time when rate has become constant (after temperature rise). Points a,
b, and c are represented in Figure 1 below with arrows. The left arrow corresponds with point a,
the middle arrow corresponds with point b and the rightmost arrow, point c.
Temperature Rise vs Time
Temperature (degrees C)
25.50
25.00
24.50
24.00
23.50
23.00
22.50
22.00
21.50
0
2
4
6
8
10
Time (min)
12
14
16
18
.
Figure 1: Temperature Rise Curve for Valine Amino Acid Trial 1. Arrows indicate temperature
measurements used in calculating the net corrected temperature rise for the Heat of Combustion
described above.
The heat of combustion, HC, for each Amino Acid trial was calculated using the
following equation:
HC 
t W  e1  e2  e3
m
(3)
where t, the net corrected temperature, has just been discussed. The variable W is the energy
equivalent of the calorimeter previously determined during standardization, and m is the mass in
grams of the sample pellet. The correction factor e1 is the correction in calories for heat of
formation of nitric acid (HNO3) Since 0.0709N alkali was used for the tiration:
e1 = c1
(4)
The correction factor e2 is the correction in calories for heat of formation of sulfuric acid
(H2SO4). Again since pure samples were used, e2 = 0. The correction factor e3, in calories, is the
heat of combutsion of the fuse wire was calculated using the equation:
e3 = (2.3)*(c3)
(5)
The values from the Heat of Combustion calculations and their uncertainties for all five
of the Valine Amino Acid trials can be found below in Table 2. The Heat of Combustion
calculations and values for the remaining two amino acids can be found in the Appendix. In
addition to the above mentioned variables found in Table 2, the variable c1 is the amount in
milliliters of standard alkali solution used in the acid titration; c2 is the percentage of sulpher in
the sample (we have used pure samples, therfore c2 = 0.0); c3 is the length if fused wire
consumed during firing in centimeters.
Trial 1
Trial 2
Trial 3
Trial 4
Trial 5
a (min) b (min) C (min)
ta (oC)
tc (oC)
+ .08
7.08
7.00
7.00
7.00
7.00
+ .005
22.120
22.260
26.060
21.250
25.950
+ .005
24.976
25.130
28.750
24.210
28.500
6.00
6.00
6.00
6.00
6.00
11.58
11.83
11.83
11.08
11.83
r1
r2
(oC/min) (oC/min)
+ .005
+ .005
0.0098
0.0018
0.0039 -0.0010
0.00
-0.0060
0.00
0.0020
-0.0086
0.00
c1
(mL)
+.005
23.15
22.90
21.55
24.60
21.70
c2
c3
(%) (cm)
+ .05
0.0 9.8
0.0 6.4
0.0 4.9
0.0 5.6
0.0 6.1
Table 2: Calculating the Heat of Combustion for Valine Amino Acid
W (cal/oC)
Trial 1
Trial 2
Trial 3
Trial 4
Trial 5
2498
2498
2498
2498
2498
t (oC)
e1 (cal)
+5.0*104 + .02
1.2180
2.84
1.2212
2.87
1.1426
2.72
1.2405
2.95
1.1051
2.56
+ .005
23.15
22.90
21.55
24.60
21.70
m (g)
e2 (cal)
e3 (cal)
0.0
0.0
0.0
0.0
0.0
+ .05
22.5
14.7
11.3
12.9
14.0
HC
HC
(kcal/mol) Uncertainty
+ (kcal/mol)
677.31
+ 13.01
684.36
+ 13.90
693.02
+ 15.05
692.82
+ 14.26
673.75
+ 14.34
Table 2: Calculating the Heat of Combustion (continued)
The Heat of Combustion for all three Amino Acids, Glycine, Valine, and Leucine were
calculated using above Equation (3), and the results of which are summarized in Table 3 below.
The experimental heat of combustion values for all three amino acids were also compared to
known literature values.
Mean HC
(kcal/mol)
Literature HC
(kcal/mol)
% Std
Dev
95% Confidence
Interval
% Deviation
Glycine
233.21
232.8
+ 0.75
+ 2.17
0.17
Valine
684.25
695.67
+ 1.28
+ 10.91
-1.64
Leucine
846.57
853.26
+ 1.36
+ 13.22
-0.78
Table 3: Accuracy, Precision and Confidence Limit Values for Heats of Combustion of Glycine,
Valine, and Leucine Amino Acids.
The values of Mean HC (AVERAGE), Standard Deviation (STDEV), % Standard
Deviation, and t (probability points of distribution, TINV) were calculated using Microsoft
Excel. The 95% Confidence Interval, or interval around the mean, was defined by the relation:
Χ + (t*s) / n
(6)
where X is the value of the mean, t is the probability points of distribution, s is the standard
deviation and n is the degrees of freedom. The Accuracy (% Deviation) was calculated using the
equation:
% Deviation = (Exp. – Lit.) / Lit.*100
(7)
where Lit. is the theoretical value from literature, and Exp. is the experimental value determined
in the laboratory.
ANALYSIS
In the plot (Figure 2) of the heats of combustion of nonpolar amino acids versus number
of CH, methylene (CH2) and methyl (CH3) groups, a direct linear relationship was seen between
the two variables. The linear relationship suggested each additional group on the side chain adds
a discrete amount of heat to the heat of combustion of the nonpolar amino acid. From the best
line fit of the plot, the equation of heat of combustion of nonpolar amino acid was found to be:
HC = 231.83 kcal/mol  3.75% CL + 152.65 kcal/mol 1.98% CL x [Total Number of CH,
Methyl and Methylene group(s)]
(8)
Each CH, methyl or methylene group on the side chain of a nonpolar amino acid was found to
increase the heat of combustion by 152.65 kcal/mol  1.98% CL. Furthermore, for a nonpolar
amino acid without the side chain groups, the heat of combustion was found to be 231.83
kcal/mol  3.75% CL, which was approx. equivalent to the heat of glycine. The average heat of
combustion of glycine was found to be 233.21 kcal/mol  0.67% STD. Thus, the heat of
combustion of a nonpolar amino acid with linear side chain could be seen as the sum of the heat
of combustion of glycine and the additional CH, methyl and methylene groups.
Heats of Combustion of Nonpolar Amino Acid v. Carbon Length**
1000
Experiment
y = 152.65x + 231.83
R2 = 0.9989
900
Heats of Combustion (kcal/mol)
800
700
600
Literature
y = 154.92x + 232.44
R2 = 1
500
400
300
200
100
0
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
Carbon Chain Length**
**Total Number of Methylene & Methyl Groups on Side Chain
Literature values
Experiment Values
Linear (Literature values)
Linear (Experiment Values)
Figure 2: Heats of Combustion of Nonpolar Amino Acid v. Side Chain Length (total number of
CH, methylene and methyl groups)
Each discrete amount of heat release from the combustion, 152.65 kcal/mol 1.98% CL,
represented the amount of heat needed to break the carbon-to-carbon single bond as well as the
carbon-to-hydrogen single bonds of methyl or methylene group. According to THEORY, each
methyl group corresponded to an additional 159.66 kcal/mol while each methylene group added
160.13 kcal/mol to the heats of combustion. The experimental value of each discrete amount of
heat was found to be highly consistent with the predicted values. It only deviated -4.39% and 4.67% from the predicted values of methyl and methylene, respectively. Furthermore, since the
heats of methyl and methylene groups were nearly equal, the apparatus was not able to
differentiate between them.
For linear side chains composed of only CH, methylene and methyl groups, the
experimental heats of combustion of the nonpolar amino acid was found to be extremely
consistent with literature values. The experimental values of the intercept and the slope from the
plot (HC v. Total Number of CH, methylene and methyl groups) only deviated from those of the
literature by -0.262% and -1.47% respectively. Furthermore, the 95% confidence limits were 
3.75% and  1.98%; indicating the high level of confidence in the experimental results.
Also, the heats of combustion of the nonpolar amino acids, glycine, valine, and leucine,
were characterized by high consistency with the literature values and precision. The percentage
deviation of glycine, valine, and leucine’s heats of combustion were +0.17%, -1.64%, and 0.78%, respectively. The percentage standard deviations of the average heats of combustion of
the aforementioned amino acids were 0.75%, 1.28%, and 1.36% respectively. Thus, all of the
standard deviations were less than 2%.
Number of CH,
Nonpolar methyl and methylene
Amino Acid
on Side Chain
Alanine
1
Proline
3
Methionine
3
Isoleucine
4
Phenylalanine
7
Tryptophan
9
Predicted HC
(kcal/mol)
387.4
689.78
689.78
842.43
1300.38
1605.68
Literature HC
(kcal/mol)
384.48
655.45
805.52
854.48
1113
1345.2
%
Deviation
-0.754
5.20
-14.4
-1.41
16.8
19.4
Table 4: Predicted HC of Other Nonpolar Amino Acids using equation [8] and Literature HC
Values
The heats of combustion of other nonpolar amino acids were predicted using equation [8]
and listed in Table 4 above. Further analysis of the results showed that the equation only applies
to nonpolar amino acids with fairly linear side chains. Also, the side chains have to be composed
with only CH, methyl and methylene groups with single bonds. The predicted heat of
combustion of alanine was highly consistent with the literature value due to the linearity of its
side chain. For isoleucine, its heat of combustion was only slightly affected by the slight
nonlinearity of its side chain. The predicted heats of combustions’ deviations from the literature
values were found to increase when the nonlinearity, double bonds, and presence of other
elements (sulfur, nitrogen) increase. Each of these factors influenced the heat of combustion in a
nonlinear manner. For proline, the deviation of the predicted value from the literature could be
accounted by the nonlinear structure of its side chain. For phenylalanine and tryptophan, the
predicted heats of combustion greatly deviated from literature due to the presence of carbon rings
and carbon-to-carbon double bonds on their side chains. For methionine, the presence of sulfur
on the side chain caused the seen above.
CONCLUSIONS
The conclusions derived from this experiment are as follows:
1. All empirical data of the heats of combustion of the tested amino acids were within the +
2% pre-stated precision range.
2. A linear relationship between the number of side chains on the tested amino acids and
their respective heats of combustion was found. Each additional C-H and C-C bond in
the side chain added 53 kcal/mol to the heat of combustion.
REFERENCES
1. Castellan, GWPhysical Chemistry, 3rd Ed., 1983. Reading, Massachusetts, AddisonWesley Pub. Co., Sections 7.21, 7.24, 7.27.
2. Robert C. Weast and Melvin J. Astle, Handbook of Chemistry & Physics, 1983 Ed. Boca
Raton, Florida, CRC Press, Inc.
3. Operating Instructions for 1108 Oxygen Combustion Bomb (205M), Oxygen Bomb
Calorimeter (204M), Pellet Press, and Mercurial Thermometer (211M). Parr Instrument
Co., Moline, IL 61265.
4. H.Y. Afeefy, J.F. Liebman, and S.E. Stein, "Neutral Thermochemical Data" in NIST
Chemistry WebBook, NIST Standard Reference, Database Number 69, Eds. W.G.
Mallard and P.J. Linstrom, February 2000, National Institute of Standards and
Technology, Gaithersburg MD, 20899 (http://webbook.nist.gov).
5. Donald Voet and Judith Voet. Biochemistry, 2nd Ed., 1995, John Wiley & Sons, New
York NY 10158