BE 210 Lab #2 - Penn Engineering



BE 210 Final Project

Bomb Calorimetry of Amino Acids

Group R3

Luis Otoya

Jeannette Ouyang

Nick Rose

Jennifer Russert

Edwin Tan

Table of Contents

I. Abstract

II. Background

III. Materials

IV. Methods

V. Results

VI. Discussion

VII. References

VIII. Appendices

Abstract

Our main objective of this lab was to determine the heat of combustion for an amino acid that plays a significant role in the biological system. Both Leucine and Valine were combusted in a standardized bomb calorimeter with energy equivalent, W, equal to 2424.69 (12.73 cal/g. Leucine and valine were chosen because they have similar structures with the exception of a CH2 group. Standardization was done by combusting pure benzoic acid in the calorimeter. After, combustion data was taken in 30 second intervals and then analyzed along with the remaining components of the post-combustion bomb giving a heat of combustion value. This was done utilizing certain equations that include an account of possible errors within the bomb calorimetry technique (explained in the background). The average heat of combustion values of Leucine and Valine were calculated to be 6510.24 ( 34.91 cal/g and 5909.34 ( 55.86 cal/g yielding only .081% error for Leucine and .440% error for Valine. Through experimental trials, we determined that the heat of combustion of Leucine is greater than that of Valine. The fact that the heat of combustion is greater than Valine is consistent with the fact that Leucine has one extra CH2 group than Valine, thus more energy is released when the extra bonds are broken. Though the amino acids were seen to release nearly twice the amount of energy of sucrose (3897.30(33.31 cal) did when they were combusted, it was concluded that sucrose was still a more efficient source of energy within the body.

Background

Bomb Calorimetry and Parr Bomb History:

Calorimetry is the basic experimental method used in thermochemistry to measure the energy effects of a process and to study the thermal properties of substances. This method of measurement dates back to 1760 when Wilcke began to play with the idea of latent heat, the mixing calorimeter, and the ice calorimetry principle. Eventually, further developments began in calorimetry with improvements in the determination of specific heat of capacity. This was seen in electric heating in the flow calorimeter in the 1870’s, the bomb calorimeter in 1881, and the aneroid calorimeter in 1910(3). Experimental procedures were done in attempt to minimize the error in the specific heat calculation and maximize the efficiency the calorimetry. Such experiments included those done by Eucken and Nernst in 1909 when they measured the specific heat capacities at low temperatures, and by Klinkhardt in 1926 when he measured the specific heats with a contact-less energy supply (3).

With the time bomb calorimetry, there came forth to be a far more versatile and more effective method than any other in calorimetric research. Further development came from scientists such as Luginin, Maler, and Hempel. Today bomb calorimetry is one of the most widely used and effective methods of physiochemical research and has lead to more than seven thousand reliable heat of combustion values for organic compounds. The calorimetric system that is utilized in the following laboratory procedures is a plain bomb calorimeter that measures and interprets the amount of heat which a gram of an amino acid takes to change the temperature of its surroundings by one degree, or in other words the specific heat of capacity of the amino acid.

The first calorimetric bomb, which is the central part of the calorimeter where the reaction takes place and where the energy is released, was invented by Berthelot in the year 1881. This bomb component is what distinguishes the bomb calorimeter from other forms of calorimeters as well as what gives it its name. Berthelots’ bomb was a hermetically sealed sleeve plated with platinum on the exterior and gold on its interior (5). Today’s calorimeter bombs have come a long way from Berthelots original. The composition of modern bombs have changed with stainless steel replacing the gold and platinum. The bomb used in the following experiments is composed of a special columbium-stabilized stainless steel (6). Improvements have also been made with the additions of such features as self-sealing covers, installed electrodes, and rotating bombs.

Bomb Calorimetry:

The standard heat of combustion of a compound is a precisely defined thermodynamic quantity, representing the energy released by burning it completely in oxygen at 298K and 1 atm. In this process, carbon, hydrogen, and nitrogen atoms in any compound are assumed to be converted to CO2(g), H2O(l), and N2(g). Combustion is the process of burning, a chemical change (oxidation) which is accompanied by the production of heat, and sometimes light. The heat of combustion of a substance is the amount of heat energy that it takes to combust one mole of the substance in as much oxygen as is necessary for the reaction to take place. The different heats of combustion for organic compounds are listed in many chemical reference books, like the following which was taken from the Handbook for Chemistry and Physics 73rd Ed.

Name O.C. Heat of Com. (cal/g)

Benzoic Acid 2426.0

Leucine 6504.9

DL-Leucine 6516.9

Sucrose 3942.1

Valine 5935.4

The heat of combustion has a biological significance in that the living system receives its energy from the combustion of organic substances that exist within that system. For an example, the human body functions through chemical changes. The body stores proteins and organic compounds in its cells throughout the body and breaks down these compounds in order to get ATP. When ATP is broken down, combusted, there is a release of energy from the breaking of its bonds. This is the energy that is used in everyday functions such as reading and exercising. The knowledge of exactly how much heat it takes to release this energy aids in understanding how systems that depend on chemical changes operate.

Calculations and Equations:

The equipment that is used to calculate such figures as the ones listed in the table above is rather technical as well as delicate. A calibration is required to account for the absorption of heat by the bomb itself.. This constant can be found through a procedure utilizing benzoic acid, which is a very pure substance and is easily combusted in the calorimeter. Throughout the duration of the individual experiments the bomb calorimeter was calibrated with benzoic acid to ensure its constancy between trials. Through the combustion of benzoic acid we can determine the energy equivalence of the calorimeter to be used in subsequent trails for in the calculating the heat of combustion of an organic compound. The equation that was used to arrive at this value from benzoic acid combustion is as follows:

W = H*m +e1 +e2 (EQN1)

t

t = net corrected temperature rise (see below)

H= heat of combustion for Benzoic acid (6318 calories/gram)

m = mass of benzoic acid sample

e1 = corrections in calories for heat of formation of nitric acid

e2 = correction in calories for heat of combustion of fuse wire

There are several important components that need to be taken into account when a substance is combusted within the oxygenated chamber. They are the formation of nitric acid, sulfuric acid, and the combustion of the fuse wire. When sulfur is burned in an oxygen bomb it reacts to form sulfur trioxide which in turn reacts with the moisture present in the bomb to become sulfuric acid. As for nitrogen, which is present in the bomb because it is a major component of air, it is oxidized and then combines with water vapors to produce nitric acid. Additionally, the combustion process uses up some of the fuse wire. This requires a certain amount of energy that needs to be accounted for in calculating the final values.

The value for e1 (formation of Nitric Acid) obtained by titrating the washings with .0709 N Alkali solution. The exact measurement in milliliter is the value used for e1. The value for e2 (combustion of the fuse wire) is obtained by multiplying the length of the fuse wire combusted by the number 2.3 (this is only applicable if the fuse wire was 45C10 Nickel Chromium Fuse wire provided by the Parr company). Finally, the value for e3 (formation of Sulfuric Acid) is obtained by multiplying the percent composition of sulfur by the number 13.7 and the mass of the pellet combusted. Since the compounds that were tested did not contain sulfur, this was not applicable.

The actual experimentation begins once energy is released by the reaction and is absorbed by the calorimeter, with data being taken as a temperature vs. time graph. In greater detail, this means once the bomb has been activated the organic compound within combusts and releases energy in the form of heat that begins to dissipate into the water surrounding the bomb. A thermometer placed in the water displays the temperature chance. Once this information is recorded, the heat of combustion, of calorific value, is calculated. The calorific value of a sample may be broadly defined as the number of heat units liberated by a unit mass of a sample when burned with oxygen in an enclosure of constant volume. The equation that is used to determine this is generally H=(Wt)/m where W, m, and t are defined below. The equation is altered to include the corrections of the sensitive calorimeter for its areas of error in order to get a more accurate value. The equation used to calculate the gross heat of combustion (Hg) is as follows:

Hg =[pic] (EQN2)

t = net corrected temperature rise (see below)

W = energy equivalent of the calorimeter - 2426 cal/(C

e1 = corrections in calories for heat of formation of nitric acid

e2 = correction in calories for heat of combustion of fuse wire

e3 = correction in calories for heat of formation of sulfuric acid

There is also corrections that need to be made when determining the value for the net temperature rise. These values are taken from a graph of time vs. temp that is plotted from the observations of temperature during the combustion. The equation used to correct the temperature rise (t) is as follows:

t = tc-ta-r1(b-a)-r2(c-b) (EQN3)

tc = temperature at time c

ta = temperature at time of firing

r1 = rate (temp/min) at which temperature was rising during 5 min period before firing

r2 = rate (temp/min) at which temperature was rising during 5 min period after time c

a = time of firing

b = time when the temperature reaches 60% of total rise

c = time at the beginning of period (after temperature rise) in which the rate of

temperature change has become constant

Biological Basis of Amino Acids in the Human Body:

The metabolic fates of amino acids can be conveniently grouped under three headings. First and most importantly, the provide the basic materials for protein synthesis in all tissues. The amount of protein synthesized daily in the body of an adult man is about 300g. Since this represents daily turnover of protein three times the intake of protein in the average diet, amino acids liberated as a result of breakdown of body protein must be extensively reutilized for synthesis of new tissue protein throughout the body. Second, amino acids provide precursors for the synthesis of numerous nitrogenous small molecules. Dietary protein provides almost all the organic nitrogen available to the body from the environment. Third, amino acids in excess of requirements undergo degradation, the nitrogen moiety eventually forming urea by a reaction sequence located exclusively in the liver. In order to reach the liver, much of this nitrogen is transported from other tissues in the form of nonessential amino acids, notably alanine and glutamine.

Amino Acids in Metabolism:

The term metabolism encompasses all the reactions that take place in the body. Everything that happens within us is part of our metabolism. The reactions of metabolism may be divided into two major categories: anabolism and catabolism. Anabolism means synthesis or “formation” reactions - the bonding together of smaller molecules to form larger ones. Catabolism means decomposition - the breaking of bonds of larger molecules to form smaller molecules. During catabolism, energy is often released and used to synthesize ATP which is then used for energy-requiring anabolic reactions.

Glucose is the main source of energy within the body. The three stages of cellular respiration - glcolysis, the Kreb’s Citric Acid Cycle, and the cytochrome (or electron) transport system - provide most of the energy for the body’s functions in the form of ATP. In the end, a total of 36 ATP molecules are produced. Although glucose is the preferred energy source for cells, proteins and fats also contain potential energy, and are alternative energy sources in certain situations. Amino acids are the subunits of proteins and the primary use for amino acids obtained from food is the synthesis of new proteins. However, excess amino acids may be used for energy production. This occurs in the liver where they are deaminated - where the amino group (NH2) is removed. The remaining portion is converted to a molecule that will fit into the Kreb’s Cycle. For example, a deaminated amino acid may be changed to a 3-carbon pyruvic acid or to a 2-carbon acetyl group. When these molecules enter the Kreb’s Cycle, the results are just the same as if they had come from glucose. The fact that excess amino acids can also be converted into glucose is important in supplying the brain especially when dietary intake of carbohydrates is low.

Biological Role of Leucine:

Leucine is an essential amino acids, one that cannot be synthesized by the body but must always be acquired from dietary sources. It is available in good concentrations in meat and dairy products, and to a lesser degree in wheat germ, brown rice, soybeans, almonds, cashews and brazil nuts, chickpeas, lentils and corn. Leucine stimulates protein synthesis in muscles, and is essential for growth. It also promotes the healing of bones, skin and muscle tissue.

Leucine, and the other carbon group (CH2) branched-chain amino acids, isoleucine and valine, are frequently deficient in the elderly, and increased body requirements can occur after trauma or surgery. These branched-chain amino acids may prevent muscle wasting in these conditions, but no studies have been done to determine if extra intake will help in muscle building in healthy individuals. Because leucine cannot be made by the body from other sources, it is important to maintain adequate amounts in the diet.

In addition, leucine, in conjunction with two other amino acids, isoleucine and valine, appear to be quite helpful in treating and in some cases even reversing hepatic encephalopathy, a form of liver damage in alcoholics. It also helps curb muscle wasting in this disease and through its actions on brain neurotransmitters, help prevent some adverse neurological effects of chronic liver disease.

A recent study shows that leucine, isoleucine and valine may be helpful in ALS, known as Lou Gehrig’s disease. This is a potentially fatal disease for which no other effective treatment has been found. A pilot study involved nine ALS patients, of whom eight benefited from supplementation with these amino acids. Also, over the one year period of the study, they retained their muscle strength and their ability to walk. In constrast, five of the nine control subjects, who received placebos, lost their ability to walk over this period.

A study reported in the British Journal of Nutrition found that a dietary excess of leucine may be a precipitating factor in causing pellagra. This effect was only apparent when the diet also provided less than adequate amounts of nicotinamide. The right handed, or D form of leucine, has been shown to have a similar effect to that of d-phenylalanine in retarding the breakdown of the natural pain killers of the body, endorphins and enkephalins.

Biological Role of Valine:

Valine is also one of the amino acids which the body cannot manufacture itself but must acquire from food sources. Valine is found in abundant quantities in most food including soy flour, raw brown rice, cottage cheese, fish, beef, lamb, chicken, almonds, brazil nuts cashews, peanuts, sesame seed, lentils, chickpeas and mushrooms. It also shows to have a stimulant effect. Healthy growth depends on it. A deficiency results in a negative hydrogen balance in the body. In addition, valine is used by bodybuilders, in conjunction with leucine and isoleucine, for muscle growth, tissue repair and as an energizer. There is little scientific evidence to support these claims, though studies have shown that these three substances might be able to help restore muscle mass in people with liver disease, injuries, or who have undergone surgery, but no studies have shown them to be effective for healthy people. Because valine cannot be produced by the body, healthy people should ensure that they are obtaining at least the recommended amount in their diet.

Materials

• Parr Instrument Model 1341 Oxygen (non-adiabatic) Bomb Calorimeter, including stirrer, precision thermometer with brackets, support rod and reading lens, calorimeter jacket with cover, and motor assembly with pulley. W=2426 cal/(C[1]

• Parr Instrument Model 1108 Oxygen Combustion Bomb

• High pressure oxygen cylinder, equipped with Parr Instrument Model 1825 filling connection for bomb.

• Parr Model 2901 Ignition Unit

• Parr Pellet Press

• Associated components for testing including sample cups, tweezers, ignition wire, stands for bomb head and calorimeter cover

• Burette with .0709 N sodium carbonate solution and methyl red indicator. Using this concentration of sodium carbonate allows correction for nitrogen equal to the mL used. To make one liter of a 0.0709 N sodium carbonate solution requires half as much Na2CO3 as does a 0.0709 M solution, 3.758 grams, since this compound has two acid-base equivalents. This solution was already pre-made. (6)

• Top-loading 5 kg capacity balance with a resolution of 0.1g.

• Gram-atic Balance, Fisher Scientific Co., Pittsburgh

• Sucrose, pre-dried with drierite. The sample must be dry to ensure that no absorbed water is included in the weighed mass. Drierite is a chemical desiccant that turns from blue to pink when its absorbing capacity is reached.

• Amino Acids: L-Leucine and DL-Valine[2]

Methods

Thermometer Inspection:

Before using the calorimeter, inspect the thermometer carefully for any mercury separations and/or entrapped gas bubbles. Examine the entire length with the reading lens provided. Use the inversion test by warming the bulb in the palm of the hand or immersed in warm water under 60(C until the mercury reaches the highest graduation. Then invert the thermometer and observe the action of the mercury thread and look for any visible bubbles. If there are bubbles refer to the operating instructions from Parr Instrument Co. for Mercurial Calorimetric Thermometers.

Standardizing the Calorimeter:

Before the sucrose can be tested in the bomb calorimeter, the heat capacity of the calorimeter must first be determined. This value represents the sum of the heat capacities of the components in the calorimeter - the metal bomb, the bucket and the water in the bucket. This is determined by burning a sample of standard material with a known heat of combustion, benzoic acid in this lab. Benzoic acid is used because it burns completely in oxygen, it is not hygroscopic, and it is readily available in a very pure form. Its heat of combustion can be determined to an uncertainty of about .01% (Hemminger 177). The acid is available in pure form and has a known heat of combustion of 6318cal/g. The conditions used in this standardization test must be followed strictly in all subsequent tests or error will result. In addition, this value will be used as a measurement of the consistency of the bomb from day to day.

Making the Pellets:

The Parr Pellet Press will be used to make the pellets. First, fill the die. Set the die and its holder on the base of the press with the beveled edge of the die cavity facing upward and with the bottom of the die resting on the flat surface in the reversible holder. Pour the charge into the die cavity. Second, compress the charge by transferring the die and its holder to the press and push the lever down to compress the charge. To obtain maximum compression, the lever should require a firm push as it moves through its full stroke. If a full stroke is not obtained, turn the anvil to lower the die until the full mechanical advantage of the press can be utilized. Conversely, if the lever moves through its full stroke without encountering sufficient resistance, raise the die until firm compression can be applied. Thirdly, reverse the die holder. Raise the lever and slide the die and its holder out of the press. Reverse the holder to bring the deep cavity under the die and return the parts to their original position. The clearance under the pinch will be limited when making thin pellets. In such cases it will be more convenient to grasp the die with one hand and slide it upward on the punch, holding it in the position while releasing the holder with the other hand. Finally, eject the pellet. Bring the lever down gently to eject the pellet into the cavity in the holder. If a thick pellet is not ejected by this stroke, turn the anvil to raise the die The pellet will then drop out freely. Remove the pellet with tweezers or forceps; reverse the holder and repeat the cycle if additional pellets are required. Weigh and record the sample.

Preparing the Oxygen Bomb:

First the fuse must be attached by fastening a 10 cm length of fuse wire between the two electrodes. Placement of the fuse wire is the critical step in the procedure. To attach the fuse to the electrodes, insert the ends of the wire into the eyelet at the end of each stem and push the cap downward to pinch the wire into place. Place the fuel capsule with its weighed sample in the electrode loop and bend the wire downward toward the surface of the charge. Bend the wire so that the loop bears against the top of the pellet firmly enough to keep it from sliding against the side of the capsule. It is important that the wire does not touch the sides of the crucible. It is recommended that you tilt the crucible in such a way that the pellet is held more firmly in place by the fuse wire. Care must be taken not to disturb the sample when moving the bomb head from the support stand to the bomb cylinder. Slide the head into the cylinder and push it down without twisting and leave the gas release valve open during this operation. Set the screw cap on the cylinder and turn it down firmly by hand as far as it will go. It is important that the cap be screwed on to a solid stop.

Next, fill the bomb with oxygen using the high pressure oxygen cylinder and the Model 1825 filling connection. Unscrew the protective cap from the tank and inspect the threads on the valve outlet to be sure they are clean and in good condition. Place the ball end of the connection into the outlet socket and draw up the union nut tightly with a wrench, keeping the 0-55 atm gage in an upright position. The pressure connection to the bomb is made with a slip connector on the oxygen hose which slides over the gas inlet fitting on the bomb head. Slide the connector onto the inlet valve body and push it down as far as it will go. If it does not slide easily, a drop of water spread around the inlet valve will lubricate the sealing rings. Close the valve on the filling connection. Then open the oxygen tank valve not more than one-quarter turn. Open the filling connection control valve slowly and watch the gage as the bomb pressure rises to 25 atm. Then close the control valve. The bomb inlet check valve will close automatically when the oxygen supply is shut off, leaving the bomb filled to the highest pressure indicated on the 0-55 atm gage. Release the residual pressure in the filling hose by pushing downward on the lever attached to the relief valve. The gage should now return to zero. If the pressure drops slowly and a large amount of gas escapes when the pressure relief valve is opened, the check valve in the bomb head is not operation properly.

Preparing the Calorimeter:

Fill the calorimeter bucket by first taring the dry bucket on a balance. Then add 2000 ( 0.5g distilled water. Keep the initial water temperature as close to room temperature as possible. (In working with different substances that tended to cause larger temperature increases, it was necessary to decrease the temperature of the water so that the temperature rise did not overshoot the limit of the thermometer.) If the distilled water is too warm, mix with the cold water in the carboy provided. Set the bucket in the calorimeter by attaching the lifting handle to the two holes in the side of the screw cap and lower the bomb into the water with its feet spanning the circular boss in the bottom of the bucket. Handle the bomb carefully during this operation so that the sample will not be disturbed. Remove the handle and shake any drops of water back into the bucket: then push the two ignition lead wires into the terminal sockets on the bomb head. Be careful not to remove any water from the bucket with the fingers. Set the cover on the jacket with the thermometer facing toward the front.

Turn the stirrer by hand to be sure that it runs freely. Then slip the drive belt onto the pulleys and start the motor. Let the stirrer run for 5 minutes to reach equilibrium before starting a measured run. At the end of this period record the time or start a timer and read the temperature to one-tenth of the smallest scale division. Read and record temperatures at one minute intervals for 5 minutes. Then at the start of the 6th minute, stand back from the calorimeter and fire the bomb by pressing the ignition button and holding it down until the indicator light goes out. Normally the light will glow for only about a ½ second but release the button within 5 seconds regardless of the light. Do not have head, hands or any parts of the body over the calorimeter when firing the bomb and continue to stand clear for 30 seconds after firing.

The bucket temperature will start to rise within 20 seconds after firing. This rise will be rapid during the first few minutes then it will become slower as the temperature approaches a stable maximum. Measure the time required to reach 60% of the total rise by estimating the temperature at the 60% point and observing the time when the rising mercury thread reaches that level. After the rapid rise period, 4-5 minutes after ignition, adjust the reading lens and record temperatures to one-tenth of the smallest scale division at thirty second intervals until the difference between successive readings has been constant for five minutes.

After the last temperature reading, stop the motor, remove the belt and lift the cover from the calorimeter. Wipe the thermometer bulb and stirrer with a clean cloth and set the cover on the A37A support stand. Lift the bomb out of the bucket. Remove the ignition leads and wipe the bomb with a clean towel. Open the knurled knob on the bomb head to release the gas pressure before attempting to remove the cap. This release should proceed slowly over a period of not less than one minute to avoid losses. After all pressure has been release, unscrew the cap. Lift the head out of the cylinder and place it on the support stand. Examine the interior of the bomb for soot or other evidence of incomplete combustion. If such evidence is found, the test will have to be discarded. Wash all interior surfaces of the bomb with a jet of DI water and collect the washings in a beaker. Remove all unburned pieces of fuse wire from the bomb electrodes and straighten them and measure their combined length in cm. Subtract this length from the initial length of 10 cm and record value. Titrate the bomb washings with a standard sodium carbonate solution using methyl red as an indicator.

Results

Standardization of the Bomb Calorimeter:

In working with a bomb calorimeter, it is important to determine the energy equivalent of the calorimeter, otherwise known as W. This process involves the combustion of benzoic acid in the calorimeter and then observing the temperature rise that occurs. The initial step in determining the constant involves creating a Temperature vs. Time graph. The graph labeled Graph 1 is the graph for the first trial of benzoic acid.

Graph 1

[pic]

In order to determine the actual temperature rise as a result of the combustion, several sources of error are considered. The graph actually contains error bars of the magnitude (0.01. The error bars are not visible because they are extremely small. The Parr Instrument Company provides a correction table for use with its thermometers. After reading the temperature off the thermometer, the corrections are applied before the graph is plotted. In determining the corrected temperature rise, the information in Table 1 is determined from the graph above:

Table 1

|Data to Read Off Graph |Data for Trial #1 of Benzoic Acid |

|Time of Firing the Bomb |5 minutes |

|Time to Reach 60 % of Total Temperature Rise |6.45 minutes |

|Initial Time When the Temperature Change Becomes Constant |12.5 minutes |

|Temperature at the Time of Firing |Degrees Celsius |

|Temperature when the (T Becomes Constant |26.906 Degrees Celsius |

|Rate at Which the Temperature Changes Before Firing |.006 Degrees/Minute |

|Rate at which the Temperature Changes After the (T Becomes Constant |0 Degrees/ Minute |

A great deal of the information in the table can be read directly off the graph or raw data table. The bomb is always fired on the fifth minute and the temperature at the time of firing is recorded. Prior to firing the bomb, the temperature is carefully measured every minute for five minutes. The stirring mechanism aids in the equilibration of the water temperature. The rate of change of the temperature is determined using a linear regression of the first five minutes of the Temperature vs. Time graph after firing the bomb. Graph 2 actually shows the regression and the number that is obtained for the rate of change. After a while the temperature change becomes constant, and this can be observed on the data table. After this temperature is reached, temperature measurements are still taken to see if any temperature change occurs after the equilibration. The rate of change after the temperature change becomes constant is determined by applying a linear regression in the same way that it was applied to determine the rate of change before firing. Graph 3 shows the rate of change after the temperature becomes constant.

Graph 2

[pic]

Graph 3

[pic]

* There is no need to apply a linear regression because the line is horizontal and therefore the rate of change or slope is zero.

At this point, the information in Table 1 is used to determine the corrected temperature rise for trial 1 of the benzoic acid. Determining this temperature rise involves using the Equation 3 from the background.

After the corrected temperature is determined, other measurements are taken to determine an accurate value for W. The amount of fuse burned in the equation represents the dissipation of energy. Therefore, the amount of fuse left is measured and subtracted from the original amount to determine the actual amount of fuse that was burned in the combustion. This number, determined in centimeters, is multiplied by the number 2.3 ( the constant that correlates with the Parr 45C10 Nickel Chromium Fuse wire used in the combustion of all substances tested in this laboratory) to yield the correction factor for the lost energy. Additionally, nitric acid formation is a common occurrence in the combustion of substances. To account for this, the washings from the bomb are titrated with 0.0709 N alkali. The volume in milliliters determined from the titration becomes the correction factor for the nitric acid formation. Table 2 shows the other figures involved in determining the W value for the calorimeter:

Table 2

|Information Needed to Determine W |Data |

|Heat of Combustion of Standard Benzoic Acid |6318 calories/gram |

|Mass of the Benzoic Acid Pellet |1.007 ( .001 grams |

|Corrected Temperature Change |2.611 Degrees Celsius |

|Correction for the Heat of Formation of Nitric Acid |8.21 ( .01 |

|Correction for the Heat of Combustion for the Fuse Wire |12.19 ( .02 |

The values in the table are next applied to equation 1. From this equation the value for W is 2437.69 calories/gram. This is the standard procedure that was carried out for each of the benzoic acid standardization trial. Table three below indicates the values for each of the trials. All of the Temperature vs. Time graphs for all of the standardization trials are available in Appendix A.

Table 3

|Trial Number |Energy Equivalent of the Calorimeter (W) in calories/gram |

|Trial # 1 |2437.69 |

|Trial # 2 |2410.26 |

|Trial # 3 |2447.28 |

|Trial # 4 |2417.06 |

|Trial # 5 |2400.01 |

|Trial # 6 |2428.94 |

|Trial # 7 |2424.50 |

|Trial # 8 |2431.74 |

|Average |2424.69 ( 12.73 |

Combustion of L-Leucine:

The next part of the experiment involved combusting the amino acid L-Leucine. The procedure was very similar to the procedure for the combustion of the benzoic acid. The only difference was that the new number that was obtained for the energy equivalent of the calorimeter was used as the W value in Equation 1. The first thing that was analyzed was the Temperature vs. Time graph. Graph 4 is the Temperature vs. Time graph for the third trial of the L-Leucine combustion.

Graph 4

[pic]

The graph above is carefully analyzed in the same way that the Temperature vs. Time graph above for the benzoic acid was analyzed. The information in Table 4 below is used to determine the corrected temperature rise (as determined using Equation 3).

Table 4

|Data to Read Off Graph |Data for Trial #3 L-Leucine |

|Time of Firing the Bomb |5 minutes |

|Time to Reach 60 % of Total Temperature Rise |6.2 minutes |

|Initial Time When the Temperature Change Becomes Constant |13.0 minutes |

|Temperature at the Time of Firing |24.981 Degrees Celsius |

|Temperature when the (T Becomes Constant |27.505 Degrees Celsius |

|Rate at Which the Temperature Changes Before Firing |.0063 Degrees/Minute |

|Rate at which the Temperature Changes After the (T Becomes Constant |.005 Degrees/ Minute |

The value for the rate of change for the temperature before the firing of the bomb is determined by applying a linear regression to the initial portion of the Temperature vs. time graph. This analysis is shown below in Graph 5. Similarly, the rate of change of the temperature after the temperature change levels off is done by applying a linear regression to the final portion of the Temperature vs. Time graph. This analysis is shown below in Graph 6. The graph actually shows that a gradual temperature rise did occur after the temperature equalized. This is possible because following the leveling off of the temperature, the water is stirred by the motor that is attached to the bomb bucket. It is a process similar to the process that occurs before the bomb is fired. As it works out, the temperature only chages occasionally after it levels off.

Graph 5

[pic]

Graph 6

[pic]

After determining the corrected temperature rise, the other variables in Equation 2 must be determined. The correction factors that are used in determining the corrections necessary when combusting L-Leucine are the same as the corrections necessary when combusting benzoic acid. The heat of combustion of the fuse wire and the formation of nitric acid are accounted for below in Table 5. The table also includes the rest of the necessary information needed to compute the heat of combustion for L-Leucine. It should be noted that when using the bomb calorimeter, it is usually necessary to correct for the formation of sulfuric acid. However, both benzoic acid and L-Leucine are sulfur-free in their chemical composition and the percentage of sulfur in the substances is zero (0%).

Table 5

|Information Necessary to Determine the Heat of Combustion for L-Leucine |Data |

|Energy Equivalent of the Calorimeter (W) |2424.69 ( 12.73 calories/gram |

|Mass of the Benzoic Acid Pellet |.920 ( .001 grams |

|Corrected Temperature Change |2.482 Degrees Celsius |

|Correction for the Heat of Formation of Nitric Acid |16.32 ( .01 |

|Correction for the Heat of Combustion for the Fuse Wire |17.25 ( .02 |

Equation 2 from the background is actually used in determining the heat of combustion for the a substance that is tested in the bomb calorimeter. The actual Heats of Combustion for L-Leucine samples that were tested are listed in Table 6 below. These (H values were computed using Equation 2.

Table 6

|Trial Number |Heat of Combustion for L-Leucine ((H) in calories/gram |

|Trial 1 |6517.74 |

|Trial 2 |6482.45 |

|Trail 3 |6506.06 |

|Trial 4 |6534.69 |

|Average |6510.23 ( 34.91 |

Combustion of DL-Valine:

The final part of the experiment involves the combustion of DL-Valine. The combustion was carried out in the exact same way as the combustion for L-Leucine. The analytical procedure for the two amino acids was also the exact same. The combustion process led to a temperature increase that is represented in the Temperature vs. Time graph below (Graph 7). This is the data for trial 4.

Graph 7

[pic]

The graph above is used to obtain data for the corrected temperature rise. Table 7 below contains the data that is used in equation 3 to solve for t.

Table 7

|Data to Read Off Graph |Data for Trial #4 for DL-Valine |

|Time of Firing the Bomb |5 minutes |

|Time to Reach 60 % of Total Temperature Rise |6.1 minutes |

|Initial Time When the Temperature Change Becomes Constant |11.5 minutes |

|Temperature at the Time of Firing |25.040 Degrees Celsius |

|Temperature when the (T Becomes Constant |27.520 Degrees Celsius |

|Rate at Which the Temperature Changes Before Firing |.0049 Degrees/Minute |

|Rate at which the Temperature Changes After the (T Becomes Constant |0 Degrees/ Minute |

The values for the rate at which the temperatures change are found by applying linear regressions to the initial and final portions of the Temperature vs. Time graph. Graph 8 is the regression for the rate of change of the temperature before firing. Graph 9 is the regression for the rate of change of temperature after the temperature levels off.

Graph 8

[pic]

Graph 9

[pic]

The additional information that is needed to compute the heat of combustion for DL-Valine is listed below in Table 8. The correction variables that are used are the same as the variables used for the combustion of benzoic acid and L-Leucine. Once all of the data needed is accumulated, the numbers are plugged into Equation 2 from the background.

Table 8

|Information Necessary to Determine the Heat of Combustion for DL- |Data |

|Valine | |

|Energy Equivalent of the Calorimeter (W) |2424.69 ( 12.73 calories/gram |

|Mass of the Benzoic Acid Pellet |1.006 ( .001 grams |

|Corrected Temperature Change |2.474 Degrees Celsius |

|Correction for the Heat of Formation of Nitric Acid |17.00 ( .01 |

|Correction for the Heat of Combustion for the Fuse Wire |16.33 ( .02 |

Finally, Table 9 shows the values for the Heat of Combustion for DL-Valine.

Table 9

|Trial Number |Heat of Combustion for DL-Valine ((H) in calories/gram |

|Trial 1 |5943.59 |

|Trial 2 |5877.74 |

|Trail 3 |5880.42 |

|Trial 4 |5935.59 |

|Average |5909.34 ( 55.86 |

Combustion of Sucrose:

Experiment 2 of the laboratory session involved the combustion of sucrose in the bomb calorimeter. The experiment was conducted two consecutive weeks, yielding a total of eight trials. The process was basically the same in determining the heat of combustion with the only difference being the value for the energy equivalent of the calorimeter. Since the experiment had to be conducted under time constraints, there was no opportunity to determine whether the given energy equivalent for the calorimeter was accurate. Upon actually testing for the coefficient with benzoic acid, it is evident that the constant was similar to, but not exactly equal to, the energy equivalent (W) value given by the Parr Instrument Co. Table 10 contains the values for the heat of combustion of sucrose.

Table 10

|Trial Number |Heat of Combustion of Sucrose in calories/gram |

|Trial # 1 |3875.48 |

|Trial # 2 |3947.17 |

|Trial # 3 |3890.18 |

|Trial # 4 |3873.45 |

|Trial # 5 |3845.29 |

|Trial # 6 |3960.97 |

|Trial # 7 |3873.45 |

|Trial # 8 |3912.02 |

|Average |3897.30 ( 33.30 |

The numbers obtained for the energy equivalent of the calorimeter, heat of combustion for L-Leucine, DL-Valine, and sucrose were accurate to within 5% error. Table 11 contains the values for the four different samples tested, the literature values, and the percent error.

Table 11

|Substance Combusted in the |Literature Value |Experimental Value |Percent Error |Standard |

|Calorimeter | | | |Deviation |

|Benzoic Acid (Used to determine the |2426 calories/ gram |2424.69 ( 12.73 calories/gram |.054 % |15.226 cal/g |

|(W) Energy Equivalent) |(W value) | | | |

|L-Leucine |6504.978 calories / gram ((H) |6510.23 ( 34.91 calories/gram |.081 % |21.939 cal/g |

| | | | | |

|DL-Valine |5935.480 calories / |5909.34 ( 55.86 calories/gram |.440 % |35.106 cal/g |

| |gram ((H) | | | |

|Sucrose |3942.100 calories / gram ((H) |3897.30 ( 33.30 calories/gram |1.13 % |39.841 cal/g |

| | | | | |

The Test for Significance

A test for significance was applied to all the numbers that were obtained experimentally to see if there was a significant variation. The results of this test were as follows:

|Substance Tested |Te value |T Value (from TINV) |Significant Difference |

|Benzoic Acid |.2433 |2.36 |no |

|L-Leucine |.4787 |3.18 |no |

|DL-Valine |1.489 |3.18 |no |

|Sucrose |3.17 |2.36 |yes |

Discussion

Discussion of Results:

To analyze the results of our experiment we used a number of programs on Excel. Our measured mean heat of combustion for sucrose was found to be 3897.30( 33.30. This ( 33.30 cal/g is the 95% confidence level of our results. All proceeding notation also represents the 95% confidence level. The standard deviation of our trials was found to be 39.841 cal/g. This magnitude may seem large but it is insignificant with regards to our mean combustion value. The accuracy of our mean value was within 1.13% of the expected value 3942.1cal/g. This shows that the bomb calorimeter is relatively accurate and precise. In addition, our methods were relatively consistent and adequately planned.

For the standardization process, benzoic acid was tested at least once per lab day to make sure that the calibration of the calorimeter stayed constant. Our average standardization value was found to be 2424.69 ( 12.73 (.525 %) cal/g with a standard deviation of 15.226 cal/g. The percent error involved with the standardization was .054 %. This low percent error was due to the fact that we used pure benzoic acid pellets provided by the Parr Instrument Company. We assigned specific jobs to each lab member to maintain as much consistency as possible. This was not done for the sucrose trials. These assignments were maintained throughout the proceeding experiments. This consistency had some part in the relatively high precision of our data shown by our standard deviation value.

The L-Leucine amino acid samples that we tested were also close to their expected value of 6504.97 cal/g obtained from the CRC handbook. The heat of combustion value we acquired was 6510.23 ( 34.91 (.536 %) cal/g with a percent error of .081 %. The standard deviation was found to be 21.939 cal/g. From the data it can be seen that our value was accurate. However the small discrepancy could be attributed to a black residue found in the crucible. The effects of this black residue will be discussed in the error analysis section.

The DL-Valine sample was chosen due to the fact that it is very structurally close to L-leucine but lacks a CH2 group. The combustion value of DL-Valine was found to be 5909.34 ( 55.86 (.945 %) cal/g with a percent error of .440 %. This low percent error again shows that our experimental value was close to the literature value which was, in this case, 5935.480 cal/g. The standard deviation was 35.106 cal/g which shows that our values were accurate and relatively precise. Again, a black residue was found in the crucible and we concluded that it was due to incompletely burned carbon or impurities.

In order to determine whether the results were significant, a test for significance was conducted. This was done by multiplying the absolute value of the difference between the experimental value an the literature value by the quotient of the square root of the number of trials and the standard deviation. This value is then compared to the value that is obtained from the TINV function from the Microsoft Excel program. If this value is less then the TINV value, then the results are not significantly different. If the number is larger then the TINV value, there is a significant difference. All of the values obtained did not show a significant difference except for the sucrose trials. The testing of the sucrose was done in the first week of lab which left a great deal of room for error. Additionally, the value for the Energy Equivalent was not tested experimentally. The significant difference in the numbers is most probably attributed to the lack of experience in using all of the instruments.

After the testing was completed we found that there was a 600.879 cal/g difference in the heat of combustion between L-Leucine and DL-Valine. Therefore the energy found in the CH2 group is approximately (within 10%) the same as this difference.

It can be seen from all of our data that bomb calorimetry is an accurate and precise method in the determination of heats of combustion. The determination of the heat of combustion of CH2 reveals yet another use of the bomb calorimeter if the structures of two substances are known. It is also apparent from our standard deviation values that our more consistent procedure in testing the benzoic acid, L-Leucine, and DL-Valine provided more precision in the experiment as opposed to our sucrose standard deviation.

Error Analysis and Error Calculations:

In performing the experiment, it was important to keep every trial as consistent as possible, thus keeping deviation between trials minimal. Therefore each member of the group performed the same tasks from week to week. Random distribution of tasks was considered but was eliminated from the procedure because it would have been difficult to explore its effects because of the limited time provided for the lab.

A number of variables may have affected the precision and accuracy of our data. With regards to the precision, measurement errors were very significant. An error of one centimeter in measuring the length of unburned fuse wire would result in a change of thermal value of 2.3 cal (6). In addition, measurement errors involving the 2000 g of water in the calorimeter bucket may have also distorted our precision. This case could involve the water lost in the insertion and removal of the oxygen bomb from the calorimeter bucket. A change of 2.8 cal would result from a 1 gram error in this measurement (6). Another error could have arisen in the titration of the nitric acid by the .0709 N sodium carbonate solution. An error of 1 mL in this measurement would result in a thermal value change of 2.8 cal (6). The measurement of the sample mass and the rate of the temperature rise could have also accounted for the deviation in precision that we experienced. These errors would have caused a change in thermal energy of 6.7 cal per error of 1 milligram and 4.8 cal per error of .002( C/min respectively (6). Therefore all of these factors could cause a maximum total error of 17.6 cal (6). Inconsistencies in these procedural sections would lead to deviation between trials. We found that we were relatively consistent in our trials and the data did experience some scatter. The standard deviation of benzoic acid was found to be 15.226 cal/g while the standard deviation of L-leucine was 21.939 cal/g. We found that 35.106 cal/g was the standard deviation for DL-valine and 39.841 cal/g was the standard deviation for sucrose.

In determining the value for the corrected temperature rise, equation 3 from the background section was used. In order to minimize the error in reading the thermometer, the Parr Instrument Company provides a table that accompanies each thermometer for the purpose of determining the error for each temperature measurement. Once the temperature is read off of the thermometer, the table was used to subtract or add the error to the number. Another problem dealing with temperature is the fact that benzoic acid rises in temperature very quickly. Room temperature was about 27( C. If this room temperature was used as the initial temperature of the water, the final temperature could have overshot the thermometer, which has an upper limit of 30( C. Therefore the water was started at an initial temperature of 25( C due to this thermometer limitation. This discrepancy in initial room temperature would increase the temperature rise because heat from the surroundings would enter the water bath in addition to the actual combustion process. However, the value would be minimal because the calorimeter is insulated against this and the metal of the water bath does not touch the walls of the calorimeter jacket. In addition, this temperature had to be duplicated throughout the experiment. Any change would effect the error involved with the experiments.

The mean and standard deviation values stated above were obtained using the commands on excel, while the value for the confidence limit involved the TINV command using the array of numbers for heat of combustion. This 95% confidence limit value for sucrose was ( 33.30 cal/g. The command yielded a value for t which was used in the equation + (t*s)/(sqrt n) ( where s is the standard deviation and is the number of measurements in the array). The value for percent error involved taking the difference between the actual and experimental value and dividing this difference by the accepted value (additionally, multiplying by 100 yields a percentage value for accuracy). To test the accuracy of our results, we found the percent error of our value from the accepted value of 3942.1 cal/g. This percent error was found to be 1.13%. The preceding calculations were utilized for the subsequent trials of amino acids and benzoic acid. The confidence limit and percent error of L-Leucine was ( 34.91 (.536 %) cal/g and .081 % respectively while they were ( 55.86 (.945 %) cal/g and .440 % respectively for DL-Valine. Finally, Benzoic acid yielded a 95% confidence limit of ( 12.73 (.525 %) cal/g and a percent error of .054 %.

With regards to the accuracy of our mean experimental value for sucrose and amino acids, a number of “experimental artifacts” (6). Because of the presence of high pressure in nearly pure oxygen contained within the bomb a number of secondary reactions might occur that would not occur in normal atmospheric conditions. Nitrogen in the air in the oxygen bomb might start a chemical reaction that creates nitric acid. This reaction is exothermic, perhaps causing a change in our thermal heat value. Heat transfer to the surroundings from the calorimeter may have also affected the accuracy of our results. Other errors including the absorption of heat by the metal calorimeter were taken care of by the calibration constant (6). In addition, the above mentioned black soot would have affected the experimental value due to the incomplete combustion of the amino acid. After speaking to professionals in the bioengineering field, we determined that this black residue could either be carbon residue that did not transform into CO2, forming soot, or impurities found in the sample. Excess carbon could be present due to the fact that 25 atm of oxygen in the bomb was not sufficient. However, this problem could not be further investigated since 25 atm is the upper safety limit of the bomb. However, we did perform before and after weighings of the residue but no change was determined by the Mettler balance. We determined the maximum possible error of the Mettler balance to be .0005 g because the balance only read to the thousandth gram. Using this error value and the fact that the heat of combustion of a mole of carbon is 7833.3 cal/g, we found the maximum possible error in enthalpy caused by the black soot to be approximately ( 3.92 cal/g. However, if this black residue is composed of impurities, it is difficult to assess the quantitative error because we do not know the identities of the impurities. However, the result in error of this black residue seemed to be minimal if it was composed of impurities, as our data was relatively close to the expected literature value.

Biological Significance of Results:

In conclusion, the Parr Bomb Calorimeter is an effective instrument in determining the heat of combustion for chemical substances. The process is facilitated by the many standards that are set for determining the error in the device. The calculated t value for the corrected temperature rise accounts for the temperature of the surroundings before and after the ignition of the bomb. The value for W (energy equivalent) accounts for the inherent error in the device while the values for e1, e2, and e3 account for the calories for the heat of formation of nitric acid, sulfuric acid, and combustion of fuse wire respectively. The determined value for the heat of combustion of sucrose was found to be 3897.30 ( 33.30 cal/g.

Also, our results brought up many interesting questions about the biochemistry of amino acids and the body. The most obvious first question is, “Why is the heat of combustion for leucine greater than that of valine?” The answer most likely lies in their bond structure. Leucine and valine are structurally identical except that leucine has an extra CH2 bond. In fact, some rough calculations show that the difference in the heats of combustion between leucine and valine is close to (within 10%) the theoretical value for a CH2 bond. Therefore, one possible explanation is that the energy needed to break this extra CH2 bond in leucine is responsible for its higher heat of combustion.

In addition, the heat of combustion of sucrose is almost half that of the L-leucine and DL-valine. So now the question arises, “Why does the body use sugars as its main source of energy as opposed to amino acids?” One reason is that amino acids are the building blocks of proteins which are very important in the proper functioning of the human body. So if the body were to use amino acids primarily, then the body would be working against itself since it would be breaking down its own framework. Also, every metabolic pathway in the body requires glucose as its initial molecule. Our bodies are equipped to breakdown a six carbon sugar to ATP through the Kreb’s cycle and the electron transport chain. Therefore, if any other compounds are to be used to produce ATP, they must first be converted into a carbohydrate. Finally, another explanation is that breaking down amino acids directly yields a great deal of nitrogen wastes, usually in the form of ammonia. So if the body were to breakdown amino acids en masse, it would counterproductively flood the bloodstream with toxins.

References

1. Blackburn, George L., Grant, John P., & Young, Vernon R. Amino Acids- Metabolism and Medical Applications. Boston, MA. John Wright PSG Inc., 1983.

2. Blaxter, Kenneth. Energy Metabolism in Animals and Man. New York, NY. Cambridge University Press, 1989.

3. Frenkel, Michael. Thermochemistry and Equilibria of Organic Compounds. New York, NY, VCH Publishers, Inc., 1993

4. Gill, Philip S. & Johnson, Julian F. Analytical Calorimetry. New York, NY. Plenum Press. 1984.

5. Hemminger, W. & Hohne, G. Calorimetry-Fundamentals and Practice. Deerfield Beach, FL, Weinheim, 1984.

6. Operating Instructions for 1108 Oxygen Combustion Bomb (205M), Oxygen Bomb Calorimeter (204M), Pellet Press, and Mercurial Thermometer (211M). Parr Instrument Company, Moline, IL 61265

7. Schepartz, Bernard. Regulation of Amino Acid in Mammals. Philadelphia, PA. W.B. Saunders Co., 1973.

8. White, Walter P. The Modern Calorimeter. New York, NY. The Chemical Catalog Company, Inc. 1928.

Websites:







Appendix A

Temperature vs. Time Graphs for Benzoic Acid Calibration Trials:

Trial 1 Trial 2

[pic] [pic]

Trial 3 Trial 4

[pic] [pic]

Trial 5 Trial 6

[pic] [pic]

Trial 7 Trial 8

[pic] [pic]

Appendix B

Temperature vs. Time Graphs for the L-Leucine Trials

Trial 1 Trial 2

[pic] [pic]

Trial 3 Trial 4

[pic] [pic]

Appendix C

Temperature vs. Time Graphs for the DL-Valine Trials

Trial 1 Trial 2

[pic] [pic]

Trial 3 Trial 4

[pic] [pic]

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[1] Parr Instrument Co. - Moline, IL 61265 - This value was eventually replaced by the value that was obtained experimentally.

[2] Sigma Chemical Co. - St. Louis, MO

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