Etiket Arşivleri: experiment

Experiment 5: Chemical Equilibrium ( Kinetics measured by Pressure )

Chemistry 102 Laboratory

Experiment 5:  Chemical Equilibrium (Kinetics measured by Pressure)

Laboratory

In this experiment you will measure the rate of decomposition of carbonic acid by measuring the rate of production of carbon dioxide in the following reaction:

H2CO3  <–>  CO2(g)  +  H2O

The equilibrium constant for this reaction, in terms of concentration, is given by the expression:

Keq  =  [CO2]/ [H2CO3]

In this experiment, however, the pressure of carbon dioxide, rather than its concentration, is to be measured.  In particular, the pressure is measured as a function of time, and at two different temperatures.  Since the equilibrium and chemical rates are both functions of temperature, two different rates will be measured at the two different temperatures.  Since this decomposition reaction is known to be first order in the concentration of the carbonic acid, the rate varies as the exponent of the absolute temperature of the solution.  For every 10o increase in temperature, the rate of the reaction will approximately increase by a factor of 2.  Equilibrium is attained when the pressure remains a constant with time.  In this experiment, you will make use of the ideal gas equation and the fact that the equilibrium constant may be stated in terms of the gas pressure of carbon dioxide as well as the concentration (mass) of carbon dioxide.

Instructions

Computer Setup

1. Obtain a Pressure Sensor and plug it into an Analog channel of the interface box.  Make sure the interface box is on, then start Science Workshop.  Use the analog plug button to select your channel and then select the Absolute pressure sensor.  Double click on the pressure sensor icon, and allow the sensor to warm up for 5 minutes.

2. Using the barometer in the Main Lab, obtain the current atmospheric pressure in mm Hg.  Convert to atm.  In the screen for senor calibration, change kPa to atm for the pressure units.  In this same screen change the low pressure known value from 300 to the atmospheric pressure you measured in “1” above.  After the current reading of the pressure stabilizes, click on read to calibrate the pressure.  You are done with this part.

3. Check to make sure the sampling rate is set for Fast at 10 Hz by looking at the Sampling Options.  Bring up a graph window by dragging its icon over that of the pressure sensor.

Getting Set Up

4. Insert the barb of a quick release connector into one end of the plastic tubing that comes with the pressure sensor.

5. Obtain a one hole rubber stopper that fits a 250 mL glass bottle or flask.

6. Place a drop of glycerin on the bottom end of the hole of the stopper.

7. Insert the glass part of an eyedropper, tip up, through the hole in the stopper; the tip must clear the top of the stopper.

8. Carefully fit the glass tip into the open end of the plastic tubing.

9. Align the quick-release connector with the connector on the PRESSURE PORT of the Pressure Sensor.

10. Push the connector onto the port and then turn the connector clockwise until it clicks.

11. DO NOT INSERT THE STOPPER IN THE BOTTLE YET!!!  The pressure sensor should still be open to the atmosphere at this point.

Recording Data

12. Obtain 100 mL of room temperature (a posted value) soda water and place it in the 250 mL flask.

13. When everything is ready, click REC to begin recording data.

14. QUICKLY insert the stopper in the bottle; make sure there is a tight seal. DO NOT shake or stir the contents of the bottle.

15. Observe the changes in pressure as the carbonic acid in the soda water decomposes in the bottle.

16. Record for 6 minutes.

17. Slowly remove the stopper from the bottle and allow the pressure readings to stabilize.  This is to determine whether or not the air pressure or calibration have changed.

18. Stop the recording.  Determine the initial rate of decomposition of carbonic acid in the same manner employed in experiment 4 (i.e. a linear fit of the data).  Also record the final pressure of the gas in the flask.

19. Obtain 100 mL of cold soda water and place it into a 250 mL flask which has been cooled on ice in an ice bath.  Leave the water and the flask in the ice bath and return to your lab station.Repeat the procedure with cold soda water.

Alpha Amylase Production ( Experiment 2 )

Alpha amylase: EC 3.2.1.1

Amylases are important hydrolase enzymes which have been widely used since many decades. These enzymes randomly cleave internal glycosidic linkages in starch molecules to hydrolyze them and yield dextrins and oligosaccharides. Among amylases α-Amylase is in maximum demand due to its wide range of applications in the industrial front. With consumers growing increasingly aware of environmental issues, industries find enzymes as a good alternative over other chemical catalysts. α-Amylase can be produced by plant or microbial sources. Due to the advantages that microbial production offers, α-Amylase from microorganisms has been focused upon and preferred to other sources for production. The ubiquitous nature, ease of production and broad spectrum of applications make α-Amylase an industrially important enzyme. This report focuses on the microbial production of α-Amylase and its applications.

 Enzyme Production

In order to cultivate a microorganisms 4 major criteria you should be controlled or manipulate

1. Temperature

2. Initial pH

3. Oxygen

4. Carbon and Nitrogen sources

In order to control the temperature, incubator or water baths could be used. Initial pH is the other parameter which is critical for microbial growth. As all microorganisms have optimum temperature, they have also an optimum pH. Many microorganisms optimum pH is neutral pH (7). The other criteria is oxygen if the microorganisms is aerobic, you should supply air to the microorganisms. The carbon and nitrogen source is another important criteria. First of all these C and N sources are food of microorganisms so they should able to digest and uptake both C and N sources. Most of the Bacillus species can synthesis and secret extracellular alpha amylase. Due to this ability of Bacillus spesies we use Bacillus in order to obtain alpha amylase. For detection and quantification of alpha amylase activity we use DNS method as mentioned below.

Production of Lactic Acid By Batch Fermentation ( Experiment 1 )

Lactic acid (2-hydroxypropanoic acid), CH3CHOHCOOH [CAS 50-21-5], is the most widely occurring hydroxycarboxylic acid. It was first discovered in 1780 by the Swedish chemist Scheele. There are two isomers of lactic acid that are present in nature, L(+) and D(-) forms. L(+)-lacticacid is biodegradable and can be metabolized by the body and this property leads the application of lactic acid in biomaterial applications. It can be used wide range of industrial areas. In food industry, it is used as acidulant, preservative and antimicrobial agent. It has been also utilized in leather, textile, pharmaceutical and cosmetic industries for many years. Lactic acid is a naturally occurring organic acid that can be produced via fermentation or chemical synthesis. By the chemical synthesis method, racemic (DL) mixture of lactic acid is produced. By microbial production method L(+) and D(-)-lactic acid can be produced according to the type of microorganism which may be homofermentative or heterofermentative.

It is present in many foods both naturally or as a product of in situ microbial fermentation, as in sauerkraut, yogurt, buttermilk, sourdough breads and many other fermented foods. The lactic acid bacteria possess a large number of metabolic properties that are responsible for their successful use as starter cultures in the commercial production of fermented dairy, meat, and vegetable products and beverages. The genus Lactobacillus represents the largest group of rod shaped organisms with in the lactic acid bacteria. Some members of this group of organisms are important in the generation of particular flavor sand in other ripening processes associated with specific cheeses.

‎Laboratory‎ > ‎Dialysis

In this experiment we observed the dialysis. The principle of dialysis is that the small molecules can penetrate through the pores of semi-permeable membrane toward the buffer solution while large molecules cannot. By using this method we separated glucose from starch and cystein from Bovine Serum Albumin (BSA). So the small molecules (glucose and cystein) were collected in buffer side and large molecules (starch and BSA) were kept in the bag. Then we tested the buffer solutions and the solutions in the bag with the reagents to decide if the separation process was completed successfully or not.

The samples taken from the buffer solution and the solution inside of the bag of starch- glucose mixture was tested with Lugol’s solution and Benedict reagent. Lugol’s solution is used to observe the presence of starch since the colour changes from yellow to blue-black in the presence of starch. The result showed that there was starch in the bag but there was not any in the buffer. This result was expected. Then Benedict reagent was used which changes colour from blue to red in the presence of glucose. The result showed that there was glucose in the buffer but there was not any in the bag. This result was also expected. So according to the results we can say that the glucose-starch separation was successful.

The samples taken from the buffer solution and the solution inside of the bag of BSA- cystein mixture was tested with Biuret reagent and Ninhydrin solution. Biuret reagent is used to observe the presence of protein (BSA) since the colour changes from blue to purple in the presence of protein. The result showed that BSA was found in both buffer and bag. This result was not expected because BSA cannot pass from the pores of membrane. So it should not be found in the buffer. The reason of that may be not to properly tying the both ends of cellulose membrane bag. Ninhydrin solution is used to observe the presence of amino acid (cystein) since the colour changes from blue to violet in the presence of amino acid. The result showed that there was cystein present in the buffer but there was not any in the bag. So we can say that cystein separation was successful.

Laboratory‎ > ‎Dialysis v1

Purpose: In this experiment, the dialysis experiment is made. Dialysis is a process that is based on the principle of osmosis, moving from an area of high concentration to a lower concentration. Therefore this laboratory is for observing the enzyme purification.

Materials: Dialysis bag, beaker, magnetic stirrer, stirring bar, water, lugol solution, benedict solution, ninhydrin solution, biuret solution, pipette, rope

Procedure: Firstly, one dialysis bag was filled with a mixture of consisting of quantities of a 2ml of %40 glucose and 2ml of %5 starch, and other dialysis bag, 2ml %5 BSA and 2ml 10% Glycine. After making this action, the content of the bag cannot escape by leaking, knotting in end of dialysis tubing. Then bags were putted into beakers contains distilled water and waited about 2 hours.

Laboratory‎ > ‎Gel Filtration Using Sephadex

In this experiment we applied gel filtration of enzyme b using sephadex as column material. This is a chromatographic technique used for protein purification. The principle is based on fractionation of proteins based on the molecular weight or size of protein molecules. In this system when the mixture of the protein is applied at the top of the gel filtration column and washed with buffer solution, depending on molecular exclusion limit of column material, the large protein molecules are excluded from the internal volume and thus leave the column first. The smaller protein molecules are absorbed by the pores of the column material and leave the column later.

We collected 24 fractions from the column but 16 of them are illustrated at the data. Because the other fractions couldn’t measured correctly. From these data we calculated the protein content, specific activity, purification fold and total activity of each fraction. The results show that 4th and 6th fractions have the higher activity. Also the protein content and total activity versus fraction number graphic is plotted. At this graphs the first peak illustrates the first eluted fraction and this is probably eluted from the void volume also from the second graph we can see that the firstly eluted fractions activity are lower.

QUESTIONS

1. Which fraction is the most active?

– 4th and 6th fractions are most active since their total activity is equal to each other and higher than the other fractions.

2. Calculate specific activity, purification factor, % recovery.

– Calculations are done at the calculation section.

3. Which fraction has the highest purity?

– 11th fraction has the highest purity since it has the highest purification fold.

Laboratory‎ > ‎Heat Treatment

In this experiment we investigated the effect of temperature on enzyme activity and protein content. After the calculations we plotted two graphs these were protein content versus temperature and total activity versus temperature. According to the graphs we can say that the total activity increases up to a point then it starts to fall down. Initially this increase is caused by the increase in temperature up to the optimum temperature for reaction to gain sufficient energy to reach the activation energy. Then the activity decreases since further increase in temperature cause denaturation of protein. According to the graph the optimum temperature is about 400C. Also at that temperature the purity of the enzyme is highest. In contrast to total activity, protein content decreases down to a point then it starts to increase.

QUESTIONS

1. State the temperature at which the activity is highest.

– Activity is highest at about 400C

2. State the temperature at which the purity is highest.

– The highest the specific activity, the highest the purity. So at 400C purity is highest.

3. What is the use of purification? Does it always necessary?

– Purification involves removing any contaminants that are present in the mixture; these may be other enzymes or proteins, carbohydrates and ash. For the some determinations it is necessary to purify enzyme from other substances in the case of determination amino acid composition, molecular weight, tertiary and quaternary structure, substrate specificity and kinetic

parameters like Km, Vmax, kcat.

4. Under which conditions a purified enzyme is required for activity determination?

Laboratory‎ > ‎Heat Treatment v2

In this experiment, Major affect of enzyme activity and stability is temperature. Since enzymes are biochemical catalysts, made up at least partially of protein, they are sensitive in varying degrees to heat. Raising temperatures of the environment generally multiplies the degree of activity by the enzyme. The most dissolved protein is denatured by heat when the temperature higher than about 50oC. Heat denaturation results in protein precipitation as a result of destruction of the secondary structure and formation of random aggregates. Because all proteins are not stable when heated in aqueous solution. The precipitate enzyme is recovered by filtration or centrifugation and dried in atm. or vacuum driers. For most commercial application cost is most important than high purity and is usually unnecessary.

When the presence of contaminating enzymes or other substance will adversely affect the product and activity. If the turbidity is observed in taken tube from ice bath, it is centrifuged. Because protein is denatured due to heat treatment and so sedimentation will occur. Therefore the sedimentation part must be removed. When we look at our results it is obviously seen that increase in temperature increases the activity of enzyme up to a level. According to our result this temperature is 40oC. In 60oC the protein content is more than in 40oC but specific activity of 60oC is less. On the other hand when we compare the velocities we can say the same thing. In 30oC velocity reaches the highest value. Finally we can say that because of protein denaturation in high temperatures, enzyme activity increases up to a level with increase in temperature.

Answers of The Questions:

1.At room temperature is highest than other temperature. Because the temperature is increased as the activity is decreased. The activity is affected for the temperature. The protein structure (interaction bond) is changed.

2.State the temp. At which the purity is highest. At 40 C the purity is highest.

3.Purification helps to elimination the contaminants, which is effect the purification. The foreign material that is not wanted in product for the pure product to be best product is changed the purification. But this is more expensive, so it is usually unnecessary for most commercial application.

4.The factor that, will affect the purification. The process condition is important for purification. If the purification is not best, the enzyme activity is not best. The unpurification enzyme is not work in process. This reason is important for working and activation enzymes.