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Page Items
* Research Assistants
* The Problem
* The Method
* Moles and molarity
* Acid/Base Titration
* Kjeldahl Method
* Personal Investigation
Research Assistant Wanted

Brother Gregory wants you to help him find the answers to questions about protein samples extracted from various parts of the Mummy of Horemheb. In these investigations you will be provided with samples of material taken from various places on the mummy. You will be asked to find out the "percent nitrogen" (%N) in these samples, and then record this information for later analysis.

First, print out your personal investigation page (below) and find out which specimens you are supposed to investigate and analyze. Carry out the experiment, gather and record your data, analyze your results and write down the appropriate numbers on your personal investigation page.

This is important. Bring your completed investigation page to the examination, if it is required on your topic schedule.




Page Items
* Research Assistants
* The Problem
* The Method
* Moles and molarity
* Acid/Base Titration
* Kjeldahl Method
* Personal Investigation
The Problem

Specimens of unknown materials have been taken from an Egyptian mummy. It is hoped that they will give some clues as to who or what killed the Pharaoh Horemheb.

One clue will be the chemical nature of the protein molecules found in these specimens. Protein molecules have characteristic amounts of nitrogen in their structures. These atoms of nitrogen are part of the amino acids which are joined together in long, polypeptide chains, to form the basic, primary structure of the final protein. The amount of nitrogen in a protein is usually written in the form "%N".

There are various ways of determining the %N in samples of protein molecules. In this investigation you will use the method devised by Johan Kjeldahl, and called the
Kjeldahl Method.

You will begin by learning about moles, how to perform a titration, and what you can learn from titrating acids and bases. Then you will carry out the Kjeldahl Method on your assigned samples from the Mummy of Horemheb.




Page Items
* Research Assistants
* The Problem
* The Method
* Moles and molarity
* Acid/Base Titration
* Kjeldahl Method
* Personal Investigation
The Method

In this method, the atoms of nitrogen found in the amino acids of proteins are first converted to the gas ammonia by digestion of the protein in strong sulfuric acid. This ammonia gas is distilled out of the digestive solution and into a trapping solution, where it dissolves and reacts. The amount of ammonia so trapped can then be determined by titration with a standard solution whose strength and composition is already known.

  • A carefully weighed sample taken from the mummy, and known to contain protein, is digested in a special type of glass flask. A very strong solution of sulfuric acid is used to breakdown the sample, release the protein and convert the nitrogen (found as part of the amino acids) into ammonia.

  • During the digestion process the sample in the flask is heated with a catalyst to ensure the total conversion of all the nitrogen into ammonia.

  • When the digestion is complete, strong alkali (usually NaOH) is added to the digestion mixture in the first flask to release the ammonia as a gas.

  • The ammonia gas is released from the digestion mixture and is passed into a "trapping" flask that already contains a carefully measured volume of a known, standard hydrochloric acid solution. The ammonia gas dissolves in the HCl solution and also neutralizes part of the acid in the trapping flask.

  • It is usual to make sure that all the ammonia is passed from the digestion flask to the trapping flask by heating the digestion flask and the NaOH solution.

  • Finally, the amount of acid left in the trapping flask, that NOT neutralized by the released ammonia, is titrated using a known, standard solution of NaOH.

  • From this final piece of data it is now possible to calculate (a) the amount of ammmonia that was distilled into the trapping flask, and hence (b) the amount of nitrogen that was released from the original sample of protein. From these numbers is it possible to calculate the amount of nitrogen in the protein.


Step One:
Page Items
* Research Assistants
* The Problem
* The Method
* Moles and molarity
* Acid/Base Titration
* Kjeldahl Method
* Personal Investigation
Understanding "moles" and molarity

As the first step in this investigation, it is necessary to understand the concept of a "mole", a value that is going to be very important later when the calculations are performed.

Definition of a mole

  1. A mole is the term, used in biology and chemistry, to define how much of a particular substance is present in a solution, a flask, a cell, a container, etc.

  2. In one mole, there are are always 6.022 x 1023 atoms/molecules/or "entities". (An "entity" can be thought of as almost anything. For example, there are 12 "entities" in a dozen eggs.).

  3. Thus "one mole of hamsters" would consist of exactly 6.022 x 1023 tiny rodents, and "one mole of protein molecules" would consist of 6.022 x 1023 polypeptides.

  1. 6.022 x 1023 is a special number named after Amedeo Avogadro, an Italian scientist, who made a very important contribution to our understanding of chemistry and the measurement of atomic weights. It is usually represented using the symbol "N".

  2. If it were possible to very carefully count out exactly 6.022 x 1023 atoms of carbon-12 and place them on an accurate balance, the weight would come to exactly 12 grams. Similarly, if it were possible to very carefully count out exactly 6.022 x 1023 atoms of nitrogen-14 they would weigh exactly 14 grams!

  3. 6.022 x 1023 molecules of hydrochloric acid (HCl) would weigh exactly 36.45 grams (35.45 from the chlorine and 1 from the hydrogen). Also 6.022 x 1023 molecules of oxygen gas would weigh 32 grams.

    These weights are all called molar masses, of the molecules or substances.

  4. Thus ...
    One "molar mass" of any substance, also known as 1 mole, is the molecular weight of that substance measured in grams.

Solutions

  1. A solution is a very special kind of mixture in which at least two different substances come together in such a way that each substance retains its own individual chemical properties.

  2. A true solution is a homogeneous mixture where all the participating ingredients exist as individual molecules (or parts of molecules, or parts of atoms such as ions).

  3. A solution usually has at least two components. These are called the solute, which is the ingredient present in the lesser amount, and the solvent, which is the ingredient that is present in the greater amount.

  1. Solvents are usually liquids, (but they don't have to be). Water is a universal solvent, and perhaps the most important one, but it is not the only possible solvent.

  2. The term concentration is used to define just how much solute is dissolved in the solvent. Dilute means that a small amount of solute is dissolved in a large amount of solvent, and concentrated means that a large amount of solute is dissolved in a small amount of solvent.

  3. Molarity is a special way of expressing the concentration of a solution.

  4. The "molarity" of a solution is determined by knowning three things:

    • the number of grams of the solute,
    • the molecular weight of the solute and
    • the liters of liquid in which it is dissolved.

    Molarity =
    (grams of solute / molecular weight) / liters of solution

  5. A one molar solution, therefore, has the molecular weight (in grams) of the solute dissolved in 1 liter of solution. This is usually written "1.0M" (where the letter M is the symbolic way of writing "moles per liter", or "mol/liter", or "mol/L").

Procedure

  1. Open the "mole" calculator which you will first use to try out a few examples of a "mole".

  2. You can use this calculator in any way you want, but here are a few examples to try first:

    • mol. wt. grams mls liquid
      23 46 1000
      23 23 100
      95 8 10
      45 4 1000

    • What did you learn?

    • In what way do the values for the molarity of the solution and the total number of moles change as you change the number of grams of solute and the total amount of liquid?

  3. Molarity and moles are important values when comparing two solutions. Which is what you are going to do next.


Step Two:

Page Items
* Research Assistants
* The Problem
* The Method
* Moles and molarity
* Acid/Base Titration
* Kjeldahl Method
* Personal Investigation
Acid/Base Titration

As the second step in this investigation you are now going to compare two solutions (an acid and a base) using a method called "titration".

In the first proceedure you are simply going to add an acid solution to a basic solution. Each solution will be of a different "strength", or concentration, or amount, and you will simply observe the relative results.

[Note: read here first, if you feel you need more information about acids and bases].

In the second proceedure you are going carry out a number of titrations in which an acid solution is carefully added to a basic solution. In each case you have to find the "end point", which is the point at which you have added just enough of the acid solution to exactly neutralize all the base that was in the original solution.

The properties of the acid solution are standardized, and fully known. So, by finding the exact amount of acid that neutralizes a known solution of base, it is possible to carry out a calculation and find out the molecular weight of the base.

Procedure #1 - Adding Acid

  1. Open the Adding Acid screen.

  2. Click on one of the selections on the left side of the page, the Bases. You can start with any of the solutions.

  3. Click on one of the selections on the right side of the page, the Acids. Once again, you can start with any of the solutions, but you should eventually test them all.

  4. Now move the slider at the bottom of the screen. As you put the pointer over the slider-control it will move. Moving the slider from the left side to the right side slowly adds some of the acid solution to the base (alkaline) solution.

  1. The yellow and red bars in the center of the screen indicate how much of the base (or acid) is left (or present) in the mixture at any one time.

  2. Try all the possible combinations of basic solutions and acid solutions and see what kind of results you get.

  3. You should at least find that a dilute solution of acid cannot neutralize a concentrated solution of base, and that a concentrated solution of acid very quickly neutralized a dilute solution of base.

  4. This exercise should help you understand what happens when you "titrate" a solution of a base using differnet solutions of an acid.

Procedure #2 - Acid/Base Titration

  1. Open the Acid/Base Titration screen.

  2. You are now in control of all the elements in a simple titration of a solution of a base against a known solution of an acid. So, you have to make a series of choices, just as you would do if you were in a traditional "wet" laboratory.

  3. Setting all the paramenters

      Bases in Flask

    • Choice of Base (in the flask): Click on the appropriate Sample #, on the left side of the screen.

    • Weight of sample (gms): if necessary, change the amount of the sample (in grams) in the appropriate box on the left side of the screen, or you can simply leave it at the default value of 0.1gm.

    • Volume of Water (mls): if necessary, change the volume of the water (in milliliters) in the appropriate box on the left side of the screen, or you can simply leave it at the default value of 10 mls.

      Acid in Buret

    • Choice of Acid (in the buret - the long tube with the tap at the bottom): Click on the appropriate Acid, on the right side of the screen.

    • It is recommended that you start with one of the hydrochloric acid - HCl reagent solutions, and save the other acid for later.

      Amount of reagent

    • Move the yellow tab beside the buret (tube) up or down by placing the cursor arrow over the tab and moving it. As you slide the yellow tab the value in the first small box will change.

    • The value in the first small box represents the volume of reagent/acid you will be adding from the buret and into the flask on each addition. Smaller values mean smaller amounts of liquid. You can change this value at any point in the experiment and at any time.

  1. Start the titration by clicking on the "tap" at the bottom of the buret. With each click you will be adding the amount of acid solution set in the box above. The total amount of liquid you have added is calculated for you and appears in the larger box in this section of the screen.

  2. On your first or second click on the tap, several things will happen at once:

    • the value for the total amount of reagent will start to change.
    • the color of the indicator dye in the flask, which is sensitive to the amounts of acid and base present, will eventually start to change color.
    • a ratio, called the "B/A ratio", will appear in the "messages" box (at the bottom-left side of the screen). This informs you of the ratio of base to acid in the flask and will help you find the point at which all the base has been neutralized by the addition of the acid.

  3. Repeatedly click on the tap at the bottom of the buret and keep adding small amounts of the acid solution to the solution of base.

  4. You are looking for the exact point at which the base is totally neutralized by the acid. You will know when this has happened in two ways:

    • the color of the indicator dye turns orange - which is the mid-way color between yellow and red. This is often very hard to see, so...
    • the "B/A ratio" becomes a value of exactly 1.0
  1. When you reach the point where the solution in the flask is exactly neutral (the acid has completely neutralized the base), record and write down all the important values, including the total amount of acid (reagent) that you have added. THIS IS IMPORTANT!

  2. To find the molecular weight of the unknown base in the sample you have just titrated you will need to use this Molarity Calculator.

  3. Using the results you obtained from your experimental titration, enter the appropriate values into the boxes on the calculator, find the "Total moles of Acid", then in a similar way find the "molarity of Base" and finaly click to find the "mol. weight of sample"

  4. Record all your findings and your results.

  5. Additional investigation: You will probably have noted that there is a choice between two different acids in this experiment, hydrochoric acid, and sulfuric acid.

  6. Using all the methods you have just practiced, now try titrating some of the same base samples, but this time with the sulfuric acid instead of the hydrochloric acid.

  7. What kind of results did you get, and what do you think they mean?


Step Three:

Page Items
* Research Assistants
* The Problem
* The Method
* Moles and molarity
* Acid/Base Titration
* Kjeldahl Method
* Personal Investigation
Kjeldahl Method for determining the %N in various protein samples

As the third step in this investigation you will now use many of the ideas and techniques that you have just discovered and practiced to find the amount (%N) of nitrogen in some of the protein samples taken from the Mummy of Horemheb.

This evidence may help you to find out what killed the Pharaoh Horemheb - but you never know!

Procedure

  1. Start by printing out your "Personal Research Investigation" (below) and finding out which of the samples you should be testing. Don't forget to write your answers on this paper and bring it to the appropriate examination.

  2. Go to the
    Kjeldahl Method - Mummy Samples
    screen.

  3. You will see a list of sampless on the left. Click on one of the ones in your Personal Research Investigation assignment.

  4. Now enter a value for the number of grams of that sample you are going to digest and release the nitrogen as ammonia. (Hint: try values in the range of 30-80 grams).

  5. Now click on the "---***Start***---" button at the bottom of the list of samples. Several things should now happen all at once - (i) your sample will be digested, and the ammonia released, - (ii) the ammonia will be trapped in the standard acid solution in the trapping flask.

  6. The trapped ammonia has neutralized part of the tapping acid, so now you must titrate the remaining acid using a standardized solution of base (the NaOH).

  7. Enter a value, in milliliters (mls), in the box under the "Equipment". This will be the amount of NaOH you will add from the buret (base) to the flask (acid) each time you click on the "---***titrate***---" button.

  8. Every click on the "---***titrate***---" button adds that amount of base to the acid, and you now have to watch the color of the indicator dye in the trapping flask.

    • While the color is RED, the solution in the flask is still acid.

    • If the color of the dye turns YELLOW, the solution in the flask has turned basic, and you have added too much of the NaOH, and must start again.

    • You are looking for the point at which the color in the flask is ORANGE, which means that the solution is exactly neutral. This is the point you want to see.

  9. When the color of the dye in the trapping flask is orange, carefully write down the total amount of base (NaOH) that you have added from the buret. This amount is shown in the information box.


Step Four: Calculation

You must now calculate the %N in the samples. This is done in three steps using the values known or determined in the experiment.

Values known or determined:

  • volume of acid (vAcid) in the trapping flask = 100ml
  • strength of the acid (mAcid) in the trapping flask = 0.03M
  • volume of base (vBase) added from buret = your results
  • strength of base (mBase) in the buret = 0.03M

  • weight (S) of protein sample = your choice


    Calculation #1:

  • moles of ammonia produced:

    molesAmmonia = molesAcid - molesBase

    molesAmmonia = (vAcid x mAcid) - (vBase x mBase)

    molesAmmonia = (100 x 0.03) - (your result x 0.03)

    Calculation #2:

  • grams of nitrogen produced:

    gramsNitrogen = molesAmmonia x molarMassNitrogen

    gramsNitrogen = molesAmmonia x 14.0067

    Calculation #3:

  • percentage of nitrogen in sample:

    %N = (gramsNitrogen / gramsSample) x 100

    %N = (gramsNitrogen / your choice) x 100


Print out your
personal investigation page

Page Items
* Research Assistants
* The Problem
* The Method
* Moles and molarity
* Acid/Base Titration
* Kjeldahl Method
Carefully enter your Seat Number or PCIN number
(e.g.
MM34, or MA56, or WA41)
in the box below, click, and print out the page that appears.

Find the answers to the questions, write your answers on the investigation page, and bring the completed assignment to the exam for grading purposes.


Personal Investigation Page

Enter your seat number or PCIN number here: -



Science@a Distance
© 2003, Professor John Blamire