Laboratory Investigation

Investigating the Properties of Certain Inorganic and Organic Substances

Prepared by Marivi Gondola Course IB Biology SL/HLa

Assessment: Data Collection, data analysis, manipulation

Objectives

Students will:

  • identify certain organic and inorganic substances by means of chemical tests
  • learn physical and chemical properties of certain inorganic substances
  • explain the importance of control (positive and negative) in biochemical tests
  • use biochemical tests to identify an unknown

Materials

Provided:

Glucose solution

Sucrose solution

Starch solution

Chloroform

Yeast suspension

Benedict’s solution in dropper bottles

Lugol’s iodine in dropper bottles

Biuret reagent

2.5% NaOH (need ~15 ml per group)

Distilled water in wash bottles

Test tubes (about 8 per group)

Diluted HCl or full-strength vinegar (Optional)

Limestone (optional)

Honey or corn syrup

Hot plate

Bunsen burner

Test tube holder or clothespin

3- 500 ml beakers

Unknown sample of food mixture

Mortar and pestle

Safety goggles

Each group must bring small amounts of food samples:

Solids samples: Apple, potato (juice), carrot (juice), bread, eggshell, cheese, meat (juice), bone (optional)

Liquid samples: Different milk samples, egg white and yolk, fruit juices, cooking oil, olive oil

Procedure

You need to design tables to appropriately record the results of each of the tests as well as any additional pertinent observation.

Need to test the unknown for procedures B-D.

Solids need to be grinded. Use 10 drops of liquid samples.

A. Calcium test. (Optional. Only if requested by teacher)

1. Place a small piece (same mass) of the following samples in a clean test tube (TT): eggshell, bone, limestone)

2. Fill a dropper, half-full with diluted HCl and add the acid to the TT

3. Observe and record the reaction after a few seconds.

What type of reaction takes place?

What happens to the samples? Are these positive or negative controls? What sample could you have included for negative control?

B. Test for reducing sugars

4 food samples (except, cheese, eggshell, bone), distilled water, starch, sucrose and glucose solution

Many monosaccharides such as glucose and fructose are reducing sugars, meaning that they possess free aldehyde (-CHO) or ketone (- C = O) groups that reduce weak oxidizing agents such as copper in Benedict’s reagent. Benedict reagent contains cupric ion with citrate in alkaline solution. This reagent identifies reducing sugars based on their ability to reduce the cupric ions (Cu2+) to cuprous oxide at a high (basic) pH. Cuprous oxide is green to reddish orange. Green indicates a small amount of reducing sugars, reddish orange indicates abundance1.

What color is produced by non reducing sugars?

  1. Use clean test tubes. Tests require a hot water bath.
  2. Place 10 drops of fluid samples or 1 cm3 of solid sample (grinded).
  3. Add 2 ml of Benedict’s solution to each tube. Place TT in hot water bath for 5 minutes and observe color changes.
  4. Record observations.

Which of the solutions acted as positive control? Negative control?

Which of the sugars tested is a reducing sugar?

Which contain more reducing sugars fruit juices or potato juices?

What does this tell you about the way sugars are stored in potatoes?

C. Test for starches

4 food samples (except, cheese, eggshell, bone), distilled water, starch, sucrose and glucose solution

I2KI distinguishes starch from simple sugars and other polysaccharides. Iodine reacts with the coiled starch polymer producing a bluish black coloration. Glycogen has a slightly different structure and produces only an intermediate color reaction2.

  1. Place material to be tested on clean TTs.
  2. Use 2-3 drops of I2KI.
  3. Record color changes in your table.

What solutions are negative controls? Positive control?

What solutions produced more intense colors?

______

1Vopovich and Moore, pp. 44

2Ibid, pp. 45

D. Test for proteins. Coagulation3. (Optional)

7 samples (except eggshell, bone): include egg albumin solution (1ml albumin+3ml distilled water), starch, sucrose, milk samples

Proteins coagulate at high temperatures.

1. Heat up the upper part of the TT until boiling. Presence of proteins is indicated by a white milky precipitate. Why?

What type of organic compound makes up albumin?

What happens to albumin when boiled?

E. Test for proteins. Biuret.

7 samples (except eggshell, bone): include honey or corn syrup, egg albumin solution (1ml albumin+3ml distilled water), starch, sucrose, milk samples, bread, potato juice, meat juice…

Proteins are made up of amino acids. Each amino acid contains an alky; group, a hydrogen atom, an amino group and a carboxyl acid attached to a central carbon. When two adjacent amino acids join the bond that forms between them is a peptide bond. This bond is identified by the Biuret reagent (1% CuSO4). Peptide bonds (C-N bonds) in proteins complex with Cu2+in Biuret reagent producing a violet color only if it complexes with four to six peptide bonds. The intensity of the color relates to the number of peptide bonds.

  1. Use 2 ml of food samples. Place samples in clean TTs.
  2. Add 2 ml of 2.5% NaOH to each tube
  3. Add three drops of Biuret reagent to each tube.
  4. Record color changes.

CAUTION Handle NaOH with care; it is toxic.

What of the solution serve as positive control?

What contains more protein, albumin or honey?

F. Test for Lipids. Solubility in polar and non polar solvents.

7 samples (except eggshell, bone) include: egg albumin solution, starch, whole milk, bread, oil samples.

1. Use 4 drops of each sample and 3 ml of solvent. Run first the solubility in water tests and then the solubility in chloroform.

What do you conclude about the solubility of your lipid samples in water and in chloroform?

Explain your results.

G. Test for Lipids. Perform Sudan IV Test4. (Vopovich and Moore, pp 49)

7 samples (except eggshell, bone) must include: distilled water, egg yolk,

whole milk, bread, vegetable oil.

TT1 salad oil + water

TT2 salad oil + Sudan IV

TT3sample + Sudan IV

TT4sample + Sudan IV

TT5sample + Sudan IV

TT6sample + Sudan IV

TT7sample + Sudan IV

______

[3]Correa and Russell pp. 24-28

4 Ibid

1. Place samples in 7 clean TTs.

2. Add five drops of Sudan IV to each of the TTs. Mix contents well.

3. Record results.

I. Test for Nucleic Acids. Perform Dische Diphenylamine Test for DNA. (Vopovich and Moore, pp 51)

7 Samples, must include the following: distilled water andyeast suspension

DNA can be identified chemically using the DischeDiphenylamine Test. Acidic conditions convert deoxiribose to a molecule that binds with diphenylamine forming a blue complex. The presence of ribose is detected by a green coloration.

CAUTION Handle DischeDiphenylamine with care; it is toxic.

  1. Place sample(s) in clean TTs.
  2. Add 2 ml of DischeDiphenylamine reagent and mix thoroughly.

Which TT acts as a negative control?

Do yeast contain more RNA than DNA?

Unknown. Identify the contents of an unknown solution.

  1. Use 2 ml of the unknown sample into each of the TTs
  2. Detect presence of reducing sugars, starch, protein, lipid, DNA of the unknown. Your unknown may contain one, none or several of these macromolecules.
  3. LEAVE ALL YOUR UTENSILS AND TABLE CLEAN BEFORE LEAVING THE ROOM.

References

Correa, J. and A. Russell. 1998 Fundamentos de Biologia: Manual de Laboratorio. Universidad de Panama.

Vopovich D. and R. Moore. 2002. Biology: Laboratory Manual. Sixth Edition. McGraw-Hill.