AICE Biology Lab Review:

Biological Molecules

B. Biological Molecules(a) carry out tests for

reducing and non-reducing sugars (including semi-quantitative use of the Benedict’s test), the iodine in potassium iodide solution test for starch, the emulsion test for lipids and the biuret test for proteins;

Benedict’s Test: Reducing & Non Reducing Sugars, Round 1

RESULTS: Aqua-blue = negative.Green to Yellow to orange = positive. Note: to detect the simple sugar sucrose, you must do round 2 test

Left to right: • Benedict's reagent

(BnR),• potato extract + BnR,• onion extract + BnR, • 5% glucose + BnR.

Testing for simple sugars: Benedict's Reagent, round 1Procedure:Add Benedicts reagent to sample, place in waterbath until just boiling.

Benedict’s Test: Reducing & Non Reducing Sugars, Round 2

A Negative result in round one DOES NOT mean an absence of carbohydrates!!

Sucrose is a non-reducing Sugar & can only be detected by doing round 2 of Benedict’s testing (Acid Hydrolysis)

Procedure: • Perform round 1 of Benedict’s Testing. Negative result indicates

either no carbohydrate OR Non-reducing Sugars (Sucrose or starch).

• How would you test for the presence of starch???• Perform Round 2 of Benedict’s Testing to see if you have sucrose if

Starch test is Negative.

Sample Procedure for Benedict’s Test for Non Reducing Sugar

• In a test tube place 5 cm3 solution, add 3 cm3 Benedict's reagent to the solution in the test tube and place the tube in the boiling water bath for five minutes.

• Add 1 cm3 dilute hydrochloric acid to the solution solution in your test tube. Note the time and place in the water bath

• After 10+ minutes, remove the tube from the water bath & cool it under the tap. Neutralize the acid by adding solid sodium bicarbonate, a little at a time, until the addition of one portion produces no fizzing.

• With a dropping pipette place 3 cm3 Benedict's solution in test tube & return to the water bath and heat for five minutes.

• A color change indicates that there was Non reducing sugar present. How would you verify that your solution was sucrose & not starch???

Benedict’s Testing: Sample Question 1

Solutions of four food substances are tested for sugars. The table shows the colours of the solutions after testing.

Which food is a non-reducing sugar?

Benedict’s Testing: Sample Question 2

Four sugar solutions were tested with a standard Benedicts solution. The table shows the colour of the solutions after testing.

What is the best interpretation of the results?

Testing for Starch IKI (Iodine in Potassium Iodide)

RESULTS: Yellow-orange = negative.Purple-black = positive.

Left to right: • IKI only, • starch solution,• starch solution +

IKI.

Emulsion Testing for LipidsSample Procedure:• Add 2cm3 fat or oil to a test tube containing 2cm3 of

absolute ethanol. Dissolve the lipid by shaking vigorously. Add an equal amount of cold water.

• Observation: A cloudy white suspension.

Basis of test:• Lipids are immiscible with

water. Adding water to a solution of the lipid in alcohol results in emulsion of tiny droplets in the water which reflect light and give a white , opalescent appearance.

Testing for Polypeptides (proteins) Biuret’s Reagent

Left to right: • Biuret's reagent (BrR), • water + BrR, • egg albumin solution, • egg albumin solution+

BrR.

RESULTS: Denim-blue = negative.

Lavender = positive.

Identify each sampleFood tests are carried out on four unknown chemicals. The

chart below shows the results of each test.

Solution Benedict’s Test

Acid hydrolysis then Benedict’s

IKI Biuret

Emulsion

A X + X X X

B X X X + X

C + X X + +

D X + + X X

(KEY: + = positive result , X = negative result)

Measuring Color in Dependent Variables• Color standards

– Carry out macromolecule test on set of solutions with KNOWN concentrations (you make these)

• Use excess of your indicator– This produces a range of colors (and you know

the concentrations)– Stand this in a test tube rack– Now test your unknown sample – Compare sample to your known concentrations

and determine the concentration of your sample

• Terms to use– Simple words: red, blue, purple, green– Qualify with “pale” or “dark”– Use +, ++, +++ to show intensity (include a

key)– State actual color…do not say “no change”

Recording Quantitative Results• Descriptions of what you see• Use simple language• Avoid terms that would be difficult to

understand (ex. yellowish-green)– Should say “this tube is darker or lighter

green than tube 1”• Never state “no change” say the color

– Example…if there was no reaction to benedicts solution, you would state, “the tube remained blue”

Tools to Measure Dependent Variable

• Colorimeter– Measures color changes– Quantitative measurements of

color intensity in solution– Good way to “improve

reliability” of experiment– Uses cuvettes that contain

solution– Deeper colors absorb more

light– Important to choose suitable

color of light to shine through (opposite of the color of solution)

Dilutions

Simple Dilution• Unit volume of liquid of

interest is combined with an appropriate volume of solvent liquid to achieve desired concentration– “1 to 5” dilution means:

• 1 unit of solute AND 4 units of solvent for a total volume of 5 units

Serial Dilution• A series of simple

dilutions which amplifies the dilution factor quickly beginning with a small quantity of material

• The solute for each step comes from the previous dilution

Concentration

• The concentration of a solution is the amount of a substance present in a given volume of solvent. concentration = amount ÷ volume

• Usually measure the amount in moles and the volume in dm3

– Concentration of a substance is usually expressed in mol dm-3

Concentration

• Concentration of a solution is the amount of stuff dissolved in a given volume of a solvent

• Most commonly given as a percentage on the Cambridge exam

1 cm3 = 1 mL

• Useful to be able to give an indication of the amount of material in solution (concentration) without needing to know molar mass

• Use percent weight / volume (% w / v) method of expressing concentration• The % w / v of a solution is defined as the number of g of material

dissolved in 100 cm3 of solvent.

Concentration as Percentage

Work These Out on Your Own…Remember…SHOW ALL WORK and UNITS

Simple Dilution• A simple dilution is one in which a unit volume of a liquid material of

interest is combined with an appropriate volume of a solvent liquid to achieve the desired concentration

• The dilution factor is the total number of unit volumes in which your material will be dissolved

• The diluted material must then be thoroughly mixed to achieve the true dilution

• Use simple dilution procedures when asked to MAKE A RANGE OF CONCENTRATIONS

• You will determine the % concertation and then show how you made them

• Hint:• 1% solution = 1 gram of something dissolved in 100 mL of water• Or 1 mL of something dissolved in 100mL of water

Making fixed volumes of specific concentrations from liquid reagents• You will need to make a specific volume of known concentration from stock

solutions• First determine what volume your different concentrations will be (V2)

– Sometimes the exam will tell you to make 10 mL samples or 20mL sample • Formula:• V = volume, C = concentration; in whatever units you are working.• (stock solution attributes) V1C1=V2C2 (new solution attributes)• Unknown is V1, so rearrange to look for your unknown• V1 = V2C2 

C1• V1 is the volume of stock solution you will need to make your new

desired concentration– You will add this volume to test tube, then add water up to make the total

volume equal to V2

Example• You have a 1% glucose stock solution, make

a range of concentrations (with a volume of 10 mL each) to test using this stock solution

• Hint: Use this formula• V1 = V2C2 

C1• First determine what concentrations you

want to make….– Let’s say… 0.75%, 0.50%, 0.25%, 0.10%

• V1= ?• V2= 10 mL• C1 = 1 g/100mL or 0.01 g/mL• C2 =range of concentrations

you use (0.5% or 0.005 g/mL)• This value will change

• A serial dilution is simply a series of simple dilutions which amplifies the dilution factor quickly beginning with a small initial quantity of material

• Source of dilution material (solute) for each step comes from the diluted material of the previous dilution step

Serial Dilution

Warm Water Bath• Materials

– Large beaker of water– Thermometer– Ring stand– Bunsen burner– Wire gauge– Thermometer clamp

• Measure temperature carefully– Use thermometer in water– Cannot touch bottom of beaker of water

or the side– Read thermometer while it is in the

water– Allow test tubes in warm water bath to

reach same temp as water• Should measure actual temp of liquid in tubes

Accuracy, Precision, Reliability• Accuracy

– How true measurement is– Depends on the calibration of instrument you are using

• Precision– Ability of the measuring instrument to give the same reading every time

you use it– Doesn’t have to be the “true” value– Repeatable, reproducible

• Reliability– Degree of trust you have in the instrument– If measurements are reliable, then you expect to get same

measurements in repeated experiment on that tool– Affected by ACCURACY and PRECSION and TYPE of measurements

being taken– Best way to deal with poor reliability: REPEAT readings several times

(multiple trials at same time)

Preparing Tables• Create a data table ALWAYS• Make table BEFORE you start• Independent Variable should be in the FIRST column• The readings/measurements you take in the next columns

– Sometimes second column will be dependent variable– Sometime you will have multiple columns because you need to calculate

the dependent variable

• Lines should be clearly drawn with a ruler and pencil• Each column must be FULLY headed including the unit of

measurement– Use a slash or brackets to denote the unit…be consistent

• length of stem/mm OR length of stem (mm)• Don’t use slash to denote “per”…use negative exponents (-1) or the word “per”• Velocity/ms-1 OR velocity/m per s

• DO NOT put units in the boxes with the actual measurements (only in headings)

Percentage of Glucose Solution (%)

Time for color change (seconds)

1.000

0.750

0.500

0.250

0.125

Preparing Tables

More on Tables

• If you have to do calculations….– Use significant figures

• Answers should have same amount of sig figs as measurements in calculations

• Keep same decimal places throughout table

Suggesting Improvements

• How could you get more valid or reliable result?– Suggest controlling certain variables better in your

experiment– You MUST state how you would better control the

variable– Examples:

• Using a thermostatically controlled warm water bath instead of Bunsen burner

• Using a colorimeter instead of color standards• DO several more repeats of experiments to get an

average

Practice Problems Set 5: #1 Simple Dilution (June 2011)

Practice Problems Set 5: #2 Serial Dilution (June 2014)

Practice Problems Set 5: #3 Serial Dilution (June 2015 Serial Dilution)

Practice Problems Set 5: #4 Serial Dilution (Nov. 2013 Simple Dilution)