AICE Biology Lab Review: Biological Molecules Prepared by Ms. Evans at GHS Lab Review ppt: Biological Molecules & Enzymes

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; Lab Review ppt: Biological Molecules & Enzymes

Benedict’s Test: Reducing & Non Reducing Sugars, Round 1 Testing for simple sugars: Benedict's Reagent, round 1 Procedure: Add Benedicts reagent to sample, place in waterbath until just boiling. Left to right: Benedict's reagent (BnR), potato extract + BnR, onion extract + BnR, 5% glucose + BnR. RESULTS: Aqua-blue = negative. Green to Yellow to orange = positive. Note: to detect the simple sugar sucrose, you must do round 2 test Lab Review ppt: Biological Molecules & Enzymes

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. Lab Review ppt: Biological Molecules & Enzymes

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??? Lab Review ppt: Biological Molecules & Enzymes

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? Lab Review ppt: Biological Molecules & Enzymes

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? Lab Review ppt: Biological Molecules & Enzymes

Testing for Starch IKI (Iodine in Potassium Iodide) Left to right: IKI only, starch solution, starch solution + IKI. RESULTS: Yellow-orange = negative. Purple-black = positive. Lab Review ppt: Biological Molecules & Enzymes

Emulsion Testing for Lipids Sample 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. Lab Review ppt: Biological Molecules & Enzymes

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. Lab Review ppt: Biological Molecules & Enzymes

Acid hydrolysis then Benedict’s Identify each sample Food 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 + B C D (KEY: + = positive result , X = negative result) Lab Review ppt: Biological Molecules & Enzymes

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 Serial 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 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

Concentration as Percentage 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.

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

Serial Dilution 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

Warm Water Bath Materials Measure temperature carefully 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 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) Preparing Tables Percentage of Glucose Solution (%) Time for color change (seconds) 1.000 0.750 0.500 0.250 0.125

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)