[Worksheet D: Method – test-tube set-up for all methods

Write down your food samples next to the numbers in the box below. Then label your test-tubes with a
number to identify what food sample is in each test-tube. You will not use all the numbers if you are testing
less than 6 food samples.

Test-tube labels:

0 = control

1 = liquid

2 = solid

3 = solid

4 = solid

5 = liquid
 Worksheet D: Method – Benedict’s test for sugars
1. Collect your equipment and label the tubes according to your completed Worksheet F, using
adhesive labels.

2. Set up a water-bath at 80°C. Monitor the temperature throughout the experiment.

Why should the temperature be monitored throughout?

So the water can be a control

3. Use a graduated pipette to add 1cm3 of distilled water to tube ‘0’. This is the control.
Why is it important to have a control sample?
So that analysis are properly performed for reliable results
4. Separate the food into solid or liquid samples.

5. Put a solid food item onto a white tile.

6. Use a scalpel or knife to cut the food into 1 piece that is about 1 cm by 1 cm in size.
7. Use a pestle and mortar to grind this piece into small crumbs.
8. Place the crumbs into an unlabelled tube; you can use a piece of folded paper to help. Pour
the crumbs onto the folded paper and collect them in the fold; then use the folded paper to
funnel the crumbs into the tube.

9. Use a graduated pipette to add 1cm3 of distilled water to the crumbs and stir using a clean
glass rod until the crumbs look roughly evenly distributed in the water.

10. Use a dropper pipette to extract the liquid and leave the solid behind.

11. Place the liquid into the correctly labeled tube; you have created a liquid food extract.
Why is a liquid food extract made? Hint: reducing sugars are soluble in water
To transfer unreacted reactants, salts, and solubles to aqueous phase
12. Follow steps 5 to 11 for all solid food samples.

13. Use a graduated pipette to add 1cm3 of the liquid food samples to the correctly labeled tubes.

14. Use a graduated pipette to add 1cm3 of Benedict’s reagent to each sample. Stir the
samples using a clean glass rod for each sample.
Why is it important to add the same volume of reagent to each sample?

To have the same rate of reaction between samples

Why do you need to stir the samples?

To make sure the sample and the reagent fully react

Why is it important to use a clean glass rod?

To avoid contamination or breakage in the substance mixed

15. Put the samples into the water-bath and leave for 2 minutes.

Why do you think the samples should be left in the water-bath for 2 minutes?

To incubate the sample at a constant temperature

16. Remove the samples from the water-bath using test-tube holders and put them into a
rack. Do not touch the hot tubes with your hands.
17. Record your observations. Compare your sample with the control, what changes can you
see? What has happened? What does this suggest?

- Sample 1 : from blue to blue which means there is no reducing sugar

- Sample 2 : from blue to dark matcha green which means there are traceable sugar
- Sample 3 : from blue to matcha green which means there are traceable sugar
- Sample 4 : from blue to yellow which means it contain low sugar
- Sample 5 : from a dark color solution to brick color which mean it has high sugar
percentage
 Worksheet D: Method – biuret test for protein
1. Collect your equipment and label the test-tubes according to your completed Worksheet F,
using adhesive labels.

2. Use a graduated pipette to add 1 cm3 of distilled water to test-tube ‘0’. This is the control.
Why is it important to have a control sample?
3. Separate the food into solid or liquid samples.

4. Put a solid food item onto a white tile.

5. Use a scalpel or knife to cut the food into 1 piece that is about 1 cm by 1 cm in
size.

6. Use a pestle and mortar to grind this piece into small crumbs.
7. Place the crumbs into an unlabelled tube; you can use a piece of folded paper to help. Pour
the crumbs onto the folded paper and collect them in the fold; then use the folded paper to
funnel the crumbs into the tube.

8. Use a graduated pipette to add 1cm3 of distilled water to the crumbs and stir using a clean
glass rod until the crumbs look roughly evenly distributed in the water.

9. Use a dropper pipette to extract the liquid and leave the solid behind.

10. Place the liquid into the correctly labeled tube; you have created a liquid food extract.

11. Follow steps 4 to 10 for all solid food samples.

12. Use a graduated pipette to add 1cm3 of the liquid food samples to the correctly labeled tubes.

13. Use a graduated pipette to add 1 cm3 of biuret reagent to each sample. Stir the samples using
a clean glass rod for each sample.

14. Leave the samples for 2 minutes.

Why do you need to leave the samples for 2 minutes?
To let the solute completely dissolve into the solvent.

15. Record your observations. Compare your sample with the control, what changes can you see?
What has happened? What does this suggest?

- Sample 1 : blue to blue with clear residue at the bottom which mean no or little protein
- Sample 2 : blue with residue to yellow with light purple which mean there is protein
- Sample 3 : blue with residue to brown
- Sample 4 : thick yellow to thick yellow
- Sample 5 : coffee brown to coffee brown
 Worksheet D: Method – iodine test for starch
1. Collect your equipment and label the test-tubes according to your completed Worksheet F, using adhesive
labels.
2. Use a graduated pipette to add 1cm3 of distilled water to tube ‘0’. This is the control.
3. Separate the food into solid or liquid samples.

4. Put a solid food item onto a white tile.

5. Use a scalpel or knife to cut the food into 1 piece that is about 1 cm by 1 cm in size.

6. Place the solid food piece into the correctly labeled test-tube.
A liquid food extract is made from solid foods when testing for other nutrients but it is not needed
when testing for starch because starch is not very soluble in water. Why might a liquid food extract be
needed when testing for other nutrients?
Because in other substance they need protein and sugar which can dissolve in water unlike starch

7. Repeat steps 4 to 6 for all solid food samples.

8. Use a graduated pipette to add 1 cm3 of the liquid food samples to the correct test-tubes. 9.
Use a graduated pipette to add 1 cm3 of iodine to each sample.
10. Stir the liquid samples gently using a clean glass rod for each sample.

11. Leave the samples for 2 minutes.

12. Record your observations. Compare your sample with the control, what changes can you see? What has
happened? What does this suggest?
- Sample 1 : turns to dark clear yellow which means there is barely any starch
- Sample 2 : turns to dark blue which means there are 0.1% of starch
- Sample 3 : turns black which means there are 1% of starch solution
- Sample 4 : turns brown which means there is starch
- Sample 5 : turns black which there is starch
 Worksheet D: Method – emulsion test for fats
1. Collect your equipment and label the test-tubes according to your completed Worksheet F, using
adhesive labels.

2. Use a graduated pipette to add 1cm3 of ethanol to tube ‘0’. This is the control.
3. Separate the food into solid or liquid samples.

4. Put a solid food item onto a white tile.

5. Use a scalpel or knife to cut the food into 1 piece that is about 1 cm by 1 cm in size.

6. Use a pestle and mortar to grind this piece into small crumbs.
7. Place the crumbs into an unlabelled tube; you can use a piece of folded paper to help. Pour the
crumbs onto the folded paper and collect them in the fold; then use the folded paper to funnel the
crumbs into the tube.

8. Use a graduated pipette to add 1cm3 of ethanol to the crumbs and stir using a clean glass rod until
the crumbs look roughly evenly distributed.

9. Use a dropper pipette to extract the liquid and leave the solid behind.
10. Place the liquid into the correctly labeled tube; you have created a liquid food extract.

Why do you think a liquid food extract is made from solid food samples?
For other nutrients, distilled water is used to make the food extract.

So substance in solids that are soluble can dissolve well

Why might ethanol have been used for fat?

Because lipids are soluble in alcohols
11. Follow steps 4 to 10 for all solid food samples.
12. Use a graduated pipette to add 1 cm3 of the liquid food samples to the correct test-tubes.
13. Use a graduated pipette to add 1 cm3 of ethanol to each liquid food sample.
14. Stir all the samples using a clean glass rod for each sample.

15. Leave the samples for 2 minutes.

16. Use a graduated pipette to add 1 cm3 of distilled water to each sample.

17. Record your observations. Compare your sample with the control, what changes can you
see? What has happened? What does this suggest?

- Sample 1 : stays clear which means no fat

- Sample 2 : turns muddy white which means not many lipids is detected
- Sample 3 : turns muddy white which means not many lipids is detected
- Sample 4 : turns milky which-yellow which means lipid is detected
- Sample 5 : still stay brown and no changes
 Hypothesis:
Benedict Test:
Results supposed to show colors and results of blue(negative), green(positive{traceable}),
orange(positive{moderate), brick red(positive{high})
Biuret Test:
Results supposed to show color of purple(positive) and blue(negative)
Iodine Test:
Results supposed to show color black(positive) and lighter brown(negative)
Ethanol Test:
Results supposed to show a layer of white liquid on top(positive)
Hypothesis for nutrients that the samples tested positive for:
Control: None, Egg white: Glucose, Koya: Glucose, protein, starch, fat, Powdered Milk: Glucose,
protein, starch, fat, Crushed Crackers: Glucose, protein, starch, fat, Soy Sauce: Glucose, protein,
starch.

Results table for Benedict’s test for sugars

Sample Observation Result

Control (0) No changes in color No Sugar

Egg White (1) blue to dark matcha Traceable sugar

green

Koya (2) Blue to green Traces of reducing sugar

Powdered Milk(3) Blue to green Traces of reducing sugar

Crushed Crackers(4) Blue to yellow Moderate level of sugar

Soy Sauce (5) Dark color solution to High Level of sugar

brick color

Results table for Biuret’s test for Protein

Sample Observation Result

Control (0) No changes in color No Protein

Egg White (1) Blue to dark blue No Protein

Koya (2) Blue to dark blue + Low protein

yellow

Powdered Milk(3) Dark blue Low protein

Crushed Crackers (4) Blue to yellow Moderate protein

Soy Sauce (5) Blue to dark brown High protein

Results table for Iodine’s test for Starch

Sample Observation Result

Control (0) No changes in color No starch

Egg White (1) Dark clear yellow No starch

Koya (2) Dark blue 0,1% starch solution

Powdered Milk(3) Black 1% starch solution

Crushed Crackers Brown Starch positive

(4)

Soy Sauce (5) Black Starch positive

Results table for Ethanol test for Fat

Sample Observation Result

Control (0) Transparent Negative

Egg White (1) Transparent Negative

Koya (2) Translucent Lipids detected

Powdered Milk (3) Translucent Lipids detected

Crushed Crackers (4) Light Yellow Positive for fats

Soy Sauce (5) Brown Negative

Interpreting and evaluating Interpreting your results

1. Look at your hypothesis. Was it correct? Use data to support your answer
Based on the results, the results color shown has different variations, this may be caused by errors during
the process and some of the final results of nutrients present may not be fully proven correct.
2. Were there any surprises in your results? Refer to your observations in your answer.
Benedict Test:
There was a lighter green precipitate on the bottom of the test tube on sample 3, on sample 5, it
shows a brown color instead of an expected color result (either blue, green, orange/yellow, brick
red)
Biuret Test:
For sample 4 and 5, it shows a unique color showing the color black and yellow, excluding the
expected results of either blue or purple, on sample 2, it shows 2 different yellow precipitate on
both above and below inside the test tube
Iodine Test:
The whole sample results shows expected results, for sample 2, it shows a blended in colour
between brownish yellow and black
Ethanol Test:
All the samples show a thin layer of Precipitate of lipid detected, but sample 4 shows the thickest
layer and yellow translucent color, meanwhile sample 5 shows a dark brown color, sample 2 and 3
shows translucent color and shows a tiny layer. This shows unexpected color results,
3. Were there any samples where it was not clear if the result of the test was positive or negative?
Refer to your observations and describe the tests and foods that had unclear results. How did
you decide if the result was positive or negative?
Benedict Test:
For sample 3, it is assumed that it has the full color green, meanwhile sample 5, which is assumed
as a “brick red” since the color tone was closest to its color to identify as high sugar level. However,
through research, brown color was one of the visual results showing it has a high sugar level.
Biuret Test:
Sample 4 was identified as positive with protein since according to research, proteins having these
amino acids give yellow color when heated with conc. HNO3. , meanwhile the black color on
sample 5 may indicate the sample has too much higher concentration than the biuret.
Iodine Test:
Sample 2 was identified as negative, since it has more tendency to the color yellow
Ethanol Test:
Sample 2 and 3 shows a thin layer, identifying a lipid detected, meanwhile sample 4 shows a little
bit thicker layer, so it is identified as positive.

Other results not mentioned identified as negative

Food sample Nutrients that sample tested positive for

Control (0) None

Egg White (1) Glucose

Koya (2) Glucose, protein, starch, fat

Powdered Milk (3) Glucose, protein, starch, fat

Crushed Crackers (4) Glucose, protein, starch, fat

Soy Sauce (5) Glucose, protein, starch

4. What can you conclude from your results?

Koya, powdered milk, crushed crackers has the most nutrient founded,with crushed
crackers have the highest concentration each of these nutrients among the 3 samples
mentioned, soy sauce only has glucose, protein, and starch, and has negative results for
fat, egg white only has glucose, and water has no nutrients
 Evaluating the data collection
1. Why is the position of the test-tube labels important?
We should position the labels on top of the test tube so when we boil the test tube, the label don’t fade and
dissolve in the water
2. Why is it OK to only use roughly the same volume of food sample each time?
If the food samples have different volume, the diffusion of the chemical substance(biuret,
benedict, etc) may spread inside the food sample with different amounts and affect the
concentration mixed in it, producing inaccurate results.
3. Why is it important to clean the pestle and mortar and glass rod between uses?
To avoid contamination or breakage in the substances mixed.
4. Why is it important to add the same number of drops of reagent to each sample?
Because if it wasn’t the same volume of a reagent, it would not represent a comparable concentration to
the solvent result,
5. Why do samples need to be mixed / stirred after the reagents have been added?
Mixing the solutions can create kinetic energy that makes the solution and the reagent
diffuse faster to combine with each particle to each other.
6. Why does Benedict’s solution need to be heated? The formation of oxides in the
solution can confirm the presence of reducing sugar. Why must each sample be
incubated at the same temperature? So all the temperatures can be controlled and
stay the same which makes a more accurate result because the oxides in the solutions
stay present, so it needs to reduce the copper and cause a red copper (I) oxide
precipitate to reduced to have clear better result

7. Why do you need to leave each sample with the reagent for two minutes?
To make sure the samples are controlled at the same temperature.

8. What is a control sample and why is it important to have one in the investigation?
Explain your answer. Control sample is a sample that is used as reference to samples
that we will analyze. Having a control sample is important because by having a control
sample,we know that the tested sample is not just a random event and has comparable
media.
9. Using your answers to the above question, can you identify any limitations to the data
you have collected? How might the limitations in the data collection have affected
your results? HINT: Look at the method and write down any issues that you can identify that
would make the results less meaningful.
During some of the test, the test tube and liquids in it didn’t mixed properly, so it can’t be
obtained fully accurate result
10. How could the method be improved to reduce the limitations? Suggest any
adaptations that would make the data more valid. Explain your suggestions.
- Use a more accurate measuring instrument (which displays the precise and accurate amount in
units) to measure the amount of samples, before carrying out the experiment.
- Make sure to keep the temperature constant throughout the entirety of the heating process, to
make sure the reaction is carried out as expected.
- Measure the time with more validity, by starting and stopping the timer at the exact starting and
ending points
.
11. Use examples from your results to suggest what is meant by the term ‘balanced diet’.
Is it the same for everyone? Give examples of when it might be different.
A balanced diet identifies as containing the right amount of nutrients that are essential. Every
one of the food samples needs certain amounts of certain nutrients in order to be immune
from probable harm. The reasons why a balanced diet is different for some food samples
include the rate at how they function. This means that, with certain foods functioning
differently, they must be ensured to have contained every needed nutrient, depending on the
food.

Reference:
https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/biuret-test