L6 Lipids Lab - Lecture notes 6 PDF

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BIOCHEMISTRY LAB Lesson 6: Lipids

LIPID SAPONIFICATION

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Soap is essential to our personal life. Soaps are made from fats, oils or their fatty acids.

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Soap is the sodium salt of long chain fatty acids which have cleansing properties in water. The most common process of making soap is saponification, in which triglycerides present in fats and oils react with alkalis (such as sodium hydroxide or potassium hydroxide) to form soap and glycerol. If sodium hydroxide is used as alkali, the soap formed is called hard soap and is used as washing soap. If potassium hydroxide is used as alkali the soap formed is soft soap and is used in some liquid hand soaps and shaving creams.

Materials ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪

Vegetable oil (castor oil, olive oil, coconut oil or palm oil) 20% sodium hydroxide solution Common salt Measuring cylinders Glass beaker (250 ml) Blue and red litmus papers Glass rod Filter funnel Filter paper Iron stand Hot plate Spatula Knife

Procedure

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If the beaker is warm, the reaction between the vegetable oil and sodium hydroxide solution is exothermic. ▪ ▪

Take a measuring cylinder. Take a beaker containing coconut oil and pour 25ml of coconut oil into the measuring cylinder.

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Take the beaker containing the mixture and place it on a hot plate. Heat the beaker using the hot plate till the mixture becomes a whitish paste.

The white suspension formed is made up of soap and glycerol. This process of formation of soap is called saponification. ▪ ▪

Remove the beaker from the flame and allow it to cool. Take a red litmus paper and dip it in the suspension formed.

Red litmus paper changes color to blue, indicating that soap suspension is basic in nature. Take a blue litmus paper and dip it in the suspension. The color of blue litmus paper remains the same, indicating that soap suspension is not acidic in nature. ▪ ▪ ▪

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Transfer it into a 250ml beaker. Take the beaker containing 20% sodium hydroxide solution. Pour 30ml 20% sodium hydroxide into another measuring cylinder. Transfer it into the beaker containing coconut oil. Vigorously stir the mixture using a glass rod. Touch the beaker from outside.

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Take 15 g common salt. Add it into the suspension and stir it well with a glass rod. After adding common salt, soap is precipitated out as a solid. Take a filter funnel and place the filter paper in it.

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BIOCHEMISTRY LAB Lesson 6: Lipids

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Fix it in a stand and place a beaker below the funnel. Pour the contents of the beaker into the funnel over a glass rod and filter the contents of the beaker. After filtration, soap is left behind in the filter paper. Transfer the soap into another filter paper using a spatula, then dry it by pressing another filter paper. Soap is prepared.

Precautions ▪ ▪ ▪

Do not touch the NaOH solution with bare hands as it may burn the skin. Do not breathe the fumes of NaOH. The mixture of oil and alkali should be stirred thoroughly. It is necessary to stir the soap solution after adding common salt to it, in order to precipitate out the soap in solid form.

The emulsion test is an example of qualitative test, this means it can test for the presence or absence of a substance but cannot tell you exactly how much of the substance is present.

Sample

Observation (after adding 2cm3 water)

Lipids present?

Proteins Liquid sample

Vegetable oil Tap water Sucrose Pasta

Solid sample

Bread Cheese

LIPID EMULSION TEST Apple Emulsion test is used for testing the presence of lipids and is relevant to both GCSE and Alevel. The emulsion test involves adding ethanol to sample followed by the addition of water. This is because lipids can’t dissolve in water due to their hydrophobic nature they can’t form hydrogen bonds with water so they can’t dissolve into it, but they can dissolve in ethanol. If lipids are present in a sample, they will dissolve in the ethanol and when water is added, the lipids will form tiny dispersed droplets in the water called an emulsion and will appear white and cloudy.

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Procedures (Liquid) PROTEINS ▪ Put a few drops of the protein suspension sample into its own labeled test tube using a dropping pipette. ▪ Add 2cm3 of ethanol using a syringe. ▪ Shake the test tube containing the protein suspension ethanol. ▪ Add 2cm3 of distilled water.

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BIOCHEMISTRY LAB Lesson 6: Lipids

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Notice that the protein suspension and ethanol is a cloudy mixture before you even added the water. Cloudiness doesn’t change even after adding the water. No lipids present.

Cloudy mixture means there are no lipids present because we are expecting to see a white emulsion appear after water is added. VEGATABLE OIL ▪ Put a few drops of vegetable oil sample into its own labeled test tube using a dropping pipette. ▪ Add 2cm3 of ethanol using a syringe. ▪ Shake the test tube containing the protein suspension ethanol. ▪ Add 2cm3 of distilled water. ▪ You can clearly see a white emulsion. ▪ Lipid is present. TAP WATER ▪ Put a few drops of tap water sample into its own labeled test tube using a dropping pipette. ▪ Add 2cm3 of ethanol using a syringe. ▪ Shake the test tube containing the protein suspension ethanol. ▪ Add 2cm3 of distilled water. ▪ No changes in the mixture, the tap water remains colorless ▪ No lipids present. SUCROSE ▪ Put a few drops of sucrose sample into its own labeled test tube using a dropping pipette. ▪ Add 2cm3 of ethanol using a syringe. ▪ Shake the test tube containing the protein suspension ethanol. ▪ Add 2cm3 of distilled water. ▪ Solution remains colorless. ▪ No lipids present.

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Procedures (Solid) PASTA ▪ Cut a piece of soaked pasta and placed it into its test tube. ▪ Add 2cm3 of ethanol using a syringe. ▪ Use a glass rod to gently crush the food sample into the ethanol. Be careful not to make a hole in the bottom of the test tube. ▪ Shake the sample and allow it to settle. ▪ Carefully pipette the ethanol from the sample and transfer it to another clean test tube leaving the solid particles behind. ▪ Add 2cm3 of distilled water. ▪ Cloudy appearance remains. ▪ No lipids present. BREAD ▪ Cut a piece of soaked pasta and placed it into its test tube. ▪ Add 2cm3 of ethanol using a syringe. ▪ Use a glass rod to gently crush the food sample into the ethanol. Be careful not to make a hole in the bottom of the test tube. ▪ Shake the sample and allow it to settle. ▪ Carefully pipette the ethanol from the sample and transfer it to another clean test tube leaving the solid particles behind. ▪ Add 2cm3 of distilled water. ▪ It turns to slightly cloudier mixture. ▪ Small quantity of lipid is present, but in a low concentration. ▪ Lipid is present.

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BIOCHEMISTRY LAB Lesson 6: Lipids

CHEESE ▪ Cut a piece of soaked pasta and placed it into its test tube. ▪ Add 2cm3 of ethanol using a syringe. ▪ Use a glass rod to gently crush the food sample into the ethanol. Be careful not to make a hole in the bottom of the test tube. ▪ Shake the sample and allow it to settle. ▪ Carefully pipette the ethanol from the sample and transfer it to another clean test tube leaving the solid particles behind. ▪ Add 2cm3 of distilled water. ▪ White emulsion is very clear. ▪ Lipids are present. APPLE ▪ Cut a piece of soaked pasta and placed it into its test tube. ▪ Add 2cm3 of ethanol using a syringe. ▪ Use a glass rod to gently crush the food sample into the ethanol. Be careful not to make a hole in the bottom of the test tube. ▪ Shake the sample and allow it to settle. ▪ Carefully pipette the ethanol from the sample and transfer it to another clean test tube leaving the solid particles behind. ▪ Add 2cm3 of distilled water. ▪ Remains clear. ▪ No lipids present.

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Observation (after adding 2cm3 water)

Lipids present?

Proteins

Cloudy

No

Vegetable oil

White emulsion

Yes

Tap water

Colorless

No

Sucrose

Colorless

No

Pasta

Cloudy

No

Bread

Becomes cloudy

Yes

Cheese

White emulsion

Yes

Apple

Colorless

No

Sample

Liquid sample

Solid sample

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