Dry Matter Determination

Lesson 1: Dry Matter

• Involves grinding bean samples to roughly 0.75 mm particle size.

• Use a desiccator, drying the desiccant at 70°C beforehand.

• Dry a ceramic cup (VCK) at 70°C to achieve constant mass and record this mass.

• To Perform: Weigh 3-5 g of bean powder into a porcelain cup (m2).

• Dry the sample at 70°C until constant mass is achieved.

• Reweigh the porcelain cup with the dried sample (m3).

• Determine dry matter mass using the formula: m4 = m3 - cup DM.

• Calculate the percentage of dry matter.

• Evaporation of water occurs when drying the sample.

• A mild thermal decomposition reaction can occur chemically if temperatures exceed 70°C.

• Original bean powder has natural colors: green, red or white.

• After drying, samples may appear paler or slightly darker due to dehydration.

• The reaction is complete when the mass remains constant across consecutive weighings (≤ 0.001 g difference).

• Use bean samples, ceramic cup, and temperature to remove water.

Lesson 2: Carbohydrates

Starch reaction to color with iodine

• Starch and iodine solution gives a blue-black solution

• Prepare 5 mL of 1% starch solution in a test tube.

• Add 2 drops of iodine solution.

• Observe the color change during and after about 5 minutes of heating.

• Let cool and observe further.

• The mixture turns blue-black when iodine solution is initially added to starch paste, indicating starch presence.

• The blue-black color disappears upon heating, thus indicating starch structure is broken, and iodine no longer interacts strongly.

• The blue-black color can reappear when cooled, as starch restructures.

• Add 5 mL of starch solution to a test tube.

• Add 2 drops of iodine solution.

• Observe color.

• Heat the test tube for about 5 minutes.

• Take out of the test tube, let cool, and observe again.

• Starch paste (colorless/opaque white) plus iodine (brown) forms blue black mixture.

• Heating causes the blue-black color disappears (colorless).

• The blue-black color may reappear on cooling.

Starch has no reducing properties

• Add 1% starch solution in the test tube.
• Add Fehling's solution (A and B in 1:1 ratio).
• Heat in a water bath for about 3 minutes.
• Observe and draw conclusions.
• Use Fehling's reagent to test the reducing properties of starch.
• If the solution contains a reducing agent, Fehling's solution turns from blue to brick red due to Cu2O precipitate formation.
• Without reducing agents, the solution remains blue.
• First add starch solution to test tube, add Fehling solution A and B, heat in boiling water for 3 minutes, and check the color.
• Starch solution is opalescent.
• Fehling's solution (A+B) is blue.
• Solution after boiling remains blue if not reducing.
• Solution after boiling forms brick red precipitate upon the presence of reducing agents

Starch Hydrolysis reaction

• Acid hydrolysis of starch, hydrolyzing starch into simpler sugars (maltose or glucose).

• Add 5 mL of 1% starch solution and 1 mL of concentrated HCl solution to a test tube, then stir well.

• Boil this solution.

• Take a drop of the hydrolyzing solution and add it to a drop of iodine solution on the watch glass plate every minute.

• Track color change.

• Keep heating and testing with iodine until the solution no longer changes color.

• Save the hydrolyzed solution for future experiments.

• Starch paste plus iodine initially turns blue-black, implying starch presence.

• The blue-black gradually fades as the starch hydrolyzes into smaller molecules like maltose or glucose.

• Heat in a water bath, checking the color every minute by dropping a drop of solution onto the iodine on the glass plate, stop when iodine no longer changes color.

• The iodine solution is no longer blue-black when the reaction ends, so the starch has been completely decomposed.

• Starch paste (colorless/opaque white) plus Iodine (brown) forms blue black solution

• Heating with HCl causes it to fade to colorless as hydrolysis completes.

• The final product monosaccharides or oligosaccharides.

Reaction With Fehling's Reagent

• Redox- reaction with Fehling's reagent (determines the presence of reducing sugars such as glucose or maltose).

• Add 2 mL of 1% glucose solution and 2 mL of neutralized hydrolyzed starch solution to separate test tubes.

• Add 3 mL of Fehling's reagent (A:B, 1:1 ratio) to both test tubes.

• Heat both test tubes in a water bath until color changes.

• Then record results.

• The solution is initially blue because of copper ions Cu2+ in Fehling's reagent.

• If reducing sugars are present, the reaction converts ions (blue) to Cu2O (brick red precipitate)during heating.

• A brick red precipitate indicates reducing sugar presence when the reaction is complete.

• Test tube 1 contains 2 mL of 1% glucose, test tube 2 contains 2 mL of neutralized hydrolyzed starch.

• Add 3 mL of Fehling's reagent ( A:B, ratio 1:1) to both test tubes.

• Heat in a double boiler and observe the color change.

• Glucose + Fehling (blue) -> Brick red precipitate (Cu2O) .

• Hydrolyzed starch (maltose, glucose) + Fehling -> Brick red precipitate (Cu2O).

• Unhydrolyzed starch + Fehling -> No precipitate, solution remains blue.

Lesson 3: Lipids

Saponification Reaction

• Related to ester hydrolysis in alkaline environment.
• Prepare two triangular flasks:
  • Tube 1 (control): distilled water and KOH.
  • Tube 2 (real sample): oil and KOH.
• Heat in mild water bath for 45 minutes with a condenser to react.
• Cool the solution.
• Add 2 mL of distilled water and 2-3 drops of phenolphthalein.
• Titrate with 0.5N HCl in alcohol until pink disappears.
• Record the volume of HCl consumed to calculate the saponification value.
• The solution is initially pink due to phenolphthalein in an alkaline environment.
• When titrating with HCl, the acid neutralizes excess KOH, solution loses its pink color.
• The reaction is complete when the solution is no longer pink, excess KOH has been neutralized.
• Prepare 2 triangle flasks with ingredients, boil in water bath for 45 minutes with a condenser, cool, add distilled water and phenolphthalein.
• Titrate with 0.5N HCl until loses pink color.
• Oil + KOH -> Glycerol + soap salt (colorless).
• Phenolphthalein -> Pink in alkaline environment.
• HCI 0.5N -> Neutralizes KOH, removes pink color.

Iodine Index

• Related to Iodine absorption reaction of double bonds in unsaturated fatty acids.
• Prepare two test tubes:
  • Tube 1 (control): distilled water, alcohol, 0.1N iodine.
  • Tube 2 (real sample): oil, alcohol, 0.1N iodine
• Shake well. leave in the dark for 30 mins to let iodine react with the double bond of unsaturated fatty acids.
• Titrate with 0.1N Na2S2O3 until the solution turns dark yellow.
• Add 1 - 2 drops of 1% starch. Blue means need for titration ,continue with Na2S2O3 until very pale straw yellow color.
• Record Na2S2O3 consumed to determine iodine index.
• Solution is initially dark yellow because the iodine has not completely reacted.
• Excess iodine is gradually reduced when titrating with Na2S2O3, yellow color gradually fades.
• Add 1% starch solution and the solution turns dark blue with excess Iodine present.
• Continue titration until the solution is very pale straw yellow (reaction ends).
• Prepare test tubes, add 0.1N iodine and shake well, let stand for 30 mins in the dark.
• Titrate with 0.1N Na2S2O3 until turns dark yellow, add 1% starch which produces a blue color .Continue titration.
• Then stop when the solution is a very pale straw yellow.
• Oil + Iodine 0.1N -> Iodine binds to double bonds in unsaturated fatty acids.
• Starch 1% -> Creates blue color with excess iodine.
• Na2S2O3 0.1N -> Reduce excess iodine, remove green color turning to straw yellow.

Lesson 4: Vitamins

Reaction to form thiochrome

• The reaction involves oxidation-reduction.

• B1 tablet is crushed and dissolved with 10 mL distilled water, filter to collect.

• Then prepare test tubes.

• Add 2 mL B1 solution, 1 mL of 15% NaOH for alkaline environment.

• Add 0.5 mL of 1% K3Fe(CN)6 and 2 mL isoamyl alcohol to extract.

• Shake well, let stand for 5 min and observe under sunlight or UV light.

• The solution has no initial color.

• Yellow or green fluorescence appears under UV light when adding K3Fe(CN)6 and NaOH if Vitamin B1 is present.

• Fluorescence intensity correlates with Vitamin B1.

• Add solution B1, 15% NaOH, 1% K3Fe(CN)6, and isoamyl alcohol to the test tube.

• Then, shake contents vigoursly.

• Lastly stand for 5 mins before observing under sunlight or UV light .

• Vitamin B1 --> Thiochrome (fluoresces blue under UV) .

• NaOH 15% --> Alkaline environment supports the reaction.

• K3Fe(CN)6 1% --> Oxidizing agent converts B1 to Thiochrome .

• Isoamyl --> Thiochrom extraction solvent.

Reaction with Diazo Reagent

• a Diazolation reaction between vitamin B1 and diazo compound.

• To carry it out, prepare test tubes.

• Add 0.5 mL Vitamin B1 solution, 0.5 mL Sulfanilic acid, 10 mL sodium nitrite and 1 mL sodium carbonate solution.

• Shake to let the reaction occur and observe the color.

• Solutions containing Vitamin B1 will produce a pink-red color using Diazo reagent.

• Solutions lacking Vitamin B1 will not change color.

• Add Vitamin B1, 1% Sulfanilic, 5% Sodium nitrite, and 10% Sodium carbonate to test tubes.

• Shake well and observe results.

• The darker the red color, the higher concentration of Vitamin B1.

• Vitamin B1: Diazo complex (pink red).

• Sulfanilic 1%: Creates Diazo reaction.

• Sodium nitrite 5%: Supports Diazo group creation.

• Sodium carbonate 10%: Alkaline environment aid reaction.

Vi-B2 Qualitative

• Vitamin B2 reduction reaction produces leuco-riboflavin.

• Weigh and dissolve 1 B2 tablet into 5 mL of distilled water, filter to collect.

• Draw the chemicals into the test tube, 0.5 - 1 mL vitamin B2 solution.

• Add acid, zinc to create reduction reaction.

• Observe changes.

• Vitamin B2 the solution has characteristics (yellow), and when HCl, zinc are added, it becomes colourless (leuco-riboflavin).

• Add a little concentrated HCl, and zinc powder to the test tube and observe results.

• Add vitamin B2 to the test tube first.

• Vitamin B2 (yellow): Leuco-riboflavin (colorless).

• Concentrated HCl: Supports reduction reaction.

• Zinc powder: Is the Vitamin B2 reducing agent.

• Re-exposure to oxygen will turn the solution yellow again.

Vi-C Quantification

• Oxidation-reduction reaction between ascorbic acid and iodine.

• Weigh 5g of fruit (grapefruit, guava, star fruit...) with vitamin C.

• Grind finely with 20mL 1% HCl, transfer to 100mL flask.

• Wash the mortar with 1% oxalic acid at least 3 times, filtering through dry filter paper.

• Conduct titration with conical flasks, add strach solution indicator

• Titrate by adding 0.01N iodine solution with 1% oxalic acids stabilizer until you see blue-violet color persists for 1 minutes.

• Added iodine does not affect the color of the solution.

• The added iodine reacts with vitamin C, thus the solution will not change color.

• Once you run out of Vitamin C, you'll know because the addition of iodine will cause a color change.

• You have reached your oxidizing point if you see a color change that lasts a minute.

• Add 10mL of vitamin C sample solution and 4mL of 1% oxalic acid to the conical flask.

• After doing this you add 3 drops of strach and titrate using 0.01N iodine until a permanent blue/violet color.

• Vitamin C (colorless) turns oxidized to dehydroascorbic acid.

• Iodine (light brown) becomes reduced to iodide ion (colorless).

• Starch (colorless) is added at the end, and if there is iodine left over a purple color can be expected.

Lesson 5: Nitrogen

Determination of total nitrogen by Kjeldahl method

• Involves inorganic chemical reaction, distillation and acid-base titration.

• Perform sample mineralization by weighing 0.5g of bean powder into a Kjeldahl tube.

• Add 5mL concentrated H2SO4 and catalyst powder (CuSO4, K2SO4), then heat until solution is clear or pale blue.

• The new product is (NH 4)2 SO4.

• Measure 20mL boric acid in receiver and pour the mineralized sample into a Kjeldahl system.

• Add 30mL 40% NaOH to distill NH3.

• Determination of NH3 via titration of (NH 4)2 BO4 with 0.1N H2SO4, and calculating protein via H2SO4 used

• Upon sample mineralization, the solution needs to be clear or light blue for the reaction to be complete.

• NH3 vapor condenses and dissolves in distillate during NH3 distillation.

• When the solution turns from green to purple during titration, this is the end result via using methyl red indicator

• In the inorganic phase, begin with: weighing 0.5g bean powder sample, add 5mL of concentrated H2SO4 and catalyst powder + heat.

• In the distillation stage: pour 20mL boric acid into the receiver, transfer the sample into the Kjeldahl system, then 30mL 40% NaOH.

• In the classification phase: 30mL 40% NaOH- Using methyl red indicator, titrate until green turns purple.

• In a Soybean flour (organic) sample you can expect, decomposition into (NH4)2SO4.

• Concentrated H2SO4 --> Dissolve the sample, creating a blue or clear solution.

• NaOH 40% = Release of NH3 from (NH4)2SO4.

• NH3 will dissolve in boric acid and form NH4OH .

• Then the H2SO4 reacts with NH4OH and change the color of methyl red indicator (blue --> purple).

Steam Distillation

• Take Soy sauce.

• Distill the sample in the distillation system.

• NH3 comes with distiller; and meets boric acid and produces NH4OH acid.

• After collecting it, litmus paper can distinguish acidity levels.

• Titrate the distilled test.

• As a result, the number is added to a solution in terms of ml.

• NH3 --> creates acid, absorbs NH3 into boric one and produces NH4HO.

• Initially, the water is purple, however changes to green with distillation.

• Check end of distillation through litmus paper, If there is a change, distillation needs repeating.

• The initial litmus paper turns to show the color of the water.

• Purple shows water, green is when water distillation.

• The specimen is put into distillation flask and add H2O to release NH3.

• Seal system to cause initial distillation.

• Lead NH3 to container to form NH4OH.

• Re-test for the end with filter paper.

• Re-titrate.

• First we have, lotus in distiller but eventually turns green from initial distillation and purple upon secondary testing of H2So4.