Garlic Nanoparticle Synthesis Laboratory Notebook

JUNE 15

Experiment: Gold Nanoparticle Synthesis using Garlic Extract Optimization Trial #1

A) Make Garlic Extract

1. Peel garlic cloves.

2. Wash garlic cloves thoroughly with tap water.

3. Rinse 5x with deionized water.

4. Place wet garlic cloves on a paper towel. Blot them dry.

5. Take 500 mL glass beaker and place it on a scale. Tare the beaker.

6. Bring the washed garlic cloves on paper towel and a clean knife/scissors to the bench.

7. Using a clean knife/pair of scissors, slice ALL of the garlic cloves into thin pieces DIRECTLY into the beaker on top of the balance.

8. Record the mass of the garlic.

9. For each gram of garlic, 5mL of milliQ water (1g/5mL ratio of garlic to milliQ water) should be gathered.

• Calculate & measure the appropriate amount of milliQ water based on the mass of your garlic into a beaker
• A graduated cylinder should be used to measure the appropriate quantity of milliQ water

10. Inside the mortar, place a small amount of the chopped garlic by scooping some out of your beaker with a metal spatula.

• The amount of garlic placed into the mortar should be sufficient to cover the bottom of the mortar.

11. Inside the mortar, pour enough milliQ water (measured in Step 9) to dampen the garlic. *Not all water measured out needs to be used in this step! Then use the pestle to crush the garlic into smaller pieces.

• The garlic should be crushed so that as much as possible is exposed to water.

12. Place a sterile glass funnel into a sterile 500mL glass bottle and place a Whatman Filter Paper 1 inside the funnel.

13. Pour crushed garlic extract from mortar into glass funnel.

14. Discard the solid garlic clumps left on the filter paper. Then cap the solution in the bottle.

• The bottle should be able to contain the filtered garlic-water solution from the previous step. i.e.: if 500mL milliQ water was added to the mortar, gather a glass bottle that can hold 500mL.

15. Label the glass bottle containing the filtered garlic extract solution.  

16. Store the garlic extract bottle at 4oC.

• Do NOT freeze the solution!

B) Make Nanoparticles using the Garlic Extract

1. Sterilize fumehood with 70% ethanol before starting

2. Turn OFF the lights around the scale, if possible, as chloroauric acid solid is light sensitive.

3. As quickly as possible, without spilling the chloroauric acid solid, use a wooden or plastic tool to transfer 0.0010192g of HAuCl4 into the weight-boat on the scale.

• Do NOT use metal scoopulas or devices to transfer the chloroauric acid.

4. Using a 200uL micropipet, measure 40uL of distilled water and add it to the measured chloroauric acid directly into the weight-boat.

• The water should visibly turn a yellow color when the water contacts the chloroauric acid solution.

5. Using the same micropipet and tip, transfer the chloroauric acid solution from the weigh-boat into a sterile 1.5mL microfuge tube.

• Wrap the microfuge tube with aluminum foil to prevent light exposure.

6. Add 50 mL and 100 mL of garlic extract (at room temperature) into seperate beakers.

7. Then go to the pH meter with the beakers containing the garlic extract mixture. Then calibrate the pH meter.

8. Add a small magnetic stir bar to the garlic solution in the beaker.

• Make sure to rinse the stir bar with distilled water.

9. Place the beaker onto a stir plate and apply minimal stirring.

10. Then add the 40uL of prepared chloroauric acid solution to the garlic solution in the beaker. Allow stirring to continue until the HAuCl4 is properly mixed in.

11. Then turn OFF the stir plate. Only then, take the pH electrode and measure the pH of solution.

• Stirring must be stopped before measuring pH to PREVENT the stir bar from cracking the pH electrode.
• The pH of solution will likely be acidic or low, since chloroauric acid is a strong acid.

12. Add NaOH (5%) solution DROPWISE SLOWLY using a new Pasteur pipet and bulb to the garlic-gold solution until a pH of ~9 while stirring on the plate.

• Stop the stirring periodically and use the pH electrode to verify pH.

13. Once the solution is at a pH of ~9, DROPWISE SLOWLY add 0.5% NaOH to the garlic-gold solution until the pH is ~10 while stirring on the plate.

• Stop the stirring periodically and use the pH electrode to verify pH.
• NOTE:a more basic, higher pH will generate smaller nanoparticles

***Caution: the extract is HIGHLY sensitive to addition of NaOH and can change pH very fast!!!

14. Remove the magnetic stir bar from solution using the magnetic rod.

15. Then take the solution in the beaker to the designated nanoparticle fumehood.

16. Set up a double boiler using a clamp and ring-stand, and the 1000mL beaker filled partly with distilled water set atop a hot plate.

• Set up the clamp so that the beaker containing the garlic-gold solution is suspended in the beaker filled with water, and partly submerged.
• Ensure that the sides of the beakers are not touching each other, to prevent hitting.

17. Set the hot plate to boil, so that the temperature of the water in the large beaker reaches 95oC. A thermometer can be borrowed, by asking an advisor.

• Then suspend the beaker containing garlic-gold solution into the water bath for ~10mins
• A color change should be observed after 7 minutes (pink), indicating nanoparticle formation. If solution is left to heat too long, the solution may appear purple and nanoparticles begin to aggregate.

18. Once a color change is seen, remove the beaker from the heat using oven-mits and place in the fume hood to cool down at room temperature.

19. Transfer the nanoparticle solution to a new scintillation vial with a plastic cap and store at room temperature.

C) Results: There was no colour change in either beakers containing varying amounts of garlic extract. I.e no gold nanoparticles formed.

D) Discussion: The reaction should have occurred within 10 minutes and there was no color changes observed after 20 minutes. This could be due to the garlic not being extracted properly. Reread the research paper on synthesizing gold nanoparticles using garlic extract and papers on how to make a plant extract in general to optimize the protocol.

JUNE 21

Experiment: Gold Nanoparticle Synthesis using Garlic Extract Optimization Trial #2

A) Make Garlic Extract: This time when making the extract, do not use the glass funnel. To extract the garlic faster and to filter it as much as possible, do the following (after step 11):

1.Set up the vacuum apparatus and connect the appropriate tubing for plant extract solution collection into a vacuum-compatible flask.

• Ensure that the size of the flask can contain the volume of plant extract solution expected.

2. Then place a buchner funnel inside the opening of the vacuum-flask.

• Ensure that the flask is stable so as not to fall over.

3. Place a piece of Whatman filter paper (0.45microns) onto the base of the buchner funnel.

4. Turn ON the vacuum pump.

5. Pour the garlic solution in the mortar onto the filtered buchner. Rinse out the rest of the mortar’s contents into the funnel using only some of the remaining fresh milliQ water (from Step 9) onto the funnel.

• Do NOT use all of the milliQ water as you will need the rest later.

6. As the vacuum is turned ON, the garlic solution is sucked into the flask while the filter paper prevents solid chunks of garlic from entering solution.

• Use a scoopula to gently scrape and stir the solid pieces of garlic while the vacuum is ON, to prevent blocking of suction on the filter paper, slowing down the process.

7. Continue to add more garlic and milliQ water to the filter-funnel for vacuum filtration until  no more weighed crushed garlic remains.

8. Discard the solid garlic clumps left on the filter paper and transfer the garlic solution filtrate from the flask into a clean 400-500mL sterile glass bottle. Then cap the solution in the bottle.

• The bottle should be able to contain the filtered garlic-water solution from the previous step. i.e.: if 500mL milliQ water was added to the mortar, gather a glass bottle that can hold 500mL.

B) Make Nanoparticles using the Garlic Extract

C) Results: There was no colour change in either beakers containing varying amounts of garlic extract. I.e no gold nanoparticles formed.

D) Discussion: The reaction should have occurred within 10 minutes and there was no changes observed after 20 minutes. All of the varying amounts of garlic extract might be too excessive for nanoparticles to form after the addition of 75mM HAuCl4 solution.

JUNE 22

Experiment: Gold Nanoparticle Synthesis using Garlic Extract Optimization Trial #3

A) Make Garlic Extract

B) Make Nanoparticles Using the Garlic Extract: Make observations of reaction times. Do not use concentrated garlic anymore. Before adding HAuCl4, dilute garlic extract with enough distilled water to achieve a 1%v/v mixture:

1. In a clean 1.5mL microfuge tube, add 1.5mL of garlic extract solution with 1000uL micropipet.

2. Centrifuge the garlic extract solution in the microfuge tube for 10 minutes at 4000 rpm (at room temperature) using the tabletop centrifuge.

3. Take a 250mL beaker and add 150mL of distilled water.

• A graduated cylinder can be used to measure the quantity of water.
• The aim is to make a 1% garlic extract solution - depending on the final volume desired, the amount of water and garlic extract should be proportionally adjusted.

4. Then add the 1.5mL of centrifuged garlic solution to the beaker containing 150mL of distilled water. Cover the beaker with aluminum foil and set aside at room temperature.

C) Results:

Inside beaker containing 100 mL of 1% garlic extract:

• After 2 minutes: a very faint pink
• After 4 minutes: light pink
• After 7 minutes: magenta pink
• After 10 minutes: magenta pink
• After 13 minutes: dark pink
• After 14 minutes: dark pink+agglomeration

Inside beaker containing 50 mL of 1% garlic extract:

• After 2 minutes: nothing
• After 4 minutes: nothing
• After 6 minutes: blueish-purple
• After 7 minutes: purple + agglomeration

D) Discussion: The color change indicates the successful synthesis of nanoparticles. The darker color changes and agglomeration occurred because the reaction was left to mix on the hot plate for too long. For all types of subsequent synthesis experiments, discontinue to mix the contents within the beaker and remove the beaker from the hot plate once a color change is observed to avoid agglomeration.

JUNE 23

Experiment: Gold Nanoparticle Synthesis using Garlic Extract Optimization Trial #4

A) Make Garlic Extract

B) Make Nanoparticles Using the Garlic Extract: vary the volume of 1% garlic extract used to mix with 40 microL of 75 mM HAuCl4 (17.5mL, 20 mL, 40 mL)

C) Results: A faint lilac color change was observed in each beaker. Reaction times: in

• 17.5mL of garlic extract: 28 minutes
• 20 mL of garlic extract:26 minutes
• 40 mL of garlic extract: 20 minutes

D) Discussion: The time required for a color change to occur takes less time as the volume of garlic extract is increasing in volume i.e as the final concentration of HAuCl4 in solution decreased. For subsequent synthesis experiments, make sure that the final concentration of HAuCl4 in solution is relatively low.

JULY 11

Experiment: Gold Nanoparticle Synthesis using Garlic Extract Optimization Trial #5

A) Make Nanoparticles Using the stored Garlic Extract (23/6/2016): vary the concentration of diluted extract between 1%-10%.

B) Results: No color changes beyond 1%.

C) Discussion: Higher concentrations of garlic extract don’t produce a successful color change after the reactions i.e the successful synthesis of nanoparticles. For subsequent synthesis experiments, use an average of 1% garlic extract concentration.

JULY 18

Experiment: Gold Nanoparticle Synthesis using Garlic Extract Optimization Trial #6

A) Make Nanoparticles Using the stored Garlic Extract(23/6/2016): vary the concentration of diluted extract to the following %v/v: 0.75%, 1%, 1.25%. Also vary the final concentration of HAuCl4: 0.15 mM and 0.25 mM.

B) Results:

For 0.15 mM HAuCl4 in

• 0.75% garlic extract: none
• 1% garlic extract: none
• 1.25% garlic extract: none

For 0.25 mM HAuCl4 in

• 0.75% garlic extract: light pink (1 hour)
• 1% garlic extract: light pink (20 minutes)
• 1.25% garlic extract: light pink (13 minutes)

C) Discussion: Nanoparticles were successfully synthesized when the concentration of HAuCl4 in solution was 0.25mM and were unsuccessful when the concentration was 0.15mM. Keep the minimum final concentrations of HAuCl4 in garlic extract for subsequent synthesis experiments at 0.25 mM. Synthesis time is also optimal when the concentration of diluted extract is near 1%, though the result for that of 0.25 mM HAuCl4 in 0.75% garlic extract shows to be as long as an hour. It might have taken so long because we accidentally diluted the wrong amount of garlic extract (too little).

JULY 21

Experiment: Gold Nanoparticle Synthesis using Garlic Extract Optimization Trial #7

A) Make Garlic Extract: Optimize filtration process:

• Filter using a strainer first
• Then filter using 0.75 micron filter paper
• Then filter using 0.45 micron filter paper
• Centrifuge for 8 minutes at 5000 rpm. Keep supernatant in a glass bottle.

B) Synthesize Nanoparticles using Garlic Extract: vary the final concentration of HAuCl4 in 20 mL of 1% garlic extract (0.25 mM, 0.35 mM, 0.45 mM, 0.55 mM, 0.65 mM)

C) Results: A faded pink color change was observed in each sample (approximately 10 minutes reaction time for each). As the concentration of HAuCl4 increases, the darker the solution appears. No agglomeration.

D) Discussion: The pink color change indicates that the synthesis of nanoparticles was successful. The experimental reaction time took approximately 10 minutes for each sample, which matches the theoretical reaction time provided by the research paper. The maximum absorbance of each sample was obtained at 530 nm, when taking uv-vis spectroscopy readings, which further indicates the successful synthesis of gold nanoparticles. Synthesis of gold nanoparticles using garlic extract has been optimized at this point.

JULY 22

Experiment: Gold Nanoparticle Synthesis using Garlic Extract STORAGE CONDITIONS Optimization Trial #1

A) Synthesize Nanoparticles using Garlic Extract: 1 mM HAuCl4 in 10 mL of 1% garlic extract.

Storage conditions:

• Refrigerator (4°C)/ shake
• Refrigerator (4°C)/ no shake
• Room temperature (24°C)/ shake
• Room temperature (24°C)/ no shake

B) Results (synthesis): Samples are a red wine color (reaction time: 10 minutes)

C) Discussion: The stored samples will be observed and compared after a period of time, in order to conclude which storage condition provides the longest shelf life of the nanoparticles.

JULY 30

Observation: Gold Nanoparticle Synthesis using Garlic Extract STORAGE CONDITIONS Optimization Trial #1

A) Results: Since their synthesis, there are no apparent changes in terms of color or agglomeration in any of the stored samples.

B) Discussion: The samples may still change over time. Continue observing any physical changes within the samples.

SEPTEMBER 15

Observation: Gold Nanoparticle Synthesis using Garlic Extract STORAGE CONDITIONS Optimization Trial #2

A) Results: All of the samples look the same. Each one has still maintained the red wine color obtained upon synthesis. Agglomerates have also been observed in each sample. There is a similar level of agglomeration in each sample as well.

B) Discussion: There are no apparent or significant differences in each sample, despite the varying storage conditions. From this point on, just store synthesized nanoparticles at room temperature within the fume hood.