De La Salle Santiago Zobel School, University Avenue, Ayala Alabang Village,

Muntinlupa City, Philippines

A Comparative Study of Anthocyanin and Chlorophyll pigments

Extracted from Solanum melongena (Eggplant) and Spinacia
Oleracea (Spinach) as Printer Ink Substitutes

Investigatory Project
Science 2 – Chemistry
Sophomore G

Submitted by:

Albano, Rufus

Arriola, Rafael

Morales, Fides

Pacis, Ted
Reforma, Bianca
Toque, Hazel

Submitted to:

Mr. Jonathan Sarza

January 9, 2015

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 CHAPTER 1

Introduction

Background of Study

Ink has been used for thousands of years in human history. Ink has allowed our kind to

perpetuate knowledge, preserve history, and communicate with one another on a highly visual

platform. Ever since, humans have innovated inks through comprehensive studies and

experiments on the coloring pigments present in these sources.

Today, ink has proven to be an essential material for business, education,

communication, and several other fields. Modern printing inks are much more complex

compared to early inks. They are composed of pH modifiers, humectants to prevent drying,

polymeric resins to bind the components, defoaming agents for the efficiency of its foam,

wetting agents, thickeners, and biocides to prevent bacterial growth. Chemicals such as carbon

black and triphenylmethane have also been standardized for use in commercial printing inks.

Additionally, a great number of households and offices are now equipped with printers to the

convenience of the people around.

Printers may be considered as a general commodity now that the world has industrialized

to the use of inkjets. However, a cartridge of ink commonly costs about $15.37, or ₱694.13, and

may contain as little as 5 ml of ink. According to David Connett, the editor of The Recycler,

printing ink manufacturers adapt to the strategies of discouraging refills in order to gain more

profit for their business. Consequently, people tend to dispose of their used ink cartridges instead

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of recycling and utilizing them for second use. About 500 million used cartridges end up in

landfills

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every year, and these would take more than 1000 years to decompose. The production of

commercial inks also pose threats to human and environmental health due to trace levels of

heavy metals such cadmium, hexavalent chromium, lead and mercury released in printing ink

manufacture.

Through this investigatory project, the researchers shall seek alternatives to printer ink in

order to save the cost and risk of buying commercial printing inks.

Spinacia olerace, commonly known as Spinach, is a regular cooking ingredient. Spinach

leaves grow in small bunches with crinkled or flat structures. Packed with vitamins and minerals,

it is an essential and a healthy vegetable. It contains chlorophyll, a mineral which is quickly

metabolized and uses to develop new red blood cells and removes cancerous substances in our

system.

However, chlorophyll has other purposes aside from health uses. Chlorophyll conducts

photosynthesis for plants. With this green pigment, the leaves absorb sunlight in order to

synthesize organic compounds to produce glucose and oxygen. It is also a food colorant and is

widely used in the culinary world in order to color various food and beverages such as pasta and

absinthe.

Eggplant, or aubergine, also known by the scientific name Solanum melongena, is a

species of nightshade vegetable, having a mild bitter taste and spongy texture. Eggplants provide

a healthy dose of vitamins and minerals, along with phytonutrients such as chlorogenic acid.

These greatly benefit the brain cell membranes, and serve as a great antioxidant.

Anthocyanin is the dominant pigment in eggplants, taking the form of nasurin. Eggplants

contain about 750 mg per 100 g, making it one of the vegetables with the highest Anthocyanin

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content. This pigment’s major function is to provide color to most flowers and fruits to attract

pollinators. Depending on their pH, they may appear red, purple, or blue. It is also used as a food

additive. They are water-soluble, which would make it possible for it to be used in paint, pen ink,

and other similar dyes.

Anthocyanin and Chlorophyll are both examples of biological pigments. These pigments

contain substances that produce color from selective color absorption through the process of

absorbing visible light.

Objectives: The study aims to

 extract Chlorophyll and Anthocyanin pigments from Spinacia olerace (Spinach) and

Solanum melongena (Eggplant)

 use these pigments as cheaper, closely accessible, and environmentally safe

alternatives to commercial printing inks

 conduct a comparative analysis between Chlorophyll and Anthocyanin based on their

performance as alternative printer inks

Statement of the Problem

 Will Anthocyanin and Chlorophyll be effective as colorants for printer ink?

 Which among the extracted pigments is a better alternative to printer ink?

 Can these natural pigments act as commercial substitutes to common printing inks?

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Hypothesis

 If the tested solutions containing these pigments satisfy the entirety of the
performance tests, then they may be used as colorants for printer ink.

 If Anthocyanin demonstrates a preferable performance over Chlorophyll, then it shall

be the better alternative for printer ink.

 If the results of the performance tests and the conducted surveys suggest positive
feedback on the marketability of these pigments, then they can act as substitutes to

commercial printing inks.

Significance of Study

Printing ink is a vital necessity to many students and professionals. Ink manufacturers

market their inks at expensive costs because of the high and inevitable demand of ink for

purposes essential to the survival of our current society. Additionally, the production of printing

ink involves various processes in order to create, package, and distribute the products. There are

several different environment, and eventually, health risks brought about by ink manufacture and

its careless disposal. From gathering the raw materials, to the production of ink, to its

distribution, consumption and finally, its disposal, the economy of printing ink has caused

dangers that go by unnoticed by most.

The sole purpose of this study is to test and investigate natural plant pigments as

commercial printing inks, and to encourage printer-owners to recycle their used ink cartridges

with cheaper and more accessible ink alternatives. The results of this investigatory project may

be used to determine if these samples would be marketable as safer and more natural printer inks.

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 Scope and Delimitation

The researchers shall only be extracting the pigments Chlorophyll and Anthocyanin from

Spinacia olerace and Solanum melongena in uniform amounts and in controlled time periods.

They shall be dissolving the extracted pigments into three different solvents: acetone and

ethanol for Chlorophyll, purified water and ethanol for Anthocyanin.

They shall inject the pigments only into empty HP ink cartridges, and print with an HP

Deskjet Ink Advantage.

The researchers shall be focusing on the performance of the two pigments as printer inks

through a series of tests and comparative analysis based on the following criteria: quality,

durability through light exposure, storage life, marketability, practicality, and flexibility of

purpose. They shall also be comparing the pigments to commercial printer inks in order to

determine its feasibility as an alternative option to the current market.

They shall conduct a marketability survey off a sample of 40 civilians, to be composed of

20 students and 20 working professionals. The survey shall be conducted within the limits of

Muntinlupa City.

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 CHAPTER II

REVIEW OF RELATED LITERATURE

In this section, we shall be summarizing previous studies and information of vital

importance to our current investigation.

A.

In 2013, researchers Almonte, Atienza, Miranda, and Simangan conducted an

investigatory project regarding the use of Alugbati berry seed juice as marker ink. Alugbati is

known to have a high Anthocyanin content and is highly recommended for ink production.

According to them, the ink produced from Alugbati was easy to make and their analysis showed

that the product was most likely the same with the features of the markers in the market.

B.

According to a study done by TUV Rheinland, over half of inkjet printer ink is thrown

away. The study revealed that almost 60% of the ink inside cartridges goes unused, meaning, the

ink efficiency level is low, considering the amount of money one has paid for.

C.

Regular commercial printer ink has significant impact to the environment around us.

According to a study conducted by the EuPIA, or the European Printing Ink Association,

disposed commercial printer ink may affect aquatic life and the atmosphere. It produces

greenhouses gases, which trap heat in the Earth and contributes to global warming.

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D.

The earliest inks were known to have been produced from iron gall, oil, ink-producing

animals, and elements such as lead and carbon, just to name a few. Fruits such as pomegranate

were used by the medieval British, while earlier civilizations used natural dyes from saffron,

turmeric, weld, and various logwoods. These are usually mixed with water stabilised with a

natural gum or other similar bases.

E.

Many scientists conducted research and experiments with different kinds of vegetables,

such as eggplants, to find out if they are acids, bases, or neutral. This determines the type of

pigments and colors they produce, which was used until the 19th century in clothes and sources of

dye in fabrics. Different types of plants produces different qualities and varieties in color and

appearance when used to create substitute commercial printer ink.

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 CHAPTER III

METHODOLOGY

Materials

 Eggplant Peels
 Pipette
 Spinach Leaves
 Mortar & Pestle
 Ethanol
 Buchner funnel
 Acetone
 Filter Paper
 Hexane
 Stirring Rod
 Distilled water
 Test Tubes (with screw caps)
 Small beaker
 Blender
 Large beaker
 Weighing Scale
 Knife
 Used HP ink cartridges
 Anhydrous sodium sulfate (NA2SO4)
 HP Deskjet Ink Advantage Printer
 Graduated Cylinder
 Typewriting Paper
 Ring Stand
 Cotton Swabs
 Micro Clamp
 Timer
 Cotton Plugs

Procedure

I. EXTRACTION

a. Extraction of Chlorophyll

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 1. Gather 1g of Spinach leaves. Rip the leaves into small pieces

and put them in the mortar. Add 2ml of acetone before grinding

it further with the pestle until it turns bright green in color.

2. Transfer the liquid from the mortar to a test tube with the use of

a pipette. Rinse your mortar with acetone.

3. Add 4.0 mL of hexane and 4.0 mL of distilled water to the

extract in the test tube, screw the cap on and start shaking the

mixture gently. Exercise caution by directing the tube away from

yourself and from other people. Leave the tube to stand in order

for the chemicals to separate.

4. Drain the water layer from the pipette into a small beaker.

5. Add another 4.0 mL of distilled water to the hexane layer in

the test tube. Once more, drain the bottom water layer into a

small beaker.

6. Separate the water and hexane by dropping microspatula

anhydrous Sodium Sulfate (Na2SO4). Add more if necessary.

7. Add a filter to the pipette by using a small cotton plug. Clamp

the pipette to a ring stand, and filter the extract into a vial. This

will get rid of the drying agent.

8. Repeat this process replacing acetone with ethanol.

b. Extraction of Anthocyanin

1. Slice the peel off the eggplant and measure about 10g.

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 2. Add the peels in a blender in order to further grind them.

3. Transfer the peels to a beaker and add acetone on a ratio of 1

part solid sample and 2 parts acetone.

4. Continuously stir the mixture for 60 minutes.

5. After 60 minutes, filter the mixture with a Buchner funnel and

filter paper into a collection beaker.

6. Dissolve the collected extract in 5ml purified water.

7. Repeat the procedure, replacing purified water with ethanol.

II. REFILLING (Procedures Sourced from Penniless Parenting)

1. Cut label at joint between top and bottom cap of the cartridge case

2. Flip back bottom cap of cartridge.

3. Squeeze sides of cartridge case and carefully wiggle out ink

bag and pump assembly from the insert lock.

4. Screw the Ball Remover screw into the center of the rubber

sealing plug, (situated in the filling hole) until a firm grip is made

and pull out the plug. (Keep the plug on the end of the Yellow

Ball Remover)

5. Put the blunt filling needle onto the syringe and fill the

syringe with ink.

6. Insert the blunt needle into the filling hole until the pink part of

the needle is pressed up against the filling hole. Be sure the needle

does not press up against the bag and punctures it.

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 7. Slowly inject with 30cc of pigments and then reseal by pushinng

back in the rubber plug. (Push in with the plug on the end of the

ball remover screw and then remove the ball remover screw

from the ball plug).

8. Remove the excess air from the ink bag and pump assembly as

follows:

a. Hold assembly at a 45° angle with outlet hole tube at

the top.

b. Insert needle of empty injector (with plunger pushed

in) into outlet hole tube.

c. Press and release pump several times which will expel air

into the syringe.

d. Pull back the plunger on the syringe to remove any air

from the outlet hole tube and then withdraw the needle.

9. Replace the ink bag assembly into the cartridge case and clip

the bottom back into position.

Source: http://www.pennilessparenting.com/2011/03/refilling-your-printer-ink- cartridges.html

III. PERFORMANCE AND COMPARISON TESTS

a. Quality / Absorption Time

1. Insert the cartridge with the Chlorophyll and Acetone solution into

the printer.

2. Choose one document to be used throughout the experiment.

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 3. Print the document on typewriting paper.

4. Start dabbing the printed document with a cotton swab in order to

check if the ink has dried. Time how long it took to dry

completely.

5. Repeat steps 3 and 4, this time printing a randomly selected photo

instead of a document. Record and take photos of the printed

copies as data for this test.

6. After doing so, repeat steps 1 to 5, using the remaining 3 ink

cartridges. Record your observations and comparisons for each

solution used for printing the document and photo. Focus on color

intensity, color stability, and absorption time.

7. Lastly, print copies of the same document and photo with the use

of a commercial printing ink. Note important observations on the

similarities and differences of the overall appearance of the

documents printed with the extracts versus the same documents

printed with commercial ink.

b. Durability Through Light Exposure

1. Place the printed documents from each of the created inks in the

following areas:

a. Outdoors (exposed to the sunlight)

If rain or any other natural or weather interference persists

during the investigation, move the samples to a dry area for

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 the time being, and then return them to their original

placement once the interruption has gone.

b. Indoors (in a dark area)

Place them drawers, folders, or any other enclosed dark

areas. Do not remove them from their place, or expose

them to light until the end of the time period.

2. The papers would be left for 30 days in their specific areas. Take

photos and record observations on the appearances and quality

of the documents before and after the specified time periods.

c. Storage Life / Reaction to Temperature

1. Set aside a portion of each ink in vials or other similar

containers, to measure around 10 ml for each.

2. Make two sets of these vials to total 8 samples.

3. Place one set in the refrigerator, while the other 4 shall remain in

room temperature.

4. Check on the inks every day 14 days. Record your observations

based on appearance, odor, and texture.

5. After 14 days, inject these inks into cartridges. Print the same

documents as you have with the freshly-made inks 14 days prior.

Record your findings.

d. Marketability

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A door-to-door survey shall be conducted in attempt to sell the product

to various students and professionals, the most frequent users of

printers. The researchers shall present them with the facts of the

investigation, and show them the samples of the printed documents

produced from the natural pigments. They shall be questioned for

feedback, comments, and ratings. The data from the survey shall be

used to determine marketability.

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 References

Almonte, A.R.J, Atienza, P.P.L., Miranda, J.C.Jr. & Simangan, J.M. (2013, February).

Alugbati Seed’s Juice as Marker Ink. Retrieved January 2, 2015 from

https://www.academia.edu/4029645/BABY_THESIS_FOR_IP_DRAFT_Autosaved_Final

Carle, R., Sadilova, E. & Stintzing, F.C. (2006, April 11/March 6). Anthocyanins, Colour and

Antioxidant Properties of Eggplant(Solanum melongena L.) and Violet Pepper (Capsicum

annuum L.) Peel Extracts. Retrieved January 2, 2015 from

http://www.researchgate.net/publication/6805743_Anthocyanins_colour_and_antioxidant_proper

ties_of_eggplant_(Solanum_melongena_L.)_and_violet_pepper_(Capsicum_annuum_L.)_peel_e

xtracts/links/0fcfd50b6f9e0e9dc1000000.pdf.

Kunjappu, J.T. (n.d). Ink chemistry. Retrieved January 4, 2015 from

http://www.rsc.org/chemistryworld/Issues/2003/March/inkchemistry.asp

Olivera, N. (2012, March). Colored Ink for Computer Printer Out of malabar nihtshade

(alugbati) Exract. Retrieved December 15, 2014 from http://jndolivera.blogspot.com/

Rich, A., & Rich, C. (n.d.). Extraction of Chlorophyll and Carotenes from Spinach and Analysis

by Thin Layer Chromatography (T. Bell, Ed.). Retrieved January 8, 2015, from

http://facstaff.bloomu.edu/pugh/experiment2.pdf

Robinson, D. (2013, February 23). Printer ink cartridges: why you're paying more but getting a

lot less. Retrieved Jaunary 5, 2014 from

http://www.theguardian.com/money/2013/feb/23/printer-ink-cartridges-paying-more-getting- less

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Anthocyanins and anthocyanidins. (n.d). Retrieved January 7, 2015 from http://www.food-

info.net/uk/colour/anthocyanin.htm

A Short History of Ink. (n.d.) Retrieved January 6, 2015, from

https://www.stinkyinkshop.co.uk/blog/a-short-history-of-ink/

Environmental Impacts of Printing Ink Manufacture. (n.d). Retrieved December 13, 2014

from http://www.eupia.org/uploads/tx_edm/2013-03

05_EuPIA_Environmental_Impact_of_Printing_Inks.pdf

HP Business InkJet SERIES. (n.d.). Retrieved January 8, 2015, from

http://www.printerfillingstation.com/Refill-Instructions/HP/H08.htm

Mutoh’s Ink Durability Test Program: Factors responsible for the degradation of a print. (n.d).

Retrieved December 15, 2014 from

http://www.mutoh.eu/Portals/0/MutohData/DocumentLibrary/Public%20Zone/Downloads/White

Paper/White%20Paper%20Mutoh%20s%20Ink%20Test%20Program.pdf

Preton Ltd, white paper: Environmental issues associated with toner and ink usage. (2010,

March). Retrieved December 15, 2014 from

http://www.preton.com/pdf/PretonSaver_envi_whitePaperFinal_1403010.pdf

Refilling your Printer Ink Cartridges. (2011, March 7). Retrieved January 8, 2015, from

http://www.pennilessparenting.com/2011/03/refilling-your-printer-ink-cartridges.html

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