DE LA SALLE UNIVERSITY -DASMARIÑAS

College of Science and Computer Studies

PHYSICAL SCIENCES DEPARTMENT
City of Dasmariñas, Cavite

GUIDELINES FOR PRE-LABORATORY AND POST-LABORATORY REQUIREMENTS

A. Introduction

Scientific writing is part of the job description of a researcher in the fields of Science, Medicine, and
Engineering. A scientific paper is a technical paper that communicates the results of significant studies,
discoveries, or inventions in these fields. It disseminates pertinent information so others in the same or related
fields can benefit from them. This document is open for validation in terms of accuracy, reliability, and
repeatability of results. Thus, a scientific paper becomes a part of creating and validating new knowledge,
techniques, discoveries, and inventions.
As a student in Chemistry laboratory, you will be required to write scientific paper based on the results of
your class experiments. This will serve as an important training ground for you as research-proficient scientists,
medical & allied health science practitioners, engineers, and educators in the future.
The training will include preparation of schematic diagram of experimental procedures as a pre-laboratory
requirement; and completion of experimental worksheets and writing of scientific paper as post-laboratory
requirements.

B. Schematic Diagram of Experimental Procedures

The writing of schematic diagram will train you in summarizing and understanding technical procedures in
doing scientific experiments. A schematic diagram is a stepwise flow chart of the experimental procedures that
can be easily understood and interpreted. The process of making this flow chart will require an active reading and
understanding of the described procedures. This will enhance your skills in interpreting technical procedures and
will train you in critiquing existing protocols so as to improve them or even make your own experimental design.
Clarity and brevity is a must in this process. These characteristics are illustrated in the sample schematic
diagram in Figure 1. It is required of every student in all Chemistry laboratory classes as a pre-laboratory output.
It is an absolute requirement before you may conduct an experiment: NO PRE-LAB MEANS NO EXPERIMENT!
This schematic diagram is previously written on the required laboratory notebook. However, in lieu of minimizing
waste of resources, it should be hand written in a single typewriting paper or on the provided schematic diagram
sheet in the upcoming new editions of your laboratory manual. The schematic diagram will also serve as the
basis of pre-laboratory discussion. This should also be consulted throughout the experimentation period for a
more efficient performance of the experiment.

C. Experimental Worksheet

The worksheet is where you write-down your pertinent observations, data, analysis and calculations
during and after experimentation. Accuracy, intellectual honesty, and neatness should be observed in completing
your worksheet. You should also research for and answer the follow-up questions and applications. Each portion
will have corresponding points for grading. Initial entry of data may be done in pencil but the final output should
always be written in black or blue pen. Raw data and calculations should always be shown to your laboratory
instructor before the end of your laboratory period for inspection and countersigning. Some parts of post-
experimental analysis may be required for accomplishment during the laboratory period as stipulated by your
instructor. However, the bulk of the analysis will serve as assignment that is due one-week after finishing the

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experiment. The worksheet is detached from the manual upon submission, but you may photocopy it prior to
submission for personal reference.

Figure 1: Sample Schematic Diagram of Experimental Procedures

D. Scientific Paper

The scientific paper is a technical paper that discusses the experimental results and their interpretations,
implications, and applications. The format that you will follow is the standard used in many international scientific
journals. The paper is accomplished individually and is due one week after finishing the experiment. It should be
computerized and printed in 8 ½ x 11 bond paper, single-spaced, and in Arial 10 font. Below is the breakdown of
the parts of a scientific paper and their description. A sample scientific paper is also given as an example.

1. Heading – the title of the experiment in bold letters (Arial 12) with the name of the author(s) in bold
letters and their affiliation(s) below.
2. Abstract – this is the study itself in a nutshell. The topic, procedures, results, and interpretations are
summarized in a concise highlighted paragraph.
3. Introduction – this gives a brief scientific background of the study and the hypothesis/assumptions of
the experiment. References are cited as numbers in parenthesis within the text and are listed in the
order that they are cited.

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4. Materials and Methods – this summarizes the materials used and the experimental procedures
conducted. The concentration and amount of each reagent and the brand & model of the instruments
used should be explicitly stated. The stepwise procedure should be mentioned in past tense.
References are cited as numbers in parenthesis within the text and listed in the order that they are
cited.
5. Results and Discussions – this section shows the experimental results and their interpretations,
implications, and applications. Pertinent data should be summarized through necessary tables and
graphs. Important reactions, equations, and mechanisms should likewise be shown. Possible
sources of errors should be analyzed and their possible remediation should be given. References are
cited as numbers in parenthesis within the text and listed in the order that they are cited.
6. References – they should be cited according to the order of mentioning in the paper. Always cite
the laboratory manual that you are using as one of your references. Reputable references include
books, laboratory manuals, thesis, scientific journals (printed and on-line), and websites of reputable
private scientific agencies & organizations, government scientific agencies, and Science departments
of universities and colleges. Wikipedia, although a very useful on-line references, is not accepted as
reliable reference because of lack of strict peer-review system. Check-out the library for available
reference materials. Do not copy any work word-for-word but paraphrase them in your own words
then properly cite the source as stipulated. Doing so is tantamount to plagiarism which is a criminal
act as per Intellectual Properties Rights (IPR) Law. Examples of proper citation are given below:

a. Textbook or Laboratory Manual

1. Clark, J.M. Experimental Biochemistry. 3rd Ed. 1994. W.H. Freeman and Company. USA
b. Scientific Journal
2. Haliwell, B.; Gutterridge, J. M.C. Oxygen toxicity, oxygen radicals, transition metals and
disease. Biochem. J. 1984, 219;1-4.
c. Website with author under an organization
3. Dollard, K. DNA Isolation from Onion. Access Excellence at National Health Museum.
2008. Retrieved 15 October 2008 from http://accessexcellnece.org/AE/AEC/AEF
1994/dollard_onionDNA.php
d. Website of an organization, no specific author
4. Genetics Science Learning Center. “Stem Cells". Learn Genetics. 2008. Retrieved 15
October 2008 from http://learn.genetics.utah.edu/content/tech/stemcells.
e. Thesis
5. De La Cruz, J. The Antioxidant Properties of Philippine Vegetables. Unpublished
Undergraduate Thesis. 2005. De La Salle University-Dasmariñas, Dasmariñas,
Cavite, Philippines.

Demonstration of Antioxidant Activity of Ascorbic Acid

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 Gideon A. Legaspi

De La Salle University-Dasmariñas
Dasmariñas, Cavite Philippines

ABSTRACT

The antioxidant activity of ascorbic acid was demonstrated through its inhibitory action on
polyphenol oxidase (PPO) enzyme. PPO catalyzes the oxidative browning of fruits and
vegetables upon exposure to atmospheric oxygen. PPO from potato (Solanum tuberosum L.)
was extracted using phosphate buffer (pH 7.40) and 0.10 M NaF. A fixed amount of the
enzyme-containing extract was added to increasing concentration of 2% catechol solution and
the absorbance was monitored at 540 nm using a UV-VIS spectrophotometer. The absorbance
of the same solution with of 2% ascorbic acid was then measured to determine if it can
significantly inhibit the oxidative browning reaction. Addition of ascorbic acid resulted to around
40% decrease in absorbance of the solutions. This demonstrates that ascorbic acid indeed
has a potent antioxidant activity. Ascorbic acid can be classified most likely as a
noncompetitive inhibitor of PPO since it is not a structural analog of the substrate and
increasing the substrate concentration does not reverse the inhibitory effect.

INTRODUCTION

The process of respiration results to the production of oxygen free radicals (oxidants) that can damage
cells by oxidizing biomolecules including lipids, proteins, and DNA. It is now established that free radical damage
leads to susceptibility to various degenerative diseases like cancer, atherosclerosis, and coronary heart diseases
(1). Antioxidants like ascorbic acid are substances that neutralize free radicals before they wreak havoc in the
cells. The antioxidant activity of ascorbic acid (vitamin C) can be demonstrated by its inhibitory action on
polyphenol oxidase (PPO). PPO is the enzyme that catalyzes the enzymatic browning of fruits and vegetables
upon exposure to atmospheric oxygen. This results to a decrease in the value of these food stuffs as it leads to
the decrease in its palatability and shelf life. It was previously demonstrated that ascorbic acid treatment is useful
in preventing the premature browning of apples under different conditions (2, 3). In this experiment PPO from
potato was used to demonstrate the antioxidant activity of ascorbic acid. If ascorbic acid can significantly
decrease the formation of quinones (brown pigments) from PPO-catalyzed oxidation of catechol, then it is indeed
a potent antioxidant that can prevent oxidative damage of cells.

MATERIALS AND METHODS

A medium sized-potato was peel and grated. Twenty grams of the grated sample was homogenized at
high speed (30 s) with ice-cold phosphate buffer, pH 7.40 (20 ml) and 0.10 M NaF (10 ml). All reagents used
were analytical grade unless otherwise stated. The homogenate was filtered with cheesecloth. The PPO-
containing filtrate was then transferred to a screw-capped tube and placed in an ice bath. The following solutions
were prepared to determine the extent of uninhibited reaction as listed in Table 1 below.

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 Table 1. Solution Preparation for the Uninhibited Reaction
Tube 1 Tube 2 Tube 3 Tube 4 Tube 5
2% Catechol 0 ml 0.20 ml 0.40 ml 0.80 ml 1.6 ml
Distilled water 2 ml 1.8 ml 1.6 ml 1.2 ml 0.40 ml

Potato extract (0.20 ml) was added to each tube at one minute interval. The tubes were incubated at room
temperature for one minute, and then the absorbance was read at 420 nm using a JASCO 580 Double-Beam UV-
VIS spectrophotometer. For the ascorbic acid-inhibited reaction, the same set of solutions was prepared as in
Table 1. The same amount of potato extract was added along with 0.20 ml of 2% ascorbic acid solution. The
same incubation conditions were applied and the absorbance was read as described before. The concentration
of catechol on each tube was computed. The absorbance against concentration was graphed for both the
uninhibited and inhibited reaction and the percent inhibition was calculated as well.

RESULTS AND DISCUSSION

PPO catalyzes the enzymatic browning in fruits and vegetables upon exposure to atmospheric oxygen.
PPO is a group-specific enzyme, which means that it catalyzes the oxidation of several substrates like catechol,
pyrogallol, L-DOPA, p-cresol, and tyrosine to different extents. Among the mentioned substrates, catechol is the
most preferred substrate for PPO as determined by its Michaelis-Menten constant (4). This is the reason why
catechol was chosen as substrate for PPO-catalyzed oxidation. Maintenance of cold conditions is necessary to
slow down the activity of the enzyme prior to mixing of the reacting components.
The conversion of catechol to the brown pigment, benzoquinone, is illustrated in Figure 1. The resulting
solution absorb light maximally at 420 nm, hence, the extent of product formation can be determined
spectrophotometrically.

Figure 1. PPO-catalyzed Oxidation of Catechol to Benzoquinone

Increasing the substrate concentration would of course result to increase in the production of
benzoquinone as shown in Table 2. Addition of ascorbic acid resulted to around 40% decrease in the oxidative
browning. Meanwhile, the graph that compares the absorbance of uninhibited and inhibited reactions is shown in
Figure 2. The results indicate that ascorbic acid indeed has potent antioxidant property.

Table 2. Absorbance of Samples in the Uninhibited and Inhibited Reactions

Catechol Conc. (M) Absorbance at 540 nm % Dec in Abs

Uninhibited Inhibited
0.007 0.37 0.23 38
0.014 0.77 0.43 44
0.029 0.92 0.60 35
0.057 1.30 0.76 42

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 Figure 2. Graph of Absorbance of Samples in the Uninhibited and Ascorbic-Inhibited
Oxidative Reactions

The structure ascorbic acid is not similar to catechol as shown in Figure 3. Therefore, it cannot be
considered as a competitive inhibitor of PPO. However, it does contain oxidizable hydroxyl groups and can
therefore act as hydrogen and/or electron donor. Previous studies have implicated that the oxidized ascorbic acid
derivative, dehydroascorbic acid, can react with the amino groups in close proximity to the active site. This
probably results to conformational changes in the enzyme that slows down the formation of the enzyme-substrate
complex or its dissociation to yield the products (4). Therefore, it can be deduced that the mode of inhibition of
ascorbic acid is noncompetitive in nature. The results support this conclusion because as can be seen in Table 2,
the % of inhibition remains more or less at 40% even if the substrate concentration is increased twice
incrementally.

Figure 3. Structure of Ascorbic acid

The antioxidant activity of ascorbic acid accounts for its health benefits to humans and its use as food grade
preservative in processed fruits and vegetables. The analogous enzyme to PPO in humans is the enzyme
tyrosinase. It catalyzes the conversion of tyrosine to melanin via a series of oxidation reactions. Melanin is the
brown-black pigment that darkens the skin, hair, and the iris. Ascorbic acid is thereby useful as active ingredient
in various whitening products as it can inhibit the formation of melanin by a mechanism similar to one described
for PPO previously.

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REFERENCES

(1) Haliwell, B.; Gutterridge, J. M.C. Oxygen toxicity, oxygen radicals, transition metals and disease. Biochem. J.
1984, 219;1-4.
(2) El-Shimi, N.M. Control of enzymatic browning in apple slices by using ascorbic acid under different conditions.
Plant Foods Hum Nutr. 1994. 43; 71-76
(3) Prestamo, G. et al. Effect of ascorbic acid against browning in apple fruit treated with high hydrostatic
pressure. J. Agric. Food Chem. 1999. 47;3541-3545.
(4) Yagur, H.; Sugiroglu, A. Partially purification and characterization of polyphenol oxidase of quince. Turk. J.
Chem. 2002. 26; 97-103.

--------------------------------------------------------------End of Sample Scientific Paper------------------------------------------------

Prepared by:

Asst. Prof. Gideon A. Legaspi, RCh, MSc

Physical Sciences Department
College of Science and Computer Studies
De La Salle University-Dasmariñas
Dasmariñas City, Cavite