INDUSTRIAL TRAINING REPORT

CHM4901

DEPARTMENT OF CHEMISTRY
FACULTY OF SCIENCE
UNIVERSITI PUTRA MALAYSIA

SYAFIQAH BINTI SHARI

MATRIC NUMBER: 157046
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TABLE OF CONTENT

Content…………………………………………………………………………………...2

Acknowledgement……………………………………………………………….….......3

1.0 Employer details

1.1 Employer contact details…………………………………………….……...4
1.2 Period of employment……………………………………………….……...4
1.3 Company background/history……………………………………….……...4
1.4 Position/Type of employment……………………………………………..4-5
1.5 Organization structure……………………………………………………….5
2.0 Project and responsibilities………………………………………………………..6-8
3.0 Highlight on project
3.1 Objective……………………………………………………………...…….9
3.2 Methodology……………………………………………….…………….9-12
3.3 Result……………………………………………………………………12-13
3.4 Calculation………………………………………………………………13-14
3.5 Discussion …………………………………………………………...…14-16
3.6 Conclusion ………………………………………………………………...16
4.0 Other activity …………………………………...…………………………......16-17
5.0 New tools and technologies……………………………………………………17-18
6.0 Suggestions for the improvement of training……………………………………..18
7.0 Conclusion ……………………………………………………………………...18-19
8.0 References……………………………………………………………………….....19

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ACKNOWLEDGEMENT

First I would like to thanks Dr. Azowa, the deputy dean of academic chemistry department’s
university Putra Malaysia for giving me the opportunity to do an internships at the abroad and
also to University Putra Malaysia to support our ticket flight and travel insurance. I am also
indebted to Faculty of Chemical lectures for their support during my practical at laboratory
Food chemistry, Biomass, Mie University. Not forgotten Dr. Hazlin our internship’s adviser
because always supports us before and after internship. The last but not least thanks to Prof.
Gwendoline Ee Cheng Lian our faculty’s supervisor.

In preparing this report, I was in asking with many people especially is my supervisor’s
Associate Prof. Takashi Mishima at Mie University. I am also very thankful to my workmates
especially in laboratory such as Toshihito Yuri, Koutatsu Sakakura, Budi Santoso and
Shigetaro Arita for their guidance, advice and also motivation. They have contributed
towards my understanding and thoughts.

For me it was valuable experience to be in laboratory of Mie University. During the

internships I learned many new things that will help me to do my final year project and to
face the working environment. Besides that, internship at the abroad is a very challenging and
I learnt to be independent at the foreign country with the different lifestyle, culture and
language.

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1.0 EMPLOYMENT DETAILS

1.1 Employer contact details

Address: Graduate School of Regional Innovation Studies, Mie University

Address: 1577 Kurimamachiya-cho, Tsu City, Mie Prefecture, Japan 514-8507
Phone: +81-59-231-9632
Fax: +81-59-231-9634
E-mail: info (a) innov.mie-u.ac.jp

1.2 Period of employment

Started on: 24 July 2013 – 24 August 2013

1.3 Company background/history and organization structure

The history of Mie University dates back to 1648, when the Toyomiyazaki-bunko was
established to the left of the Outer shrine of Ise Yamada. In August 1874, the Watarai
Prefectural Normal School was established at this site. Industrial training for chemical
students was placed at the section of The Graduate School of Regional Innovation fosters
individuals who can assume leadership roles in the regional community, connected with
regional industries centred in Mie Prefecture, and based on the regional industrial world
and the academic-industry alliance model, to implement interdisciplinary research that is
necessary to overcome growth constraints in the regional businesses. Based on this theory
of education and research, this school also develops students into individuals who can plan
and manage research and development.

1.4Position/Type of employment

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I was trained as trainee and being supervised by Associate Prof. Takashi Mishima (R&D),
Food chemistry, Biomass laboratory. The description of work is on project and the scope of
work is handling of equipments such as High-performance liquid chromatography (HPLC)
analysis with different column, UV-1800 UV-Vis Spectrophotometer

1.5 Organization
1.5.1 President of Mie University

Atsumasa Uchida M.D.Ph.D

1.5.2 The organization in the laboratory

Assoc. Prof. Takashi Mishima

BUDI SANTOSO KOUTATSU SAKAKURA TOSHIHITO YURI SHIGETARO ARITA

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2.0 TRAINING
2.1 Activity and responsibilities

There are many activities or project that should be involved and be responsible towards the
works given. The following table is a list of the types of projects which I was involved in
during the course of my industrial training and the responsibilities and extent of my
involvement in each. From this course I was learn to handle many instruments, how to used
apparatus in laboratory and do the experiment accurately. All of the data were collected, then
recorded and analyze by using the instrument depend on what type of our substances. Besides
that, we also involved any outdoor activities such as science show with primary school.

Activity Responsibility
1. Used pipetman correctly  Measure the volume of distilled
water by using pipetman.
 Pipetman have many types with
different measurement.
 Weight the mass of distilled water
by using electronic balance.
 Decimal place is 0.0001g
 Calculate the weight average and

ELECTRONIC
coefficient of variation by using
PIPETMAN
BALANCE excel

2. Determination of reducing and  The standard solution was

nonreducing sugar using phenol-sulfuric prepared for Glucose, Maltose,
acid Galacturonic acid, Fructose and
Glucosamine
 Measure the absorbance when
react with the phenol-sulphuric
acid

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3. Solubility Amygdalin with different  Solvent: Distilled water,100%

solvent Ethanol and 50% Ethanol
 Solute : Amygdalin in powder
form
 The solubility of Amygdalin was
observe
 observe the absorbance by using
UV-1800 UV-Vis
Spectrophotometer
 Plot graph absorbance versus
wavelength
4. Run HPLC by using AsahiPak NH2P5O-4E  Mobile phase: 70% and 80% of
column for all standard sugar Acetonitrile.
 Kept for 1 day before run the
HPLC.
 Make the dilution of all Standard
sugar for Glucose, Fructose,
Sucrose, Maltose and Cellobiose.
 Dilute standard sugar with
different concentration :
10.0,0.1,0.01,0.00 mg/mL
 Sample was putted in glass bottle.
 Run the HPLC 30 minutes for
each sample
 Collect the data
 Sketch the graph concentration
sugar versus Area of peak by
using excel

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5. Prepare PTC Amino acid  PTC Amino acid need to dry

CENTRIFUGE EVAPORATION  Use centrifuge evaporation to dry
VACUUM PUMP V700 the sample.
SPIN DRYER
 Sample was putted in the eppen
tube.
 Handle the experiment in the fume
chamber.
 Make sure all the solution totally
mixed before add the mobile
TAITEC present mixer
phase.
 Shake the solution by using
blender

6. Run HPLC by using Wakosil –PTC  Set up theHPLC

column for amino acid  HPLC must be stabilize before
analyze the sample
 Run the sample.
 Analyze for each sample- 47
minutes.
 Collect the data and the peak of
the sample.
 Compare the area of peak for each
sample.
 Repeat the step above for
Glutamic acid and Aspartic acid.
 Dilute both samples with the
different concentration.

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3.0 Highlight on project

3.1 Objective
The objectives for this project are
i. Analysis the Standard sugar content in the vegetables and fruits
ii. Analysis the Amino acid content in the vegetables and fruits

3.2 Methodology

3.2.1 Extracted the fruits and vegetables for sugar and Amino acid measurement.

The fruit and vegetables got from the Mie University Farm near the Tsu City. The fruits and
vegetables that would use must be fresh for measure the standard sugar and Amino acid
content. All fruits and vegetables contain the different concentration of sugar and Amino acid.
There are five standard sugar will be measure are Glucose, Fructose, Sucrose, Maltose and
Cellobiose. While for Amino acid content are Glutamic acid and Aspartic acid. Firstly, the
fruits and vegetables must be extracted before do the analysing.

i. Make sure all the samples must be weight and record.

ii. The sample was cut into small pieces about 2~3g and then weight of each sample
must be record.
iii. Cut’s sample was put in the eppen tube.
iv. Distilled water was added three time of sample’s weight.
v. Use polyron to homogenize the samples and it take long time to blend the sample.
vi. Handle the equipment carefully because it is dangerous.
vii. The samples need to centrifuge 3000 rpm for 10 minutes to separate residue and
extracted solution
viii. Supernatant was filter by using filter paper number two for HPLC
ix. 1.0mL of extracted solution was collected and put in the eppen tube.
x. Keep the solution in the refrigerator.

3.2.2 Standard sugar measurement.

All the standard sugar with different concentration is already measure. The standard curve of
concentration (mg/mL) versus area will use to measure the concentration standard sugar for
each sample. The concentrations for each standard sugar are 0.1mg/mL,0.5mg/mL,1.0mg/mL

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and 5.0mg/mL. The concentration of extracted solution for each sample is unknowns. Before
run the HPLC , the concentration of each sample must be know because if the sample have a
high concentration ,the HPLC could not analyse the sugar content in the sample.

a) Thus, the sample must be reacting with Phenol-sulphuric acid to know the absorbance
each sample.
i. Prepare 10 times and 100 time dilution for each sample.
ii. 500µL of 5% phenol and 2500µL of 96% sulphuric acid was added.
iii. Handle the experiment in the fume cupboard
iv. The solution will turn to brown color and boil vigorously.
v. Leave the solution for 30 minutes until cold.
vi. Measure the absorbance by using the Biochrom WPA CO7500
Colorimeter
vii. Record all data
b) Dilution for HPLC
viii. 100 time dilution is most suitable concentration for run the HPLC.
ix. Dilute the sample with 50% of Acetonitrile for HPLC and fill in the small
glass bottle.
x. Before run the sample make sure that temperature and pressure is stable.
xi. The condition of HPLC are
a. Flow rate is 1min/mL
b. Use the AsahiPak NH2P5O column
c. Analyse for 30 minutes each sample
d. Use 80% of Acetonitrile as mobile phase
e. Detector type is RI
xii. Collect the data and calculate the concentration of standard sugar for each
sample from the peak area.
c) Preparation of mobile phase
i. 1L of 80% of Acetonitrile will be prepare
ii. 800mL of Acetonitrile is adding into 200mL of distilled water.
iii. The solution must be shake to homogenize
iv. Kept the solution in bottle for 1 day before use because Acetonitrile difficult
to mix with distilled water.

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3.2.3. Amino acid measurement

There are many types of Amino acid but for this project only Glutamic acid and Aspartic acid
content will be measure. Both Amino acid is important for our taste but not for healthy
because people will prefer to eat something that have taste. Tasty food is a main role in
quality of life and it will make people enjoyment during eating. From the standard curve
(concentration of Amino acid versus peak area) that has been plotted, the concentration for
both Amino acids can be calculated for all samples. PTC Amino acid must be prepare before
analyse the Amino acid contents by using HPLC.

a) Prepare solution A and B

 3mL of solution A
i. Ethanol: Deionized water:Triethylamine(TEA)=
(800µL:800µL:400µL)
ii. The solution was mixed by using TAITEC present mixer.
 3mL of solution B
i. Ethanol: Deionized water: TEA: Phenyl Isothiocyanate (PITC) = 2100
µL:300 µL:300µL:300 µL
ii. Mixed up the mixture.
b) Preparation of mobile phase A and B
 1000mL of mobile phase A
i. 960mL of Sodium Acetate was prepared (pH6.0)
ii. 60mL of Acetonitrile was added into Sodium Acetate
iii. Kept the mixture solution in the bottle 1 day before use.
 1000mL of mobile phase B
i. 400mL of Sodium Acetate was prepared (pH6.0)
ii. 600mL of Acetonitrile was added into Sodium Acetate.
iii. Kept the mixture solution in the bottle 1 day before use.

c) Preparation of PTC Amino acid

i. 50µL of extracted solution was taken and put in the eppen tube.
ii. The sample must be dry by using the a centrifugal evaporator until the solution is
dried.

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iii. 100µL of solution A was added and mixed

iv. The mixture sample was dried by using centrifugal evaporator
v. 100µL of solution was added in the same eppen tube and dry it
vi. The solution must be totally dry before adding 200µL of mobile phase A
vii. the mixture was mixed and filtrate by using membrane filter (0.45µm)
viii. the sample solution was kept in the freezer

d) Run the HPLC

 The condition of HPLC:
i. Use wakosil-PTC column
ii. Flow rate is 0.8mL/min
iii. Detector type is 254nm
iv. Analyse 47 min for each sample
 Collect the data and measure the concentration of Glutamic acid and Aspartic
acid.

3.3 Result

3.3.1 Standard sugar analysis

a) Concentration of standard sugar

1.60000
Concentration of standard sugar
concentration(mg/mL)

1.40000
1.20000
1.00000
0.80000
0.60000
0.40000
0.20000
0.00000 Glucose conc.
Tomato (Biggest)
Cucumber 2

Tomato (Big)
Cucumber 1

Cucumber 3

Chili (Paprika)

Tomato (Long)
Chili (Long)

Eggplant (Long/thin)
Melon
Eggplant (white)

Eggplant (Striped)

Gourd (Green)
Chili (Small)

Tomato(small/black)

Eggplant (Normal)
Tomato (Small/red)
Chili (Small,Big)

Tomato (Small/yellow)

Gourd (Yellow)
Eggplant (Small/green)

Fructose conc.
Sucrose conc.

Sample

3.3.2 Amino acid analysis

a) The result of concentration of Amino acid for each simple.

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6.000
Concentration of Amino acid
5.000
4.000
3.000
concentration(mg/L)

2.000
1.000
0.000

Glutamic acid Aspartic acid

3.4 Calculations
3.4.1 Standard sugar content

The concentration of standard sugar will calculate from the standard curve of standard sugar.
From the graph of concentration standard sugar versus area of peak, the concentration of
sugar content for vegetables and fruit can be calculated.

Standard sugar Standard curve

Glucose y = 5E-06x + 0.0499
Fructose y = 5E-06x + 0.0476
Sucrose y = 5E-06x + 0.1101
Maltose y = 6E-06x + 0.1405
Cellobiose y = 1E-05x - 0.4109

Sample 1: cucumber 1

The area of peak from chromatogram of sample 1 is

Glucose: 104307 and Sucrose: 129218

The concentration of Glucose y = 5E-06X + 0.0499

*X is the value of area of peak.

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y = 5E-06(104307) + 0.0499

= 0.57144 mg/ mL

The formula for ratio of concentration is

Ratio of concentration:

= mg/mL

=0.22857mg/mL

*calculations for standard sugar and Amino acid are same for all samples.

3.5 Discussion
This project we need to analyse the standard sugar and Amino acid content in the vegetables
and fruits by using High-performance liquid chromatography (HPLC). The fruit and
vegetable had been collected at the Mie University Farm. The sample must be extract to get
their supernatant. HPLC formerly referred to as high-pressure liquid chromatography is
a chromatographic technique used to separate the components in a mixture, to identify each
component, and to quantify each component. In general, the method involves a liquid sample
being passed over a solid adsorbent material packed into a column using a flow of liquid
solvent.

There are 21 types of sample had been analyse. We used AsahiPak NH2P5O column and
80% of Acetonitrile as a mobile phase to measure the standard sugar. Mobile phase is the
most important parameter in reversed-phase HPLC. Type of mobile phase used may have a
big effect on the retention time. Retention time is the amount of time elapsed from the
injection of a sample into the chromatographic system to the recording of the peak maximum
of the component in the chromatogram. It can promote or suppress an ionization of the
analyse molecules, and it also can shield an accessible residual silanol or any other active
adsorption canters on the adsorbent surface. Proper selection of the mobile phase is the
second most important step in the development of the separation method. In this project we
choose 80% of Acetonitrile because the peak of standard sugar found is sharper and narrow
compare 70% of Acetonitrile. The shape of peak is an important because it will shows the

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concentration of each standard sugar. The flow rate is 1 mL /min and the detector for analyse
sugar contents is ultraviolet (UV). Besides that, the solvent that use for dilution also play a
main role in this analysis. The peak sample with solvent 50% of Acetonitrile is more sharp
and narrow. Thus this peak is more accurate quantisation.

The concentration of standard sugar of each had been calculated from standard curve of
Glucose, Fructose, Sucrose, Maltose and Cellobiose. All the sample contain of Glucose and
Fructose while for Sucrose only for long and thin Eggplant and green gourd. The highest
concentration of Glucose contents is red small tomato and the lowest contents are yellow
gourd. For the highest Fructose concentration is small red tomato and the lowest is white
eggplant. If the vegetables and fruit have the high total sugar content, the taste will be sweet.
From the result of total concentration of sugar, the small green tomato the highest
concentration of sugar and the white eggplant show the lowest sugar content. For striped
eggplant and green gourd is also show the high sugar content because it contains Sucrose
comparing the other sample only contains Glucose and Fructose. The retention time for
Glucose, Sucrose, Frucrose, Maltose and cellobiose is 11.2 min.8.6 min, 17.8 min, 21.5 min
and 24.5 min. For all samples do not have peak on 21.5 min and 24.5 min.

The second part of our analysis is measure the Amino acid content. There a lot of Amino
acid but in this project we only measure Glutamic acid and Aspartic acid content. From the
result both the Amino acid had found in the all sample but their concentration is different.
We need to prepare the PTC Amino acid before analysis all the sample by using the HPLC.
Each sample must react with Phenyl Isothiocyanate (PITC) to be able to detect by HPLC.In
this analysis wacosil column will be use to analyse the Amino acid content. The sample must
be analysing for 47 minutes each sample. The flow rate is 0.8ml/min and the detector is
reflective index (RI). The RI is a measure of molecule’s ability to deflect lighting a flowing
mobile phase in a flow cell relative to a static mobile phase contained in a reference flow cell.
We need to analyse Amino acid mixture at first and at the end of the experiment because to
detect the retention time of Amino acid is change or not. The wacosil column are use two
mobile phases and it has the different concentration. From the result, the peak of Aspartic
acid will be appearing at time 3.99 minutes and Glutamic acid is at 4.22 minutes. The
retention time of Amino acid will be same if we not change their mobile phase. All samples
contain both of Amino acid but their concentration is different for all the samples. The

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highest concentration of Glutamic acid and Aspartic acid is chilli paprika and long tomato.
Therefore, both this sample tastier compares the other sample. While the lowest this Amino
acid is cucumber number two. As we know Glutamic acid is usually used for flavour in the
food and it is no side effects have been noted with this amino acid. There are few type of
tomato that had been analyzed and the result show that for the small tomato the
Concentration of Glutamic acid and Aspartic acid is more higher compare to the biggest
tomato. This is because of the biggest tomato has a higher volume of water. That’s why the
concentration of both Amino acids is lower compare to the smaller size. The highest total
concentration of Amino acid is chili paprika. People will be interested to eat chili paprika
because it is tastier compare cucumber number two because is tasteless.

3.6 Conclusion

From the analysis of sugar and Amino acid content by using the HPLC, their concentration
will be known for each sample. For the sample that has a higher sugar content the taste will
be sweeter and for Amino acid the sample is tastier and suitable used as flavor in the food.

4.0 Other Activity

During in an industrial training, the others work that I done is join the science show with

primary school. We had done three experiments to show to them

a. Air box gun with dry ice.

i. Make a hole at the box and dry ice was put in the box. All side of box was

cover by scotch tape and tap at the both side of the box. Then, we can see the

blow smoke will come out from the hole of the box

b. Balloon in the liquid nitrogen

i. Make a puppy from the balloon and the balloon was put into the liquid

nitrogen and then balloon will be compress immediately. It will turn to normal

shape when put at outside.

c. Color change

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i. Mix the Glucose, distilled water, Sodium hydroxide and indigo carmine and

shake the solution. It will change from green to red and to yellow. The

solution will turn back to green color when it static.

Air box gun with dry ice Balloon in liquid nitrogen Colour change

5.0 New Tools and Technologies

There are many new tools and high technologies that I used during the internships. The
equipment is more accurate and easy to handle. For example at the below:

1. Eppendoft tube

There are many sizes of eppendoft tube and capped plastic tubes. It is usually use for non
toxicity reagent. For the sizes range about 0.5-1.5mL it will thrown way after use. It is easy to
used compare put in the test tube and easy to keep because has a cap. For eppendoft tube with
yellow caps usually the size around 15mL until 50mL and it will reuse.

2. Pipetman

Pipetman is used for measure the small volume of liquid around 10µL to 1000µL. it is more
accurate and easy to use compare with pipette. Besides that, it will avoid the solution become
contaminate because after use the tube it will thrown out. Furthermore, the pipetman are
durable because it made from plastic.

3. Distilled water equipment

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Distilled water is water that has many of its impurities removed through distillation.
Distillation involves boiling the water and then condensing the steam into a clean container.
We used distilled water because it does not contain any ion compare tap water. When used
the HPLC, distilled water also play a main role. We need to filter the distilled water before
used for HPLC. In our laboratory it provides the equipment that produces distilled water and
already filters the distilled water.

4. Syringe’s filter

Supernatants are difficult to filter by using normal filter paper. Thus, by using syringe’s filter
the solution that will get from filtration is totally clear and suitable for HPLC. It will filter the
entire residue in the supernatants.

6.0 Suggestions for the Improvement of Training

The suggestion is to extend the industrial training more than three month because some of
company more prefer to accept the student will do the industrial more than three month.
Besides that, student also will learn many things and more experience that we will get from
the internship. Especially for the student do the internship at the abroad two month is not
enough. We only handle one project because we do not enough time to do another project. If
we do more than three month probability to get the scholarship during the internship is high
because internship at the abroad use more money compare at our country. Besides that,
student should be providing to do internship at the abroad because they will expose to
different and high technologies, management styles and achieve more knowledge.
Furthermore, it will gain career experience while gaining international experience that will be
helpful when applied work in the country. They also can improve their language skill and self
confidents. Besides that, student should be preparing their knowledge before entering the
laboratory.

7.0 Conclusion.

The experience and knowledge during the internship at Graduate School of Regional
Innovation Studies, Mie University it was great. This organization has a superb work culture,
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great minds and very high quality of work. At the laboratory, they provide many instrument
and their student will conduct the entire instrument by their own. During the internship, I
was introduced and learned to handle equipment such as HPLC for measure the standard
sugar and Amino acid contents, UV-1800 UV-Vis Spectrophotometer and Biochrom WPA
CO7500 Colorimeter for measure the absorbance and other equipment. The knowledge and
skills get from the internship will be use to apply a work and do the final year project.
Working with people from different country was a rare chance and it was another opportunity
to make friends and share ideas. During the internship I saw that Japanese people really
hardworking and very helpful. They will help us until the problem is solved. Besides that,
when student do the internship at the abroad they not only get the knowledge during working
but they also will learnt how to be independent and survive at the foreign country with
different culture, lifestyle and language.

8.0 References

i. L.R. Snyder, J.J Kirkland, and J.W. Dolan, Introduction to Modern Liquid
Chromatography, John Wiley & Sons, New York, 2009.
ii. Skoog, Holler, and Neiman. Principles of Instrumental Analysis. 5th edition.
Orlando: Harcourt Brace & Co., 1998.
iii. http://polymer.ustc.edu.cn/xwxx_20/xw/201109/P020110906263097048536.pdf
iv. http://hydrology1.nmsu.edu/teaching/soil698/student_material/HPLCHP1090/hpl
ccol.htm
v. Xiang, Y.; Liu Y. and Lee M.L. (2006). "Ultrahigh pressure liquid
chromatography using elevated temperature". Journal of Chromatography
A 1104 (1–2): 198–202.
vi. http://www.chemguide.co.uk/analysis/chromatography/hplc.html
vii. http://www.shimadzu.com/an/hplc/support/lib/lctalk/53/53intro.html
viii. http://cmdr.ubc.ca/bobh/methods/phenolsulphuricacidcarbohydrateassay.html
ix. http://en.09635.com/pd/4894058/UV-VIS-Spectrophotometer-UV-1800-.htm

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