Indones. J. Nat. Pigm., Vol. 02, No.

1 (2020), 17–20

Simple Photometer Development For Educational Purposes in Natural

Pigment Analysis

Jovine Marcella Kurniawana, and Marcelinus A.S. Adhiwibawaa*

a
Ma Chung Research Center for Photosynthetic Pigments, Universitas Ma Chung, Malang 65151, Jawa Timur,
Indonesia
*Corresponding Authors: marcelinus.setya@machung.ac.id

Article History: Received 24 February 2020, Revised 04 March 2020, Accepted 04 March 2020, Available Online 06 March 2020
Abstract
In this paper, we will discuss the making of the simple photometer instrument to measure the absorbance of natural pigment crude
extract. To make simple photometer, cuvette holder was printed using 3D Printing Machine, the development of simple photometer
instrument also uses some components such as LED light (633 nm) as the light source, Photodiode-TSL250 as a detector and a
variable resistor to set the initial intensity of the light source. In this development, Arduino Uno was also used as USB data
acquisition device to capture the signal from the instrument. The results of sample measurements between simple photometer
instrument and UV-Vis 1800 spectrophotometer showed 98% regression coefficient of determination and simple photometer has a
LOD and LOQ absorbance at 0.0267 and 0.0344. Simple photometer instrument was able to show a similar response as a
commercial instrument. Through the development of simple photometer, the student can easily understand the working principles
of a spectrophotometer in natural pigment analysis that difficult to studied in detailed before. The process of making a simple
photometer will be very useful for the learning process of students.

Simple photometer development: A simple photometer prototype

consisting of a light source(LSR), cuvette holder(C), light
sensor(LSN) and microcontroller(M) can be used as a medium for
learning instrumentation in detail. Students can build their own
instruments with low costs in a short time. It is expected that by
interacting in the development of a simple photometer students can
understand the work process of a spectrophotometer in natural
pigment analysis.

Prototype of simple photometer instrument

© 2020 MRCPP Publishing. All rights reserved.
http://doi.org/10.33479/ijnp.2020.02.1.17
Keywords: Photometer, Natural Pigment, Arduino Uno

INTRODUCTION that experiences maximum absorption, while

Spectroscopy is an analytical method that used to test
material based on the light emitted, absorbed or reflected
by the material [1,2]. The instrument used in
spectroscopy is a spectrophotometer [4,5]. One type of
spectrophotometer is a visible light spectrophotometer
[6,7,8]. The tool can be used to determine the
characteristics of a material in solution, in the form of
maximum absorption wavelength, absorbance value and
concentration of material in solution, by giving light
radiation to the solution and then measuring the intensity
of the light absorbed by the solution[9,10,11]. Radiation
can be polychromatic and monochromatic light;
Polychromatic light is used to select the spectrum of light
Adhiwibawa, et al. (2020)
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monochromatic light can make data accurate because it
has a narrow wavelength range [12,13,14,15].
The importance of spectrophotometers in the
analysis is often not followed by knowledge of how the
spectrophotometer works. This development was
conducted with a principle that resembles visible light
spectrophotometers, using simple tools and materials
such as LEDs, cuvette holders, light sensors, Arduino
Uno and computers [16,17,18,19]. Furthermore, in this
study students can take measurements of natural
pigments using the Lambert-Beer principle on a simple
photometer instrument.
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 Indones. J. Nat. Pigm., Vol. 02, No. 1 (2020), 17–20
EXPERIMENTAL
Cuvette Holder Design
Cuvette holder design was created with the 123D
software. Before designing the cuvette holder,
measurements of length, width, and height cuvette to be
used was made. To place the light source and the light
detector precisely it is also necessary to measure the
hole position in a straight parallel line. After getting the
appropriate design, then the design was printed into
three-dimensional cuvette holder with a three-
dimensional printer. Cuvette holder was printed using a
black colored material in order to avoid light reflection
that may affect the absorbance values of the
compound[20].
LED lamp as the light source
LED was used as a light source in the simple
photometer, In this experiment the wavelength of LED is
633nm. This light source is used as the sample to be
measured is crude extracts of Spirulina sp which have
been known to contain chlorophyll pigment. The green
color of the pigment can absorb red light at wavelength
region around 600-700 nm so that light with a wavelength
of 633 nm will be suitable for use.
The Light Detector IC-TSL250
The detector is an important part of the photometer
development. The detectors will capture the light signal
and will transform the light signal into an electrical
current. The higher the intensity of light is detected, the
greater the current generated. In the experiment, IC-
TSL250 was used as the detector.
Arduino Uno Microcontroller
Arduino Uno is a microcontroller that can be used to
convert analog current signals into digital signals. Analog
pins from Arduino Uno connected to IC-TSL250. Then
Arduino Uno ADC converted analog current signal that
displayed on Arduino IDE by Using PC[21,22].
Instrument Schematic of Simple Photometer
Once all the equipment is ready, the photometer
instrument coupled with a scheme as shown below:
Natural Pigment Sample Preparation
A total of 0.1 grams of Spirulina sp powder was weighed,
CaCO3 and Sodium Ascorbate was added as a
neutralizing agent and antioxidants. Samples were
extracted with 10 ml of 100% methanol solvent,
homogenized with a vortex for 2 minutes, sonicated two
times for 2 minutes, and centrifuged for 2 minutes at a
speed of 10,000 rpm. The extraction is done in duplicate,
and the supernatant obtained is stored in a vial and
diluted at least 25 times as the stock solution for
absorbance measurement using the simple photometer.
Adhiwibawa, et al. (2020)
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Absorbance measurements and Method Validation
Absorbance measurement was conducted by
determining the initial intensity of LED light. After that, the
solvent absorbance also measured to make sure the
absorbance of the sample is not affected by the solvent
that used. Then, the absorbance of the sample was
measured. Measurement of the absorbance can be done
by using the equation:
Vsample is a sample light signal detected by Arduino Uno,
Vled is a an initial light source signal and T is light
transmittance. Samples were also measured with a
commercial spectrophotometer Shimadzu UV-1800 as
instrument performance comparison.
LOD (limit of detection) and LOQ (limit of Quantification)
was also conducted to know the sensitivity of the simple
photometer. The limit of detection (LOD) is the lowest
concentration of the sample can be measured and limits
of quantitative measurement (LOQ) is the lowest
concentration of the sample can be measured
absorbance by means of acceptable precision and
accuracy in statistics[23]. Here is the equation used to
determine the sensitivity:
Sensitivity : LOD or LOQ
k : 3 to 10 for the LOD and LOQ
SD Blank :The standard deviation of the
absorbance of the blank
μ : Average absorbance of the blank
RESULTS AND DISCUSSION
Design of Simple Photometers
The design of the photometer using the 123D application
has resulted in a functional cuvette holder model.
Educational photometer consists of LED light sources,
cuvette holders, light detectors and microcontrollers.
Educational photometer measurements consist of solvent
and sample measurements. Making a photometer in
accordance with a single beam system. This simple
spectrophotometry system contained IC TSL-250 which
was installed on the cuvette holder. Educational
photometer has dimensions of 5.5 × 1 × 5 cm.
Educational photometers operate at 5 Volts with a
current of 30 mA. The LED light source uses a
wavelength of 663 nm adjusted to the chlorophyll light
absorption as the target compound. Black cuvette holder
to reduce interference from outside light. The light signal
from the TSL-250 IC sensor is processed by Arduino
UNO and displayed at the computer layer using the
Arduino IDE software interface. The picture below is a
simple photometer instrument that used to measure the
absorbance of Spirulina sp crude extract sample with
various concentration.
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 Indones. J. Nat. Pigm., Vol. 02, No. 1 (2020), 17–20

Figure 1. Simple Photometer Prototype

Quantitative measurement of Simple

Photometers
Table 1 presents observation data obtained by using
simple photometer, where absorbance shows a positive
trend along with an increase in sample concentration.
The intensity value of the photometer instrument has Figure 2. Regression between simple photometer and
been converted from the digital number to voltage. The commercial instrument
absorbance results obtained by the photometer show a
difference with the absorbance results of commercial
spectrophotometers. This is influenced by the differences
in the light source and the detector used by the device,
the difference in the wavelength of the light source, and
the inaccuracy in diluting stock solutions.

Table 1. Simple Photometer Observation Data

Vled Vsample
C T (%) A
(mV) (mV)
0% 1002.35 949.64 94.74 0.02
40% 1002.35 816.91 81,50 0.08
50% 1002.35 788.60 78.68 0.10
60% 1002.35 730.04 72.83 0.13
70% 1002.35 695.88 69.43 0.15 Figure 3. Regression of simple photometer and
commercial instrument for different concentration
80% 1002.35 651.96 65.04 0.18
90% 1002.35 520.20 51.90 0.28 After measuring the absorbance of the sample, the next
100% 1002.35 476.28 47.52 0.32 step is the validation of the photometer which is
conducted by measuring the sensitivity of the instrument.
LOD and LOQ are obtained by calculating the average
However, if the regression coefficient of determination absorbance of the blank and its standard deviation. Table
between the absorbance results of the two instruments is 2 present LOD and LOQ calculation.
calculated, then the obtained R2 is 98%. This shows that
Table 2. Simple Photometer LOD and LOQ Calculation
the absorbance measurement using a photometer has a
linear relationship to a commercial spectrophotometer.
The average absorbance of the
Regression between simple photometer and commercial
blank 0.0234
instrument present in Figure 2.
The standard deviation of the
Figure 3 presents a regression curve for measuring absorbance of the blank 1.095×10-3
simple and commercial photometers. The R2 value LOD (Absorbance) 0.0267
generated from the linear regression equation using a
LOQ (Absorbance) 0.0344
simple photometer is 0.9269 while using a commercial
photometer is 0.9745. The low value of R2 simple
photometer shows that the data obtained is less
In table 2, it can be seen that the detection limit or the
accurate. Some factors which are thought to cause
lowest sample concentration that can be detected by a
inaccuracies in measurement are errors in sample
photometer instrument is 2.67%. This means that if the
dilution, inaccurate adjustment of the initial intensity of Spirulina sp crude extract sample is less than 2.67%, the
LEDs which are regulated using a variable resistor, and photometer instrument detector is not able to detect its
different light sources between a simple photometer and
presence. For the LOQ, the simple photometer
a commercial photometer.
instrument has a minimum quantification or concentration
limit that can be detected by the instrument and is
statistically trusted at 3.44%.
Adhiwibawa, et al. (2020)
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 Indones. J. Nat. Pigm., Vol. 02, No. 1 (2020), 17–20

CONCLUSION
The results of sample measurements between simple
photometer instrument and UV-Vis 1800
spectrophotometer showed 98% coefficient of
determination and simple photometer has a LOD and
LOQ absorbance at 0.0267 and 0.0344. Simple
photometer instrument was able to show a similar
response as a commercial instrument. Through the
development of simple photometer, the students could
easily understand the working principles of a
spectrophotometer in natural pigment analysis that
difficult to studied in detailed before.
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Abstrak
Pada jurnal ini akan dibahas mengenai pembuatan instrumen fotometer sederhana untuk mengukur absorbansi molekul, mulai dari pembuatan
tempat kuvet menggunakan teknologi 3D Printing, menyusun instrumen fotometer menggunakan lampu LED (633 nm) sebagai sumber
cahaya, Photodiode-TSL250 sebagai detektor dan variabel resistor untuk mengatur intensitas awal sumber cahaya. Selain itu akan dibahas
pula mengenai penggunaan alat Arduino Uno sebagai USB Data Acquisition Devices untuk menangkap sinyal dalam bentuk tegangan dari
instrumen yang dibuat, dimana akan dihubungkan juga dengan penggunaan software Arduino sebagai media untuk menampilkan hasil.
Sebagai uji coba instrumen yang telah dibuat, dilakukan pengukuran absorbansi pada ekstrak pigmen kasar Spirulina dengan menggunakan
persen konsentrasi yang berbeda-beda. Hasil pengukuran absorbansi yang didapat dibandingkan dengan instrumen Spektrofotometer
komersial Shimadzu UV-1800 sehingga dapat dilakukan validasi metode terhadap instrumen fotometer buatan sendiri.

Kata kunci: fotometer, pigmen alami, arduino uno

Kata kunci: kata kunci 1, kata kunci 2 (sampai kata kunci kelima)

Adhiwibawa, et al. (2020)

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