The Differences of the Result of Copper Test Using UV-Vis

Spectrophotometry with Neocuproine Complexing Agent and AAS

Indah Sari

, Rima Merdaliani

, Pra Dian Mariadi

and Rosnita Sebayang

Forensic Science Depertement, University of Airlangga, Airlangga Street, Surabaya, Indonesia

Lecture, University of Catholic Musi Charitas, Palembang, Indonesia

Keywords: Copper, UV-Vis Spectrophotometry, Atomic Absorption Spectrophotometry

Abstract: Copper is a hazardous heavy metal and often pollutes the environment that may degrade the water quality.

This study is a comparative study, conducted in Baristand Industry Laboratory of Palembang. The samples

are SRM (Standard Reference Material) based on the standard working ranges that are made from pure

copper products from NIST (National Institute of Standards and Technology). The samples are assigned into

three points 0.5 ppm; 1 ppm; and 2 ppm from the standard working range. Data of the study differences of

copper examination results using UV-Vis Spectrophotometry with neocuproine complexing agent and

Atomic Absorption Spectrophotometry were analyzed by independent t-test. Levels of the three samples of

UV-Vis Spectrophotometry method were 0.5021 ppm; 1.0298 ppm; and 2.0109 ppm, respectively while

level of the three samples of Atomic Absorption Spectrophotometry method were 0.4803 ppm; 0.9957 ppm;

and 2.0024 ppm, respectively. Result of independent t-test exhibited the sig (2-tailed) value of p = 0.974

with the average difference of 0.0214667. Obtained p value was p > 0.05. Based on this study, it can be

concluded that there are no difference of copper examination results using UV-Vis Spectrophotometry with

neocuproine complexing agent and Atomic Absorption Spectrophotometry.

1. INTRODUCTION

Health laboratory is a health facility that performs

measurement, determination and testing of human-

derived materials or non-human derived materials

for the determination of diseases, health conditions

or factors that may affect the health individual and

society according to KEPMENKES RI No: 364/

MENKES/SK/III/2003.

Laboratory services in Indonesia are currently

being held in various types and levels of services,

such as in Puskesmas (Government Primary Health

Care Service) Laboratories, Regency/Municipal

Health Laboratories, Regency/Municipal Hospitals,

Public and Private Hospitals, Private Clinical

Laboratories, Central Laboratory of Health (BBLK)

and Health Laboratory Center (BLK) according to

KEPMENKES RI No: 1792/MENKES/SK/XII/

2010.

The types of health laboratories based on

services consists of clinical and public health

laboratories. Public health laboratory is a laboratory

which conducts examination services in the field of

microbiology, physics, chemistry and or other fields

related to public health interest and environmental

health, especially to support prevention of disease

and improvement of public health in accordance to

KEPMENKES RI No: 364/MENKES/SK/III/2003.

The chemical field consists of Aluminium (Al),

Iron (Fe), Hardness, Chloride (Cl), Manganese

(Mn), pH, Zinc (Zn), Sulphate (SO4), Ammonia

(NH3) and Copper (Cu) level assessment. Cu plays

an important role in the formation of red blood cells,

release of iron from the tissues, formation of bone

and central nervous system and other connective

tissues. Cu is also a component of certain enzymes.

United States of America assigned a safe ingested

level of Copper as 1.5 - 3.0 mg a day (Almatsier,

2009). Excess amount of copper will cause gastric

irritation, capillary blood vessel damage, damage of

liver, kidney and nerve tissues resulting in

depression (Windri, 2011).

Techniques used for metal examinations include

Atomic Absorption Spectrophotometry (AAS) and

UV-Vis Spectrophotometry. AAS is an expensive

sophisticated tool and not all laboratories possess it

while UV-Vis Spectrophotometry is the most

convenient technique due to its availability of

Sari, I., Merdaliani, R., Mariadi, P. and Sebayang, R.

The Differences of the Result of Copper Test Using UV-Vis Spectrophotometry with Neocuproine Complexing Agent and AAS.

DOI: 10.5220/0007542703510355

In Proceedings of the 2nd International Conference Postgraduate School (ICPS 2018), pages 351-355

ISBN: 978-989-758-348-3

351

instrumentation, simplicity, speed, precision,

accuracy, and low cost (Tehrani et al., 2014).

Copper determination can be conducted by

atomic absorption spectrophotometry (AAS).

Several studies had determined Cu levels in samples

such as Windri (2011), Listiowati et al., (2011) and

Sa'adah and Winata (2010) that examinied Cu levels

in samples using AAS.

Another method in addition to AAS is UV-Vis

Spectrophotometry which use a variety of

complexing agents. Applicable complexing agents

are 6- (2-Methoxynaphthyl) -2,3-dihydro-1,2,4-

triazine-3-thione that was carried out by Tehrani et

al. (2014), Ligan (2- [6-Nitro -2-benzothiazolylazo]

-4-hydroxy benzoic acid) that was carried out by

Jreo (2015) and neocuproine that was carried out by

Itnawita and Bali (2012).

Copper (Cu) examination using UV-Vis

Spectrophotometry with neocuproine complexing

agent (American Public Health Association) and

Atomic Absorption Spectrophotometry (SNI 06-

6989.6-2004) are well established and standardized

methods. Validation is usually performed for newly

manufactured and developed analytical methods,

whereas for available and standardized methods (e.g.

from AOAC, ASTM, and others) in exception for

the first time use in certain laboratories, validation is

usually unnecessary, only verification is required.

Verification of analysis method is a method

validation measure, an assessment of certain

parameters aimed to prove that the parameters meet

the requirements for its application. Parameter

verification methods include: linearity, detection and

quantitative limit, accuracy, and precision

(Anggraini, 2016). Based on description above, the

authors aim to find the difference of copper (Cu)

examination results using UV-Vis

Spectrophotometry with neocuproine complexing

agents and Atomic Absorption Spectrophotometry

(AAS) in which method verification will be

performed.

2. SUBJECT AND METHODS

This study was conducted in Balai Riset and

Standarisasi Industri (BARISTAND) from April 09

to May 03, 2017. The samples in this study were

made using SRM based on the standard working

range that were made from pure Copper (Cu)

products from NIST (National Institute of Standards

and Technology). The standard working range of

UV-Vis Spectrophotometry were 0.0; 0.4; 1,2; 2.0;

2.8; and 3.6 ppm (Standard Method, 2005) while

Atomic Absorption Spectrophotometry were 0.0

ppm; 0.2 ppm; 0.5 ppm; 1 ppm; 2 ppm; 3 ppm and 4

ppm (SNI 06-6989.6-2004). Water samples were

taken at three points 0.5 ppm; 1.0 ppm; and 2.0 ppm.

This study is a comparative study. The

examination methods used in this study is UV-Vis

Spectrophotometry with neocuproine complexing

agent and Atomic Absorption Spectrophotometry

(AAS).

3. RESULTS AND DISCUSSION

3.1 Verification of UV-Vis

Spectrophotometry and AAS

Methods

Method verification is a reconfirmation measure by

testing a method by completing objective evidences,

whether the methods meet the established

requirements and fit the objectives.

3.1.1 Determination of Wavelength on UV-

Vis Spectrophotometry

Determination of wavelength was conducted by

measuring the absorbance of copper standard

solution of 2 ppm concentration in the wavelength

range of 400 – 550 nm. The results of maximum

wavelength measurements were presented in Figure

Figure 1: UV-Vis Spectrophotometry Wavelength Curve

Based on above figure of wavelength spectrum, a

wavelength of 456 nm was obtained. Wavelength

range for copper examination using UV-Vis

spectrophotometry with a neocuproine complexing

agent is 450 – 460 nm (American Public Health

Association).

ICPS 2018 - 2nd International Conference Postgraduate School

352

3.1.2 Linearity

Method linearity is used to determine the standard

capability, so it can prove a linear relationship

between the analytical concentration and the

detector response (Wardani, 2012).

Linearity test is obtained by making standard

curve of copper examination conducted by making a

series of copper standard solution with various

concentrations of UV-Vis Spectrophotometry at 0;

0.4; 1,2; 2; 2.8; and 3.6 ppm whereas of AAS at 0;

0.2; 0.5; 1; 2; 3; and 4 ppm, made from a 1000 ppm

solution. Solutions were diluted to 20 ppm for UV-

Vis Spectrophotometry and 10 ppm for AAS then

each standard solution was read on UV-Vis

Spectrophotometer and AAS devices. The results of

the copper linearity curve using UV-Vis

Spectrophotometry can be seen in Figure 2 below.

Figure 2: Standard Curve of UV-Vis Spectrophotometry

Figure 3: Standard Curve of AAS

The absorbance value seen on the standard curve

of copper examination using UV-Vis

Spectrophotometry was y= 0,136x with linear

regression of r= 0.995. The absorbance value seen

on the standard curve of copper examination using

AAS was y= 0.119x with linear regression of r=

0.999. Both linear regression values (r) have met the

established requirements of r > 0.995 (Wardani,

2012).

3.1.3 Accuracy

Meticulousness is expressed as a percent return of

added analytics and the value of precision can be

expressed by percent recovery (Wardani, 2012). The

accuracy test was conducted by adding 1 ppm

concentration of copper standard solution into 1 ppm

copper sample, subsequently read using UV-Vis

Spectrophotometer and AAS % recovery then

calculated.

Figure 4: Copper Accuracy Examination

The average % recovery with 7 times

measurements is 95.98% in UV-Vis

Spectrophotometry and 95.57% in AAS. The result

of this retrieval test has met the precision of the

predetermined requirements. The recovery results

for the analytic in1 ppm matrix (%) sample, the

accepted recovery (%) was in the range of 80 -

110% (Wardani, 2012). Thus, the results of this

retrieval tests on both methods have met the

prescribed conditions based on the acceptable

recovery range.

3.1.4 Precision

Precision is a measure indicating the degree of

conformity among individual test results, measured

by distribution of individual results from the mean if

the procedure is applied repeatedly to the samples

taken from a homogeneous mixture. Precision is

measured based on standard deviation or relative

standard deviation (Riyanto, 2014). Precision test

was conducted by measuring standard copper

solution of 2 ppm concentration 7 times. The

precision test was obtained by calculating the value

of % relative standard deviation (% RSD).

y = 0.136x

R² = 0.995

0,2

0,4

0,6

01234

Abs.

Conc. (ppm)

Standard Curve of UV-Vis

Spectrophotometry

y = 0,1192x

R² = 0,9995

-0,2000

0,0000

0,2000

0,4000

0,6000

0246

Abs.

Conc. (ppm)

Standard Curve of AAS

1234567

UV-Vis Spectrophotometry

101,1393,84%91,93%90,55%100,7492,39%101,28

AAS

95,87%97,23%95,37%95,96%95,11%94,86%94,60%

80,00%

85,00%

90,00%

95,00%

100,00%

105,00%

% Recovery

Accuracy

The Differences of the Result of Copper Test Using UV-Vis Spectrophotometry with Neocuproine Complexing Agent and AAS

353

Figure 5: Precision of copper examination

Obtained %RSD was 1.12% in UV-Vis

Spectrophotometry and 1.92% in AAS. The result of

this precision tests indicated that both methods have

met the precision criteria. Precision criteria are given

if the method provide a relative standard deviation

(RSD) or a coefficient of variation (CV) of 2% or

less (Riyanto, 2014). The results of the second

precision test of this method showed that the

obtained accuracy is precise i.e. 1% < RSD ≤2%

(Wardani, 2012).

3.1.5 LOD and LOQ

LOD is the smallest amount of detectable analytic in

the sample and still provide a significant response

compared to the blank, while LOQ is the smallest

amount of analytic in the samples that still meet the

accuracy and precision criteria and quantifiable with

good accuracy and precision (Wardani, 2012).

LOD and LOQ tests were performed by

measuring the absorbances of the smallest copper

solution of 0.4 ppm for UV-Vis Spectrophotometry

and 0.2 for AAS. LOD and LOQ values were

calculated from regression equation of the obtained

copper standard calibration curve.

Figure 6: LOD and LOQ of copper examination

Obtained LOD and LOQ values in UV-Vis

Spectrophotometry were 0.0216 and 0.0720,

respectively while in AAS were 0.0144 and 0.0481.

Level below 0.0216 was no longer detectable by

UV-Vis Spectrophotometry and level below 0.0144

was no longer detectable by AAS, it is known that

AAS has smaller LOD than UV-Vis

Spectrophotometry so that AAS remains capable of

detecting very small samples. The LOD and LOQ

values met the specified requirements as no sample

levels were below the LOD and LOQ values.

3.2 Copper Examination on Samples

Examination of copper in this study was conducted

using two methods: UV-Vis Spectrophotometry with

neocuproine complexing agent and AAS using

aquadest solvent. Copper examination was

conducted simultaneously on the same day and time

for each sample, using a double beem UV-Vis

Spectrophotometry and AAS, as well as glass

apparatus including a separating funnel used for

extraction in UV-Vis Spectrophotometry.

Samples made from SRM were heated by adding

concentrated nitric acid performed in a fume hood.

After heating in the Spectrophotometry UV-Vis

method, neocuproine was added then extracted using

chloroform and the extract was subsequently diluted

using methanol, whereas in the AAS method, the

sample filtered using filter paper and diluted using

aquadest. Sample examination results of UV-Vis

Spectrophotometry and AAS were shown in figure 7

UV-Vis

Spectrophotometry

AAS

% RSD

1,12% 1,92%

0,00%

0,50%

1,00%

1,50%

2,00%

2,50%

Precision

LOD LOQ

UV-Vis

Spectrophotometr

0,0261 0,0720

AAS

0,0144 0,0481

0,01

0,02

0,03

0,04

0,05

0,06

0,07

0,08

LOD and LOQ

ICPS 2018 - 2nd International Conference Postgraduate School

354

Figure 7: Copper examination result of samples

Above figure showed that the results of copper

examination on the three samples using UV-Vis

Spectrophotometry and AAS methods exhibited

relatively no different results.

4. CONCLUSIONS

There are no difference in copper (Cu) examination

results using UV-Vis Spectrophotometry with

neocuproine complexing agent and AAS.

5. SUGGESTION

A one-time extraction and two-time extraction

differences are required in copper examination using

UV-Vis Spectrophotometry with neocuproine

complexing agent. Copper examinations using

samples taken from nature, for example: river water,

well water and wastewater are necessary.

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Sample 1 Sample 2 Sample 3

UV-Vis Spectrophotometry

0,5021 1,0298 2,0109

AAS

0,4803 0,9957 2,0024

0,5

1,5

2,5

Abs.

Cooper Examination Result of Samples

The Differences of the Result of Copper Test Using UV-Vis Spectrophotometry with Neocuproine Complexing Agent and AAS

355