Web of Science (Emerging Sources Citation Index), ISC

Document Type : Original Research Article


Department of Chemistry, College of Science, University of Baghdad, Baghdad, Iraq


A highly accurate, simple, sensitive and fast developed method was used for the determination of copper (II) ion using a homemade NAG-ADF-300-2 system with continuous flow injection analysis system. This method is based on reaction between copper (II) ion and rubeanic acid to obtain a black precipitate for the ion-pair complex. Turbidity was measured via the attenuated of incident light by precipitated at 0-180°. Optimum parameters were studied to increase the sensitivity for newly developed methodology of copper (II) ion -rubeanic acid system. The linear range for calibration graph was 1-25 mmol.L-1 for cell A and 1-30 mmol.L-1 for cell B, and LOQ 27.0538 µg /78.5 µL for cell A and 18.5539 µg /78.5 µL for cell B respectively with correlation coefficient (r) 0.9979 for cell A and 0.9992 for cell B, RSD % was lower than 0.5%, (n=8) for the determination of copper (II) ion at concentration 18, 23 mmol.L-1 for cell A and 12, 19 mmol.L-1 for cell B, respectively. A comparison was made between the newly developed method with UV-Spectrophotometric method as a classical method by tow type of statistical analysis for comparison the means and variance from different methods. The comparison of data revealed that long distance chasing photometer (NAG-ADF-300-2) was the choice with excellent extended detection, wide application and more sensitive.

Graphical Abstract

Attenuation of incident light sources (two flow tubes in one geometrical flow cell assembly: First with eleven sources while the second is covered by six sources) in CFIA for the determination of Copper (II) ion


Main Subjects


The alloys of copper are utilized in car radiators, warm exchangers, domestic warming frameworks and boards for retaining sun powered vitality. Copper (II) ion may be a naturally dynamic metal; its compounds influence crucial movement of plant and creature life forms. Copper is one of basic elements required for typical human digestion system [1]. Copper (II) ion is known to play noteworthy part in organic frameworks additionally as a pharmaceutical agent [2]. Its antibacterial properties have been known for thousands of a long time. Manufactured copper (II) complexes have been detailed to act as a potential anticancer and cancer repressing specialists and number of copper complexes have been found to be dynamic both in vitro and in vivo [3,4]. Hence it is essential to control that the substance of copper (II) ions particles within the natural objects is inside the reasonable concentrations.

Several methods depend on continuous flow injection analysis [5-12]. Different spectrophotometric methods have been proposed for the determination of copper substance of the different samples [13-15] and several analytical methods have been reported for determination of copper (II) ion including, HPLC-MSIS-ICP-OES [16], voltammetry [17], FAAS [18], capillary zone electrophoresis [19], and Ion-selective electrode [20].

In this work using Continuous flow injection analysis method, was measured via precipitation of copper (II) ion by rubeanic acid, a black precipitate product was obtained which was determined at angle 0-1800 using homemade long-distance chasing photometer (NAG-ADF-300-2) [21].

Chemicals and apparatus

Reagents and chemicals

All chemicals were used of the highest purity available and all the solutions dissolved by distilled water. A standard solution of 250 mmol.L-1 of Cu(NO3)2.3H2O (BDH), molecular weight 241.60 g.mol-1, was prepared by weight of 6.04 g and dissolving in 100 mL conical flask. A series of rubeanic acid (BDH) solutions were prepared from the dilution of standard solution 50 mmol.L-1 with distilled water. Two alloys samples preparation for the conducted research High purity copper (B.C.S.NO.197e) (99.95%) and low alloy steel (B.C.S.NO.255/1) (0.75%): 1 g of first alloy and 10 g of second alloy were weighed, transferred to 400 mL beaker followed by the addition of 1:1 HCL: HNO3 then heated gently with constant agitation until all brown fume was ceased. The remainder was diluted to 100 mL. The washed filter paper was washed few times, and the solution was collected in volumetric flask (100 mL).


Using the manifold homemade NAG-ADF-300-2 instrument is a multi-purpose photometric device including the offer of multi measurement individually or simultaneously or combine or separated whether 0-180  or 0-90  This applies to clear solution or colored or precipitated reaction product whether colloidal or crystalline colored or white or even clear precipitate. It is a long-distance chasing photometer as a flow cell with 300 mm as a distance with 2 mm as a path length to chaise and to accumulate the output resulted from attenuation of incident light 0-180° and diverged or fluorescence light at 0-90° via a flow cell. The first flow cell is of 110 mm length covered with 11 white snow LED (WSLED) followed by uncovered distance of 100 mm length then attached to another with 2 solar cell at each side of (0-1800 and 0-900) cell (cell no. 2) which was covered by 6 WSLED and a single photo cell (solar) of 60 mm length at each side was used with peristaltic pump (Ismatec, Switzerland) and six-port medium pressure injection valve (IDEX Corporation, USA) with sample loop (1 mm i.e. Teflon, variable length). Potentiometric recorder was used to estimate the output signals (Siemens, Germany). UV-Vis. Spectrophotometric (RF-1501, Shimadzu, Japan) was use for classical methods.


Using a manifold of two lines coupled NAG-ADF-300-2 instrument to determine Copper (II) ion via its reaction with rubeanic acid as shown in Figure 1. It is composed of two lines. The first line is the carrier stream of ammonium chloride (3.8 mL.min-1) flow rate will pass through the injection valve to carry the sample segment (78.5 µL for both cell) of 20 mmol.L-1 initial concentration Copper (II) ion to mix with second line at flow rate 3.8 mL.min-1 by Y-junction point that carries the reagent of rubeanic acid (17 mmol.L-1) for cell A and cell B before it is introduced to the NAG-ADF-300-2 analyzer, and then leading to measure using both cell A and cell B. The reaction product is black particles from the ion pair complex. The obtained signals which resulted from the attenuation of the incident light by the precipitating particles are agglomerated at 0-180°. It can be noticed that the results obtained from cell A are higher in sensitivity than the output signals from cell B. The higher sensitivity (i.e.; 46% enhanced signal (S/N-YZ (mV) compared with cell A) might be attributed to the completion of the formation of particulate, rearrangement of precipitated particulate or re-crystallization effect which might be occurs in the cell B.

Scheme 1 shows a proposed expected mechanism for the reaction of copper (II) ion with rubeanic acid in aqueous medium [22,23].

Results and discussion

Optimization of variables

Chemical variables

Rubeanic acid concentration

A series of the rubeanic acid solution ranging from 5-20 mmol.L-1 were prepared using 78.5 μL sample volume that was injected into the carrier stream line (distilled water). 20 mmol.L-1 of copper (II) ion was injected with 3 mL.min-1 flow rate for carrier stream line and reagent line. The increase of rubeanic acid concentration leads to an increase in the peak height expressed as an attenuation of incident light reaching 17 mmol.L-1 obtaining highest sensitivity; this can be attributed to the nature of formed particulate (e.g., Colloidal, crystalline, or suspension) and its surfaces, also its tendency to obscure the direct light to the detector. Dealing with higher concentration (i.e.,> 17 mmol.L-1) leads to decrease of peak heights that were obtained from both cells. This might have been probably caused by the increase of accumulation of particulate to prevent the optical fiber phenomenon, which might occur in the measuring flow cell that will increase the light intensity. These results were provided through with slope – intercept method as segment a4 –a6 (Figure 2) was the choice thus 17 mmol.L-1 falls for both cells.

Effect of different media (Selected salts and acids)

Copper (II) ion 20 mmL.L-1 reaction with  rubeanic acid 17 mmol.L-1 for both cells, 78.5 μL sample volume and 3 mL.min-1 flow rate for two line (rubeanic acid line & carrier stream line)  to form black color precipitate was studied in different medium at 50 mmol.L-1 concentration (ammonium acetate, ammonium chloride, sodium nitrate, sodium sulfate, sodium chloride and potassium chloride ) as a salt media, as well as the use of  50 mmol.L-1 concentration (hydrochloric acid and sulphuric acid) as an acid media in addition to distilled water used as a carrier stream. From Figure 3, it was noticed that all media solution (salts & acids) used for cell A leads to an increase of (S/N)- response due to the effect of formation of tiny solid particulate that might be the cause to decrease of inter spatial distances and an increase of attenuation of incident light, while for cell B was noticed that there is no clear significant effect from the data response obtained except ammonium chloride and ammonium acetate. In general, salts and its negative radicals helps to reconstruct and the accumulation of precipitated particulate which increase the prevention of light and then increase peak height. The same effect was noticed when we use NH4Cl while acids in general helps peptize, precipitate and scatter them causing the spectral filtration which in turn causes decrease in sensitivity; therefore, acids were avoided. NH4Cl was used as a transferring medium to improve the sensitivity of measurement to determination Cu (II) ion.

Effect of [NH4Cl]

The effect of NH4Cl was investigated by using Copper (II) ion (20 mmol.L-1)- Rubeanic acid (17 mmol.L-1) system for both cells, 78.5 μL sample volume at 3 mL.min-1 flow rate for carrier stream and reagent. Variable concentrations of ammonium chloride at ranging 10-60 mmol.L-1 as a carrier stream were used. From Figure 4, it was noticed that an increase of ammonium chloride concentration for the range 30-50 mmol.L-1 leads to an increase the output signal for both cells. This might be attributed to the ability or the tendency of the salt in growing the nuclei to crystals followed by the formation of compact granules with each other, thus causing the surface area that obscure the generated light which coming from W.S.LEDS i.e., it makes a more compact barrier compared with the rest of the solution. When dealing with concentration more than 50 mmL.L-1, a decrease in peak heights was noticed. This could be attributed to the dispersion of precipitate to a smaller tiny scattered without affecting on obstruction of incident light and this is called spectral filtering. A 50 mmol.L-1 was chosen as the most favorite concentration to be used on all over this work for both cells.

Physical variables

Flow rate

Variable of flow rates (0.5-5) mL.min-1 for two lines (50 mmol.L-1 of NH4Cl solution as a carrier stream and  Rubeanic acid  as reagent line ) for cell A and cell B was used at copper(II) ion (20 mmol.L-1)-rubeanic acid (17 mmol.L-1) –NH4Cl 50 mmol.L-1 system, 78.5 μL sample volume for both cells. The obtained response profile is shown in Figure 5. It can be observed the obtained responses are distorted especially at low flow rates, in the meantime, enough time is available for successive growth of crystals to large particulate which in turn will be affected by the motion of the peristaltic pump in case of relaxed and pressed mode. In case of pressed mode, the formed precipitated particulate will be pressed causing an accumulative compactness leading to obscure the incident light and distorted for the obtained responses, while at high speed, a more uniform response is due the unavailability of enough time for crystal growth. Also, no spreading of precipitated particulate on a wider tube length causing loose of sensitivity and response time will suffer from extension. Therefore, the 3.8 mL.min-1 flow rate for both cells will be the choice within it to obtain a regular response.

Sample volume

Sample volumes variations (40-281) μL were studied at optimum flow rate 3.8 mL.min-1 for both lines (NH4Cl line & reagent line), with selected concentration (20 mmol.L-1) of copper (II) ion, rubeanic acid of (17 mmol.L-1) and NH4Cl of 50 mmol.L-1 were used. It can be seen from the reported results in Table 1 there is an increase in sensitivity with the increase of sample segment (loop) up to 78.5 μL and obtaining symmetric responses reflects the form of crystal formed. Here the crystals are spherical; in addition to aid in moving with the carrier stream, the speed will be higher (more than) and falls within the effect of convection. While at volume larger than 78.5 μL., responses are lower and a width at the apex and base width that will be obtained from cell A. This might be attributed to the nature of formed crystals and its morphology i.e., non-spherical form which causes a slow movement and falls on the effect of diffusion. This effect will cause the transparency for certain or some of the particulate and obtaining spectral filtering; therefore, the responses are smooth and losses in sensitivity are minimized. Based on the above statement, and in order to compromise with the economy of sample usage, 78.5 μL was chosen as it could give the highest sensitivity.

Delay reaction coil

In general, the aim of this study was to decide whether a reaction (direct attachment) or delay coil necessary for maximum reaction rate or precipitate particulate formation or growing to form a dense of particulate or granules to increase a high sensitivity and taking into account a compromise should be made between reaction completion and avoiding excessive dilution. Effect of variable coil lengths of 10,15, 25 and 30 cm were studied which was attached after Y- junction point, conducted on the use of copper (II) ion (20 mmol.L-1) – rubeanic acid - (17 mmol.L-1)- NH4Cl (50 mmol.L-1) system, 78.5 μL sample volume and 3.8 mL.min-1 flow rate for both cells. The increase of reaction coil volume for both cells (Cell A & Cell B) led to decrease of response height with increase of base width (ΔtB); these might be attributed to diffuse and dispersion of precipitate particles, causing increase of the dispersion regions and leading to decrease in the (S/N) energy transducer response which in turn gave less sensitivity of response. Table 2 tabulates all these results. In addition to the tabulated results of doing the precipitation reaction in a dynamic system, the presence of delay reaction coils will give an enough time to growth crystals in addition to increasing the effect of dilution and dispersion of the large particulate on a wider area causing the loss of sensitivity, also measurements of time will be longer. Due the presence of water with the body of precipitated particles, photons will suffer from internal refraction and dispersion leading to divergence of incident light and decrease of sensitivity. Therefore, there is no need for delay reaction coils and direct attachment is regarded as the most suitable for both cells.

The study of the applied voltage expressed as an intensity used for supplying the White Snow Light Emitting Diodes (WSLEDs) in NAG-ADF-300-2 analyzer

A study was carried out to determine the effect of intensity of incident light of the irradiation sources on the (S/N)- response of the energy transducer response via the selector switch. Using the control of the light intensity knob (they are two), the increase will be maximized clockwise at four stages (level of irradiation I=1-2-3-4 i.e., 3.0 VDC, 3.2 VDC, 3.4 VDC and 3.5 VDC, respectively. In addition to the off position, an indication of LED is controlled electrically for intensity operation i.e., indication lamp for cell A attached with eleven sources of WLED plus and indication lamp for cell B parallelly attached with the six WLED.  This study was conducted with physical and chemical parameters achieved in previous sections which were kept constants for both cells. 3.4 VDC is the optimum for both cells (Even 11 WLED were used in cell A while 6 WLED for cell B), It was noticed from figure 6 that a selection of position 3 (i.e.; I=3) was very convenient intensity for cell A (cell no.1) (larger number of the selector switch means more light intensity) while position 2 (I=2) of the selector switch was a convenient intensity for cell B (cell no.2).

Variation of concentration with obtained response leading to calibration range

The steps involved concentration (measured as tsec (dmm) as the x- axis versus the response YZi (mV)) and this falls in the range i.e., the extent to which or the limit between which variation is possible. i.e., obtaining a peak which mean a mountain with a pointed summit, and this prevents noise (electrical disturbance). A series of solutions ranging 1-50 mmol.L-1 were prepared leading to the scatter plot as shown in Figure 7A & B in which a correlation of YZ (mV) versus tsec (dmm) of 0.9333 and 0.9420 with coefficient of determination of 0.8711 and 0.8882 is given and the chosen equation represents 87.11% and 88.82% of the obtained results for cell A and cell B consequently which is fair enough. Therefore, both cells were able to distinguish various ranges of concentration. Table 3 summarizes all results for both cells (A & B). Ordinary spectrophotometer will never reach this level of concentration. If under any circumstances higher concentration was required for analyte dilution, it can be used to make the responses with the range available in the text above.

Three approaches were used to describe the limit of detection (L.o.D). Any one of the described definition could be used depending on the practical research need. While Limit of quantitative (L.o.Q) will be decided by the research to decide the comfortable concentration to deal with and always L.o.Q is higher than L.o.D (Table 4).

To study the efficiency of the newly developed methodology using NAG-ADF-300-2 analyzer for determination of copper (II) ion, it was compared with an available literature method of UV-spectrophotometric which was based on the measurements of absorbance for the range of concentration 1-37 mmol.L-1 at λmax= 300 nm [24] using quartz cell. The scatter plot and the best linear range extended from 1-27 mmol.L-1 with (r) of 0.9979 and % capital R square= 99.59 %, n=12 (no. of measurements). Table 5 shows the variable data treatments. The limit of detection was 0.01 mmol.L-1 equivalent to 0.6355 μg/sample.

Assessment of NAG-ADF-300-2 analyzer using both cells for the determination of copper (II) ion in alloys

A comparative study was done between the developed method and conventional method to determine copper in two types of alloy from different manufactures with different contents. On this basis, the quoted value is the absolute reference value that will be compared with the values from the table 6 A & B. The values of the copper (II) ion were obtained using copper (II) ion – rubeanic acid (17 mmol.L-1) -NH4Cl (50 mmol.L-1) system, flow rate 3.8 mL.min-1 of each line, 78.5 μL sample volume for cell A and cell B respectively, in addition to direct attachment delay reaction coil for cell A and cell B, I=3 for cell A and I= 2 for cell B which form a black precipitate. The standard addition method for both methods (i.e., developed method and classical spectrophotometric method) were applied by preparing a series of solutions  from sample no.1 via transferring 1.27 mL of (0.157  mol.L-1) to each volumetric flask (25 mL), followed by the addition of (0, 0.5, 1, 2 and 3 mL) from 100 mmol.L-1 of standard solution to obtain 0, 2, 4, 8 and 12 mmol.L-1 and 16.95 mL from sample no.2 of (0.0118 mol.L-1) to volumetric flask (25 mL), followed by the addition of (0, 0.5, 1, 2 and 3 mL) from 100 mmol.L-1 of standard solution to obtain 0, 2, 4, 8 and 12 mmol.L-1. Table 6B represents the form of two types of statistical analysis for comparison the means and variance from different methods.

The first comparison was based on the one-way ANOVA (F-test) [25,26], which was carried out at α = 0.05 (95 % confidence level) for compare between four different methods (i.e., Cell A, Cell B (using NAG-ADF 300-2), UV- spectrophotometry and reference method).

This test (i.e., ANOVA) (Table 6. B and scheme 2 summed up the obtained results) depends on the calculated F- value for comparing three or more means. The first estimate is called between group variance while second estimate is based on the within variance.

The hypothesis should be used as follows:



H0 (Null hypothesis):  Cell A =  Cell B =  UV-spectrophotometry =  reference quoted method                   

A gainst

H1 (Alterative hypothesis): At least one mean is different from the other

i.e.,  Cell A  Cell B    UV-spectrophotometry    reference quoted method



From the results obtained that were tabulated in Table 6B (Column 5), the kind of two samples of alloys shows no significant difference between the means since the value of Fcal (5374.6940) >> Ftab (5.99); therefore, null hypothesis will be accepted and the alternative hypothesis will be rejected.

As for the second comparison, we used paired t-test to compare the newly developed method i.e.; NAG-ADF-300-2 analyzer for both cells with the classical spectrophotometric method at λmax = 300 nm. The obtained results summed up in Table 6.B (column 6) and scheme 2, in which a hypothesis can be estimated as follows:

Null hypothesis: There is no significant difference between the means obtained from both methods


i.e., H0 = i (NAG-ADF-300-2 analyser)= i( classical – spectrophotometry)


(for either of the two alloys)


Alternative hypothesis: There is a significant difference in the means of both method for two samples


i.e., H1 : i (NAG-ADF-300-2 analyser) I (classical – spectrophotometry)


for either of the two alloys

And, from the results, it was concluded that there was no significant difference between the two methods at 95% confidence level as the calculated t- value (tcal= 0.9855 (cell A), 0.9956 (cell B)) is less than critical t- value (12.706).

On this basis, two methods (cell A& cell B) were added to determine the copper (II) ion in addition to the traditional method.


The unchallenged precision and accuracy gained in this research work using NAG-ADF-300-2. Shows that an attenuation method that can be used to determination Copper (II) ion in a well trusted measurements, which is the complete agreement with the cited values. No other turbidimetric method is available in the literature that can follow the same mode of working with the simplicity of the manifold used. Therefore, an alternative method is available with extended linearity and excellent L.o.D. 


I would like to express my deepest gratitude to Professor Issam M.A. Shakir for his appreciable advice, important comments, support and encouragement.


Nagham S. Turkey: https://orcid.org/0000-0002-6562-0636


How to cite this article: Ghadah F. Hussein*, Nagham S. Turkey.  Attenuation of incident light sources (two flow tubes in one geometrical flow cell assembly: First with eleven sources while the second is covered by six sources) in CFIA for the determination of Copper (II) ion. Eurasian Chemical Communications, 2021, 3(11), 763-778. Link:  http://www.echemcom.com/article_137170.html


Copyright © 2021 by SPC (Sami Publishing Company) + is an open access article distributed under the Creative Commons Attribution License(CC BY)  license  (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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