Lab–Built Semi-automated Stop–Flow System for Spectrophotometric Phosphate Determination in different water samples

 

Mohammad Th. K. Al-Balaawi¹, K. H. Al-Sowdani²

¹Marine Chemistry Department, Marine Science Center, University of Basra, Iraq.

²Chemistry Department, Education College for Pure Sciences, University of Basra, Iraq.

 

ABSTRACT:

A lab built semi-automated stopped-flow system was employed for spectrophotometric determination of phosphate in different water samples. The construction system consists made software programs. The first one was UNO type used to control and manipulates a home-made injection-pump stepper-motor type to control the reaction-time in the flow cell. The other one was Mega type and which was used as data-logger to manipulate and recording the results as stopped-flow peaks by using Microsoft Excel 2010 program. The linearity was up to 2 µg/ml with regression coefficient of 0.9960. The detection limit was 0.05 µg/ml and the samples can be analyzed at rate exceeding 45 samples/h.

 

 

 

INTRODUCTION:

There are many techniques were used for spectrophometric determination phosphate^1,2,3. Flow injection analysis is the most frequently used techniques. A stopped – flow, which is a modern techniques driven from flow injection analysis⁴. This mode of flow analysis is based on stopping the carrier flow and let the reaction to take place in the measuring flow cell. The flow is stopped by shut down the propelling pump or by means of valve. Stopped–flow system has many advantages as simple, easy to handle, flexible in application, inexpensive and allowed better measurements sensitivity⁵.

 

Currently, a wide variety of advance technologies have been applied in the field of analytical chemistry⁶. Open sources platforms such as Ardniuo micro-controllers have gained considerable attention from chemists, due to their low cost, integrated development interfaces and does not require expert knowledge^3,7,8.

 

 

 

 

So, thought of combing the advantages of stopped-flow technique as sensitive, selective and low consumption of reagent and sample with two Arduinos were supplied with suitable software. First one was Uno tybe to efficient, reproducible control the home-made injection pump stepper motor type and to stop the chemical reaction spontaneously in the flow cell were the reaction take place with high precision. The second one was Mega types which used as a datalogger to manipulate and recording the results of the stopped flow system as a peak height using Excel program 2010⁴.

 

It was decided to construction and build up a semi-automated stopped-flow system which can readily be assembled from inexpensive components and evaluated the system for phosphate determination.

 

To the best of our knowledge such as that lab-built semi-automated stopped-flow system equipped with Arduino micro control platform and suitable software have not previously been applied for phosphate or other substance in Basra University.

 

MATERIAL AND METHODS:

Reagents and samples:

During analytical application with lab- build stopping flow system deionized distilled water was used throughout and all reagent employed were analytical grade unless otherwise stated.

 

The value of measurement as peak height were the average of three successive measurements.

 

A stock phosphate solution of 100 µg/ml was prepared by dissolving 0.1432 g of anhydrous hydrogen dipotassium phosphate in 1L of water.

 

The working standard solution were prepared appropriate dilution of the stock solution with water. 0.3M ammonium molybdate (BDH) solution was prepared by dissolving 185.4 g in 500 ml of water. 0.02 M Ascorbic acid   (BDH) was prepared by dissolving 0.875g of ascorbic acid in 250 ml of water.

 

0.02 M potassium antimony tartrate was prepared by dissolving 13.36 g in 1L of water. Water samples were taken from deferentplaces of Basra area, filtered by 0.45µm membrane filter.

Instrumentation:

Fig. 1, A and B shows the lab-build stopped flow system which consist.

 

 

Figure (1, A): Shows the lab build

 

 

Figure (1, B): Component of Stop-flow System

 

Procedure:

Grasshoff ⁹method was used in this work for phosphate determination by the lab-build stopped–Flow System. Fig. (2) Shows the flow chart of the program running in the system. After adjusting the wave length of the spectrophotometer on 880nm.The Arduino type Uno used to control simultaneously start of injection pump to wash the manifold spontaneously for 10 sec. , with refilled reagent in the 60 ml. plastic syringe after the injection of (180 µL) the sample manually through injection valve (Rhyeoyne, catali, California) mixed reagent stream. Then the home-made injection pump type stepper motor stopped when the reaction zone of sample and reagents react at the flow measuring cell. The other Arduino type Mega to recording the signals as peak height with aid Microsoft Excel 2010 program. After that injection pump restart to wish the flow system and restart for other samples.

 

 

Fig. (2): Flow Chart of the program running in the Stop-Flow System

RESULTS AN DISCUSSION:

Fig. (3) Shows the manifold of the system used for phosphate determination, which clearly indicated the most crucial and novel part in this system is to use the role of two micro controllers. The first Arduino type UNO was programmed by Arduino software V1.8.3, which used to control the movement and provided by power supply (12V). The home-made injection-pump type stepper motor (Nema17). In order to determine the phosphate through using the micro control to control the starting and fill up the injection pump. The micro control unit equipped with variable resistance 50 ohm (Fig.2) which supply the system with 1024 analog signal value which can make each signal as order to let the injection stepper motor work in suitable style. In this lab-built only (10-100) range analog signal was fixed in order to order to stepper motor to start and (101-200) range for fill the injection-pump with reagent. The injection-pump consist mainly from the stepper-motor Fig. with relatively high moment and the most significant properties is the motor which a can be adjust precisely the volume of injected reagents by the number of steps.

 

The moment selection for this stepper-motor spend on the number of syringes used in this system. It is possible to use two 60 ml volume plastic syringes or only one syringe filled with mixture of the two reagent which is used in this system. The sample is injected by the injection valve and propelled to the measuring flow cell, by the carrier stream in the spectrophotometer (U.V 303 Apel) and the absorbance measured at 880 nm. This spectrophotometer contain an out-pw to the analog signal which proportional to the absorption valve of the colored complex in the measuring cell. This analog signal convert to Digital signal through a micro control Arduino type Mega which work as data logger to mat and recording the data by lab tab equipped with Microsoft Excell 2010¹⁰.

 

 

Fig. (3): Manifold of the stop-Flow System

The Optimum conditions for phosphate Determination by the proposed system:

The lab-build semi-automated stopped flow system as showing in Fig. (1) was used to optimize the variable that effecting the peak height by carrying out series of experimental to establish the optimum analytical condition that in influence the peak height. Table (1) lists all optimum results which was used in subsequent work.

 

Table (1): The Optimum conditions for determination of phosphate

 

Calibration curve:

Under the established conditions listed in Table (1) a calibration curve for phosphate was obtained Fig. (4). It is linear over the range 0.125-2.0 µg/ml. phosphate. The linear curve has regression coefficient 0.9962and detection limit was 0.05 µg/ml. and the 3.8 R.S.D %.

 

 

Figure (4): Shows the Calibration curve

 

Accuracy:

In order to establish the validity and the accuracy of Lab-built system for the determination of Phosphate in water one standard and two representatives samples were examined by using standard additions method⁵ listed in table 2. The same batch of samples were analyzed by classical manual method¹¹. The recoveries and RSD% were calculated (Table 2). The value of RSD% for reproducibility obtained were 1.37% which clearly indicated that lab-built stopped-flow system has a high reproducibly which can be used for phosphate determination in different water sample (Fig.5).

 

 

Table (2): Determination of Phosphate in Standard and representative samples using standard additions method.

Claimed conc. (µg/ml.) of Phosphate	Determination by Lab-Build Stopped-Flow method			Determination by Classical method
	Found (µg/ml.)	Recovery	R.S.D%*	Found (µg/ml.)	Recovery	R.S.D%
50 ppm (Riccachemical)	51.55	96.9	0.82	50.05	95.2%	0.93
0.8 ppm	0.81	101.25	0.94	0.79	98.4%	1.07
1.2 ppm	1.18	98	0.78	1.19	97.7%	0.87

*For Triple successive stopped-flow peaks.

 

 

Figure (5): The reproducibility of height peaks of 1.5 µg/ml of Phosphate

 

Applications:

The proposed system was used successfully for determination of phosphate in different water samples Table (3) list the results obtained by using addition standard method to avoid all the possible interference. The results were in the range (0.74-1.35 µg/ml.) which mean that the all water samples not polluted¹².

 

Table (3): Sites of Water samples

Sample	Concentration Of Phosphate (µg/ml)	RSD%
Tap water -Qebla	0.70	1.069
Tap water–Karamat Ali	0.74	1.654
Bottled water–Seraj water	NA	--
Bottled water-Naba'a Al-Basra	NA	--
Garave river	0.78	1.699
Arab Gulf	0.35	1.450
Aquarium-Basra science Center	1.35	1.707
Al-Aiz river-Basra	0.75	1.006
Tiger river-Qurna-Basra	0.650	1.778

 

CONCLUSION:

Assembling a lab-build semi-automated stopped-flow system forma few available, simple and low price components. For the first time microcontroller (Adrianotype Uno and Mega) supplied with a suitable software were used in our laboratory as controller to the proposed system especially the stepper-moto3r and data logger to recording the data, respectively. The lab-build system offer simple, sensitive, reproducible means for phosphate determination.

 

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Received on 12.10.2018           Modified on 17.11.2018

Accepted on 18.12.2018         © RJPT All right reserved