Assessment of long distance chasing photometer (NAG-ADF-300-2) by estimating the drug atenolol with ammonium molybdate via continuous flow injection analysis

Atenolol was used with ammonium molybdate to prove the efficiency, reliability and repeatability of the long distance chasing photometer (NAG-ADF-300-2) using continuous flow injection analysis. The method is based on reaction between atenolol and ammonium molybdate in an aqueous medium to obtain a dark brown precipitate. Optimum parameters was studied to increase the sensitivity for developed method. A linear range for calibration graph was 0.1-3.5 mmol/L for cell A and 0.3-3.5 mmol/L for cell B, and LOD 133.1680 ng/100 μL and 532.6720 ng/100 μL for cell A and cell B respectively with correlation coefficient (r) 0.9910 for cell A and 0.9901 for cell B, RSD% was lower than 1%, (n=8) for the determination of atenolol at concentration (0.5, 0.7 and 5) mmol/L respectively. The results were compared with classical method UV-Spectrophotometric at λ max=270 nm using the standard addition method via the use of t-test, at 95% confidence level. The comparison of data explain that long distance chasing photometer (NAG-ADF300-2) is the choice with excellent extended detection and wide application.

In this work, study and determination of atenolol with ammonium molybdate the obtained resultant signals which resulted from the attenuation of the incident light on particulate surfaces of molybdenum dioxide (i.e.; the precipitated particulate is dark brown in colour of MoO 2 ). using a new long distance chasing photometer for 300 mm length with 2 mm path length to chase and to accumulate output resulted from attenuated incident

Materials and Methods:
Reagents and chemicals All chemicals were used of analytical-reagent and all the solutions dissolved by distilled water. A standard solution of 50 mmol/L of atenolol, molecular weight 266.336 g/mol, was prepared by dissolving 1.3317 g in 100 ml. A series of ammonium molybdate solutions were prepared from the dilution of standard solution 10 mmol/L with distilled water.

Apparatus
A homemade NAG-ADF-300-2 is a long distance chasing photometer as a flow cell will have 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 0 and diverged or fluorescence light at 0-90 0 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 (0-180 0 and 0-90 0 ) cell (cell number 2) which is 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 to estimate the output signals (Siemens, Germany). UV-Spectrophotometric (RF-1501, shimadzu, Japan) was use for classical methods.

Methodology
Using a manifold of two lines coupled with NAG-ADF-300-2 instrument to determination of atenolol via It's reaction with ammonium molybdate as shown in fig.2. The first line as a carrier stream at 3.6 ml/min flow rate (distilled water) passing through the injection valve to carry the sample segment of atenolol (5 mmol/L) to meet with the ammonium molybdate (1 mmol/L) in the second line at 5.8 ml/min flow rate at a Y-junction point before it is introduced to the NAG-ADF-300-2 analyser.
The obtained resultant signals which resulted from the attenuation of the incident light on particulate surfaces of molybdenum dioxide (i.e.; the precipitated particulate is dark brown in colour of MoO 2 ). It can be noticed that the result obtained from cell A are higher in sensitivity than the cell B output signals.
The higher no. of WSLED S and solar cell detection available for cell A gives most probably the reason of this higher sensitivity compared with reference to cell B. Scheme 1. Shows a proposed expected mechanism for the reaction of atenolol with ammonium molybdate in aqueous medium (26,32).

Atenolol
Ammonium  (6 WSLED) with in between distance of 100 mm).An arbitrary selected intensity was put into work (an intensity of indication approximate of selector switch for cell A was on no.3. while it was on no.2 for cell B which was based on preliminary experiment).These numbers reflect the 0-1-2-3-4 intensities which were varied a according to nature and type of reaction carried out. On the basis of obtained responses profile (Fig.2) other necessary chemical and physical parameters were carried out, which describe the full detailed study supported by the recorded of Y Z (mV ) -t min (d) cm response . Figure 3. A and B shows the intensity (I) of the response for either cells from I=1 to I=4.The optimum intensity of the measuring cell A, I=3 and I=2 for cell B which was adopted in subsequent studies.

Optimization of variable
Chemicals parameters (mainly concentration of reagent and type of carrier stream for the atenolol with ammonium molybdate system) as well as physical parameters: sample volume, flow rate were studied using two lines manifold system (Fig.2).

-Chemical variables -Ammonium molybdate concentration
Variables concentration of precipitating of Ammonium molybdate 0.5-7 mmol/L were prepared of 100 μl sample volume was injected through the carrier stream (distilled water). 5 mmol/L concentration of atenolol was injected with 3.2, 4.6 mL/min flow rate for carrier stream and reagent respectively. In addition to I=3 for cell A, I=2 for cell B. It was found that an increase in peak height expressed as an attenuation of incident light with increase of ammonium molybdate concentration.
It is possible that might be attributed to the increase of coloured precipitate particulate which in turn work on attenuation of part of the incident irradiation light plus it's absorption due to its coloration. In addition to the decrease in the resultant intensity due to its penetration to the precipitate particulate suffering many internal reflection and refraction which in turn to attenuate the incoming penetrating light toward the detection area. While at higher concentration (i. e > 1 mmol/L) might lead to increase agglomeration of precipitated particulate and an increase of inter spatial distances which help to increase penetration light toward the detector and a decrease in the response height. Therefore 1mmol/L was selected as optimum concentration for either cells. The results were summarized in Table 1.

-Effect of different salt
The reaction of atenolol (5mmol/L) with ammonium molybdate (1mmol/L) was studied in different media (water, ammonium acetate, potassium nitrate and sodium nitrate) at 10 mmol/L concentration in addition to aqueous medium as a carrier stream. It was noticed for Fig.4. A, B that the studied; salt causes to a decrease of S/N-response; this might be attributed leads to its effect in an increasing the agglomeration i.e.; increase the density of aggregates and compactness with each other than increase the intensity of transmitted light as there will be more vacant spaces in between agglomerates of particulate. On this basis; it 'were (the salts) cancelled throughout this work and distilled water as a carrier stream in the next studied.

-Effect of variable concentration of sodium hydroxide
Variable concentration of sodium hydroxide at ranging 2-50 mmol/L were used to dilute the prepared solutions via the use of atenolol (5mmol/L)ammonium molybdate (1 mmol/L) system , 100 μl sample volume at 3.2 ml/min and 4.6 ml/min flow rate for carrier stream and reagent solution respectively. It was noticed that an increase of NaOH solution causes a decrease in S/N-transducer response, most probably due to friability (flakiness) of precipitated particulate; then more room will be available in not obstructing or attenuated light source effect; or the prevention of the oxidation process for atenolol drug with an output of not forming MoO 2 precipitate. Therefore dilution of the prepared drug solution will be done by distilled water not else. The set of data were summarized in Table 2.

-Physical variables -Flow rate
Flow rate study was carried out using the unit cell of two lines as shown in Fig.45.A to assess the Y Z (mV )-t min (d) cm response profile. It was noticed ( Fig. 5. A). that an increase in S/N-response profile from cell A & cell B up to 3.6 ml/min for carrier stream. This may be attributed to an increased opportunity for the crystal that are formed to grow up relatively; while there is a time lag difference between cells used.
At high speed (i.e.; more than 3.6 ml/min (carrier stream)) does not offer an a time lag period causing increase attenuation of incident light that is measured by the detector. So at high speed, it is noticed that a decrease in response height might be attributed to incomplete or immature precipitation. Therefore; 3.6 & 5.8 ml/min flow rate for carrier stream and reagent respectively for either cells (Fig.5.B and C). [NaOH] mmol/L -Sample volume A sample volume will represent the analyte concentration to be determined. Variable length of Teflon tube ranging (2.6-25.5) cm of diameter (D) 1 mm that is equivalent to  µL of sample volume. Therefore and on this basis a clear response profile is required for the chosen reaction intended to be carried out.

Attenuation of incident light expressed as an average peak heights (n=3) Ῡ Z i(mV) RSD% Confidence interval at (95%) Ῡ Z i(mV) ±t 0.05/2,n-1 σ n-1 /√n
Large volume is not a recommended methodology in CFIA. As this kind of volume will form by local concentration for the precipitate that can dissolve i.e.; at least part of it due to the continuous flow of carrier stream that will cause the k.s.p of the precipitate > than the multiplication of the concentration of reacted species raised to power of its sharing strength of contribution.
Low sample volume will represent a quick transit of reaction product in front the detector (solar cells which are not of fast response detection compared to PMT that can detected at Nano sec. level).Therefore a very small sample segment might be not a convenient level of sample volume. A compromise was made in this study to choose 100 µL as a suitable most convenient of sample size level.
By this broadening as well as disturbed Y Z(mV) -t min (d) cm response were avoided to obtain trustable more confident; a way of interference caused by precipitated aggregate that might cause a delayed movement of reacting product. The obtained results tabulated in Table 3.

-Effect of reaction loop length
Effect of including a coil (reaction, delay, exchange, adsorption, or reduction and oxidation (i. e change of valence of selected reaction ions)) in the flowgram in CFIA used throughout this research work; as part of the designed unit for determination of atenolol. The reaction coil of any kind whether it is made of glass or Teflon as turns (depending on length, loop diameter, and tube diameter used).Glass is preferred material due to its ability that it can be cleaned easily via pumping a certain cleaning solvent to remove precipitated particulate (usually at high concentration of reactants or even sedimentation of precipitate particulate due to high molecular weight of precipitated reaction product or precipitated reagent that is used (e.g. change of valence). Usually any addition of extra length to the manifold will increase dispersion via different process (e.g. convection at high speed of flow rate or diffusion at low speed of flow rate).
On this basis reaction coils are avoided unless it serve the purpose of using or verifying its use. The volume of the reaction coil can be found as it is a cylinder of cross section diameter of ϕ =2r (where r

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= radius; i.e. tube radius). A volume of a cylinder =πr 2 L (where L= length of the used tube for e. g is ϕ=1, the r=0.5 mm for 100 mm length. The volume will be equal to 3.14(0.05 cm) 2 × 20 cm=0.157 cm 3 =157 µL. So, it was found that using different length of reaction coil (variable volume) will be introduced leading to dilution of sample segment reaction product which that ladies' to an even distribution when used of a very high volume, will lead to a highly dispersed of precipitate which cause a weaker signal (or even undetected signal). Therefore a compromise of using a convenient reaction coil length ( fig. 6. A,B and C) and in watching the results of measurement shows that a direct measurement with no coils in the manifold system will the way of the system works i.e., better with very good excellent output, in addition to trust ability of S/N response and Y Z(mV) -t min (d) cm response profile. Figure 6. A shows a kind of response.

-Study of the optimum intensity used for While Snow Light Emitting Diodes (WSLEDS) in NAG-ADF-300-2 analyser
A study was conducted for the effect of intensity of incident light for the irradiation sources on the S/Nresponse of the energy transducer via the selector switch (C.F front panel diagram of NAG-ADF-300-2.The selector switch gives 0-1-2-3-4 i.e.; four choices plus the off position for both cell individually controlled. It was noticed from a selection of 3 position (i.e.; I=3) was very convenient intensity for cell no.1 (cell A) (larger number of the selector switch means more light intensity).while position 2 (I=2) of the selector switch was a convenient intensity. It was same intensity chosen in first of atenolol study of assessment. The higher intensity (I=3) for cell A refers to that precipitated particulate that is formed are line particles and dense; therefore high light intensity is required. While it, was not necessary to use high light intensity for cell B due to conglomeration of precipitated particulates and inter spaces are formed. Therefore, a low intensity of height is required for cell B. The results summed up in table 4.
So, it was concluded that the intensity that was chosen at the beginning (at preliminary experiments) remained as it was.  Table 4. Effect of intensity on attenuation of incident light expressed as an average peak heights (mV) For determination of atenolol (5 mmol/L) -ammonium molybdate (1 mmol/L) system, speed of Recorder 60 cm/hr., flow rate of the carrier stream 3.6 ml/min and sample volume 100 µL.

-Estimating the linear dynamic range from scatter plot for the variation of atenolol versus S/N Energy transducer response
Using the optimum chemical and physical parameters; a series of atenolol solutions (0.1-20 mmol/L) were prepared this will represent the xaxis (Independent variable). The attenuation of incident light that was measured gave the following S/N energy transducer responses as Y here represent the dependent variable as shown in Fig.7; in which, the height of response increased when the analyte of concentration is increased. The directly proportional up to 9 mmol/L and 7 mmol/L for cell A and cell B respectively (Fig.8.A, B) between variation precipitate particulate formation and concentration might be attributed to increase of many such as: internal reflection, refraction, absorbance and diverged light from within the precipitated particles when the beam of light diffused inside of particles. In addition to abstraction of light by the precipitated particulate; while gives at the end for all these factors combined all together, the measurement that is due to was only at 0-180 0 angle. Fig. 8    While increasing the concentration more than 9 and 7 mmol/L for cell A and cell B respectively causing that the signal (S/N) energy transducer independent on concentration, It is might be attributed to agglomeration of particulate and increase of inter spatial distances which lead to increase of transmitted light toward the detector.

-Limit of Detection
In general terms, the L.O.D of an analyte may be described as that: concentration which gives an instrument signal y significantly different from the blank or back ground signal. This description gives the analyst a good deal of freedom to decide the x The last two methods are an output of a linear regression graph treatments where the obtained (real) results are subjected to statistical treatments, these method can be used as an approximate indication but should not unless otherwise defined. A study was carried out to calculate the limit of detection of atenolol-ammonium molybdate (1 mmol/L) system through three methods as tabulated in Table 5.

-Repeatability
The relative standard deviation expressed as percentage which is equally to the repeatability of the measurement. A repeated measurements for eight successive injections were measured at fixed concentrations of atenolol for three concentrations were used 0.5, 0.7, 5 mmol/L respectively in optimum parameters. The obtained results is tabulated in Table 6 is shown of repeatability at 0.5, 0.7, 5 mmol/L respectively. In addition to study of repeatability with minimum of the RSD% which equal to 1%.

-Classical method of UV-Spectrophotometric
The assessment evaluation of the new developed methodology (i.e.; NAG-ADF-300-2 analyser) for the determination of atenolol using atenololammonium molybdate (1 mmol/L) system was compared with the available literature method, namely UV-Spectrophotometric method (33) which was based on the measurements of absorbance for the range of concentration 0.01-6 mmol/L at λ max = 270 nm using quartz cell. Table 7 shows the variable data treatments. The detection limit was 0.005 mmol/L (5 µmol/L) equivalent to 1.3317 µg / sample.

Table 7. Different ranges for the atenolol concentration versus absorbance using spectrophotometer (Classical method)
Type of mode

-Assessment of NAG -ADF-300 -2 analyser using two cell and multi solar cells for the Determination of atenolol in drugs
The newly developed methodology (NAG-ADF-300-2) was used for the determination of atenolol in three different samples of drugs from three different of companies (Atenolol, Bristol, UK, 100 mg),(Vascoten, medochemie, Cyprus, 100 mg) and (Nova ten, Ajanta, India, 100 mg).
The continuous flow injection analysis used of homemade NAG-ADF-300-2 which that mean a long distance chasing photometer for 300 mm length with 2mm path length to chase and accumulate output response from attenuation of incident light at 0-180 0 via the use of two cells of 110 mm (cell A) and 60 mm length (cell B) and was compared with UV-spectrophotometric method via the measurement at λ max =270 nm.
A   (4.303) at confidence level 95% and degree of freedom =2; Null hypothesis will be reject and accepting the alternative hypothesis; these mean that there is a significant difference between the quoted active ingredient value and the measured value. One this base; the newly developed method can be used equally well as standard reference methods. Another obtained t cal -value indicate that there was no significant different between the newly developed method and claimed method by the company as calculated tvalue is less than tabulated tvalue. So, the newly method can be used as an alternative analysis method for the determination of atenolol in different drugs.
Second test: Using paired t -test at α = 0.05 (2tailed) for the comparison of developed method using NAG-ADF-300-2 analyser and classical method using shimadzu (UV-1800 double beam) spectrophotometer as shown in Table 8. B (column 6).Taking into the consideration that all drugs from different companies are the same population i.e.; neglecting individual differences between one manufacturer and another. Assumption Null hypothesis H o : µ NAG-ADF-300-2 analyser =µ UV-SP. There is no significant difference between the mean of different two methods. An alternative hypothesis: There is a significant difference between the mean of classical method and NAG-ADF-300-2 analyser i.e.; Alternative H 1 : µ NAG-ADF-300-2 analyser ≠ µ UV-SP. The obtained results indicate clearly that there was no significant differences between newly developed method and UV-spectrophotometric (classical method) at 95% (α = 0.05) confidence level as the calculated t cal (3.996 and 0.4053) is less than t tab (4.303) for each cell (i.e.; cell A & cell B) for the determination of atenolol in pharmaceutical drugs as shown in Table 8. B (column 6).   µ: quoted value, ѿ: practical content mg, x d: average of difference between two type of method (developed& classical), n (no. of sample) = 3, σ n-1: standard deviation, ѿ i: practically weight in mg, t 0.05/2,2 =4.303

Conclusion:
The assessment of long distance chasing photometer (NAG-ADF-300-2) through this research work was applied using comparison between NAG-ADF-300-2 analyser with classical UV-Spectrophotometric method. It was recognized that a narrower range is obtained with UV-Spectrophotometric, while a wider range was the characteristic of NAG-ADF-300-2 analyser. A long distance chasing photometer (NAG-ADF-300-2) is the choice with excellent extended detection and a wider applicability. In the future using a new long distance chasing photometer as a flow cell will have 300 mm as a distance with 2 mm as a path length to chaise and to accumulate the output resulted from Attenuation and the Diverged or Fluorescence light at 0-90 via two flow cells of 110 mm and 60 mm length (NAG-ADF-300-2) for study and determination of some selected drugs.