Potentiometric titration for the high precision determination of active components in six types of chemical disinfectants

Chemical disinfectants effectively kill pathogenic microorganisms, eliminating routes of transmission for infectious diseases. Accurate quantification of the active ingredients can help make a more effective use of disinfectants. In this study, the active ingredients in six different types of chemical disinfectants were systematically quantified with great precision and accuracy using potentiometric titration. The coefficient of variations fell in the range of 0.04%-0.46%. The recovery rates of samples were all above 95% and the extended uncertainty was 0.32g/L. This method can be broadly applied to the analysis of disinfectants in the future.


Introduction
Chemical disinfectants effectively kill or remove pathogenic microorganisms, blocking disease transmission routes [1]. Commonly used chemical disinfectants include chlorine compounds, iodine, oxidants, aldehydes, alcohols, and quaternary ammonium salts [2]. The efficacy of these chemical disinfectants to eliminate microbes can be affected by many factors [3], of which the content of the active component of the disinfectant is clearly the most critical one. The most commonly used techniques for determining the content of chemicals include volumetric analysis, spectrophotometry, gas chromatography, and high-performance liquid chromatography [4][5][6][7]. Nevertheless, using any of the above methods for systematic measurement of the components in a chemical disinfectant can be problematic due to certain limitations of the techniques, such as large error range, difficulty of operation, susceptibility to interference, and the different properties of the various chemical components contained within each disinfectant. In this article, potentiometric titration [8]is used to determine the effective content of general chemical disinfectants from six different categories. The method offers many advantages, including simplicity, speed, easier end-point determination and higher accuracy. The testing and results of potentiometric titration are discussed in the following sections.

Instruments and reagents
A. Instruments. Titrations were performed using an 809 Titrando automatic potentiometric titrator, a burette drive and a magnetic stirrer. PLOS  These samples were all used prior to the expiration date.

Electrode measurements
Composite platinum (Pt) electrodes or a monomer platinum (Pt) indicator electrode and reference electrode were used to measure available chlorine, available iodine and hydrogen peroxide content.A composite water phase pH electrode(containing a pH indicator electrode and a Ag/ AgCl reference electrode)or an aqueous phase pH-indicating electrode and reference electrode monomer were used to measure glutaraldehyde content.A composite non-aqueous phase pH electrode (containing a pH indicator electrode and a Ag/AgCl reference electrode)was used to measure the chlorhexidine content. Finally, a surfactant aqueous phase titration indicator electrode and a reference electrode were used to measure benzalkonium bromide content.

Test method
A. Ambient temperature and humidity. The ambient temperature was 20˚C-25˚C,and the relative humidity was 45%-85% for these studies.
B. Principle. The indicator electrode and the reference electrode (or the reference and an indicator electrode included as a composite electrode) were immersed in the same solution, in which the reference electrode was maintained at a constant; then,the indicator electrode was immersed in the test substance. When the titration approached the equivalence point, small changes in the activity of the test substance solution elicited a dramatic change to the indicator electrode, and the largest change detected in indicator electrode potential was considered the end point of titration.
C. Titration mode, control program design, and installation. The Tiamo operating procedure software was installed before installing and debugging the automatic potentiometric titrator.Prior to using the instrument,titration mode was selected,and the standard detection method for test items and parameters were entered into the control program. The test project and the indicators for samples were transferred to the method bar and bar usage before conducting the titration,allowing the database to generate results automatically.

Method for determination
A.Capacity Analysis. This method was performed in accordance with the Ministry of Health of the People's Republic of China's"Disinfection Technical Specifications" (2002) [9].
Method details: (1) The electrode and the titration mode were chosen according to the type of sample.(2) Sample pretreatment: 10 times the amount of solid (powder, tablet) chemical disinfectant required for analysis was obtained, and,the appropriate amount of sample for accurate determination after grinding was weighed. The liquid chemical disinfectant was shaken until reaching a uniform state and then analyzed with or without dilution.
(3) The sample information, standard titrant concentration and formula were entered as the input into the device,and the content of the effective component of the sample was measured.(4)The database then generated the results automatically.
C. Determination of the titration equivalence point. A peak maximum appears during the titration procedure when the threshold value is exceeded, and the jump point, or the titration equivalence point, is identified by taking the first derivative of the curve.The titration equivalence point corresponding to the volume of titrant recorded (a titration curve example is shown in Fig 1) is transferred to the formula for further calculations.
D. Calculation of results. The content of the test substance was determined using the chemical reaction formula and the amount of titrant consumed according to formulas (1) and (2).The result was averaged across six experiments. Liquid chemical disinfectant effective content Solid chemical disinfectant effective content For formulas (1) and (2): X = substance content (g/L or %), C = standard titration solution concentration (mol/L),VST = corrected volume for the standard titration (mL), k = coefficient (g), V = test substance sampling volume (mL), and m = test substance sampling mass(g).

Evaluation and methods
A. Precision assessment. The measurements were compared with those obtained by the manual chemical titration method (i.e., the volumetric analysis)The precision of the method was assessed by the coefficient of variation (CV) according to formulas (3) and (4), which should not be greater than 1.0%.
in which S = standard deviation, X i = single measured value, X t = average of measurements, S = the sum of absolute values, CV = coefficient of variation, and n = number of measurements. B. Linearity test. For the six types of chemical disinfectants that were analyzed,available chlorine(3.35-42.97g/L), available iodine (2.29-11.43g/L),hydrogen peroxide (10.48-34.94g/ L),glutaraldehyde (4.57-19.99g/L), benzalkonium bromide(0.83-8.50g/L)and chlorhexidine acetate(7.74%-93.41%)were determined by titration with sodium thiosulfate (0.1mol/L), potassium permanganate(0.02mol/L),sulfuric acid (0.25mol/L), perchloric acid (0.1mol/L) and sodium tetraphenylborate (0.02mol/L), respectively. The measured chemical disinfectant composition was plotted as the horizontal axis, and the consumption of titration liquid volume was plotted as the vertical axis. A linear regression analysis was performed to evaluate the reproducibility of the method. The linear regression equation: y = a+bx (y: titrant consumption, a: constant, b: slope, x: content or sample volume).
C. Accuracy assessment. The accuracy of the method was measured by the recovery of standard substance added (P) as calculated by Eq (5), which should be>95%.
in which P = spiked recoveries, c1 = sample background concentration, c2 = sample spiked concentration, and c3 = plus scalar. D. Uncertainty assessment. The main sources of uncertainty of this analysis included sample volume(μ 1 ), standard solution preparation(μ 2 ), repeated measurements (μ 3 )and interpolation using the standard curve(μ 4 ). Glutaraldehyde disinfectant was selected as a substance to estimate the upper bound of the uncertainty of this method because of the complexity of its composition.
Mathematical model in which x = Glutaraldehyde content(g/L), c = concentration of sulfuric acid titrant(mol/L), Vst = corrected volume of sulfuric acid titrant(ml), and 0.1001 is the conversion factor.

Statistical analysis
The data from the experiments were analyzed with the SAS9.0 statistics software. Results from two groups were compared using independent sample T-test and correlation analysis. The difference was considered to be statistically significant with a P 0.05.

Accuracy and precision of potentiometric titration
The results of potentiometric titration and direct titration were shown in Table 1. there were no significant differences between the two methods (P>0.05).This indicated that the analysis of chemical disinfectants by potentiometric titration agrees with the current National Standard (direct titration). the CVs for the effective content in chemical disinfectants determined by potentiometric titration were lower than those obtained using the other method, except for the bromogeramine disinfectant, suggesting that potentiometric titration has a superior precision direct titration. acetate, y = 0.0479x + 0.1192, R 2 = 0.9998, and for benzalkoniumbromide, y = 3.1069x + 0.0734, R 2 = 0.9999. The above coefficients were all greater than 0.999, suggesting a strong linearity of the potentiometric titration method in analyzing the active ingredients of all these disinfectants.

Recovery tests of standard substance (sample) addition
The accuracy of potentiometric titration was assessed by the recovery rates of standard substance (sample).If the recovery was between 90% and 110%,the method was considered reliable. The data in Table 3 show that the recovery rates for all of the disinfectants were in the range of 95%-104%, demonstrating the high reliability of this method.

Uncertainty measurements for glutaraldehyde disinfectant
A. Relative uncertainty caused by sampling volume (μ 1 ). By sampling 10L with a 10mL single scale line straw, the relative uncertainty (μ 1 ) caused by the sampling volume (v 1 )was the result of a reading value variation from the scale line(μ 1.1 ) and the volume variation of the scale due to a temperature change(μ 1.2 ).
1. The allowable error in a 10mL single scale line straw was 0.1mL, with a uniform distribution of κ ¼ ffiffi ffi 3 p , then:  Six kinds of chemical disinfectants were determined by potentiometric titration 2. Set temperature variation was 3˚C (ΔT = 3,expansion coefficient of water α = 2×10 −4 /˚C, with a uniform distribution of k ¼ ffiffi ffi 3 p , then: The same standard solution was titrated 6 times,and the results are shown in Table 4. Table 3. The recovery test results of standard substance (sample) addition in six types of chemical disinfectants.

F. Expanded uncertainty (U) and the result of sample concentration (C).
Setting the coverage factor k = 2, the extended uncertainty: The result is expressed as follows: x" x AE UU ¼ ð20:03 AE 0:32Þg=L G. By analyzing and calculating the uncertainties for the glutaraldehyde disinfectant,all of the partial uncertainties were very low(<0.60%),and the value for the expanded uncertainty was 0.32g/L.This suggests that potentiometric titration measurements are of high quality.

Discussion
In classical chemical titration (volumetric analysis), the endpoint is based on the color change of an indicator. If the sample being tested is colored or turbid, it is difficult to estimate the titration endpoint with a reliable indicator. In contrast, potentiometric titration relies on abrupt changes in electrode potential to determine the endpoint. The ion concentration being evaluated often varies by orders of magnitude and generates abrupt changes in the electrode potential when the endpoint is approached. The content of the samples can then be calculated from titrant consumption.
In this article, potentiometric titration was adopted to quantify the effective contents of 6 different chemical disinfectants. Compared to standard chemical titration,this method offers many advantages, including simplicity, speed, ease of end-point determination, accuracy, stability, and the ability to overcome a number of confounding factors. Therefore,potentiometric titration is suitable for the fast and accurate determination of 1) effective chlorine/effective iodine/hydrogen peroxide content using the selective composite platinum (Pt) electrode or monomer platinum (Pt) indicator electrode combined with reference electrode, 2) glutaraldehyde content using the composite aqueous phase acid-base electrode (containing pH indicator electrode and Ag/AgCl Reference electrode) or aqueous phase acid-base monomer indicator electrode combined with reference electrode, 3) chlorhexidine acetate using the composite non-aqueous phase acid-base electrode (containing pH indicator electrode and Ag/AgCl reference electrode), and 4) benzalkonium bromide using the surfactant aqueous phase titration indicator electrode and the reference electrode. This method is particularly adaptable to determining the content of certain bactericidal active ingredients in compound chemical disinfectants.
This method has robust precision for all the disinfectants tested, with the CVs ranging from 0.04% to 0.46% Furthermore, our tests also show that the potentiometric titration method yields results that agree well with the current National Standard (direct titration) while having outstanding accuracy as indicated by high the recovery rates (greater than 95% for all samples tested).
The results of the linearity tests shows that potentiometric titration maintains an excellent linearity for a wide concentration range of the analyte. Moreover, the results also demonstrate good robustness.The value for expanded uncertainty was only 0.32g/L. This work represents the first analysis of various chemical disinfectants for systematic determination of the effective contents using potentiometric titration. The robustness and simplicity of this method will prepare it for a broad application in the analysis of disinfectants in the future.