Description:PRINCIPLE
[0006] The phenol detection kit design is based on the reactivity of phenol to the Folin-Ciocalteu (FC) reagent under alkaline conditions (pH 10, adjusted by sodium carbonate). FC owes electron-transfer reaction in which phenolic species donate electrons and FC reagent acts as an oxidant. The reduction of phosphotungstic and phosphomolybdic acids causes a color shift from yellow to blue and the intensity of color is directly proportional to the concentration of phenolic compounds. The blue color complexes formed during reaction can be detected using a spectrophotometer at 750 nm.
SPECIFICATIONS
[0007] The on-site detection kit for phenol is comprised of:
Reagents: The kit contains three components:
1. Folin-Ciocalteu (FC) reagent: 0.5 mL of reagent is used for 1 mL of sample
2. Sodium carbonate (20%): 200 g of sodium carbonate is dissolved in distilled water and final volume is made to 1 L in one mL of sample 1.5 mL of sodium carbonate (20%) is required.
3. Distilled water in 1 mL of sample 7 mL of distilled water is required.
Micropipette (1 and 10 mL): Both micropipettes are required to measure the samples as well as reagents for on-site analysis.
Cuvette: A quartz cuvette of 1 cm pathlength is required which is painted with ceramic white paint on all the surfaces except its one transparent side.
Comparison chart: A comparison chart was prepared to compare the developed color and estimate the phenol concentration.
PREPARATION OF COMPARISON CHART
[0008] A color difference chart was prepared to compare the developed color and estimate the phenol concentration.
[0009] The chart was prepared using different concentrations of gallic acid (5-100 ppm) using same concentration and quantity of reagents.
The scheme of gallic acid standard preparation is given in table 1. The experiment was done in triplicate.
[00010] After adding all the reagents, the absorbance of developed color was noted at 750 nm using a UV-vis spectrophotometer (Shimadzu) and a standard curve was formulated whose R2 was 0.997 as shown in figure.
[00011] Then, these reaction mixtures were analyzed for their color values using a Hunter LAB Colorimeter (Colorflex EZ). A ring and plate assembly was used in which ring ensures the depth of filled liquid up to 1 cm which is exactly equal to the pathlength of the cuvette. On the other hand, plate provides a white background to the liquid under analysis. Five values were observed using colorimeter, i.e., L*(lightness/darkness), a* (redness/greenness), b* (yellowness/blueness), C* (Chroma), hº (hue angle) as depicted in table 2. Real colors were obtained using these values with Hunter LAB easy match software. The obtained color was converted into the form of a reference/comparison chart as given in figure 2.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] The illustrated embodiments of the subject matter will be understood by reference to the drawings that are consistent with the subject matter as claimed herein,
Figure 1: Standard Calibration Curve of Gallic Acid (5-100 ppm) for Phenol Concentration Estimation in Wastewater
Figure 2. Color difference chart prepared using different concentrations of gallic acid (5-100 ppm)
Figure 3: Color Comparison Chart for Phenol Concentration Estimation in Wastewater Using the Detection Kit
DIRECTION OF USE
[00013] Collect the polluted water sample.
• Filter the water with a whatman no. 1 filter paper.
• Take 1 mL of sample and add 0.5 mL of FC reagent, 1.5 mL of sodium carbonate (20%), and 7 mL of distilled water to it.
• Fill the sample in cuvette which is painted with ceramic white paint on all the surfaces except its one transparent side.
• Compare the colour developed with sample.
• The chart can estimate the phenolics with an accuracy of 5 PPM.
Table 1: The scheme of gallic acid standard preparation
Gallic acid (µg/mL) Volume of standard gallic acid (100 µg/mL) µL Volume of DW (µL) Total volume of Standard (mL) FC reagent (mL) Sodium carbonate (20%) (mL) Distilled Water (mL) Readings at 750 nm
R1 R2 R3
0 0 1000 1 0.5 1.5 7 - - -
5 50 950 1 0.5 1.5 7 0.041 0.055 0.057
10 100 900 1 0.5 1.5 7 0.109 0.108 0.104
15 150 850 1 0.5 1.5 7 0.168 0.166 0.146
20 200 800 1 0.5 1.5 7 0.242 0.215 0.242
25 250 750 1 0.5 1.5 7 0.289 0.272 0.294
30 300 700 1 0.5 1.5 7 0.344 0.337 0.349
35 350 650 1 0.5 1.5 7 0.388 0.395 0.398
40 400 600 1 0.5 1.5 7 0.411 0.433 0.444
45 450 550 1 0.5 1.5 7 0.502 0.497 0.498
50 500 500 1 0.5 1.5 7 0.572 0.566 0.550
55 550 450 1 0.5 1.5 7 0.629 0.631 0.620
60 600 400 1 0.5 1.5 7 0.654 0.681 0.618
65 650 350 1 0.5 1.5 7 0.739 0.728 0.721
70 700 300 1 0.5 1.5 7 0.75 0.777 0.771
75 750 250 1 0.5 1.5 7 0.818 0.820 0.879
80 800 200 1 0.5 1.5 7 0.901 0.920 0.895
85 850 150 1 0.5 1.5 7 0.925 0.943 0.966
90 900 100 1 0.5 1.5 7 1.002 1.000 0.995
95 950 50 1 0.5 1.5 7 1.026 1.008 1.060
100 1000 0 1 0.5 1.5 7 1.087 1.086 1.070

Table 2: L*(lightness/darkness), a* (redness/greenness), b* (yellowness/blueness), C* (Chroma), hº (hue angle) for different concentrations of gallic acid.
µg/mL L* a* b* C* hº
0 67.76 -0.96 -0.75 1.22 217.91
5 61.71 -2.16 -3.53 4.14 238.47
10 59.48 -2.50 -4.59 5.22 241.43
15 55.05 -2.93 -6.70 7.31 246.35
20 50.17 -3.17 -9.26 9.79 251.08
25 45.54 -3.33 -11.34 11.82 253.62
30 41.60 -3.13 -13.15 13.52 256.62
35 37.56 -2.86 -14.99 15.26 259.20
40 33.60 -2.31 -16.75 16.57 261.23
45 27.31 -0.63 -19.67 19.31 268.17
50 24.11 0.40 -20.92 20.92 271.10
55 20.97 1.35 -22.25 22.29 273.47
60 19.10 1.92 -21.90 21.98 275.01
65 16.21 3.17 -22.97 23.19 277.86
70 13.47 4.26 -23.48 23.86 280.29
75 12.39 4.74 -22.37 22.87 281.97
80 9.25 6.10 -23.21 24.55 285.13
85 7.25 6.91 -23.05 24.06 286.68
90 5.75 7.02 -23.06 24.11 286.94
95 4.72 6.58 -21.56 22.54 286.98
100 3.97 6.21 -20.61 21.52 286.78 , Claims:I/ WE CLAIM:
1. An On-Site Detection Kit for Measuring Phenol Concentration in Wastewater, comprising:
• A Folin-Ciocalteu (FC) reagent for facilitating a colorimetric reaction;
• A sodium carbonate solution (20%) for adjusting the sample pH to alkaline conditions;
• Distilled water for diluting the wastewater sample;
• Whatman No. 1 filter paper for filter the water solution;
• A micropipette for precise measurement of reagents and the sample;
• A quartz cuvette with a ceramic white-painted surface for enhanced color intensity evaluation;
• A comparison chart for estimating phenol concentration based on observed color intensity; and
• A standardized protocol for preparation and usage to enable rapid, reliable, and accurate detection of phenols.
2. The Kit of Claim 1, wherein the FC reagent is used in a volume of 0.5 mL for every 1 mL of water sample, the sodium carbonate solution is used in a volume of 1.5 mL per mL of water sample, and distilled water is added at a volume of 7 mL per mL of sample.
3. The Kit of Claim 1, further comprising a comparison chart developed using a standard curve of gallic acid concentrations ranging from 5 to 100 ppm, wherein the intensity of the developed blue color is correlated with the phenol concentration in the sample.
4. The Kit of Claim 1, wherein the quartz cuvette includes all surfaces coated with ceramic white paint except for one transparent side, enabling enhanced color contrast and improved accuracy during visual comparison with the reference chart.
5. A Method for Detecting Phenol in Wastewater Using the Kit of Claim 1, comprising the steps of:
• Collecting a wastewater sample and filtering it through Whatman No. 1 filter paper;
• Measuring 1 mL of the filtered sample and mixing it with 0.5 mL of the FC reagent, 1.5 mL of the sodium carbonate solution (20%), and 7 mL of distilled water;
• Filling the prepared mixture into the quartz cuvette; and
• Observing the resultant color change and comparing it with the reference chart to determine the phenol concentration.
6. The Method of Claim 5, wherein the developed color comparison chart enables estimation of phenol concentrations with an accuracy of ±5 ppm.
7. The Kit of Claim 1, adaptable for use in detecting phenolic substances in various water sources, including agricultural runoff, industrial effluents, and sewage, without requiring specialized laboratory equipment.
8. The Kit of Claim 1, wherein the comparison chart is calibrated and standardized using UV-visible spectrophotometric analysis at 750 nm, providing a high correlation coefficient (R² value of 0.997) for phenol concentration estimation.
9. The Kit of Claim 1, characterized by a portable, user-friendly, and cost-effective design, enabling rapid on-site detection of phenols in contaminated water sources and reducing dependency on extensive laboratory procedures.