CHAPTER is the instrument which measure intensity

CHAPTER 3
MATERAL AND METHODS
3.1 Ultraviolet C (UV-C) Lamp:
UV-C lamp have shorter wavelength (100-280 nm) radiations and widely used for disinfection of air from microbial pollutants (Chelah et al., 2012). UV-C 254 nm lamp irradiations avoid fruit and vegetables deterioration. Exposure of UV-C lamp irradiations provides resistance against pathogens. Effects of UV-C lamp irradiations has been studied on soluble sugar and organic acids (Erkan et al., 2001). Irradiations of UV-C lamp were bombarded on DCF sodium solution and its oxidation is measured by using spectrophotometer.

Fig. 3.1: UVGL-58 Handheld UV Lamp
3.2 Ultraviolet-Visible Spectrophotometry:
UV-Visible spectrophotometry technique is used for pharmaceutical analysis. It measures the UV /Visible radiations absorbed by a substances in solutions. UV-Visible spectrophotometer is the instrument which measure intensity of two beams in UV visible region. It follows Beer Lambert law which states that absorbance is directly proportional to concentration (Behera et al., 2012). First lambda max for DCF sodium was found and then Beer Lambert law was verified by using different concentration of DCF sodium.

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Fig: 3.2 SPECORD 210 PLUS Spectrophotometer
3.3 High Performance Liquid Chromatography (HPLC):
HPLC is a form of column chromatograph, used to identify, separate and quantitative analysis of compounds. HPLC consist of a column containing packing material (stationary phase), pump that moves the mobile phase in the column and detector which tells about retention time of molecules. Retention time is varies and depends upon stationary phase, molecules and solvents used. Sample is introduced in a small quantity in mobile phase and retreated by interaction of physical or chemical properties with stationary phase. Retardation time depends upon analyte, mobile and stationary phase. The time during which analyte comes out is known as retention time. Methanol and acetonitrile are most commonly used solvents. HPLC was used for mineralization and byproduct study of DCF sodium after irradiations of UV-C lamp.

Fig: 3.3 Agilent Technologies 1100 Series
3.3.1 Types of HPLC:
Following types of HPLC used in analysis:
3.3.1.1 Normal Phase HPLC:
In this type separation takes place on the basis of polarity and stationary phase is polar and mobile phase is non polar.
3.3.1.2 Reversed Phase HPLC:
In reversed phase HPLC stationary phase is non-polar and mobile phase is polar.
3.3.1.3 Size Exclusion Chromatography:
It is also known as gel permeation chromatography. It separates particles on the basis of size. This technique is used for molecular weight determination of polysaccharides and protein and amino acids.
3.3.1.4 Ion Exchange HPLC:
It is helpful in water purifying. In this technique retention time is based on attraction among solute and charges sites bound to stationary phase.
3.3.1.5 Bio-Affinity HPLC:
In this technique separation takes place on the basis of reversible interaction of protein with ligands. It gives high purification in single step (Malviya et al., 2010).
3.3.2 Detector:
UV-Visible detector is most commonly used in detector in HPLC and absorbance is measured by light transmitted through detector cell by Beer’s Law:
A = log (Io/I) = ? b c
A = absorbance
I = intensity of transmitted light
e = molar extinction coefficient of sample
b = path length of cell
c = concentration of sample
UV absorbance takes place due to ?-?*, n-? and n-?* transitions (Swartz et al., 2010).
3.4 High Performance Liquid Chromatography-Mass Spectrometry (HPLC-MS):
High performance liquid chromatography (HPLC) is combined with mass spectrometry is a measurement method for gathering information about compounds present in extracts. It is key tool in biochemical pathway analysis. Combination of liquid chromatography with mass spectrometry (HPLC-MS) was widely used in chemical fields with high impact.

Fig: 3.4 Schematic Diagram of HPLC-MS
In HPLC-MS method peak detectors are important. Various software, programs tool box applications, programs used for HPLC –MS analysis. Peak identification obtained in HPLC-MS by using home build data base, but public data base also used (Urban et al., 2012).
3.5 Gas Chromatography (GC):
This processes is discovered by Martin and Synge in 1941. It was applied first time for fatty acids separation in 1951. After this technique is developed more rapidly and controlled by computer. Gas chromatography tells us about uses of large diameter column for purification of large quantities of materials by sample collection at column outlet. GC is important and widely used in analytical chemistry. GC is most successfully used for analysis of chemicals (Zuo et al., 2013).
GC is very useful technique for isolation and characterization of volatile components and detector is nondestructive (Prabu et al., 2010). Gas chromatography is unique technique. First time it was also used for analysis of gases and vapours. This technique is used for product identification and also coupled with mass spectrometer for positive detection of peaks on chromatogram.
3.5.1 Principle of GC:
The principle of gas chromatography is affinity of compound is greater for stationary phase and compound retained by column before elution and detection. Therefore, heart of GC is column through which components separation takes place and it also control the gas flow. Volatility of analyte influenced by temperature so column is placed in thermostatically controlled oven. Unlike other chromatography types there is no interaction between mobile phase and analyte molecules, it just transport analyte through column. Faster GC is more beneficial option. Analysis time decreases so sample output increase and operational cost reduced. By changing parameters such as thin film of stationary phase, short length and smaller inner diameter of column, reduction in analysis time can be achieved (Bhardwa et al., 2016).

Fig: 3.5 Schematic Diagram of GC
3.6 Gas Chromatography-Mass Spectrometry (GC-MS):
This technique is excessively used in pharmaceutical industries. It is also a part of pharmaceutical biotechnology, medicinal chemistry, and pharmaceutical analysis (Hadi et al., 2017). GC-MS is a safer process and easily available in health centers as compared to other methods which are expensive such as LC-MS. This technique is also preferred for quantitative determination (Jalali et al, 2016).

Fig: 3.6 Schematic Diagram of GC-MS
GC-MS applied for medicinal plants analysis, analysis of non-polar components, fatty acids, lipid, and alkaloids. Cost of GC-MS equipment decreases but reliability increased. GC-MS is used for detection and measurements of contaminants, spoilage of food, oil, ghee that are harmful and should be controlled and checked by government agencies. It is used for analysis of organic solvents, impurities in styrene, inorganic gases and allergens in cosmetics. GC-MS also used for characterization of formic acid in acetic acid for utilization in industries (Rubaye et al., 2017).

Fig: 3.7 7010 Agilent Technologies GC-MS
Total Organic Carbon (TOC) Analysis:
There are two types of carbon present in water. Total organic carbon (TOC) and inorganic carbon (IC). These two forms of carbon are known ac (TC) and they relate as:
TOC= TC-IC
Combustion tube is filled with oxidant catalyst and Sample is introduced in a tube. Firstly, carbon is convert into CO2 by combustion of TOC and TC (Florescu et al., 2013).
TOC analyzer have high temperature range for combustion. Firstly name, date, number and type of sample enter in log book and then TOC analyzer turned on then indicator change to initialized (Reckhow, 2012).

Fig: 3.8 Shimadzu TOC-LCSH/CSN
3.7 Actinometry:
Chemical actinometry is most common method used for the determination of radiant flux in photochemical studies. Actinometers have the ability to measure the photon flux in irradiated cell. The use of actinometer based on accuracy, sensitivity, linear dynamic range and easy to use. During photo process study occurred in water, the actinometer must be aqueous based to reduced refractive index differences. Potassium ferrioxalate actinometry is a common method used for the determination of monochromatic radiant fluxes. Potassium ferrioxalate actinometer used for the determination of radiant flux from UV to visible wavelengths (Jankowski et al., 1999). Incident light flux chemical actinometry is obtained by measurement of rate from photochemical reactions of well-known quantum yield. Uranyl oxalate actinometer has been used to quantify the intensity of UV-C lamp used for destruction of water pollutants (Miron et al., 2000).
3.8 pH Meter:
The pH meter (Benchtop pH/v meter) was employed for accurate pH measurement of sample solutions before and after irradiation.
3.9 Reagents used:
Diclofenac sodium which is target pollutant in our study was purchased from Fisher Scientific. The chemicals like H2O2, PS, iron sulfate and oxone were purchased from Sigma Aldrich.
3.9.1 Standard Solution of DCF Sodium:
An accurately weighed amount of DCF sodium was dissolved in 500 ml volume of distilled water for preparation of 2 mM stock solution. By diluting it, solutions of different concentrations were prepared and were used for degradation study.
3.9.2 Oxidants:
Solutions of different concentrations of H2O2, PS and oxone were used.
3.9.3 Catalysts:
Solution of iron sulphate was prepared and its effect was studied on degradation of DCF sodium.

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