UV SPECTROPHOTOMETER: PRACTICAL CONSIDERATIONS

This article deals with UV Spectroscopy its application (practical) and its mode. There are various modes of UV spectrophotometer namely photometric, spectrum, quantitation, kinetics, time scan, multi-component and bio-method. Photometric measures absorbance or % transmittance of a sample at arbitrary wavelengths. Spectrum Scans a wavelength range to measure the absorbance and % transmittance of a sample as a function of wavelength. Single beam energy measurement can also be performed. Data processing such as peak detection, smoothing, and mathematical calculation may be applied to the measured spectrum. Quantitation creates a calibration curve from a standard sample and quantitates an unknown sample. Kinetics Calculates enzyme activity from the time dependent change in absorbance. Time scan This function is used to measure the change in the rate of absorbance, transmittance, or energy in the fixed wavelength. Multi-component Enables samples with up to 8 constituent components to be measured and quantitated. In the Bio-method Mode, DNA Quantitation and Protein measurement are done. These modes are used for


Introduction
UV Spectroscopy is the measurement of the attenuation of a beam of light after it passes it through a sample or after reflection from a sample surface. Absorption measurements can be at a single wavelength or over an extended spectral range.
The region beyond red is infra-red whereas region beyond violet is ultra-violet. The wavelength range of UV radiation starts at blue end of visible light (4000 Å) and ends at 2000 Å 1 .
There are various modes of UV Spectrophotometer for the analysis of sample. The following are:

SPECTRUM
The spectrum mode is used to measure spectra. Measure the absorbance and transmittance spectra of the sample by performing the wavelength scan. Changes in the sample can be tracked using repeated scans 2 . This mode also allows you to measure the energy of a light source using its single beam.

Application 1
Identification of compounds.
UV spectroscopy can characterize those types of compounds which absorb UV radiation. It is done by comparing the absorption spectrum with the spectra of known compounds 15 .

QUANTITATION
The quantitation mode is used to quantify an unknown sample by creating a calibration curve from the standard sample. Various combinations of wavelength and calibration curve factor and firstto-third order regression calibration are possible. The following four types of measurement methods are available according to the number of wavelengths used: • One-wavelength method: The sample is quantified using its absorbance at a single wavelength. The operation procedures in this chapter mainly describe this method. • Two-wavelength method: The absorbance at a wavelength other than the quantitation wavelength is used to eliminate the interfering components and contaminants. • Three-wavelength method: The absorbance of two wavelengths is used to eliminate the interfering components. • Derivative quantitation: The derivative value (1st through 4th orders) for the spectrum at the quantitation wavelength is used. The calibration curve is created using one of the following three methods 3 : • K factor method: In the equation, "concentration = K x absorbance + B", K and B are predetermined.

Kinetic Rate
The kinetics rate measurement mode is used to measure the change in the rate of absorbance for one wavelength and obtain the change in quantity of absorbance per rate interval. A discriminant is used to determine whether the absorbance is being changed linearly and to display the linearity (L) or the non-linearity (N). The discriminant is as follows. Linearity (L) is determined by whether the ratio of the rate of change of absorbance during one cycle and the rate of change of one cycle before are within the proportion (%) set in the criteria 2 .

TIME-SCAN
The time scan mode is used to measure the change in the rate of absorbance (Abs), transmittance (%T), or energy (E) in the specified arbitrary wavelength 6 .
Application 1: Time scan graph of hydrogen peroxide.
In this study hydrogen peroxide was kept at 50 ○ C for 10 minutes after that it runs for 150 seconds. As time passes, there is decrease in absorbance which tells that at high temperature the absorbance of hydrogen peroxide increase 7 .

MULTI-COMPONENT
The multi-component quantitation mode is the mode in which the concentration of each constituent component is determined by using the absorption spectrum of the mixed sample with pure standards or standards made up of multiple constituent components 8 .
1) Mixed samples with up to 8 constituent components can be quantitated.
2) In addition to using pure samples of each constituent component as the standard samples, a mixed sample in which the concentration of each constituent component is known may also be used. The effects of interference among the various constituent components can be minimized by using a mixed sample as the standard sample.
3) Parameter files for this mode store measurement parameters and standard sample data, making their file size relatively large. Therefore, they can only be saved by using a USB memory device.
In addition, a spectrum saved in memory can be loaded and used as the standard sample or unknown sample data. However, unlike other parameter files, these can only be saved one at a time.
4) The measurement wavelengths can be set at uniform intervals or randomly set 9 .

BIO-METHOD
The Bio-method mode allows you to obtain the DNA and protein concentrations with various quantitation methods 2 .

Concentration of NaCl
Different concentration of NaCl were prepared and the spectrum was run between 400-200nm 10 . UV spectroscopy can characterize those types of compounds which absorb UV radiation. It is done by comparing the absorption spectrum with the spectra of known compounds. 10ppm Ibuprofen solution was prepared in hexane, ethyl acetate and methanol and spectra were taken 11 .

Determination of total organic carbon
The sample is oxidised to produce carbon dioxide which is then measured by a detection system. Aquatic humic water divided into two portions and kept at ph ≤ 2 and other at ph between 5-7and sit capped for 30 minutes in cold place and 40μL HCl, H 2 SO 4 were added and spectrum was run between 400-200nm 12 .    7. Monitoring of Bio-processes Protein standards were made up using BSA and phosphate buffer, in the range 0.0 to 1.0 g l−1 at 0.1 g l−1 intervals. A 0.1 ml sample (i.e. calibration standards and actual samples) was pipetted into a 4.0 ml plastic cuvette, and then 3.0 ml dye reagent added. The solution was left for 10 to 15 min and then the absorbance measured at 595 nm 16 .
8. DNA extracted from human saliva 0.4 ml of DNA extraction buffer was added to tubes containing 0.5 ml of saliva sample. e. 0.02 ml Protease K was added to all the tubes to digest the cellular material and proteins. Centrifuge tubes were placed at 50-60 C in an incubator for 3 h for fresh samples and for dry samples kept at 50-60 C in an incubator overnight. To collect the DNA the tube was centrifuged at 14,000·g for 10 min. 0.2 ml of DNA wash was added to the pellet and centrifuged at 10,000·g for 10 min. 50 ll of TE buffer was added to the pellet, incubated at room temperature for 10-15 min and then stored in a refrigerator at 20 C. Quantity of DNA was calculated by taking the absorbance of samples at 260 nm. Concentration of DNA calculated from absorbances for fresh samples ranged from 125 to 795 lg/ml and for dry samples ranged from 15 to 85 lg/ml 17 . 9. Para-Nitrophenol on Raney Alloy Stock concentration of para nitrophenol (PNP) 272 ppm was prepared in aqueous solution. For the sonication process, the reactor cell volume was kept at 5 ml. 50%Ni-50%Al raney alloy (100 mg) which was suspended in 3.62 mL of deionized water and sonicated for 5 minutes. 40ppm, 50ppm and 75ppm PNP solution was prepared and stirred for 15-30-45 minutes at 30 °C and centrifuged for 3 minutes at 14500 rpm. The spectra was run between 4500-350 cm -1 18 .

Caffeine in coffee beans
The solution was stirred for one hour using magnetic stirrer and heated gently to remove caffeine and filtered by a glass filter 22 .   The CR-39 (C12H18O7) detectors of (2 × 2) cm2 size were cut from a sheet of 0.1 cm thickness. These detectors were then exposed to neutrons from two different sources. Each of the detectors was etched in successive time intervals in an aqueous solution of 6 M NaOH at (70 ± 1) •C. The detectors were studied with the help of the spectrophotometer and etched up to 570 min in 14 different time intervals starting from 10 min 25 .   Tetrakis (4-N-dimethylaminophenyl) porphyrin (H2TDMAPP, 1) and manganese tetrakis(4-Ndimethylaminophenyl) porphyrin (MnIIITDMAPP, 1a) were prepared according to the reported work [21]. In N2 atmosphere, into a three-neck round-bottom bottle were added DMF (50 mL), H2TDMAPP (1, 1.0 g), and CH3I (3.0 g, excess). The resulting mixture was stirred at 120 •C for 1 h. After cooled to room temperature, the mixture was added with diethyl ether to precipitate a solid, which was thoroughly washed with acetone and diethyl ether. The collected red-brown solid was then dried in vacuo to yield the product of tetrakis  20. Determination of chemical characteristics of olive oils 89 samples of olive oil from two harvest years (2015,2016) were used for analysis. The first derivative was obtained after performing the FTIR in presence of silica gel in n-hexane. Second derivative was obtained after performing FTIR in presence of silica gel and n-hexane at 80 ○ C. third derivative was obtained after performing FTIR in presence of second derivative with MSc and SNV 29 .

Writing age of black roller and gel inks
Sample is extracted in a vial with 1 mL methanol containing ethyl benzoate (ethyl benzoate is used as external comparative matter) for 20 min to maximize the amount of all available volatile components of ink. After extraction, 2 mL of every sample is removed and analyzed by GC. The residual solution of extraction is extracted secondarily with 0.9 mL DMF for 20 min to maximize the amount of all available dye components of ink and analysed in UV spectrophotometer 30 .

CONCLUSION
It can be concluded that UV spectrophotometer can be used for many purposes. It contains various modes like Photometric, Kinetic, Kinetic rate, Spectrum, Time-Scan, Bio-method, Quantitation and Multi-component. Most commonly mode used are Photometric and Spectrum. Photometric is used for calculating the absorbance at a particular wavelength which is the ƛ max of that particular molecule. It gives the exact absorbance of molecule at that particular wavelength. In case of Spectrum, the sample is run in a range (generally 400-200nm is used) a graph is shown which gives the absorbance of sample. Spectrum and Photometric is used for the known concentration. Quantitation is used for plotting the calibration curve of unknown concentration. Kinetics is used to measure the change in absorbance as a function of time, and obtains its enzymatic values. Time scan is used to measure change over time in photometric value at a specific wavelength. Bio-method is used to quantify DNA and protein by using different method.