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33 !"#"$#%&'

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47 READ OUT MODULES

DC Signals produced in spectrophotometers are amplified and the voltage is read on analog meters, recorders, voltmeters or on computer displays.

High gain amplifiers are subject to significant drifts and offset errors. The presence of low noise restricts the signal to noise ratio.

Therefore the signal is modified by AC amplifier and converted back to DC output by a demodulator or rectifier. Sometimes the modulation is performed by interrupting the beam of by a rotating fan or a chopper. The rotating chopper blocks the beam physically. Therefore the radiation alternates between full intensity and total blackout resulting in a square at the chopping frequency.

Alternatively the light sources can be pulsed electronically to produce the same effect.

An AC Amplifier tuned to alternate in phase with the chopper passes the sinusoidal signal during the positive half cycle and blocks it during the negative half cycle. This is a form of rectification. Usually a reference signal is provided by the chopper to drive a switch. The reference signal is of the same frequency and has a fixed phase relationship with the analytical signal.

Synchronous demodulation results in a DC signal that can be sent through a low pass filter to provide the final dc output.

The tuned amplifier, synchronous demodulator, reference input and the low pass filter are integrated into a single electronic module called as

“lock – in amplifier”. Such amplifiers allow recovery of signals which are otherwise obscured by noise.

Some lock-in amplifiers directly pass the sinusoidal wave during the first half cycle and invert it during the other half cycle to provide a fluctuating DC signal. Such a signal is relatively easy to filter with a low pass filter. Such devices are called analog multipliers and used extensively in synchronous demodulators.

A lock-in amplifier is generally relatively free of noise because extraneous signals of different and phase are rejected by the system. MODULATOR DESIGNS EFFECT OF MODULATION OF DC SIGNAL SOFTWARE METHODS

A variety computer programs are available to enhance the signal to noise ratio. These include various types of averaging , digital filtering, Fourier transformation and correlation techniques which are applicable to non-periodic or irregular wave forms such as absorption , signals with no reference wave and periodic signals.

These include: i) Ensemble averaging ii) Box car averaging iii) Digital filtering iv) Correlation Methods a) Raw Spectrum (b) Quadratic 5 point smooth curve (c) Fourth degree 13 point smooth curve (d) Tenth degree 77 point smooth curve For more information i) G.Holic and G.M.Hieftje, ‘Contemporary topics in Analytical and clinical ’, D.M.Herculic et al., eds., Vol 3, pp 153 – 216 New York Plenum Press, 1978 ii) G.M.Hieftje and G.Horlick American laboratory 1981, 13 (3), 76. COMMERCIAL INSTRUMENTS

A number of colorimeters and spectrophotometers are available commercially. Their prices range from a few thousand rupees to several lakhs depending on the type of analysis and sophistication. At the low end are filter and colour comparators, and at the higher end are uv-visible-near spectrophotometers fitted with computer controlled instrument operations and microprocessors for data handling. COLOUR COMPARATORS

These are relatively inexpensive non-scanning filter photometers useful for dedicated analysis of only a few analytical parameters. The colour development is obtained by adding the specified quantity of the reagent to the sample which can be compared with a predetermined colour chart. The concentration is also marked on the colour chart. Colour comparators are adequate for rough estimates of the analyte for process monitoring or field studies or for specific analysis such as chlorine in swimming pools, gold estimation in jewelry etc.

FILTER PHOTOMETERS

Colorimeters or filter photometers are single beam, rugged direct reading instruments useful for visible range. They use a tungsten or LED as a source, a to collimate, a filter for selection and a barrier layer or a as the transducer. The read out is in or transmittance mode or in terms of concentration.

The 100% transmittance (or zero absorbance) is adjusted with the or the reference solution or simply by changing the voltage applied to the lamp. In modern instruments reference signal is stored in memory. Then the reference is removed and sample is inserted in the light path. The ratio of the sample signal to that of reference is computed and the absorbance is calculated and displayed. FILTER LED BASED SINGLE BEAM SPECTROPHOTOMETERS

In general the term spectrophotometer is used for visible (350 – 800 nm), uv-visible (190 – 900 nm) or uv – visible – near IR (190 – 3000 nm). Single beam grating instruments are relatively inexpensive, rugged and readily portable. These are used for quantitative analysis and scanning.

The most celebrated filter photometer is spectronic 20 which was introduced in the mid 1950s. Modified versions of this instrument employing concave gratings and microprocessors are still being offered. As in filter photometers, here also wavelength scan is performed with the reference solution and stored in the computer memory. Then the samples are scanned and ratioed. The output options include log absorbance, transmittance, derivative spectra, overlaid spectra, repetitive scans, peak location, peak height and peak area, kinetic measurements, flow through cells etc.

Single beam instruments have the inherent advantages of high energy throughput, low signal to noise ratios and simple sample compartments. Their disadvantage is the baseline stability. DOUBLE BEAM SPECTROPHOTOMETERS In double beam spectrophotometers the monochromatic radiation is split into two equal radiant power beams. One beam passes through the sample and other through a reference solution or blank.

The output of the reference beam is kept constant by:

a) Employing a feed back loop to regulate sensitivity via dynode voltage or

b) Controlling slit width by means of servomotors or

c) By measuring the ratio of P/Po continuously In these instruments uv or visible radiation enters the Czerney - Turner configuration. The radiation is collected in the photodiode. As long as the intensities are identical the amplifier has a dc output. Any difference in the intensities results in an AC signal at the chopping frequency. The unbalanced signal gives an ac output which is amplified and used to drive an optical attenuator into or out of the reference. The servomotor can also be connected to a recorder pen which provides the scan. Alternatively the servomotor can be digitized and computer output of the scans may be obtained.

QUANTITATIVE ANALYSIS BY

1. METHOD DEVELOPMENT

In principle any coloured solution can be subjected to chemical analysis by spectrophotometry. Numerous reagents react selectively with non absorbing species to give products that absorb strongly in the and visible regions. A vast literature exists which detail reactions of this type. Typical inorganic regents include: thiocyanate ion for Fe, Co and Mo.

Hydrogen peroxide for Ti, V, Cr Iodide ion for Bi, Pd, Td Organic chelating agents: DOTC for Cn, Dithiozone for Pb, 1,10-Phenanthroline for Fe, DMG for Ni etc. Typical method development procedure involves: i) Determination of the wavelength for maximum absorption . ii) Optimization of the experimental variables for completion of the reaction. These include: pH, reagent concentrations, , stability of the coloured product, effect of high electrolyte concentrations, stoichiometry of the complex etc. iii) Preparation of the calibration curve and v) Evaluation of the interfering substances, matrix effect, and other ionic species. vii) Determination of molar absorptivity, Sandell sensitivity, statistical evaluation of the bias. vi) Applications in diverse matrices. SIMULTANEOUS SPECTROPHOTOMETRIC ANALYSIS

Two dissimilar having different !max and " if present in a given mixture can be analysed if their are additive.

The procedure involves measurement of the total

absorbance of the mixture at both !max . Then two simultaneous equations can be written ,

C1("1)!1 + C2("2)!1 = A!1

C1("2)!2 + C2("2)!2 = A!2

The equations are solved for C1 and C2. Theoretically any number of components can be determined by setting up simultaneous equations. But at best 3 component systems are the limit. The !max of the components must differ by at least 30 nm. DIFFERENTIAL OR EXPANDED SCALE

In spectrophotometry the analog scale varies from zero absorbance to 100% absorbance. The latter is an artificial requirement. Therefore when reference solution transmits more than another standard, scale expansion techniques can be used to increase the precision. Three cases result: i. High absorbance method. ii. Trace analysis method. iii. Maximum precision method.

DERIVATIVE

In derivative spectrophotometry spectra are obtained by plotting the first or higher order derivative of the absorbance with respect to wavelength as a function of wavelength.

In a derivative spectrum the ability to detect and to measure minor spectral features is considerably enhanced. The enhanced spectral features can distinguish between very similar spectra and follow subtle changes in a spectrum.

Derivative spectrophotometry is also quite useful for the simultaneous determination of two or more components in mixtures. Absorption spectra of the analyte can be extracted from turbid solutions. Examples: i) Determination of amounts of Mn and Zn in mixtures by complexion with 5,8-dihydroxy - 1,4 - napthoquinone. ii) Tryptophan, tyrosine and phenyl alanine contain

aromatic side chains with !max in 240 – 300 nm region. These sharp peaks are not apparent in spectra of typical preparations but observed in first and second derivative spectra. iii) Derivative methods are widely applied to pharmaceutical preparations and to vitamin mixtures.

PHOTOMETRIC TITRATIONS

Photometric titrations are useful for locating the equivalence point of a titration provided the titrant or titrand or one of the reaction products absorbs the radiation. The titration curve is a plot of the absorbance corrected for volumetric changes as a function of the titrant. The end point is obtained by extrapolating the linear portions of the curve. Photometric titrations can be automated to record a fixed absorbance or by taking the second derivative.

a) Arsenic (III) with bromate – bromide; Bromine absorbs b) Copper (II) with EDTA; Cu (II) – EDTA complex absorbs c) P – tolnidine in butanol with perchloric acid; P – tolidine colour disappears d) Coloured titrant reacting with the titrand to produce coloured product absorbing at the same wavelength e) Bromination of a stuff f) Formation of two successive complexes KINETIC METHODS In kinetic methods absorbance of the solutions are measured as a function of time. Therefore the measurements represent ‘dynamic ’ before attaining equilibrium regime. A variety of chemical reactions can be characterized in this way. For example,

1. Determination of trace quantities of iodide in the reaction [I]! 2Ce(IV) + As(III) Ce (III) + As (V)

2. Determination of as catalysts

3.Determination of ascorbic acid in phosphomolybdenum reaction

KINETICS OF REACTIONS Kinetic methods can be classified into two types. Differential methods compute the rate of reaction from the slope of the absorbance Vs time curve and relate it to the analyte concentration.

Integral methods use an integrated form of the rate equation and determine the concentration of the analyte from the absorbance changes that occur over various time intervals. Curve fitting methods are used to fit a mathematical mode to absorbance versus time curve and compute the parameters of the model including the analyte concentration. Kinetic methods based on the reactions with half greater than about 10 seconds are amenable for spectrophotometric follow up. Temperature control of to ± 0.10C required. In many commercial instruments accessories are available as modules that can be attached to spectrophotometers for kinetic measurements.

For reactions with half lives of less than 10 seconds, stopped flow mixing technique can be adopted. In this technique streams of reagent and sample are mixed rapidly and the flow of mixed solution is stopped downstream suddenly. The reaction is then monitored. COMPOSITION OF COMPLEX IONS

The most common techniques used for complex ion studies are :

1. Method of continuous variation

2. The mole ratio method and

3. The slope ratio method COMPOSITION OF A COMPLEX YOE’S MOLE RATIO METHOD FLOW INJECTION ANALYSIS

FIA (’’) involves injection of a measured amount of liquid sample into a moving non segmented stream of reagent. As the injected sample plus reagent carrier proceeds down a narrow tube propelled by a constant flow pump the sample reacts to form a coloured complex under dynamic flow conditions which passes through a flow cell attached to the spectrophotometer continuously. A sharp signal develops followed by a trailing edge. The peak height is proportional to the concentration of the analyte. The sample peak can be detected in about 15 sec after injection. Since the system operates under dynamic conditions the sample clears out of the reactor in 15 seconds and be ready for another analysis. The whole system can be made very compact.

Samples as small as 30 #l and reagent volumes of less than 1 ml are possible.

TURBIDIMETRY AND NEPHLOMETRY

The scope of spectrophotometry can be greatly expanded if the optical inhomogeneity in a sample can be reproducibly measured.

The presence of suspended matter in stable systems, thermal density fluctuations, colloidal particles, etc., introduce optical inhomogeneity in an otherwise homogeneous medium that results in elastic scattering (measured at the same wavelength). The scattered radiation is preferentially absorbed rather than transmitted through the sample.

The intensity of the parallel and perpendicularly polarized components of the electromagnetic radiation is a function of the relative refractive index, the size of the particle, the angle of observation relative to the incident radiation and the concentration of the scatterer.

The size parameter ($ = 2%r/!) determines the phase distribution of the scattered radiation around the scattering center.

The scattering envelope is symmetrical when the size parameter is smaller than one – tenth of the wavelength and the refractive index of the particle Is almost same as that of the surrounding medium (). As the size parameter reaches one fourth the wavelength of the incident radiation, scattering is concentrated in the forward direction.

If the particles are larger than the wavelength of the incident radiation, scattered intensity would be a very complex function of the angle of scattering, but still a large amount of it scattered in the forward direction. This is known as Mie scattering. For very large particles, scattering does not depend upon the wavelength but depends upon the particle geometry, molecular absorption, sample concentration and size distribution and sometimes even in the physical design of the instrument used for measurement. Therefore the results are essentially empirical and exhibit variance in the data even though same calibration standard is used. There are two methods for measuring the turbidity of a sample, in the same axis and perpendicular to the incident radiation. When measurements are performed in the forward direction analogous to absorbance, it is called turbidimetry. When the scattered radiation is measured at right angles to the direction of the incident beam, it is called ‘Nephelometry’. Usually nephelometric measurements are preferred for samples having transmittance greater than 90% as in and waste water analysis. At 900 the concentration is least sensitive to variation in particle size and simple optical systems can be used for the determination that is relatively free of stray radiation.

When a very narrow beam of radiation strikes the surface of the sample at a very small angle, part of the reflected beam strikes the water surface and escapes into a light trap. The remaining portion enters the sample at 450 angle. If particle turbidity is present, then radiation scattering occurs and some of the scattered radiation reaches the detector. Such an instrument can detect turbidities upto 0.01 NTU. In turbidimetry small amounts of insoluble compounds are prepared in a state of aggregation so that moderately stable suspensions are obtained. When light is passed through such a suspension, the fraction of the energy dissipated by absorption, reflection and refraction, scattering etc., is ratioed with respect to the incident radiation and displayed as absorption or transmittance.

Beer’s law is obeyed for such suspensions up to a particular concentration and analysis can be performed just like a spectrophotometry. Since the results for turbidity are at best empirical. Construction of calibration curves is recommended for every analysis owing to the uncertainties in reproducibility. The experimental conditions should also be exactly reproducible as far as possible.

In turbidimetry the following conditions need to be adhered to:

1. Concentrations of substances participating in the reaction

2. Order of mixing and rate of mixing

3. Use of protective colloids such as gelatin, , dextrin, polyvinyl alcohol etc

4. Presence of other salts and

5. Temperature In theory, any precipitation reaction can be converted into a turbidimetric procedure provided they can be prepared in a reproducible manner. Excellent results in sub PPB levels have been obtained for:

• The determination of BaSO4 using glycerol-alcohol as stabilizer

• The determination of phosphate as strychnine phosphomolybdate

• The determination of using Nessler’s reagent Nephelometers and turbidimeters resemble filter photometers in instrumentation. They are also available as accessories to ultraviolet and visible spectrophotometers. Such instruments provide a practical way to continuously monitor turbidity in water, determination of the clarity of beverages and pharmaceutical preparations, product quality control, boiler feed and condensates etc., SPECTRA OF

Quite often in the real world, turbid liquids, powders opaque and translucent substances, glossy and semi-glossy painted surfaces are encountered. The reflection of the incident radiation falling on such compounds can be measured with a specular reflector available as a standard accessory to spectrophotometers. Since every contact of a light beam with a particle results in specular reflection at every few millionth of a centimeter any surface appears uniformly bright in all directions. This is referred as sheen or gloss. This is the diffuse component of any component that is coloured otherwise it appears . The whiteness and increase as the diameter of the component decreases. The reflectance of a substance follows a semi-empirical relationship,

3 S = log Po/P = k bcd D4+$!4 where d is the particle diameter, ! is the wavelength, c is the concentration, b is the path length, and k is a geometric factor, D is a method constant . For using reflectance a mgo standard is employed as a reference and all other measurements are made with reference to this standard.

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