Spectrophotometer Instrumentation

Absorption filter Interference filter Prisms Gratings 

Barrier-layer cells Phototubes Photomultiplier tubes 

Most modem ultraviolet/visible spectrophotometers are double beam instmments which generally covers the range between about 200 nm and 800 nm. In these instmments the monochromated beam of radiation, from tungsten and deuterium lamp sources is divided into two identical beams of equal intensity, one of which passes through the reference cell and other through the sample cell. 

The signal for the absorption of contents of the reference cell is automatically electronically subtracted from that of the sample cell giving a net signal corresponding to the absorption for the components in the sample solution. The instruments also possess digital display for the instantaneous reading of the absorbance values as these are measured. 

When the sample absorbs light, its intensity is lowered. Thus the photo electronic cells will receive an intense beam from the reference cell and a weak beam from the sample cell. This results in the generation of pulsating or alternating currents which flow from the photoelectric cells to the electronic amplifier. The amplifier is coupled to a small servo motors which drives an optical wedge into the reference beam rmtil the photo electric cell receive light of equal intensities from the sample as well as the reference beams. 

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As with any spectrophotometer instrument, the light intensity is determined at each wavelength using a photomultiplier tube. Emission measurements can be made over a wide dynamic range, which is an interesting feature because elements with widely different concentrations or sensitivity can be measured in a single sample solution. 

Fundamentals of spectrophotometer readings In order to best understand the use of UV/visible spectrophotometry and a spectrophotometer instrument it is important to first understand the data that can be derived from the use of this technique/equipment. The fundamental outcome from the use of a spectrophotometer is a measure of transmittance or absorbance. As the names suggest, transmittance (T) is the amount of light that is transmitted through the sample solution whereas absorbance (A) is a measure of light absorbed by the sample solution. Modern instmments can provide readings of both transmittance and absorbance (or its reciprocal 1/A) normally the primary reading for most bio-analytical applications is absorbance. However, it is important to appreciate that transmittance and absorbance are related by fundamental equations. 

When using an ultraviolet/visible spectrophotometer, instrument parameters will include wavelength and slit width. If the wavelength is changed then the measured absorbance of the sample solution is likely to change. 

Digest a 0.25-g tobacco sample at 80 °C overnight in 8.0 ml of nitric acid perchloric acid = 9 1. Continue heating the mixture to fumes and take to dryness. Dissolve the residue in 25ml of IN HCI. Dilute to appropriate concentration with distilled water. Analyze the solution at 422.7 nm in an atomic absorption spectrophotometer (Instrumentation Laboratory Sll) using a nitrous oxide-acetylene flame. 

Block diagram for a single-beam fixed-wavelength spectrophotometer with photo of a typical instrument. 

Two common detectors, which also are independent instruments, are Fourier transform infrared spectrophotometers (FT-IR) and mass spectrometers (MS). In GC-FT-IR, effluent from the column flows through an optical cell constructed... 

The methods dependent upon measurement of an electrical property, and those based upon determination of the extent to which radiation is absorbed or upon assessment of the intensity of emitted radiation, all require the use of a suitable instrument, e.g. polarograph, spectrophotometer, etc., and in consequence such methods are referred to as instrumental methods . Instrumental methods are usually much faster than purely chemical procedures, they are normally applicable at concentrations far too small to be amenable to determination by classical methods, and they find wide application in industry. In most cases a microcomputer can be interfaced to the instrument so that absorption curves, polarograms, titration curves, etc., can be plotted automatically, and in fact, by the incorporation of appropriate servo-mechanisms, the whole analytical process may, in suitable cases, be completely automated. 

In spectrophotometric analysis a source of radiation is used that extends into the ultraviolet region of the spectrum. From this, definite wavelengths of radiation are chosen possessing a bandwidth of less than 1 nm. This process necessitates the use of a more complicated and consequently more expensive instrument. The instrument employed for this purpose is a spectrophotometer. 

When a spectrophotometer is used it is unnecessary to make comparison with solutions of known concentration. With such an instrument the intensity of the transmitted light or, better, the ratio I,/I0 (the transmittance) is found directly at a known thickness /. By varying / and c the validity of the Beer-Lambert Law, equation (9), can be tested and the value of may be evaluated. When the latter is known, the concentration cx of an unknown solution can be calculated from the formula ... 

Double-beam spectrophotometers. Most modern general-purpose ultraviolet/ visible spectrophotometers are double-beam instruments which cover the range between about 200 and 800 nm by a continuous automatic scanning process producing the spectrum as a pen trace on calibrated chart paper. 

Visual methods have been virtually displaced for most determinations by methods depending upon the use of photoelectric cells (filter photometers or absorptiometers, and spectrophotometers), thus leading to reduction of the experimental errors of colorimetric determinations. The so-called photoelectric colorimeter is a comparatively inexpensive instrument, and should be available in every laboratory. The use of spectrophotometers has enabled determinations to be extended into the ultraviolet region of the spectrum, whilst the use of chart recorders means that the analyst is not limited to working at a single fixed wavelength. 

The colour filters used with absorptiometers should be examined from time to time by measuring the absorbance in a spectrophotometer if the results depart markedly from those expected for the filter, it should be replaced. Normally a filter as supplied with the instrument will be used, but if necessary, filters from the Wratten range supplied by Kodak Ltd or from the Spectrum Filter or Bright Spectrum Filter series supplied by Ilford Ltd may be used. 

A special titration cell is necessary which completely fills the cell compartment of the spectrophotometer. One shown in Fig. 17.24 can be made from 5 mm Perspex sheet, cemented together with special Perspex cement, and with dimensions suitable for the instrument to be used. Since Perspex is opaque to ultraviolet light, two openings are made in the cell to accommodate circular quartz windows 23 mm in diameter and 1.5 mm thick the windows are inserted in such a way that the beam of monochromatic light passes through their centres... 

Detection of the infrared signal is, of course, of prime importance. A range of detectors is available for this purpose, the type used in any particular instrument depending upon the type and quality of the spectrophotometer. 

All infrared spectrophotometers are provided with chart recorders which will present the complete infrared spectrum on a single continuous sheet, usually with wavelength and wavenumber scales shown for the abscissa and with absorbance and percentage transmittance as the ordinates. More advanced instruments also possess visual display units on which the spectra can be displayed as they are recorded and on which they can be compared with earlier spectra previously obtained or with spectra drawn from an extensive library held in a computer memory. These modern developments have all led to quantitative infrared spectrophotometry being a much more viable and useful analytical procedure than it was just a few years ago. 

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