INSTRUMENTS FOR NEPHELOMETRY AND TURBIDIMETRY
Nephelometric and turbidimetric measurements may be made with a fairly reasonable accuracy and precision by using either standard instruments available commercially or by improvising other similar de-vices. A brief description of such available means shall be discussed below :
In general, nephelometric measurements essentially require an instrument with a photocell placed in position so that it may receive selectively the scattered light rather than the transmitted light. As this principle and geometry also hold good specifically to fluorimeters; and, therefore, these can be employed as nephelometers by selecting proper filters.
The following instruments are used invariably for nephelometric measurements, namely :
In actual practice, the so called ‘visual’ nephelometer (comparator type) have been more or less super-seded by the photoelectric instruments Nevertheless, a Duboscq Colorimeter with a slight modification may be used conveniently for nephelometric analysis, for instance :
(a) the path-of-light should be arranged in such a fashion that the light enters the side of the cups at right angles to the plungers rather than through the bottoms,
(b) clear-glass-tube with opaque bottoms are to be used instead of the normal cups,
(c) the glass-plungers are precisely fitted with opaque sleeves, and
(d) the light that enters at right angles to the clear-glass-tubes should be monitored carefully so as to achieve an equal-illumination on either sides.
Now, a standard suspension is placed in one clear-glass-tube, and the unknown solution is treated exactly in an identical fashion and placed in the other clear-glass-tube. Finally, the dividing line existing between the two fields in the eye-piece (Figure 20.2) must be distinctly thin and sharp, and it must disappear when the two fields are matched properly.
The Duboscq Colorimeter should always be maintained meticulously neat and clean. The clear-glass-tubes and the plungers are either rinsed with distilled water or with the solution to be measured.
First of all, it is necessary to ensure that the readings are zero when the plungers just touch the bottoms of the clear-glass-tubes. Now, the standard solution is placed in one clear-glass-tube, whereas an equal volume of the solution in question (unknown) in the other ; bearing in mind the fact that the clear-glass-tubes should never be filled above their respective shoulders.
Subsequently, set the unknown solution at a scale reading of 10.0 mm and simultaneously adjust the standard until the fields are matched equally. Perform at least five similar adjustments with the clear-glass-tube (A) containing the standard solution, and calculate the mean value. Care should always be taken that the plungers (B) always remain below the surface of the liquid. However, it is advised to visualize the match-point from above and below :
Assuming Beer’s Law holds good the concentration of the solution in question (unknown) may be determined by the help of the following expression :
where, l1 = Average readings for the clear-glass-tube having the solutions of known concentration,
l2 = Average reading for the clear-glass-tube having the solution of the unknown concentration,
c1 = Concentration of the known solution, and
c2 = Concentration of the unknown solution.
It may, however, be observed that if l2 = 10.0, the standard scale when multiplied by 10 shall give the percentage concentration of the sample in terms of the standard.
The most important characteristic feature of a nephelometer is the ‘reflector’ that has been specifi-cally designed so as to collect the light which has undergone scattering by the particles present in a turbid or cloudy solution. A typical nephelometer is illustrated in Figure : 20.3, below :
Following are the different parts of a nephelometer :
A = A light source,
B = A sensitive micro-ammeter,
C = Filter wheel with a series of colour filters,
D = An annular photocell,
E = A reflector to collect the scattered light,
F = A test tube, and
G = A metal test tube cover to exclude extraneous light.
The test solution (sample) is placed in a test tube (F) that has been duly rested on a light source (A) as exhibited in Figure 20.3. The scattered light caused by the particles in a turbid or cloudy solution is immedi-ately directed by the reflector (E) on to an annular photocell (D). A series of standard colour filters are usually provided in the form of a filter-wheel (C) so as to facilitate analysis of coloured solutions ; taking care that the filter chosen must be similar to colour to that of the solution. The current generated after passing through the photocell (i.e., light energy is being converted to electrical energy) is recorded by a sensitive micro-ammeter (B). The test tube is provided with a metallic cover (G) to get rid of any extraneous light. Usually a nephelometer is provided with zero-setting controls, sensitivity adjusting device and a set of previ-ously matched test tubes.
In fact, either visual or photoelectric colorimeters may be satisfactorily employed as turbidimeters. However, the use of the blue filter normally enhances the sensitivity appreciably. It has been observed that the light transmitted by a turbid solution does not normally obey the Beer-Lambert Law accurately and pre-cisely. Therefore, as an usual practice it is advisable to construct a ‘calibration curve’ by employing several standard solutions. The concentration of the unknown solution may be read off directly from the above calibra-tion curve as is done in the case of colorimetric assays.