Construction of a calibration graph

You must first prepare the dishes – volumetric flasks, test tubes, pipettes, reagents and solutions.

Figure 14 – Graph for the determination of nitrites

To construct a calibration curve, prepare a series of standard solutions covering the range of measured concentrations of the test substance according to the procedure.

The calibration graph is built on 5-6 series of scales; the number of concentrations in each scale should be at least 5. Sharply different values of optical density are not taken into account. From the rest, the arithmetic mean value for each concentration is calculated and a graph of the dependence of the optical density on the concentration of the substance is plotted (Figure 14).

The approximate size of the graph is 20-25×30 cm; the line should pass through the origin at an angle of approximately 45°. In the ideal case, all points are located on a straight line, usually some points are located on a straight line, some are above and below it, the points seem to alternate.

The graph should indicate the conditions of photometry: the number of the filter or wavelength (nm); cuvette size (mm); photometry time. In addition, the date of construction of the calibration curve must be indicated. The calibration curve should be checked periodically for 2-3 concentrations.

The most common instruments are the FEK-56M photoelectric colorimeter, the KFO single-beam photoelectric colorimeter, the KFK-2 concentration photoelectric colorimeter (Figure 15), the KFK-2MP with the Electronics microprocessor system, and the SF-46 spectrophotometer (Table 2).

Table 2 – Technical data of photometric instruments

Device brand Spectral range, nm Number of filters
FEK-56M 315-630
CFD 400-700
KFK-2 (KFK-2MP) 315-980 And
SF-46 190-1100

The reliability of the measurement results when working on photoelectric colorimeters and spectrophotometers is ensured by the correct installation and operation of the devices. Therefore, it is possible to start measurements only after carefully reading the description of the device device and the rules for its operation.


Method of work on the photoelectric concentration colorimeter KFK-2 :

Preparation for work. The device is connected to the network 15 minutes before the start of measurements. During warming up, the cell compartment should be open (in this case, the shutter in front of the photodetectors blocks the light beam). Then install the required color filter and set the “sensitivity” knob to position “1” (when measuring with filters 315, 364, 400, 440, 490, 540 nm, indicated on the front panel of the device with black numbers, the “sensitivity” knob is set to position “1”, “2”, “3”, also indicated on the front panel with black numbers). When measuring with light filters 590, 670, 750, 870, 980 nm, indicated on the front panel of the device with red numbers, the “sensitivity” knob is set to one of the positions “1”, “2”, “3”, indicated on the front side of the panel also in red numbers. The handle “installation 100 rough” is transferred to the leftmost position. Before measurements and when switching photodetectors, it is necessary to check that the pointer of the device is set to “O” on the transmittance scale “T” with the cell compartment open. When moving the arrow from the zero position, it is brought to zero using the “zero” potentiometer, brought under the slot.

Determination of optical density. Cuvettes with solvent (or control solution) and test solutions are installed in the cuvette compartment. First, a light beam is passed through a cuvette with a solvent (or control solution). Close the lid of the cuvette compartment. The “sensitivity” and “setting 100 coarse” and “fine” knobs set the reading to 100 on the scale of the device. Then, by turning the knob, replace the cuvette with the solvent (or control solution) with the cuvette with the test solution. Take readings on the instrument scale in units of optical density. The measurement is carried out 3-5 times, and the final value of the optical density is determined as the arithmetic mean.

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