Photometry - The traps waiting for you in the preparation and execution of assays

  • Fig.1: Different forms of tests
  • Fig.2a: Pipette 1,0ml of a sample into reaction cell, do not mix.
  • Fig.2b: b: Using a pipette add 1,0 ml NO3-1K, screw on lid and mix. Careful! The cell will get hot.
  • Fig.2c: Reaction time 10 minutes.
  • Fig.2d: place the cell in the cell slot. Place it so that the markings on the cell face the photometer.

From planning the experiments, buying and setting up the necessary apparatus and reagents, through general storage, taking samples and their transport and storage, to starting the practical analysis, execution, data collection and analysis, and documentation - there are many steps which determine whether the results fulfil the demands. If only one link in this chain is flawed, then the results will be flawed too.


It sounds as if there is an endless number of complicated possibilities for making mistakes, but in fact it is quite easy to obtain good results. Over 99% of all incorrect measurements can be put down to the user. It’s all about mistakes in the handling and poor preparation. 


The Correct Assay

If the absorption maximum of a substance is within the range of the photometer a measurement can be taken directly. If it is outside the range of the device being used it is necessary to carry out a chemical colour reaction which will provide a product that has an absorption maximum within the measurable range. The reduction in the colouring of a sample through a reaction can also be used. As colour tests mostly lie within the range of light visible to man, it is possible to make a rough estimate regarding the sought substance, with the aid of chromaticity diagrams. However, different lighting conditions, changes in the chromaticity diagrams due to age or UV rays and individual characteristics of the experimenter can cause significant deviations in the test results. As the reagents are not exactly cheap it makes sense to purchase a photometer that will ensure good test results. Particularly when the tests being carried out are routine. Otherwise one goes to a lot of trouble to meticulously prepare an assay as one would for an exact analysis only to obtain a rough estimate. The lab work is the same for both processes. Colour tests for many analytes are available in several forms. For quick and simple tests in the field where one is exposed to the elements it makes sense to use powder tests which come in portion bags. These are very small, which means one can take enough assays along. Pre-prepared tests which are already in the test cell are recommended for high throughput with highly efficient personnel. Bulk packs of assays are less expensive and useful for average throughput and if there is plenty of personnel. 


Tips for Reagent Tests

It is always advisable to read the instructions. One should choose an assay test in which expected area of concentration lies in the middle of the area of concentration covered by the assay. It should definitely not be close to the lower limit. According to Good Laboratory Practise (GLP) every assay should be carried out and measured two or three times. If there are deviations in the results the average can be used.  


Storage of Assays

Assays should always be stored under cool and dark conditions. The containers should be well sealed. In field work one should only take as many assays as one needs. Transporting in high temperatures, in a car for example, can affect the readings considerably or even make the assay unusable.  


Taking Samples and Filtration

Immediately after the sample has been taken, for example when testing water from the environment, change processes begin in the sample which have an influence on the readings. Parameters such as nitrate or concentrations of ammonia change because of the biological degradation processes as does the pH value. Immediately after taking the sample its temperature changes, which can have an influence on the speed of the degradation process and also the speed of the reaction of the assay. If the assays cannot be carried out directly after taking the sample it makes sense to filter and then cool the sample in order to remove any bacteria that are part of these processes. Using a syringe filter for the filtration is the only reliable method and is to be preferred to simple folded filters or other ordinary paper filters. In some tests more serious mistakes can occur when preserving samples with acidification as the colour reaction is pH dependent. If acidification is necessary, sulphuric or phosphoric acid are recommended. Interfering ions can also distort the readings. As an example, when determining the COD level the chlorine from hydrochloric acid can cause incorrect readings. 


Pipetting and Preparing the Samples

During the production of a dilution series for a calibration curve or when setting up a colour reaction, pipetting errors can ruin everything. When using an adjustable pipette it is important to pay attention to the exact setting of the volume to be pipetted. One must also ensure that the correct pipette tip is used and that it fits tightly (see cross media strip). Make sure the cells are clean. Dried water drop marks or stubborn remains of reagents will distort the reading. Depending on the assay it may be necessary to use cleaning agents for the cells. The instructions in the cell packaging often have the information needed. Even fingerprints can contribute to inaccurate readings. The cell itself also needs to be appropriate. Glass cells can be used for the visible range of light. However, as glass absorbs in the UV range, quartz cells should be used for this range. When using apparatus that has different slots for the various sizes of cell the length of the beam path should be taken into consideration.


Carrying Out the Test

The actual execution of the test is usually quite simple. If one follows the instructions even unpractised personnel can easily carry out correct readings. Instructions using pictograms make it easy. The temperature of the sample, the reagents and the room do need to be kept in mind. Chemical reactions like colour reactions depend on temperature. As a rule the reaction times stated in the user guidelines are valid for a sample with a temperature of 20°C. The reaction time doubles for every 10 degrees lower.    



Dr. Arne Kusserow


G.I.T. Laboratory Journal

Wiley-VCH Verlag




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