Test kits for water analysis
Test kits for water analysis

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Test kits for water analysis

2.3 Photometric tests

The colour of a solution and hence the amount of visible light it absorbs, is related to its concentration. This is expressed as the Beer-Lambert law (often shortened to Beer's law).

A = ε c l
Equation label: (3)

where c is the concentration of the solute, l is the pathlength of the cell, usually one centimetre, and ε is the molar absorptivity (or molar extinction coefficient), a measure of how strongly a chemical species absorbs light at a particular wavelength and in a particular solvent.

In the laboratory the absorbance of a coloured solution is measured using a UV-visible spectrometer or simpler fixed wavelength colourimeter. Video 3 demonstrates a spectrometer in use.

Download this video clip.Video player: Video 3
Skip transcript: Video 3 UV-visible spectrophotometry.

Transcript: Video 3 UV-visible spectrophotometry.

Spectroscopy in the ultraviolet and visible region is widely used in analytical chemistry. We measure the intensity of light being absorbed by a sample using a spectrophotometer - there's a range of types having varying degrees of complexity.
On top of our spectrophotometer you'll notice a row of sample cells - or cuvettes - these are made of quartz, glass and plastic. It sounds obvious to say, but it's important that light can pass through the material the cell is made of. Quartz cells, which are the most expensive, must be used for measurements in the UV - below about 300 nm. Glass or plastic just wouldn't be any good as these materials absorb UV light, but in the visible region, they're fine. Cheap, disposable plastic cuvettes are routinely used when your solvent is water, or alcohol. However, for most other organic solvents glass cells are used.
The cells shown here all have an optical path length of 10 mm. In fact most spectrometers are designed to take cells of these dimensions, although longer path length cells are available. Typically there's two optically transparent faces opposite each other, and two frosted opaque faces, and you should always handle the cells by these frosted faces. Grease, dirt or scratches will adversely affect your spectrum. Also, bear in mind the solvent you use must not absorb significantly over the wavelength range you're studying.
Looking again at our spectrophotometer, this is of the double beam variety. Inside, light is split into two beams, one passes through a cell containing the sample and the other through a reference cell.
The reference should contain all the same reagents as the test cell, apart from the substance being measured. Often the reference is simply the solvent, such as water. So effectively we're measuring two spectra at the same time, our baseline spectrum is then subtracted to leave the spectrum of whatever you're measuring.
Looking inside the sample compartment, you can see it's split into two sections to accommodate the sample and the reference cells. You'll notice the 'R' and 'S' labels.
Now let's run a spectrum. Our sample is a dilute solution of potassium chromate in water. We start by washing out the cells. The reference cell is washed with deionised water, the sample cell with the sample solution. We then fill the reference cell, and the sample cell. It's important to make sure the cells are filled sufficiently. If not the meniscus may be low enough to cause refraction of the light beam. Also make sure the solution isn't cloudy - suspended solids will scatter the light beam. If your solution is cloudy, then centrifuging is advisable. Next, by wiping with soft tissue, we make sure the optical faces are clean - and there's no bubbles in the solution. If you do happen to see bubbles then gently tap the cell on the bench to remove them.
We then load the cells into the sample compartment. The cells must be positioned correctly, so the light passes through the optical faces. We then set the wavelength range over which we wish to scan. And click to run the spectrum. Notice the wavelength range in this case is between 250 and 500 nm.
Looking at the bottom right-hand corner you can see how the absorbance of the solution changes as a function of wavelength.
Remember our test solution is yellow and this spectrum shows as expected, it is strongly absorbing in the blue region. There is also a second peak in the ultraviolet. Finally, the computer optimises the spectrum on the screen
End transcript: Video 3 UV-visible spectrophotometry.
Video 3 UV-visible spectrophotometry.
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In the field, however, the need for convenience and portability means less complex devices called photometers are used. These miniaturised spectrometers tend to use light emitting diodes (LEDs) as an excitation source. LEDs have many advantages: they emit narrow band radiation compared with a tungsten light source and because they are more efficient at producing visible light their energy consumption for a given light intensity is much lower. This makes them ideal for a battery-powered field instrument. Photometers also contain interference filters for wavelength selection and detection of light intensity is usually by a photodiode. They use a microprocessor to produce an instant concentration output displaying mg l1.

Portable photometers are pre-programmed with test methods and the sample cell is often barcoded so the correct method is selected. Calibration is carried out by the manufacturers who also supply traceable reference standards for on-site checking. Photometers also correct for turbidity (discussed in detail in Section 4.1).

Photometers are available that measure multiple analytes or just one. The sample is contained within an optical grade glass cell, and a typical procedure involves adding measured doses of reagent to the sample, using a dose-metered dropper, a pipette or dissolving tablets. The solution is then left for a prescribed period for the colour to develop, and is then inserted directly into the photometer to obtain a reading - standard practice for colourimetric measurement.

There is a linear relationship between the colour of the solution (absorbance) and the concentration of analyte but this isn't necessarily the case over the whole concentration range you might wish to measure (Figure 5). A 'flattening out' is common at high concentration, and you must make sure your unknown value falls within the linear range on the curve. Modern photometers have this range electronically stored.

If the concentration of a particular analyte is outside the linear range of the test method, dilution is necessary, and, as we discussed earlier, your measured concentration value must be multiplied by the appropriate dilution factor.

This is where test strips come into their own. They can be used as a preliminary test, i.e. an indication of concentration of your analyte to tell you if it is within the working range of your instrument or in the non-linear region.

Figure 5  The working range of a photometer.

Question 7

We have shown the graph passing through the origin in Figure 5. In reality such a graph becomes non-linear at low concentration values, why is this?


As concentration is progressively lowered you will reach a point known as the limit of quantification, which is the lowest concentration of analyte that can be determined with an acceptable level of uncertainty; this defines the lower limit of the linear range. This is to be distinguished from the limit of detection which is the lowest concentration of analyte that can be detected but not necessarily quantified.


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