The Basics of Photometric Measurement

Part 1: Principles, Optics, AQA, Test Kits - Introduction in the basics of photometric measurement

Photometric measurement procedures are among the most important measurement methods, along with pH measurement. They are used in research and teaching to investigate new substances or enzymatic and biochemical processes. In the environmental field, they are used to monitor environmental influences. In quality control they are used for food and beverages. The methodology ranges from absorption/transmission measurement and concentration determination to scanning and kinetics measurement. For decades, WTW has simplified the complex measurement process of photometry. For the user, this means convenient and fast measurement through automatic measurement settings and analytical quality control (AQC).

Phos (Greek) for light

Photometry is a measurement method to analyse (aqueous) solutions by means of a light source.
Light (physical) is a spectra of electromagnetic waves, divided into different ranges: Visible light (white light) ranges from approx. 380 – 780 nm

Photometric / Colorimetric Analysis:

Determination of substances by their specific colour reaction and light absorbance in dependance of their chemical properties at a specific wavelength.

Introduction – Light Sources and Optics

Specific Wavelengths are obtained by

Different light sources:

• LEDs = lowest power consumption, lower light intensity
• Tungsten (white light halogene lamp) for VIS range
• Xenon (UV-VIS) => Flash lamp with long life span
• Deuterium (UV) => special lamp, expensive

Unterschiedliche Optiken:

• Monochromators
• Polychromators
• Filters
• LED

Optics: Filter and LED Photometer

Filter photometer with reference beam: photoLab® S6/S12

LED + optical Filter – single beam: photoFlex® Series

Monochromator of photoLab® 7000 Series

Measurement modes

What type of measurement is performed in with a photometer?
3 Measurement modes of photometric analysis and their relation

1. Transmission T(%): Ratio of light intensity after cuvette (I) and before (I_o)
2. Absorbance: Abs = - log10 (T ) or „extinction of light“ passing the cuvette
3. Concentration: quantitative analysis of a substance (mg/l, ppm,…) at a defined wavelength based on a calibration curve

Transmission measurement

Transmission is the ratio of passed light I / initial light I₀:

Transmission measurement is also being used to measure turbidity at 180° angle (unit FAU, e.g. for quality control) and for turbidity correction in concentration measurement.

Absorbance Measurement

Absorbance = „Extinction of light“:
Each substance has a specific spectra with absorbance peak(s).
Run spectrum to define maximum or optimal peak = wavelength definition for concentration measurement

Concentration measurement:

Konzentrationsmessung:
Measurement at specific wavelength, obtained by either matching LED, optical filters from white light or mono-chromator

Relation %T : Absorbance : Concentration
Transmission measurement:

The transmission of a sample varies exponentially with thickness and concentration

Absorbance measurement:

Absorbance of a sample is proportional to thickness of the sample and concentration

100      10      1      0,1      Transmission (T%)               -            Logarithmic correlation
0          1        2      3         Absorbance A = -log₁₀ (T)    -            linear correlation
0          4        8      12      Concentration (mg/l)              -            linear correlation

Relation %T : Absorbance : Concentration

Lambert-Beer’s law
Experiments by BOUGUER (1698–1758) and LAMBERT (1728–1777) showed that the absorbance is dependent on the thickness of the absorbing layer of the cell used. The relationship between the absorbance and the concentration of the analyte in question was discovered by BEER (1825–1863). The combination of these two natural laws led to the derivation of Lambert-Beer’s law, which can be described in the form of the following equation:

            
E = ελ· c · d
ελ = molar absorptivity, in l/mol x cm
d = Path length of the cell, in cm
c = Concentration of the analyte, in mol/l

Source: Operating Instructions of photoLab® S12, Part 1: General Information (www.wtw.com)

Concentration Measurement

The correlation of absorbance/concentration is determined by setting up a characteristic calibration curve for each substance (parameter).
The chemical reaction must be known:
Dilution series with defined concentrations, measured at defined and cuvette size (pathlength)

Characteristic (calibration) curve
Unknown sample concentration can be „read“ from the curve!

Methods/ programs in photometers contain all data and compute result automatically, including various cuvette sizes. Barcoded test kits additionally call up the respective method=program.

Method data / Program for each parameter

Programmed data for comfortable concentration measurement are consisting in:

1. matching the absorbance for determination
2. Reagent blank E₀ = coloration of reagent
3. Slope of calibration curve for calculation
4. Citation & unit (e.g. NO₃-N mg/l)
    Factors for citation & unit switch (e.g. NO₃; mmol/l)
5. Sample blank (e.g. coloration of the sample) is not included!
   „E_sample“ comes on top of E₀ : individually for sample, for small volumes mostly negligible

Prerequisites for concentration measurement

1. Coloured solution contains dissolved dye
2. Absorption of light leads to a coloration(complementary to λ)
3. Color intensity is correlating with concentration
4. The chemical reaction of analyte leads to building or disappearance (e.g. COD 4-40 mg/l) of dye in defined reaction time
5. Reaction must be selective for the analyte – no cross reaction with other disturbing substances
6. The developed dye must be stable for time of measurement => e.g. reading within 10 minutes after reaction time w/o color deterioration (see analytical instructions)

Instrument Check – AQA

Self calibration and warm up time especially for kinetics and spectra

AutoCheck:
photoLab® levels meter vs air in the background

Zeroing/Baseline:
correcting the meter to „E₀“ => especially after transport or in changing conditions (temperature…) meters E₀ „drift“ some mAbs => readings become inaccurate (mostly too high)

AQA Tools:
1. Optical or liquid filters
2. Color solutions, e.g. PhotoCheck®
3. Selected unscratched zero cuvettes
4. Control standards of the substance

FAQ Commercial test kits in brief

Measurement range

The range depends on the equipment, the reaction has detection limits. MR-values are reaching approx. ± 2 - 2,5 Abs (test dependent!)

At the lower end detection limits and tolerances of the procedure have the biggest influence on accuracy of readings: Limitation of chemical procedure, confidence interval and accuracy are often at the lower limit.

Note: Scratches, pipette errors and other errors in the handling of the measuring instruments additionally influence the accuracy of the measurement results and lead to a higher inaccuracy!

Measure in the middle of the MR, if possible!

FAQ – The importance of “photometric Zero”

Performing a zero (see manuals!)

LED meters, e.g. pHotoFlex® Series
Portable meters require a zero due to changing conditions, transportation and optics.

Filter photometers, e.g. photoLab® S12
In lab with stable conditions, slow drift and often stabilizing reference beam requires less zeroing.

Spectrophotometers, e.g. photoLab® 7000 Series
Zero/base line is required for many functions of spectral tasks, concentration mode is similar to filter photometers with reference beam.

Influence of meter drift:  Zeroing
Package leaflet of many tests show sensitivity by correlating absorbance A (E=A) to mg/l.

Influence can be seen directly:
For COD test 14560, 4-40 mg/l COD, an absorbance of 10 mE means 0.4 mg/l COD. 10 mE drift without zeroing means 0.4 mg/l or 10% evitable miss-reading in the low end!

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Spektralphotometer in der Routineanalytik

Digital and mobile photometric measurement on the road?

No problem - our experts explain to you which two essential factors are important. Learn more about the suitability or the correct handling of the devices and sensors as well as the reagents.

Photometry tips and tricks for practice

On 36 pages, you will find in the practical manual for photometry, in addition to many other tips and experience values of our experts for practice, for example, the 10 most important questions and answers about plausible measured values in photometry.

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