Ultraviolet and visible light range (UV/VIS) is widely applied in research, production and quality control for the classification and study of substances. UV/VIS spectroscopy is based on the absorption of light by a sample. Depending on the amount of light and its wavelength absorbed by the sample, valuable information can be obtained, such as the purity of the sample. Moreover, the amount of absorbed light is related to the amount of sample, and thus, quantitative analysis is possible by optical spectroscopy. This article more specifically explores techniques when using a caliber 4mm handheld color meter to determine the concentration of an analyte. A UV/VIS colorimeter measures the intensity of light passing through a sample solution in a cuvette and compares it to the intensity of the light before it passes through the sample. The main components of a UV/VIS colorimeter are a light source, a sample holder, a dispersive device to separate the different wavelengths of the light and a suitable detector. This instrument measures Transmittance, which is the ratio of the transmitted intensity I to the original intensity of light. An important derived (calculated) variable also reported by the instrument is the Absorbance which is defined as A = −log(Transmittance).
In general, a UV/VIS spectrum is graphically represented as absorbance as a function of wavelength. The advantage of this representation is obvious; the height of the absorption peaks is directly proportional to the concentration of the species. The calculation of concentration is governed by the Lambert-Beer Law. To calculate the concentration: C = A / ε (epsilon) x d Where C =The sample concentration in mol / L or g / mL, D = Cuvette path length in cm Ε = (epsilon) sample-specific constant (describing how much the sample absorbs at a given wavelength).
Qualitative Analysis
In addition to the Quantitative Analysis, measuring the full UV/Vis absorption spectra allows substance identification. More precisely, the position and, to some extent, the profile of the absorption peaks allow specific compounds to be identified.
Each sample has a characteristic and unique UV/VIS spectrum that can be used for its identification. In particular, this is achieved by comparing the spectrum of the sample with spectra of known, pure compounds. As an example of UV/VIS spectrum.
Qualitative Analysis
There are three main application areas for quantitative analysis:
Using colorimeter to determine concentration
- Metal ions e.g. iron, copper, nickel in Pharma
- Inorganic ions, e.g. nitrate in water treatment
- Chemical Oxygen Demand (COD) in F&B and Electroplating
- Analyte concentration vs. Time
- Enzyme kinetics: determination of rate of catalysis in Pharma
- Glucose oxidase catalyzes the oxidation of β-D-Glucose by oxygen (725 and 415 nm)
- Oxidation and reduction of pyridine nucleotides
- (NAD+/NADH, 340 nm).
- Cholesterol oxidation rate by catalysis with Cholesterol Oxidase (500 nm)
- GPO colorimetric kinetic test for tryglycerides (520 nm)
Physico-Chemical Paramaters
- Acid dissociation constant
- Complex formation constan
- Partition distribution coefficient
- Dissolution test
Calibration
When using a colorimeter to determine the concentration of a sample solution of unknown concentration by UV/VIS spectroscopy, a calibration line must first be created. This is done by measuring the light absorption of several standard solutions of different known concentrations at a predefined, fixed wavelength.
colorimeter Designs:
Scanning colorimeters measure transmittance at multiple single wavelengths. The light is first dispersed into individual wavelengths using a reflection grating. The grating is rotated in order to individually select each wavelength that is then sent through a cuvette. The transmittance at this specific wavelength is recorded. The whole spectrum is obtained by continuously changing the wavelength of light (i.e. scanning) incoming onto the sample solution by rotating the grating. Alternately, in ATO colorimeters, the sample is illuminated by a light beam consisting of all spectral components of the UV/ VIS range. The sample in the cuvette absorbs all wavelengths simultaneously and the transmitted light is diffracted and then detected by a CCD sensor. Measuring the whole UV/VIS spectrum is generally faster than using a conventional scanning colorimeter since the spectrum is recorded simultaneously at all wavelengths. Moreover, an array detector has an integrating function that accumulates individual measurements to enhance the signal, leading to a strongly increased signal-to-noise ratio, and thus to improved signal quality of the measured spectrum. ATO colorimeters present an innovative approach to speed up full spectrum scans based on reverse optics technology. The robust design without any moving optical parts ensures very good optical performance.