Diego A. Sampietro*, Melina A. Sgariglia, Jose R. Soberón, Emma N. Quiroga and Marta A. Vattuone
Colorimetry, the measurement of the absorption of visible light, allows the first qualitative and quantitative estimation of the phytochemicals present in a plant, soil or water sample. Colorimetric methods allow the assessment of an unknown colour in reference to known colours (Thomas, 1996). In practice, they provide qualitative or semiquantitative estimations rather than quantitative determinations. Early colorimetric techniques consisted of the visual comparison between the colour in a sample and that of several permanent colour standards, which could be the same substance at known concentrations. The use of the human eye as a colour detector, however, includes a subjective perception reducing measurement reproducibility. The instrumental progress in visible spectroscopic techniques allows the development of reliable and sensitive instruments. Colorimeters and spectrophotometers, photoelectrically measure the amount of coloured light absorbed by a coloured sample with respect to a "blank" or colourless sample with reference. Modern spectrophotometers split the incident light into different wavelengths and measure the intensity of that radiation. Colorimetric measurements often involve the reaction of colourless substances under analysis with specific reagents (Hagerman, 2002). This allows the formation of coloured complexes with absorbance maxima at wavelengths comprised in the visible light. In this chapter, general reactions of colour
Authors' address: Instituto de Estudios Vegetales "Dr. A. R. Sampietro", Facultad de Bioquímica, Química y Farmacia, Universidad Nacional de Tucumán, España 2903, 4000, San Miguel de Tucumán, Tucumán, Argentina.
*Corresponding author: E-mail: [email protected]
development are provided for the qualitative and quantitative determination of natural products in a sample.
2. GENERAL COLORIMETRIC PRINCIPLES 2.1 Beer-Lambert Law
Electromagnetic radiation can be characterized as a wave with frequency (v) and wavelength (A). This wave has energy (E) proportional to its frequency and is given by the equation:
where, h is Plank's constant and q is light velocity. When a wave of radiation finds a substance and the energy is absorbed, a molecule of the substance is promoted to an excited state (Thomas, 1996). The absorption of visible light generally excites electrons of a molecule from a ground electronic state to an excited one.
The amount of light absorbed by a substance in a solution is quantitatively related to its concentration. This relationship is expressed by the Beer-Lambert Law (Fig. 1):
Logio (Iq/J) = -logio T = abc = A or Iq/I = 1/T = 10abc where, Iq is the intensity of incident light, I is the intensity of transmitted light, T is the transmittance, a is the absorptivity or extinction coefficient, b is the path length through an absorbing solution, c is the concentration of the absorbing substance, and A is the absorbance (or optical density in old literature). Absorptivity is constant for a given compound and is a function of the wavelength. When c is expressed in moles per L and b is expressed in centimeters, the constant is known as the molar absorptivity (e) and is expressed in L per mole per centimeter (L mol-1 cm-1).
Beer-Lambert Law expresses a linear relationship between the absorbance and the concentration of a given solution, if the length of the path and the
wavelength of light are kept constant. This enables determination of the concentration of a substance through the measure of the transmittance or absorbance.
Deviations from the linear relationship between concentration and absorbance often occur and can lead to errors in the application of Beer-Lambert Law (Fig. 2). High concentrations of substances in the solution, the use of radiant energy that is not monochromatic or conditions within the solution causing shifts in the chemical equilibrium, are common deviation sources (Brown, 1997). Linear relationship can be evaluated by building a calibration curve, where the absorbance of standard solutions is plotted as a function of their concentrations. If the absorbance of an unknown sample is then measured, the concentration of the absorbing component can be determined from this graph. In general, transmittance should be in the range of 10-90% (absorbance 1.0-0.1) to avoid high instrumental errors.
Figure 2 Deviation from Beer-Lambert Law
Colorimetric analysis is often performed using a spectrophotometer (Brown, 1997). The cheapest spectrophotometers are single beam instruments (Fig. 3A). They have a broadband lamp as the light source. A wavelength selector narrows the wavelength range to a typical band width of 20 nm. This light then passes through one compartment with a typical path length of 1 cm and the transmittance is measured by a photodetector. The instrument is calibrated to zero when a shutter is lowered to 0% transmittance (or infinite absorbance). Then, a blank (a reference solution) is placed in the cell and the instrument is adjusted to 0 absorbance (100% transmittance). A double beam instrument operates similarly, but the beam is split to pass through a reference cell containing the blank and the sample cell, simultaneously (Fig. 3B). A blank is kept in the reference cell for all standardization and sample measurements, as the instrument reports the difference in transmittance or absorbance measured from the two cells.
Most modern instruments allow computer interfaces for data-logging or automated operation. Also, flow-through cells may be used for continuous measurements. Fibre optic units with modern instruments are computerized and have sophisticated optical charged coupled device (CCD) arrays to monitor the emission intensity at many wavelengths simultaneously. Statistical correlations are used to generate highly selective methods and to reduce interference levels.
CCD detectors are becoming quite popular, as complete UV-Vis spectra can be obtained in seconds, in situ sensing is available and computer interfacing is enhanced by digital electronics.
Some natural products are coloured substances and can be directly analyzed through colorimetry. Natural colourless or weakly coloured compounds, however, must be converted to more coloured derivatives. The reaction for colour formation may include enzymatic conversion, chemical modification of a substance to produce coloured products or formation of complexes between a substance and a colour-forming reagent. Several colour forming reagents are commercially available for analysis of many substances.
I. Chromogenic reagents usually react with a mixture of compounds belonging to the (correspond to the) same chemical class or many related chemical classes. Colour reactions of these compounds are controlled by the different kinetics producing variations in colour development (Fig. 4) and reducing the accuracy of the colorimetric method for quantitative analysis. Standard compounds should be selected according to past knowledge of the main representative molecules that are known components in a sample. Construction of several calibration curves with different standard compounds help to validate the use of a colorimetric method for quantitative analysis.
II. A sample composition should be evaluated using several colorimetric methods (different chemical, reactions). Although water is the solvent of many colorimetric reactions, it interferes with some of them, hence is replaced by organic solvents. For this reason, the solvent used in the samples must be the same as that used to dissolve the standard compounds.
III. Colorimetric measures of a sample must be in the range of absorbances comprised in the calibration curve. Extreme absorbance values (too high or low) must be avoided. This ensures that the colorimetric measurements will be as per norms of Beer-Lambert Law. Little changes in reagents used in a colorimetric method can modify the slope of the calibration curve. Hence, calibration curves should be built each time a colorimetric method is applied for sample analysis.
IV. Sometimes components of the sample solution absorb at the wavelength used for colorimetric measurements or react with the chromogenic reagent increasing colour development. Partial isolation of the compounds of interest before colorimetric measurements aid in overcoming or reducing this kind of interference. Absorbance can also be read in the sample solution before and after colour development at the same wavelength or at the wavelength of maximum absorbance of interfering compounds. These values are further used to correct the absorbance lectures of the colorimetric method. General UV-Vis analysis can help to check if major interferences from other compounds are detected in the sample.
3. COLORIMETRIC METHODS 3.1 Phenolic Compounds
Experiment 1: Total Phenolic Compounds (Folin Ciocalteu Reagent)
The Folin Ciocalteu reagent is used to quantify concentrations of easily oxidizable compounds, such as phenols, by colour changes accompanying a redox reaction. The hydroxylated compounds reduce Cu2+ to Cu+ in an alkaline medium, which in turn reduces the phosphotungsten-phosphomolybdic acid of the Folin Ciocalteu reagent to form an intensely blue complex (Singleton et al., 1999). This complex is often quantified spectrophotometrically at 750 nm. A lithium sulfate salt is also a component of the reagent added to reduce the amounts of precipitate that can appear when high concentrations of reagent are used to increase the assay's reactivity. Folin Ciocalteu reagent has been widely used in phytochemistry and chemical ecology for preliminary analysis of phenolic compounds (Blum et al., 1991).
Assay tubes, Erlenmeyer flasks and pipettes; analytical balance with 0.01 mg sensitivity; spectrophotometer or filter photometer capable of measurement at 750 nm, equipped with a 1 cm path length microcuvette; vortex mixer.
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