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When light falls on a substance, a part of the light that interacts with a substance is absorbed, and the remaining light is either reflected or transmitted through the substance. Colorimetric is used for the detection of dyes, food colourant in which the wavelength of light that different coloured dyes absorb varies. The majority of dyes are conjugated substances with alternate double and single bonds, and they generally absorb light in the visible spectrum. It is used for the food colourant studies due to the long history and widespread use of dyes in retail goods, the spectroscopic identification of food colourings appears to be a viable methodology.
Department of Pharmaceutical Chemistry, Sri Ramachandra Faculty of Pharmacy, SRIHER (DU), Chennai, India
Nithya Sermugapandian
Department of Pharmacology, Sri Ramachandra Faculty of Pharmacy, SRIHER (DU), Chennai, India
Sumithra Mohan
Department of Pharmacology, SRM College of Pharmacy, SRMIST, Kattankulathur, India
Manikandan Krishna
Department of Pharmaceutical Analysis, SRM College of Pharmacy, SRMIST, Kattankulathur, India
*Address all correspondence to: soniapharm68@gmail.com
1. Introduction
When light falls on a substance, a portion of the light that interacts with a substance is absorbed, and the remaining light is either reflected or transmitted through the substance. Visible light is reflected by objects that we perceive as having colour. The wavelength of light that is reflected determines the colour of the material that we can perceive [1]. A material that seems blue to us will reflect light in the visual spectrum’s blue region (430–480 nm). The same substance absorbs light that is complementary to the light that is reflected, according to the colour wheel. As a result, the blue material absorbs light in the visible spectrum’s orange band (590–630 nm). Because not all chemicals absorb in the visible area, they appear colourless to the human eye.
Colour measurements are a way to quantify the values of the hues that humans perceive. Measurements of colour are affected by lighting, an object’s spectral properties, and the human eye’s spectral sensitivity properties. A colour value can be estimated if the spectrum reflection of the item is known since the spectral distribution of the illumination and the spectral sensitivity characteristics (colour-matching function) of the eye are established in the JIS standards. (The computation can use spectral transmittance if the light can flow through the item. However, the explanations that follow employ spectral reflectance.) To further clarify, the JIS standard calculates the spectral distribution of the illumination and the colour-matching function under a number of different situations in Figure 1.
A UV–Visible (UV–Vis) spectrophotometer is used to experimentally test light absorption. This device makes use of a light source a particular monochromator turns into a range of wavelengths that control over a sample and towards a detector on the spare end. If the organic chemical is a solid, a solvent is essential since the samples must be in a liquid. The container warned to carry this solution is a cuvette. The cuvette may be formed of quartz crystal, glass, or plastic and have a certain path length depending on the sample. The distance that light must travel over the sample is measured in terms of path length. A sample blank of only the solvent is needed because it will also absorb light. In order to display just the absorbance induced by the sample, the instrument can remove the background spectrum of the solvent from the absorbance spectrum of the sample component. The percentage of the primitive light that passes through the sample is known as the transmittance, or T. The irradiance, or efficiency per second per unit area, of the light beam, just before it hit the sample is P0 in this case. P denotes the intensity of the light beam that hits the detector [3]. Due to the sample’s absorption of some light, P is normally lower than P0.
Absorbance is a negative log of transmittance.
A=logP0P=−logTE2
The value of absorbance ranges from 0 (no absorption) to 2 (99% absorption). When no light is absorbed, P0 equals P and transmittance equals one. As a result, absorption is zero. If 90% of the light is absorbed, 10% is transmitted, and T=0.1. This yields an absorbance of one. If 99% of the light is absorbed, then 1% of the light is transmitted T=0.01, and absorbance equals 2.
The resulting spectrum is a plot of absorbance versus wavelength. This range for a UV–Vis spectrophotometer is between 200 and 800 nm.
The transmittance and absorbance of a certain substance are proportional to its concentration in solution. The Beer–Lambert law describes this relationship.
A=εclE3
The sample absorbance is equal to the product of the chemical concentration, route length, and molar attenuation coefficient. This coefficient is specific to each molecule and varies with wavelength. However, if the wavelength is maintained fixed, the molar attenuation coefficient remains constant regardless of concentration changes [3]. The wavelength with the maximum absorbance of the sample, known as max, will likewise have the highest molar attenuation coefficient (Table 1).
The wavelength of light that different coloured dyes absorb varies. The majority of dyes are conjugated substances with alternate double and single bonds, and they generally absorb light in the visible spectrum.
The conjugated portion of the dye molecule might be lengthy, indicating a high degree of conjugation with numerous alternating double and single bonds, or extremely short, indicating a low degree of conjugation with few alternating double and single bonds. It’s not necessary for these alternating double bonds to merely be between two carbons. The carbonyl groups and the double bonds between carbon and oxygen can be examples of these conjugated bonds. The amount of conjugation affects how much light the chemical can absorb at each wavelength. For instance, substances that have higher levels of conjugation absorb light at longer wavelengths than substances that have lower levels of conjugation.
Delocalized electrons inhabit molecular orbitals according to molecular orbital theory. The highest energy orbital with an electron is known as the highest occupied molecular orbital or HOMO. The lowest energy orbital without an electron is known as a LUMO or the slightest unoccupied molecular orbital. The energy difference between the HOMO and LUMO is often quite big in molecules with little to no conjugation [4]. However, the efficiency difference between the HOMO and LUMO is lower for conjugated molecules.
The molecule must absorb light with energy equivalent to the initiative gap between the two orbitals in order to excite a transition of an electron from one energy level to another, or from the HOMO to the LUMO. For this reason, higher energy light—such as UV light—is necessary to excite an electron in molecules with a wide energy gap. However, because dyes have a narrower energy gap, an electron must be excited by lower-energy light, like visible light.
ΔEHOMO−LUMO=hcλE4
For this reason, higher energy light—such as UV light—is necessary to excite an electron in molecules with a wide energy gap. However, because dyes have a narrower energy gap, an electron must be excited by lower-energy light, like visible light. Remember that the relationship between light’s energy and wavelength is inverse [5]. Therefore, light with more energy has shorter wavelengths than light with lower energy, which has longer wavelengths.
5.1 Natural dyes
The term “natural dyes” refers to colourants (including dyes and pigments) that are derived from plant or animal matter without the use of chemicals. Although there are some known vat, solvent, pigment, and acid types, they are primarily mordant dyes. Natural dyes are used to colour clothing, food, medicine, and cosmetics. Additionally, very little amounts of dyes are used to colour paper, leather, shoe polish, wood, cane, candles, etc.
In the past, only natural sources were used to create dyes. But because dyes obtained from comparable plants or other natural sources are affected and subjected to the whims of climate, soil, cultivation techniques, etc., natural dyes suffer from some inherent drawbacks of standardised application and the standardisation of the dye itself. In order for natural dyes to be genuinely commercialised and to compete favourably with synthetic dyes, standardisation techniques are crucial and play a huge role. Indian culture has a long history of employing natural dyes. Traditional skilled craftspeople in the nation have been manufacturing natural dyed textiles for centuries in numerous villages. When used alone, natural dyes have several restrictions on their colour fastness and brilliance. However, they provide vibrant and quick hues when used with metallic mordants. Although using metallic mordants is not always environmentally benign, the pollution issues they cause are of very low order and are easily resolved. Therefore, “Mild Chemistry” can be used to obtain practically identical results in place of environmentally harmful technology for the production of colours.
This chapter discusses the self-association of certain dyes and divides the phenomenon into two categories: H- and J aggregates. The aggregation processes of the following dyes, which have been the subject of spectroscopic studies: rhodamine B, rhodamine 6G, Neutral Red, Nile Blue A, Safranine T, Thionine, Methylene blue, Methylene green, thiazole orange, and TO-3, were discussed. One of the characteristics of dyes in solution is their capacity for self-association, which many colours exhibit. Aggregation is a phenomena that changes the characteristics of dyes as well as their coloristic and photophysical properties. Due to high intermolecular attractive forces between the molecules, the aggregation phenomena in solution or at the solid–liquid interface is a frequently encountered event in dye chemistry. The ionic dyes have a well-known propensity to congregate in diluted solutions, resulting in the formation of dimers and occasionally even higher order aggregates. In this scenario, factors including dye concentration, dye structure, ionic strengths, temperature, and the presence of organic solvents all have a significant impact on the molecular nature of the dye. Although the structure and behaviour of dyes are highly idiosyncratic, there are some fundamental guidelines for aggregation that have been established. It may rise with an increase in dye concentration or ionic strength; fall with an increase in temperature or the addition of organic solvents; fall with the addition of ionic solubilising groups to the dye structure; fall with the addition of long alkyl chains; fall with an increase in aggregation due to higher hydrophobic interaction in solution.
In both fundamental science and technological applications including optical memory, organic solar cells, and organic light emitting diodes, dye aggregates have played a significant role [6, 7, 8]. Among all synthetic dyes, xanthene dyes are among the earliest and most widely used. They were used for food colouring and clothing, among other things [9]. The unique photophysics characteristics of these kinds of molecules are the reason for their numerous and expanding applications in physics and chemistry. Because of their high time-zero anisotropy, photostability, and red emission, they are used as fluorescent protein probes in detecting protein orientation in biology and as probes in biochemistry to monitor membrane fusion, determine the distance between aggregations, and detect protein orientation.
It was discovered in the 1930s that a group of dyes known as cyanines have a high propensity to aggregate in polar solvents, and this group has since been the focus of numerous studies that primarily analyse the effects of concentration on absorption and emission spectra. The dimerization process, which takes place in a specific concentration range and occurs before the development of more complex aggregates, was given the most attention by the bulk of the authors. Spectral sensitization in photography, size-enhanced nonlinear optical polarizabilities, sensitization of semi-conductor materials, etc. are just a few technologically interesting processes that include organised assemblies of cyanine dyes as molecular functional units. In these situations, aggregates develop at the air-water interface on solid surfaces or in monomolecular layers, where the packing of the chromophores is aided by particular dye-substrate interactions.
Measure the object’s spectral reflectance before using a UV–VIS spectrophotometer to measure colour. The colour is expressed as a numeric number using calculations based on the spectrum distribution of the illumination, the object’s measured spectral reflectance, and the colour-matching function. To acquire colour measurement values when the spectral reflectance spectrum is measured, the colour measurement programme stores illumination spectral distributions and colour-matching function values [10]. The XYZ tristimulus values serve as the foundation for measuring colour. “Methods of Colour Measurement -- Reflecting and Transmitting Objects” determines the XYZ tristimulus values.
A colour specification system is a way to define colours as numerical values, whereas a colour difference is a way to express how different hues are from one another [11]. The Uniform Colour Space (UCS), which is more closely aligned with the human visual system, is used in calculations to describe colour difference values mathematically. A common UCS colour specification system is the L*a*b* colour space. A* and b* stand for hue and saturation, whereas L* stands for brightness. The technique of computation in the L*a*b* colour system is shown in JIS Z 8729, “Colour specification -- CIELAB and CIELUV colour spaces.” The L*a*b* value for each colour of the object (sample) is used to determine the colour difference. Using “Colour specification -- Colour differences of object colours,” the colour difference E*ab in the L*a*b* colour system is calculated.
To scrutinise the absorption properties of fluorescein, −carotene, and indigo dye using UV–Visible absorption spectroscopy, besides known as UV–Vis spectroscopy. A compound between a light source and a photodetector in order to perform UV–Vis spectroscopy. When light’s wavelengths match the energies required to excite a molecule’s electrons, it will absorb that light and scatter or transmit the rest.
The fluctuation in the number of photons of each wavelength that reach the detector is represented by an absorption spectrum. There are fewer photons of that wavelength detected when the absorbance is higher. A spectrum of the true solvent, whichever the device determines then subtracts from the spectrum of the dye solving to only display the dye’s absorbance information.
The colours in a fume hood use -carotene and indigo solutions like hexane and dimethylformamide, respectively. To get solvent on thin nitrile gloves, such as DMF or hexane, change them immediately [12].
Typically, a cuvette will have two see-through sides and two artificial, opaque sides. The cuvette’s lucent sides can easily get dirty, thus it’s best to always hold it by the rough sides.
Examine a fluorescein-water mixture for flaws. About three-quarters of your cuvette should be filled with deionised water to create your solvent blank.
Use a laboratory wipe to clean the cuvette’s see-through sides. If you notice any air bubbles in your cuvette, you can get rid of them by giving it a light tap.
Finally, open the spectrometer’s sample cavity and remove the fragile cuvette. Dust and fingerprints can skew results by either absorbing or reflecting light.
Place the cuvette into the representative holder with the lucent side facing the light. Put a heavy lock on the isolation room door. Scan your cuvette as the solvent blank using the spectrometer’s software as directed. The absorbance range should be from 200 to 800 nm.
When the scan is finished, you should remove any large drops of water by emptying the cuvette and the associated compressed air.
Then, take it to the common hood and fill the cuvette three-quarters of the way with the 3 mM fluorescein solution using a clean Pasteur pipette.
Before bringing the cuvette to the spectrometer, make sure there are no air bubbles in it and pop them if there are. Before inserting the cuvette into the sample chamber, make sure the see-through sides are spotless. The sample cuvette should be scanned.
When the scan is complete, figure out which wavelengths fluorescein was able to absorb. Maximum absorbance occurs at a specific wavelength, denoted by “max.”
Write down the absorbance peaks’ wavelengths and the full absorption spectrum, then store this information.
Then, grab a clean cuvette and fill it three-quarters of the way with hexane to use as a blank solvent.
The cuvette should be placed in the spectrometer after any air bubbles have been removed and the transparent sides have been cleaned. Perform a solvent blank scan using the cuvette and an absorbance scanner set to 200–800 nm.
Remove any remaining fluorescein from your cuvette by emptying it into the aqueous trash and rinsing it thoroughly with water.
Next, you’ll need a hexane solvent blank to analyse beta-carotene. After removing any trace of water from the cuvette, fill it three quarters of the way with pure hexane.
Remove any trapped air, wipe off. Place the cuvette in the light path of the spectrometer. As if the cuvette were a solvent blank, perform a scan with the absorbance set at 200–800 nm.
When the scan is finished, discard the cuvette and blow it with compressed air to eliminate any residual microorganisms.
Fill the cuvette 3/4 of the way with the 3 mM -carotene solution and return it to the spectrometer using a good pipette.
Remove the cuvette’s lucent sides, insert it into the spectrometer, and perform a scan. Notate in your lab book how long in wavelength each peak was.
Now, let us have a look at the indigo dye. Since the indigo solution is in DMF, you’ll need to dispose of the used cuvette in the organics bin and flush any residual beta-carotene or hexane from it with pure DMF [13]. Then, you’ll fill the cuvette up to three-quarters of its capacity with pure DMF, replace it in the spectrophotometer, and make sure that your scan settings have not changed. Once the solvent blank scan is complete, you can toss the cuvette’s contents into the compost bin and blow dry the remaining moisture.
Pipette 3/4 full of the given 3 mM indigo dye solution into the cuvette, and then scan it as your sample. Document your absorbance results and store them for further use.
Finally, after rinsing it out with acetone, discard the cuvette’s contents into the compost or garbage.
8.1 Measurement using UV visible spectroscopy
Fluorescein, beta-carotene, and indigo dye are studied using absorption spectroscopy, also known as UV–Vis absorption spectroscopy. In UV–Vis spectroscopy, a solution is placed between a light source and a photodetector. The wavelengths of light required to activate their electrons will be absorbed, and the remaining light will be diffused or transmitted [14].
An absorption spectrum depicts the fluctuation in the number of photons of each wavelength that reach the detector.
When the absorbance is high, fewer photons of that wavelength are picked up by the detector. To get the dye absorbance information, you need a spectrum of the pure solvent, which the instrument will subtract from the spectrum of the dye solution. An example of what is called a solvent blank [15]. The -carotene and indigo solutions are prepared in a fume hood using hexane and dimethylformamide, respectively.
Typically, cuvettes have two clear sides and two rough or opaque sides. To keep the clear sides clean, always hold the cuvette by the textured sides.
Examine a fluorescein solution in water. Fill your cuvette 3/4 full with deionised water to create your solvent blank.
Clean the clear sides of the cuvette using a lab wipe. Inspect the cuvette for air bubbles and tap it slightly to get rid of them.
After that, take the cuvette to the spectrometer. Open the spectrometer’s sample chamber, then wash your cuvette once again. Smudges and dust can reflect or absorb light, which might result in inaccurate statistics. Now, position the cuvette in the sample holder so that its transparent sides are facing the light source. Completely shut the sample room door. Set up a scan of absorbance between 200 and 800 nm in your spectrometer’s software, using your cuvette as the solvent blank.
After the scan is finished, drain the cuvette and use compressed air to blast large water drops away.
After that, take it to the common hood and add the provided 3 M to the cuvette until it is 3/4 filled using Pasteur pipette.
Before taking the cuvette to the spectrometer, detect air bubbles and get rid of them if necessary. Recall to before placing the cuvette into the sample chamber, clean the cuvette’s transparent sides one last time. The next step is to scan the cuvette.
After the scan is finished, note the wavelengths that fluorescein absorbed. The frequency of the wavelength with the greatest absorption is referred to as max.
In your lab notebook record and save the data after noting the wavelengths of the absorbance peaks and the total absorption range.
Empty the cuvette into the watery trash and rinse it to eliminate any remaining fluorescein.
To analyse - carotene next, you’ll require a blank for hexane solvent. Before adding pure hexane to the cuvette, rinse it several times with hexane to get rid of any water that may still be present.
Clear the cuvette of any air bubbles and place it inside the spectrometer. Ensure that the scan is adjusted to absorbance between 200 and 800 nm. The cuvette will then be scanned as a solvent blank at nm and then scan the cuvette as a solvent blank.
After the scan is completed, Dump the cuvette into the organic waste after the scan is finished, then pressurise the air to dry it.
Using a clean pipette, fill the cuvette 3/4 of the way with the supplied 3 M -carotene solution before returning to the spectrometer.
Clean the clear sides of the cuvette, place it in the spectrometer, and use it as your sample to be scanned. Write the wavelengths of the peak in your lab notebook and save the data.
Examine the indigo dye right now. Because the indigo solution is in DMF, place your cuvette in the organic trash. Then, thoroughly rinse away any remaining hexane and -carotene with pure DMF. Fill the cuvette 3/4 full with pure DMF, place it in the spectrometer, and repeat the previous scan conditions. After the solvent blank scan, immerse the cuvette in organic waste and dry it with compressed air. The provided 3 M indigo dye solution into the cuvette until it is 3/4 filled, then scan it as your sample.
Finally, rinse your cuvette with acetone after emptying it into the organic garbage.
8.2 Applications
8.2.1 Applications of UV spectroscopy: Identification of dyes
A “colour wheel” is created by arranging several hues in a circle. One potential solution to this is depicted in the diagram. Complementary colours are those that are settled opposite one another on the colour wheel [16]. Colours that complement one other include blue and yellow, red and cyan, green and magenta, and red and indigo. White light is created by combining two light hues that are complementary to one another in Figure 2.
Figure 2.
Complementary colours in colour wheel.
8.2.2 Natural dyes extraction
Anthocyanin is a natural pigment found in purple cabbage (Brassica oleracea var. capitata f. rubra). This dye is made up of hydroxyl and carbonyl groups. Spinach (Spinaciaoleracea) includes natural chlorophyll pigments called Chlorophyll a and Chlorophyll b. Anthocyanin and chlorophyll pigments are extracted from purple cabbage and spinach by cutting them into small pieces and mashing them into a paste with a mortar [17]. The samples are then placed separately in an ultrasonic cleaner for 15 minutes at a frequency of 37 Hz in the ‘degas’ mode at 30°C. The coloration of the samples is separated by centrifugal force at 2500 rpm for 30 minutes. The pH of the produced anthocyanin and chlorophyll dyes in ethanolic solution is 7.03 and 7.2, respectively [18]. Now, in a 1:1 ratio, combine the extraction of purple cabbage juice and spinach juice, and then add Ethanol to the mixture. After 10 minutes, sieve the mixture. The pH of the produced dye is 7.15.
8.2.3 Spectral studies of food colourants
Due to the long history and widespread use of dyes in retail goods, the spectroscopic identification of food colorings appears to be a viable methodology. The spectrum information for frequently used dyes that is necessary as a result is presented for instructional purposes. The analysis of food colorings in four distinct lemonades and chocolate beans, both qualitatively and quantitatively, has been used to educate students to key analytical methods such sample preparation and the elimination of confounding variables. In the cases of tartrazine and curcumin, these analyses also show the method’s limitations in the visible light spectrum. Quantitative studies can study typical concentration calculations by using Lambert–Beer–Bouguer’s Law in various variations.
Most people begin eating with their eyes. Consumers today expect food to look as appetising as possible, which can be achieved by adding extra or artificial colouring. Plant extracts or saffron were utilised as natural dyes [17]. Prior to industrialisation, this form of natural food colouring was out of reach for the average person. Under favourable economic circumstances, it was necessary to recolor food with dyes due to the somewhat unpleasant colour shift that occurred during the processing of food.
Because of their affordable production costs, greater colouring capabilities, and practical application in products, these new artificial food dyes have replaced both dangerous inorganic salts and natural colourants. Self-service supermarkets, mass production, and transparent packaging have all increased, making it necessary to standardise the Food colour schemes were established. Contrary to what people might think, many of these are made synthetically. We investigated carotenes, chlorophylls, and curcumin in diet in this work.
It is necessary to determine attenuation coefficients for systems with practical relevance - like lemonade or candies - when teaching quantitative analysis by spectroscopy. For support, extensive experimental data pertaining to attenuation coefficients of food dyes are provided as supplementary materials. Below are the results of spectroscopic analysis of dyes such as Allura red, Amaranth, Apocarotenal, Azorubine, Beta carotene, Brilliant blue, Caspasnthin, Curcumin, Carmine, Erythrosine, Green S, Indigo carmine, and Lutein.
8.2.4 Calibration of dyes - β-carotene calibration curve
UV–Vis spectroscopy was used by comparing the absorbances of five different concentrations of -carotene solutions, it was possible to link absorbance intensity and concentration for this compound. Make an absorbance vs. -carotene concentration calibration curve, and then determine the equation that describes the connection [18].
Hexane should be used as your solvent blank in a clean cuvette that is 3/4 full.
Clear the cuvette’s clear sides of any air bubbles, then place it in the spectrometer. Set up an absorbance scan with the cuvette serving as the solvent blank between 200 and 800 nm.
Dump the cuvette into the organic waste after the scan is finished, then pressurise the air to dry it.
Pour the 1.9 M -carotene solution, which has the lowest concentration, into the cuvette using a clean pipette.
Fill the cuvette 3/4 full with the 1.9 M -carotene solution, which has the lowest concentration, using a clean pipette.
Before placing the cuvette into the spectrometer and scanning it as a sample, clean the sides of the cuvette.
Display the absorbance strength of the peak at 450 nm after the scan is finished. In your lab notebook, write the absorbance for the 1.9 M solution.
Submerge the cuvette in the organic waste, followed by a hexane rinse and air drying.
Using a clean pipette, add the 3.7 M -carotene solution to the cuvette until it is 3/4 full. Before placing the cuvette into the spectrometer and scanning it as another sample, clean its sides.
After the scan is finished, the peak’s absorbance at 450 nm is calculated. Rep for the remaining choices in order of decreasing concentration. Save your work after you are done.
Finally, immerse the cuvette in organic waste and clean it with acetone.
The second half uses the absorbances of five distinct solutions containing various concentrations of -carotene, UV–Vis spectroscopy was used to establish a relationship between absorbance intensity and concentration. The equation expressing that relationship is derived from a calibration curve of absorbance versus -carotene concentration in Table 2.
Create a table with columns for -carotene concentration and absorbance at 450 nm in your lab notebook to start.
After the scan is complete, empty the cuvette into the organic garbage and blow it dry.
Use a clean pipette to dispense the 1.9 M -carotene solution, the lowest concentration, into the cuvette to a depth of 3/4 filled.
Concentration (μM)
Absorbance at λmax (450 nm)
1
1.9
2
3.7
3
7.5
4
11
5
15
Table 2.
Absorbance of β-carotene.
Trim the cuvette’s sides, place it in the spectrometer, and scan the sample there.
Display the peak at 450 nm’s absorbance intensity after the scan is complete. This should be noted as the absorbance for the 1.9 M solution in your lab notebook.
Next, pour the contents of the cuvette into the organic waste, wash it with hexane, and then seal it with air pressure.
Use a clean pipette to dispense the 3.7 M -carotene solution into the cuvette until it is 3/4 full. Clean the cuvette’s sides before placing it in the spectrometer and scanning it like a different sample.
After the scan is complete, note the peak’s 450 nm absorbance. Working from lowest to greatest concentration, repeat this procedure for the remaining solutions. When finished, save your data.
Afterward, rinse the cuvette with acetone after emptying it into the organic garbage.
8.2.4.1 Results
Blue and purple light is also absorbed by -carotene. In hexane, beta-carotene has a maximum absorbance of 450 nm and another significant peak at 478 nm. -carotene appears orange in part because of the intense absorption of purple light.
DMF’s indigo absorbs red, orange, yellow, and UV light, with a clear peak at 611 nm. In order to get its distinctive colour, indigo dye reflects mainly blue and purple light.
The concentration calibration curve for -carotene. Plot the congregations of the liquids versus the -carotene absorbance values at 450 nm from the second part of the experiment. Execute a trend line next, and then locate the linear equation that best describes the data. In this equation, y represents absorbance, x represents concentration, and the slope is uniform due to the conforming molar attenuation coefficient and the slope factor. Path length, in accordance with the Beer–Lambert law. Fill in the -carotene absorbance at 450 nm from the first fraction of the lab and rearrange it to solve for concentration. Nearly 3 M/L of concentration must have been computed. Complementary Colours.
8.2.4.2 Interpretation for dyes
The wavelength at which fluorescein absorbs blue and purple light in water is 490 nm. Red light is not absorbed by it; it only takes a little amount of yellow and green light. Fluorescein solids are red, while fluorescein liquids are often yellow to green. Blue and purple light are also absorbed by -carotene [19]. In hexane, the greatest absorbance of -carotene is 450 nm, with another big peak at 478 nm. Because of due to a high amount of purple light absorption, −carotene appears orange. With a distinct peak at 611 nm, indigo absorbs UV light as well as red, orange, and yellow light. Dye indigo as a result, reflects mostly blue and purple light, giving it its distinctive colour.
For the -carotene concentration calibration curve, Plot the -carotene absorbance values at 450 nm vs. the solution concentrations [19].
Next, use a trend line to identify the linear equation that best matches the data. In this equation, y is absorbance, x is concentration, and the slope is the product of the applicable molar attenuation coefficient and the pathlength, according to the Beer Lambert law., according to the Beer–Lambert law.
8.2.5 Spectroscopic analysis of methylene blue
In the textile industry, Methylene Blue (MB) is widely used to dye wool, cotton, and silk [6]. It can also be used to dye specific body tissues and fluids before or during surgery. The visible light absorption spectra analysis confirmed that methylene blue has high order methylene blue aggregate without any preconception about the aggregation order.
Since Colorimetric is widely used not only in the pharmaceuticals but also used as food colourant, dyes and natural dyes estimation.
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Written By
Sonia Karuppaiah, Nithya Sermugapandian, Sumithra Mohan and Manikandan Krishna
Submitted: 03 June 2023Reviewed: 24 July 2023Published: 30 October 2023
Open access peer-reviewed chapter
