Fundamentals and Applications of Colorimetric Analysis

## **Chapter 2** Fundamentals of Colorimetry

*Yagya Kumari Shrestha and Shree Krishna Shrestha*

#### **Abstract**

There are several kinds of analytical techniques following the principle of photometry in which colorimetry comes under absorption photometry. The colorimetry is commonly used analytical technique involved in quantitative estimation of color i.e. it is utilized to find out concentration of the colored substance in the sample solution e.g. water, biological samples at visible spectrum of light (380–780 nm). The colorimeter is an instrument in which this technique is used. It is also called absorptiometer. A substance must be colorful or should have property of forming chromogens through the addition of reagents which will absorb light according to their color intensity to be measured. The intensity of the color is in proportion to the concentration of colored compound. Most of the analytical techniques used in our clinical laboratory presently are based on this photometric principles in which absorbed, transmitted or emitted light are measured. When intensity at different wavelength on the whole range of electromagnetic spectrum is measured, it is called spectrophotometry. Smartphone accessories have been evolved to allow the simple, quick reproducible values of the molecules.

**Keywords:** Beer's and Lambert's law, chromogen, colorimetry, photometry, standard curve

#### **1. Introduction**

Colorimetry is a type of photometry which is basically considered as the techniques in which light is detected and also detects changes in its intensity. The root word "photo," means light. A photometer is a machine measuring the energy of electromagnetic wave in the range of infrared radiation to ultra-violet radiation, including the visible part of the electromagnetic spectrum (**Figure 1**). It changes light into electric current by using a photocell. Colorimetry is referred if the measured light is in the visible range of the electromagnetic radiation. In this method a beam of light from a light source is allowed to pass via sample holder containing the analyte in the solution, the intensity of light transmitted will be less than the light passing through sample in the cuvette. The absorbed light is in proportion to the concentration of the analyte. The color of the sample is an intrinsic characteristic of the solution or it can be evolved by the addition of suitable reagents. The absorption of the sample is compared to that of standards from which the concentration of test sample can be calculated [1–3].

Photometric methods can be divided into two catagories, visual and physical. The physical photometry is commonly practiced.

**Figure 1.** *Visible electromagnetic spectrum.*

There are various kinds of analytical techniques that are based upon photometric principles. In Spectrophotometry, atomic absorption, turbidometry etc. absorbed or transmitted light is measured while in flame emission photometry emitted light is measured.

#### **2. History**

Scientist James Clerk Maxwell from Scotland, invented colorbox in 1860 which was the primitive colorimeter featuring a prism that assisted in control of red, green, and blue light beams separately to match the color in the sample. Later Louis Jules Duboscq (1817–86), a French optical instrument maker, designed Duboscq colorimeter in 1854 (**Figure 2**). It was one of the early colorimeters to be one of the popular. It allowed for the coexistent color comparison of two liquids. Duboscq explained an improvised variety to the French Academy of Sciences in 1868. A Duboscq colorimeter calculates the concentration of a substance through a visual comparison of the color intensity of the compound against that of a standard solution. This method of identification was revolutionary when first introduced, but the colorimeter was replaced by the emergence of the more precise spectrophotometer in the early 1940s. Bausch & Lomb then remodeled the first Duboscq colorimeter (**Figure 3**).

Later photoelectric colorimetry became widely known in the mid-1930s, and in 1938 William Henry Summerson brought up colorimeter containing a photocell. There is fascinating background on use of photocells which were mainly used by Germans. After the world war was stopped, an English pupil Arthur Evans was acquainted to a

#### **Figure 2.**

*Duboscq colorimeter, by F. Hellige and Co., Freiburg, Germany, 1901–1914.*

leading medical researcher Dr. Rose of the Hammersmith Hospital, (UK) was looking for inventing a device which would enable him calculating the intensity of a color in a solution thereby calculating the concentration of an element in it. Colorimetry was already in use widely as analytical technique in medical, scientific and industrial laboratories which was performed by matching the solution under direct visual examination to known standards. Evan's EEL company started to produce first selenium photocell colorimeters (the model 2) commercially and were distributed all over the world. It gave both accurate and reliable results. Later the company was bought by Corning which was the pioneer company to produce pH sensitive glass for pH electrodes which are still popular in medical and industrial laboratories. However, new colorimeters do not use selenium photocells. This pioneer company is still gaining it's momentum in this field.

Later Klett Bio Colorimeters were made for clinical laboratory work. This Klett colorimeter includes standard color disks made of glass, which can be used in place of standard solutions in color comparisons [4–6].

#### **2.1 Terminologies**

**Light**: It is an electromagnetic radiation (EMR). It is composed of γ rays, X rays, ultra violet rays, visible rays, infrared rays, radio waves and micro waves. Of the components only those visible to eye is known as white light [7–10].

#### **Figure 3.**

*Bausch & Lomb Duboscq Colorimeter.*

**Photons:** Light is energy which exists in the form of bundles or as discrete packets of quanta called photons. The energy (E) of EMR is directly proportional to its frequency (u) and velocity (c) but inversely proportional to its wavelength (λ).

Thus, E α uBut, u ¼ c*=*λSo, E α c*=*λE ¼ hu ¼ h c*=*λ

where, h = plank's constant, c is the velocity of light in vacuum, 3 � <sup>10</sup><sup>8</sup> m/sec. EMR is produced by events at the molecular, atomic, or nuclear level. Some of the events which give rise to EMR are oscillations of nuclei and electrons in electrical or magnetic fields, molecular bending and vibrations, excitation of orbital electrons, ejection of an inner orbital electron and rearrangement of the other electrons, nuclear break-up etc. The radiation they emit will have different wavelengths as each of these events differ in terms of the energy involved (**Table 1**). Thus, a full spectrum of EMR will be produced. Their magnitude of electrical vector and magnetic vector are denoted by the symbol **E and H** respectively. A beam of light from a light source consists of many randomly oriented plane polarized components being propagated in the same direction (**Figure 4**).

**Polychromatic light:** A beam of waves from a light source consists of several wavelength and is known as polychromatic.

**Monochromatic light:** A beam of waves in which all the rays have the same wavelength is known as monochromatic.

**Spectrum**: When EMR allowed to pass through a prism, all the 7 rays are dispersed into an orderly pattern called spectrum and can be measured by a spectrometer. The components have different wavelength and frequency. It can be classified as emission spectrum & absorption spectrum. When radiation is directly examined by a spectrometer the resultant pattern of wavelength is called emission spectrum. If a radiation is examined after interacting with an absorbing medium it is called absorption spectrum. Emission and absorption spectrum may be either line or band type. Spectrum produced by atoms in gaseous state appears as lines and it is called line or atomic spectrum. Spectrum produced by molecules in solution or crystal appears as bands

#### **Figure 4.**

*Electromagnetic radiation wave of which is propagating transversally in space and time consists of the electric (E) and magnetic (H) field vectors directed perpendicular to each other.*


**Table 1.**

*Effect of absorption of electromagnetic radiation on the molecules.*

and it is referred as band or molecular spectrum. Emission spectrum are of line & band emission spectrum while Absorption spectrum are of line & band absorption spectrum.

**Spectroscopy:** It is a technique which is used for quantification, characterization and structural analysis of a substance based on interaction of light with that substance. Interaction of light with a substance may result in different phenomena and generate a specific analytical signal. The signal is detected by a detector and recorded by a digital recorder in the form of a spectrum. The spectrum can be correlated with the amount, structure, property and functional group of the sample.

#### a. Emission spectrum

It is characteristic of the emitting atom or molecule in excited state. Line emission spectrum appears as fine bright lines on a dark background. e.g. line emission spectrum of sodium atoms excited by flame. Band emission spectrum appears as broad bright bands. e.g. Band emission spectrum of Ca salts heated in a flame.

#### b. Absorption spectrum

It is characteristic of the emitting atom or molecule in ground state. Line absorption spectrum appears as dark lines on a bright background e.g. line absorption spectrum of sodium atoms irradiated by white light. Band absorption spectrum appears as broad dark bands e.g. Band absorption spectrum of iodine vapor irradiated with white light (**Figure 5**).

**Figure 5.** *EMR spectrum.*

**Frequency:** When radiation is propagated into a medium, it oscillates. Number of oscillations occurring per second is called as the frequency (**u**) of the radiation. It is expressed as Hertz (**Hz**).

**Wave number:** Number of waves propagating per centimeter of the medium is called its wave number (**σ**) or **ū**.

**Wavelength**: The distance between two successive peaks of a wave is known as wavelength (**λ**) (**Figure 5**). The wavelength of a light wave determine its color or visibility. For e.g. light wave with λ between 380 and 780 nm is visible to human eye and therefore called visible light or white light. Visible light has a range of colors which are abbreviated in mnemonic VIBGYOR.

**Velocity**: Distance traveled by the light wave per second is called its velocity (**c**). It is expressed as cm/sec.

#### **3. Parts of colorimeter**

The parts of the colorimetry are as follows [3, 7, 9–11]:


4.**Filters or monochromator:** Only light of required wavelength is passed through filter or monochromator while light of other wavelengths are absorbed.

**Filter:** It permits only monochromatic light to pass through while absorbs unwanted ones. It is usually made up of colored glass or dyed gelatin. This is a means of selecting light of narrow wavelength λ (50 nm or more). A green filter allows green color to pass through while rest are absorbed. The color of filter used is always complementary to color of solution.


**Monochromators:** These are better and more efficient than filters in converting a polychromatic light into monochromatic light. It is usually found in spectrophotometer. Monochromators are of various types:

I.Prism: It is either of refractive type prism or reflective type prism.

II.Grating: It may be diffraction grating or transmission grating.

5.**Cuvette/sample cell/sample holder:** They are specially designed tubes made up of optical glass (borosilicate or quartz) to hold the colored sample for measurement in colorimeter for accurate reading. There are square, rectangular or round shaped cuvettes. It is used to hold samples of fixed optical pathlength which is usually 1 cm. Its capacity is 3–4 ml or even lesser in case of micro cuvettes. The colored solution in cuvette absorbs the complementary color (**Table 2**). It is made of material that does not absorb light of wavelength range of the interest.


#### **Table 2.**

*Colors of solutions and their complementary filters.*

**Color wheel**: The Color of Light *absorbed* and *observed* passing through the compound are complementary. Complementary colors lie across the diameter on the color wheel and combine to form "white light", so the color of a compound seen by the eye is the complement of the color of light absorbed by a colored compound.


### **4. Principle of colorimetry**

**Principle of colorimetry**: When a beam of monochromatic light passes through a colored solution, the coloring substances absorbs a portion of the light & the rest is transmitted. Absorption of light is related to the color intensity. The color intensity will be proportional to the concentration of the chemical (analyte) responsible for producing the color [7–11].

**Absorbance (A):** When light passes through a medium, the ratio of log of intensity of incident light to the intensity of transmitted light is called as absorbance or optical density (OD). Previously it was called extinction.

**Transmittance (%T):** When light passes through a medium, the ratio of intensity of the transmitted light to the intensity of incident light is called as transmittance.

**Path length (l):** The internal cross length of the cuvette through which light passes is called as path length. Usually it is 1 cm.

**Absorbance maxima:** Wavelength of maximum absorbance is known as absorbance maxima. It is denoted by λmax. The wavelength at which a substance shows maximum absorbance is called absorption maximum or λmax (**Figure 6**).

The amount of light is absorbed or transmitted by a colored solution in accordance with 2 laws.


#### **Figure 6.**

*Showing relationship between absorbance, absorptivity, pathlength and concentration where, io = incident light, I = transmitted light, a = absorbance, c = concentration of absorbing compound, l = pathlength, ε = molar absorptivity.*

**Figure 7.** *Beer's law.*

> solution depends on pathlength of cuvette or thickness or depth of colored solution **Aαl** (**Figures 8** and **9**).

#### **Relationship between absorbance (A) and transmittance (T):** Relationship between (A) & (T) i.e.

Io ¼ A þ I A ¼ Io–I*:* By definition, A ¼ log Io*=*I*:* A ¼ � log I*=*Io*:* A ¼ � log T As T ð Þ ¼ I*=*Io *:* A ¼ log 1*=*T*:* A ¼ log 100*=*ð Þ %T to take into percentage multiply by 100*:* A ¼ log 100– log%T As log a ð Þ *=*b ¼ Log a–Log b *:* A ¼ 2 � log%T As log 100 ð Þ ¼ 2 *:*

**Combined Beer's – Lambert's law:** It is denoted as, amount of light transmitted through a colored solution decreases exponentially with increase in concentration of colored solution & increase in the pathlength of cuvette or thickness of the colored solution.

$$\mathbf{Aac} \text{ or } \mathbf{A} = \mathbf{k} \text{ c} \tag{1}$$

**Figure 8.** *Lambert's law.*

**Figure 9.**

*Relationship between absorbance (a) and transmittance (T).*

Where, k = linear absorption coefficient of the absorbing material.

$$\mathbf{A} \mathbf{d} \mathbf{l} = \mathbf{k}' \mathbf{l} \tag{2}$$

Where, kʹ = absorptivity constant.

Combinely from Beer-Lambert law both k & kʹ can be merged to form a single constant a & hence, combined equation can be expressed as **A** ¼ **acl** (**Figure 6**)

Where,

A = absorbance.

a = proportionality constant defined as absorptivity.

c = concentration of the absorbing compound sample in solution usually expressed in grams per liter.

l = pathlength of the sample (cm).

**A = acl**, this equation forms the basis of quantitative analysis by absorption photometry. **A** values have no units, hence, the units for **a** are the reciprocal of those for **l**, when **l** is **1 cm** and **c** is expressed in moles per liter the symbol **ε** (epsilon) is substituted for the constand **a**.

**A** ¼ **εcl**,

where,

**ε** = molar absorptivity/ molar absorption coefficient/molar extinction coefficient (Lmol�<sup>1</sup> cm�<sup>1</sup> ).

**Figure 10.** *Working principle of colorimetry.*

The value for molar absorptivity **ε** is constant for a given compound at a given wavelength under prescribed conditions of solvent, temperature, pH, etc. It is a measurement of how strongly a chemical substance absorbs light at a given wavelength. For any particular molecular type, absorptivity changes as wavelength of radiation changes.

Let,

AT = Absorbance of test AS = Absorbance of standard. CS = Concentration of standard CT = concentration of test. AT ¼ kxCT x L and AS ¼ kʹ x CS x L*:* AT*=*AS ¼ ð Þ kxCT x L *=*ð Þ kʹ x CS x L *:*

AT*=*AS ¼ CT*=*CSÞ*:*

Therefore,

**CT** ¼ **AT***=***AS**� **CS**

i.e. Concentration of test = Absorbance of the test X concentration of a standard/ Absorbance of the standard.

Therefore, above equation states that optical density (OD) is proportional to the sample concentration if the pathlength is constant. It follows then, that if the OD of a standard solution is known, then an unknown sample concentration can be calculated from its OD by applying the above formula. The standard chosen should have an optical density nearer to the OD of the unknown. The most accurate way of measuring the concentration in the unknown sample is through preparation of a calibrated curve or standard graph from a number of standards (**Figure 10**).

#### **4.1 Preparation of standard graph and evaluation of unknown**

A calibration curve is prepared by plotting known concentrations of a given substance against absorbance at a particular wavelength (usually the λmax). The relationship between absorbance and concentration is linear if the Beer's law is obeyed (**Figure 11**).

y ¼ mx*:*

Where, y = A, x = concentration and m = ab.

Unknown concentration of a substance is measured by comparing the absorbance of unknown to the absorbance of known concentrations of the same substance measured under exactly identical conditions (same pathlength l usually 1 cm, same wavelength or the same band of wavelength, same temperature, same solvent and same instrument etc).

A plot of absorbance values against known concentrations of a substance is known as a standard graph or a calibration curve and standard curve should always be linear. The linearity of the curve represents that it follows Beer's-Lambert law.

**Figure 11.** *Preparation of standard graph and evaluation of unknown.*

**Figure 12.**

*Evaluation of unknown concentration of FAD from standard graph.*

From this graph the unknown concentration of flavin adenine dinucleotide (FAD) can be read off (**Figure 12**).

#### **5. Linearity limit**

It is the range of concentrations of an analyte which the instrument can measure linearly beyond which the instrument does not response definetly.

**Importance of knowing linearity limit**: In biochemistry and other allied sciences, the linearity limit of each test is to be known. It is different for different kits of same parameter. Reading beyond this range is not reliable. In such condition, the sample is diluted and retested and multiplied by the dilution factor. In conclusion calibration curve or standard graph can be used to detect limit of linearity of a test.

#### **6. How to draw standard curve?**

Standard curves are defined as a graphs with absorbance or % transmittance plotted on the y axis, and increasing concentrations of standard along the X axis. If the Beer's law is followed, the resulting line representing absorbance vs. concentration will be straight.

A standard curve is constructed after obtaining the (%T) or (A) readings from a number of solutions of known concentration used in a reaction or procedure.

For example, Let us draw standard curve of glucose:

The glucose can be estimated even by plotting graph. This method is helpful to learn more about pipetting and plotting a graph with different concentration of glucose ranging from 50 to 400 mg.


Followed by measurement of the absorbance of those standards in colorimeter.

After the readings are obtained each is plotted on semi-log graph paper (% transmittance) or linear graph paper (absorbance) against the corresponding concentration. If the procedure follows Beer's Law, the points plotted will generally lie such that a straight line can be drawn through them. The concentration of controls and other unknowns (patient samples) can be determined by locating their %T or A reading on the line, then dropping an imaginary line down from that point to intersect the concentration axis.

Once a standard curve is developed for a particular test method on a particular spectrophotometer, it should be checked periodically to determine that it is still good.

A new curve should be constructed when there is a change in reagent lot numbers, methodology or procedure, an instrument parameter (change bulb, optics cleaned, etc.),

Once the curve is drawn, a number of things must be considered to determine its acceptability. The majority of the curve's points should be on or close to the line. There could be many reasons for a point not being on the line. Whether or not the curve passes through the point of origin (the "0"), varies with the procedure. If Beer's law is followed and the procedure is linear at the lower concentrations, the curve's line generally goes through the zero.

Always a standard curve should be prepared to confirm that Beer's law is applied to the analysis being carried out. The curve should be linear expectantly. There are several factors that may cause deviations from linearity which refers to deviations from Beer's law. Following factors may cause deviations from Beer's law:

**Imperfect monochromacy or polychromatic radiation:** The most common factor for deviation from linearity in most colorimeters is the use of a band of wavelengths to measure absorbance. Beer-lambert's law is applicable for monochromatic radiation only.

Other factors which includes followings:

**High concentration of chromophores**: According to the Beer-Lambert law absorbance (A) is linearly proportional to the concentration of chromophores. There may be deviation in samples with high sample concentrations (>10 mMol of solute) & high absorbance. The intensity of light at choosen wavelength (Iλ) should be 10 times higher than the intensity of the stray light (Istray) to reduce deviation from linearity [2]. In addition, the chromophore molecules may dimerize at high concentration and the absorption spectra of the dimers are not the same as that of the monomers. If the spectra differ, the absorption coefficient will differ leading to a positive or negative deviation (**Figure 13**).

**Polymerization**: It results into the spectral shift. This wavelength at which molar absorption coefficient is not changed is called the isobestic point.

**Aggregation:** Moreover, high concentration may also lead to aggregation. The intensity of the radiation reaching the detector is decreased because large aggregates scatter light. In such a case a positive deviation will be seen. Aggregation can also lead to electronic interactions that can either decrease or increase the absorption coefficient. At high concentrations, chemical events (association, polymerization,

**Figure 13.** *Deviation from Beer-Lambert law.*

*Fundamentals of Colorimetry DOI: http://dx.doi.org/10.5772/intechopen.112344*

dissociation, pH change, interaction with the solvent to produce a product with different absorption characteristics) which may lead to a change in the chemical composition of the solution. Consequently, a deviation from linearity will result.

**Low concentrations of chromophores:** Deviation may also occur at low concentrations. Proteins are known to denature at low concentrations and, the denatured product has an absorption spectrum that is different from the native protein.

Apart from imperfect monochromacy, other instrumental drawbacks may also result in deviations from Beer's law. Stray radiation reaching the detector changes sensitivity of the detector and power fluctuations of the radiation source and detector amplification system also cause deviation.

Besides above factors, the following factors can also cause deviations from the Beer's law:


#### **7. Variety of applications**


In serum sample: colorimetry may be applied for


In urine:

1.Determination of primaquine metabolites in urine.

*Fundamentals of Colorimetry DOI: http://dx.doi.org/10.5772/intechopen.112344*

2.Determination of urinary chloride.


In CSF: colorometry is applied for

1.protein determination in CSF.

2.chloride determination in CSF.

3. glucose determination in CSF.

In water:

1. Iron

2.Chloride

Plant material:

1.Phosphorus estimation

### **8. Pre-requisitive for good colorimetry**


#### **9. Advantages and disadvantages of colorimetry**

**Advantages:** Colorimetry is cheap and useful for quantitative determination of colored substances. It is portable. Since it has limited manual operations it is easy to handle.

**Disadvantages:** It can not be used for those substances which do not possess color. Errors in inferences can be seen from alike color of interfering compounds. Moreover, very elevated concentrations cannot be measured hence is less sensitive. The filters available are of only narrow range. Unstable light source has possibility of errors since there is change in the light intensities.

#### **10. Case study**

• Under aseptic condition total 2 ml of blood from every participant was drawn which was placed in Ethylenediaminetetraacetic acid (EDTA) test tube at school and transpoted to Biochemistry laboratory of Nepal Medical College and Teaching Hospital (NMCTH) in cold blood box and hemoglobin was estimated on the same day [12].

• Hemoglobin (Hb) estimation by Drabkin method was performed at Biochemistry laboratory.

**Principle:** Hemoglobin (Hb) + Ferriccyanide ! Methhaemoglobin ! Cyanomethhaemoglobin (stable red compound) which is measured colorimetrically, it's intensity is directly in proportion to amount of Hb.



**Table 3.**

*Mean, Range and Median of haemoglobin of the participants enrolled in the study.*

**Discussion:** Hb was found to be ranged from 6.0 to 22.80 g/dl, mean � SD of Hb being 12.63 � 2.91 g/dl.

#### **11. Advances in colorimetry**

The accessibility of smartphones and the speedy development of different mobile applications is having a great effect in our daily life [13]. Nevertheless, till now, smartphones have been rarely applied to other sciences and their applications in this research field may provide an immense leap in terms of routine sample analysis with an accessible, fast, simple and low-cost strategy. Smartphone cameras produces images using a red, green, and blue (RGB) color code. Based on this code, simple analysis of these digital pictures to quantify color changes is done with the help of different applications. These differences in color have been used for the quantification of various analytes in liquid suspensions, both in simple solutions and direct sampling matrices [14]. In many cases, a reagent is used to produce a colored compound through the reaction with a particular analyte, and the color of the newly formed analyte-reagent complex is measured [15].

Smartphones are embedded with an optical chemical or biosensing base via the use of an attached lighting control enclosure that is fitted to the device and the assay

#### *Fundamentals of Colorimetry DOI: http://dx.doi.org/10.5772/intechopen.112344*

platform that may be strip, cassette, cuvette etc. In this configuration, the smartphone can function as an illumination source (Light Emitting Diode), a signal detector, and a signal processor. Based on the conditions of the assay, various lenses, filters, diffraction gratings, and alternative light or power sources may be incorporated within the attached enclosure to intensify signal detection from colorimetric, fluorescence-based, chemi/bioluminescence-based, and scattering-based assays [16, 17].

Pharmaceutical compounds can be determined in dosage forms and biological matrices by using various colorimetric reagents with the help of spectrophotometric and chromatographic methods. The colorimetric methods have more sensitivity than that of ultraviolet spectroscopic methods. Moreover colorimetric methods measure light of longer wavelengths which decreases effects of interfering excipients. Most of the procedures are inexpensive and reagents easily available. There is a great scope for development of new reagents and new colorimetric methods [18].

There has been a focus on newly developed chemosensors on systems that permit for portable, on-site testing of the target analytes without requiring high yield, costly laboratory instrumentation [19, 20]. It is challenging to design a portable chemosensors to maintain high selectivity, sensitivity, and wide applicability in the efficient measurement of various analytes especially that are similar in structure and size. The Levine group has developed sensitive and selective fluorescence-based systems for analyte detection by using the property of cyclodextrin to act as a supramolecular scaffold that aids proximity-induced, highly analyte-specific interactions between an analyte of interest and a high-quantum yield fluorophore [21–25].

Recently there has been developments in the the field of optofluidics making use of light matter interactions in integrated devices with significantly improved features [26]. The newly developed microfluidic device integrated in optofluidic system has been introduced. The sensitivity of measuring concentration of sample by optofluidic system is better than that of other methods [27].

#### **Author details**

Yagya Kumari Shrestha<sup>1</sup> \* and Shree Krishna Shrestha<sup>2</sup>

1 KIST Medical College and Teaching Hospital (KISTMCTH), Nepal

2 National Trauma Center, National Academy of Medical Science (NAMS), Nepal

\*Address all correspondence to: dr.yagyashrestha@gmail.com

© 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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### **Chapter 3**

## Application of UV-Visible Spectrophotometric Colour Analysis in Different Natural Product Identification

*Sonia Karuppaiah, Nithya Sermugapandian, Sumithra Mohan and Manikandan Krishna*

#### **Abstract**

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.

**Keywords:** colorimetric, dyes, food colourant, natural dyes, reflected

#### **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

**Figure 1.** *What is colour measurement?*

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**.

#### **2. Light**

Light is defined by its energy, E, and its wavelength, λ*:* Here, c is the speed of light and h is Planck's constant [2].

$$E = \frac{hc}{\lambda} \tag{1}$$

The relationship between light's energy and wavelength is inverse. Therefore, light with increased energy has a shorter wavelength.

#### **3. Spectrophotometer**

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.

*Application of UV-Visible Spectrophotometric Colour Analysis in Different Natural Product… DOI: http://dx.doi.org/10.5772/intechopen.112636*

$$A = \log\left(\frac{P\_0}{P}\right) = -\log T \tag{2}$$

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 ¼ 0*:*1*:* This yields an absorbance of one. If 99% of the light is absorbed, then 1% of the light is transmitted Tð Þ ¼ 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.

#### **4. Beer–Lambert law**

The transmittance and absorbance of a certain substance are proportional to its concentration in solution. The Beer–Lambert law describes this relationship.

$$A = \text{el} \tag{3}$$

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**).

#### **5. Dyes**

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.


**Table 1.** *Absorbance and λmax of dyes.*

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.

$$
\Delta E\_{(HOMO-LUMO)} = \frac{hc}{\lambda} \tag{4}
$$

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

#### *Application of UV-Visible Spectrophotometric Colour Analysis in Different Natural Product… DOI: http://dx.doi.org/10.5772/intechopen.112636*

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 [6–8]. 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 semiconductor 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 dyesubstrate interactions.

#### **6. Measurement of colour**

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.

#### **7. Colour difference**

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.

#### **8. Absorbance wavelength comparison**

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.


*Application of UV-Visible Spectrophotometric Colour Analysis in Different Natural Product… DOI: http://dx.doi.org/10.5772/intechopen.112636*


#### **8.1 Measurement using UV visible spectroscopy**


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.


• 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**.

#### *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

**Figure 2.** *Complementary colours in colour wheel.*

#### *Application of UV-Visible Spectrophotometric Colour Analysis in Different Natural Product… DOI: http://dx.doi.org/10.5772/intechopen.112636*

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].


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**.


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.


*Application of UV-Visible Spectrophotometric Colour Analysis in Different Natural Product… DOI: http://dx.doi.org/10.5772/intechopen.112636*


#### *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.


#### *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.

#### **9. Conclusion**

Since Colorimetric is widely used not only in the pharmaceuticals but also used as food colourant, dyes and natural dyes estimation.

### **Author details**

Sonia Karuppaiah<sup>1</sup> \*, Nithya Sermugapandian<sup>2</sup> , Sumithra Mohan3 and Manikandan Krishna<sup>4</sup>

1 Department of Pharmaceutical Chemistry, Sri Ramachandra Faculty of Pharmacy, SRIHER (DU), Chennai, India

2 Department of Pharmacology, Sri Ramachandra Faculty of Pharmacy, SRIHER (DU), Chennai, India

3 Department of Pharmacology, SRM College of Pharmacy, SRMIST, Kattankulathur, India

4 Department of Pharmaceutical Analysis, SRM College of Pharmacy, SRMIST, Kattankulathur, India

\*Address all correspondence to: soniapharm68@gmail.com

© 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Application of UV-Visible Spectrophotometric Colour Analysis in Different Natural Product… DOI: http://dx.doi.org/10.5772/intechopen.112636*

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Section 3
