Tuesday, March 31, 2020

Advantages and disadvantages of reversed-phase chromatography

The reversed-phase chromatography is a type of HPLC chromatography; it is working on the principle of hydrophobic interactions and is one of the most popular techniques, as it applies to a wide variety of analytes. The RP- chromatography is a commonly used separation technique, in which the molecules are separated based on polarity. The polar mobile phase and a non-polar stationary phase are used in RP-chromatography. The more hydrophobic molecules are the more strongly attaches to the column and the more volume of solvent required to elute the molecule.  Hence, the retention times are longer for non-polar components, while polar compounds are more readily elute from the stationary phase.
The advantages of reversed-phase chromatography are as follows.
  • It is an economical method compared to other chromatographic techniques.
  • RP-HPLC allows water to be used in the composition of the mobile phase with other solvents.
  • Another advantage of using reversed-phase chromatography is that it provides accurate results with small amounts of sample.
  • RP-chromatography also has the advantage of being able to use pH selectivity to improve the separation.
  • The hydrophobic stationary phase in reverse-phase columns works well for the retention of most organic molecules.
  • In RP-chromatography we can use pH selectivity to get better separations.
  • About 75 percent of all HPLC methods use reversed-phase chromatography.
The disadvantages of reversed-phase chromatography are as follows.
  • Water-insoluble compounds and amines can be more difficult to analyze.
  • In RP-HPLC need to create pressure.
  • It requires technical capability and skill to handle the system.
  • The silica of the reversed-phase column can be dissolution at pH > ~7.5.
  • The eluted sample from the column cannot be recovered.
  • Additional techniques are needed to confirm the identity of the analytes.

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Thursday, March 19, 2020

Principle and Application of Polarimetry

What is a polarimeter?

Polarimetry can be defined as the analysis of the rotation of polarized light by transparent components. Polarimetry is a responsive, non-destructive technique to measure the optical activity of organic and inorganic compounds. If linearly polarized light rotates when passing, it is assumed that a compound is optically active. The concentration of the chiral molecules and the molecular composition of the material determine the amount of optical rotation. The direction and extent of rotation are useful for qualitative and quantitative analysis and are also useful for the elucidation of chemical composition. Optical behavior is the ability to polarize light rotation in the determination of certain compounds.

Polarimeter principle:

The basic operation principle of a polarimeter includes a source that produces light with a specifically prepared linear polarization state, typically by going through a polarizer. The light is transmitted by an optically active sample that often rotates the polarization direction. After going through the sample, another measure of the changed angle of the polarization by transferring light through analyze, that can be rotated around the beam axis. The easiest technique is to move the analyzer to the place for which the transmission of optical power is at its lowest.

Working principle of polarimeter:

Polarimetry determines the rotation of polarized light as it travels through an optically active sample solution. A polarimeter includes a source of polarized light, filter, sample cell, analyzer, and a detector to measure the rotation angle. The determined rotation can be used to measure the concentrations of the sample particularly sugar, vitamins, peptides, and volatile oils.

Types of polarimeter:

Different types of polarimeters are mentioned below.
Manual polarimeter, fully-automatic, semi-automatic polarimeter, quartz-wedge polarimeter, biquartz polarimeter, lippich polarimeter, and Laurent’s half shade polarimeter, etc.

Polarimeter procedure:

Before beginning a polarimeter experiment, we must recognize the various components necessary to execute the process. The optical activity is calculated by the Polarimetry that consists of the source, filter, sample cell, analyzer, and detector.

Source: Sodium vapor lamps are usually used to produce wavelengths greater than 450 nm.
Filter: The filter is used to absorbing the undesired radiation of polychromatic light into monochromatic light.
Sample cell: Sample cells are used to place the sample in the sample compartment, there is made up of glass and has long tube-shaped.
Analyzer: It is mostly employed to analyze the samples when they rotate to the right or left side of the plane of polarized light.
Detector: The photomultiplier tube is broadly used for wavelength detection. The optically active component present in the solution rotates the plane-polarized light either in the clockwise direction or anti-clockwise direction. After that analyze calculates the angle of rotation and the detector detects it.

Experimental procedure of polarimeter:

  • Prepare the sample solutions and standard solutions as you required.
  • Switch the instrument to turn on and warm up for around 5 minutes.
  • Rotate the Polaroid wheel so that display reads “ZERO”.
  • Put the Polarimeter tube inside the instrument and close the cover.
  • Rotate the wheel back and forth to display minimal intensity. Note down the readings of degrees (X1) and don’t disturb the system.
  • Then take out the tube and refill it with the test solution, and put it back in its place. The intensity of the sample will rise due to the rotation of the plane of polarization.
  • Rotate the wheel in the direction of decreasing intensity until the intensity reaches the minima and note down the readings of degrees (X2).

Polarimeter applications:

  • Polarimetry is used to determine the specific rotation and optical rotation of products such as amino acids, cocaine, antibiotics, dextrose, carbohydrate, analgesics, vitamins, steroids, sugars, serums, diuretics, and codeine, etc.
  • For structural determination, it can be used. In this application, changes in optical rotation about the substance chemical change are estimated.
  • It is used to ensure product quality by determining the purity and concentration of compounds in sugar-based food, syrups, and cereals
  • The applications of polarimeter consist, quantitative and qualitative analysis of optically active components.
  • Polarimetry is used in the analysis of fragrance, flavor, and essential oil manufacturer
  • Chiral compounds can also be determined by polarimetry.

Advantages of polarimeter:

  • Polarimetry is an easy technique to operate and needs no experts to handle.
  • It is not affected by changes in laser intensity.
  • The temperature and pH within the eye remain stable.
  • The advanced version of the polarimeter has a broad wavelength emission range over conventional light sources.
  • The analysis is very easy and rapid, which means this a cost-effective technique.

Disadvantages of polarimeter:

  • To determine the specific and optical rotation of a substance, it required large sample volumes with high concentrations.
  • The major disadvantage of polarimetry is that the only optically active components can be analyzed by polarimetry.
  • It has low sensitivity compared to other techniques.
  • It is very sensitive to motion and scattering.

Commonly asked questions on polarimeter are as follows.

What is Polarimeter?
A polarimeter is an analytical tool that is used to determine the angle of rotation due to the passing of polarized light through the optically active molecule.

How does a polarimeter measure optical rotation?
A polarimeter is a tool that determines the angle of rotation bypassing the source of polarized light through the optically active material.

Which light source is used in the polarimeter?
Sodium (Na) vapor lamp is used in a polarimeter as a light source, since it produces monochromatic light and with high-energy output

What is the major advantage of a polarimeter?
The main advantage of a polarimeter is its analysis of a wide range of compounds.
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Saturday, March 14, 2020

Principle and Application of Partition Chromatography

What is Partition Chromatography?

Partition chromatography is a type of chromatography in which the analytes of a sample mixture distribute more likely in two liquids, due to differences in partition coefficients. In partition chromatography, both the mobile phase and the stationary phase are in the same phase and in which the analytes are separated. Therefore, the analytes are preferably distributed in any phase.


Partition Chromatography Principle:

The separation of analytics from a sample solution is done by the method of partitioning the analytes between two phases. Partition chromatography is a basic principle used in many different methods such as gas chromatography, paper chromatography, high-performance liquid chromatography, and thin-layer chromatography (TLC). Partition chromatography is usually understood as a means of solute partitioning using the separation of two liquid phases. The process of separating the sample mixture of compounds passes through a solid stationary phase in which the components travel with a mobile phase. The mobile phase travels from the stationary phase and separates the components according to the affinity towards the stationary phase.

Partition Chromatography Procedure:

  • For an easy understanding of partition chromatography, here is the procedure for conducting paper partition chromatography.
  • Select a suitable type of development: It is determined based on solvent, paper complexity, mixture, etc. Ascending paper chromatography is widely used as it is simple to do. It is also simpler to use and the chromatograms are rapidly obtained.
  • Choosing a proper filter paper (stationary phase): Filter paper is selected based on pores size and quality of the analyte.
  • Preparation of sample: Sample preparation involves dissolving the component in a suitable solvent used in the process of producing mobile phases.
  • Spot the sample on paper: Apply the sample mixture to the appropriate position on the paper using a capillary tube.
  • Development of chromatogram: Using the chromatographic jar, the development of the chromatogram is observed by immersing the paper into the solvent or mobile phase. The mobile phase runs over the test sample by capillary action.
  • Paper drying and the identification of compounds: Once the chromatograms are developed then the paper is dried by an air dryer. Paper with a different band of molecules can be examined in the UV cabinet and Rf values are determined.

Partition Chromatography Applications:

  • Paper chromatography has various applications. Some applications are mentioned below.
  • It is used as a qualitative analytical chemistry method to detect and separate color mixtures including pigments.
  • Partition chromatography is used to separate and identify proteins, nucleic acids, sugars, glycosides, lipids, alkaloids, and other biomolecules.
  • Partition chromatography is used for the isolation and identification of amino acids.
  • It is used to separate polar and non-polar molecules.
  • It can be used to test the purity of pharmaceuticals and to monitor the chemical synthesis reaction.
  • It can be used in forensic research for investigations and criminal trials.
  • It is also used for DNA/RNA sequencing.
  • It is used to detect pollutants in beverages and food products.

Types of Partition Chromatography:

Their are two types of partition chromatography are available, liquid-liquid chromatography and gas-liquid chromatography.
Liquid-liquid Chromatography: In this type of partition chromatography a sheet of absorbent paper is used instead of an adsorption column. The analytes are separated based on their differential migratory velocity. When chromatograms are separated, they are stained to make them visible.

Gas-liquid Chromatography: It is a type of partition chromatography in which the sample mixture is separated by an inert gas with a tube. The tube is packed with finely divided inert solids which are coated with nonvolatile oil. The migration of each analyte occurs at a rate that is determined by both its oil solubility and vapor pressure.

The Advantages of Partition Chromatography are as Follows.

  • The advantages of partition chromatography include a simple to operate and an inexpensive separation technique.
  • It has high efficiency.
  • The partition chromatography method can separate organic as well as inorganic compounds.
  • This gives accurate results compared to other chromatography techniques.
  • It separates compounds in a short period.

The Disadvantages of Partition Chromatography are as Follows.

  • The data cannot be stored long, which is the major disadvantage of partition chromatography
  • Sometimes high amounts of solvents are required for separation.
  • Automation made more complicated and expensive.

Difference between partition and adsorption chromatography:

The major difference between partition and adsorption chromatography is that the partition chromatography separates the analytes by partition, while adsorption chromatography separates analytes by adsorption.

Commonly asked questions on chromatography are as follows.

What is partition chromatography in chemistry?
It is a separation technique whereby the component mixture is more likely to distribute into two liquid phases due to variation in the partition coefficient in the stationary phase during mobile phase flow.

What is the principle of partition chromatography?
The principle of partition chromatography, which consists of two phases, a mobile phase and another are stationary phase, and the sample is separated amongst the two phases based on affinity at each phage.

What are the two main types of chromatography?
Liquid chromatography (LC) and gas chromatography (GC) are the two main types of chromatography.

What is the purpose of column chromatography?
The main objective of column chromatography is to separate the components with different molecular structures.

Why must the TLC chamber be covered?
During the development of the chromatogram, it is significant to have covered the chamber, since that the solvent vapor in the chamber will saturate the air, the mobile phase does not evaporate and the chromatogram develops correctly. This is the main reason to cover the chromatography chamber.


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Wednesday, March 11, 2020

Principle and Applications of Fluorimetry

What is Fluorometry?

Fluorometry is a type of spectroscopy and is also called fluorescence spectroscopy. It is used to identify and determine the analyte concentrations in a sample.
The mechanisms involve the excitation of an ultraviolet light beam in the molecules of a specific analyte and enable them to emit visible light. At a wavelength, it is the molecular absorption of light energy and its almost instantaneous re-emission at a longer, different wavelength.
Several molecules are inherently fluorescent, and others need to be modified for fluorescence.


Principle of Fluorometry:

The phosphorescence and fluorescence are processes of the photon emission, which arise from electronically excited states throughout molecular relaxation. These photonic mechanisms cause the polyatomic fluorescent molecules (fluorophores) between vibrational and electronic states. Fluorophores play an essential role in the fluorescence spectroscopy. 
Fluorophores are the components that produce fluorescence in molecules. The samples that have been excited electronically after absorption of UV (200 nm to 400 nm), visible (400 nm to 800 nm), or NIR (700 nm to 1100 nm) radiation. The excitation method is very rapid from the ground state to the excited state on the order of 10 to 15 seconds. 
The molecule is quickly relaxed after excitation, to the lowest vibrational point of the excited electronic state. The quick process of vibrational relaxation takes place on the time scale of femtoseconds to picoseconds. The emission and excitation spectra of fluorescence, respectively, reflect the vibrational level structures in the ground and the excited electronic states.

The different electronic states in fluorimetry are as follows.
Singlet excited state: It is a state in which the electrons from the opposite spin are unpaired out.
Triplet state: It is a state in which unpaired electrons of the identical spin are present
Doublet state: It is a state which contains unpaired electrons.
Singlet ground state: It is a state in which all electrons are paired within a molecule.

Applications of Fluorescence Spectroscopy:

  • The application of fluorometry is significant as a potent and valuable tool for studying the physical and chemical behavior of macro-molecules.
  • Fluorescence spectroscopy used in environmental analysis.
  • Fluorescence spectroscopy is used where the sample is scared and complex to process.
  • Fluorescence spectroscopy used in food analysis.
  • It is used to determine several types of analytes in serum.
  • Each form of fluorescence activity is to assist to apply fluorescent probes in polymer systems.
  • Fluorescence spectroscopy used in dairy processing.

The Advantages of Fluorescence Spectroscopy are as Follows.

  • Because of the unique optical properties of the molecules, it has high precision.
  • Fluorescence spectroscopy can be used for the quantitation of fluorescent species.
  • This can calculate the decay time, fluorescence intensity, and concentration of the component.
  • This method is less expensive compared with other methods.
  • It has the capability of rapid and rapid diagnosis.

The Disadvantages of Fluorescence Spectroscopy are as Follows.

  • The major disadvantage of fluorescence spectroscopy is that it can only analyze fluorescent molecules.
  • It has limitations associated with loss of photostability and recognition capability
  • Fluorophores have a short lifespan.
  • It is also susceptible to the auto-fluorescence of the solution.
  • It can susceptible to interference due to changes in sample pH and oxygen levels.

Difference between spectrofluorometer and spectrophotometer:

The major difference between spectrofluorometer and spectrophotometer is that spectrofluorometer is used to determine the fluorescence of the analytes, whereas spectrophotometer is used to determine the intensity of electromagnetic radiation.

Commonly asked questions on fluorometry are as follows.

What is fluorescence spectroscopy?
Fluorescence spectroscopy is a tool for determining fluorescence of the components, often as a means of measuring the nature of the substance that emits fluorescence.

What is the basic principle of fluorescence spectroscopy?
A molecule is absorbed and excited by incidental electromagnetic radiation. In its excited state, it is unstable, and by emitting radiation, it returns to the ground state.

What are the types of fluorometer?
The filter fluorometer and the spectrofluorometer are the two basic types of the fluorometer.

What is the major advantage of fluorescence spectroscopy?
The high sensitivity and its very low detection limit are the major advantages of fluorescence spectroscopy.



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Tuesday, March 10, 2020

Principle and Procedure of Conductometry

What is Conductometric Titration?

Conductometric titration is a kind of titration wherein the electrolytic conductivity of a sample mixture is determined continuously with the addition of a reactant. The equivalence point is the situation in which the conductivity of the reaction mixture undergoes a sudden change. The electrical conductivity of the sample solution is reliant on the number of free ions in the sample and the charge parallel to each of these ions. Conductometry has an important application in analytical chemistry, where a standard technique is conductometric titration.


Principle of Conductometry:

To determination of the ability of a substance to carry out the electric current, conductivity is used. Siemens "g" conductance is the basic unit and that is the reciprocal of resistance and which is determined in ohms. The electrical conductivity of the sample is proportional to the number of ions present in it, and thus the conductivity calculation of the solution will provide a reading.

The working principle by which conductivity is calculated in the simple system. Two conductivity plates are located in the solution and potentially applied to them and, thereafter, the current is calculated (generally ac voltage). From the current and voltage values, the conductivity can be found. Precise and constant conductivity can be measured by multiplying the conductivity by the electrode cell.

This constant conductivity is determined by the formula given below,
Gt = Gt cal { 1 + a (t - t cal) }
Where Gt is conductivity at any temperature in °C
G tcal is conductivity at a temperature of calibration in °C
a is the temperature coefficient of the solution
t cal is the temperature of calibration.

This adjustment can be done manually or automatically by calculating the temperature coefficient of the solution with the advanced conductivity meters. Automatic temperature compensation with specific temperature sensors is necessary for reliable measurement of conductance, and the sample and standard solution should be calibrated with the same temperature.

Types of Conductometric Titration:

There are generally five types of conductometric titration, mentioned as follows.
1. Acid-base titration: It is a quantitative analysis process to determine the acid or base concentration, by accurately neutralizing it by a standard solution with a recognized concentration.
2. Precipitation titration: Precipitation titration is a titrimetric method that involves the formation of precipitates throughout the process of titration.
3. Replacement titration: The metal can be determined by this method when direct or back titration does not provide a precise endpoint or when there are no appropriate indicators for molecules.
4. Redox titration (oxidation-reduction): It is a type of conductometry titration, redox titration is a reduction by an oxidizing agent. Normally, this titration involves a potentiometer or a redox indicator.
5. Complexometric titration: In this titration, the color complex is used to determine the endpoint, this is a type of volumetric analysis.

Procedure of Conductometric Titration:

  • Take 30 ml of 0.001 M HCL in a beaker (100ml).
  • Fill the burette with NaOH solution.
  • Switch on the conductivity meter.
  • Clean the conductivity cell and temperature probe with distilled water and wipe gently with tissue paper.
  • Immerse the conductivity cell in the HCL solution.
  • Press the conductivity key.
  • The device will display conductance.
  • By continuous mixing, add NaOH in HCl.
  • Note down the readings.
  • Plot the graph of conductance versus volume of NaOH.
  • Switch off the instrument and clean the conductivity cell and temperature probe.

Applications of Conductometric Titration:

  • Conductometry is also used to find the concentration of salinity and total dissolved solids (TDS).
  • The purity of water can be determined by conductometry.
  • The solubility of sparsely soluble salts such as lead sulfate, sulfate, and barium is determined by conductivity.
  • The alkalinity of freshwater can be determined by this method.
  • Conductometric titration has several applications in precipitation titration, redox titrations, acid-base titration, and complex titrations.
  • It can be used for tracing microorganisms.
  • Conductometry is used to monitor the progress of a chemical reaction.
  • The chemical equilibrium in ionic reactions can be determined by it.

The Advantages of Conductometric Titration are as Follows.

  • Conductometric titration is simple and has wide selectivity.
  • It is also suitable for complex samples, such as turbid suspension, low concentrations, and colored solutions, etc.
  • It gives precise results with minimal errors.
  • The conductometric titration is also suitable for weak acids and more dilute solutions.
  • The endpoint of some samples (colored or turbid) cannot be seen with the naked eye. In this case, conductometric titration is used.
  • The temperature remains constant throughout the process.

The Disadvantages of the Conductometric Titration are as Follows.

  • It is not possible to measure samples with high concentrations by this method.
  • It is less accurate than other methods.
  • Changes in salt levels can increase the conductivity of the solution.
  • Only a limited number of redox titrations can be performed.

Commonly asked questions on conductometric titration are as follows.

What is conductometry?
Conductometry is the determination of electrolytic conductivity to observe the progress of a chemical reaction. It is generally used to measure the total conduction of a solution.

What is the basic principle of conductometric titration?
The main principle involved in conductometric titration is that the movement of ions produces electrical conductivity. The ions movement mainly relies on the concentration of ions

What are the types of conductometric titrations?
Acid-base titration, redox (oxidation-reduction) titration, complexometric titration precipitation titration, and replacement titration are the types of conductometric titration.

What is the major advantage of conductometric titration?
The major advantage of conductometric titration is that it can determine colored or turbid solutions and can work with many diluted solutions and weak acids.

What is the major difference between conductometric titration and potentiometric titration?
The main difference between conductometric and potentiometric titration is that conductometric titration determines the electrolytic conductivity of the sample solution, while potentiometric titrations determine the potential of the sample solution.


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Sunday, March 8, 2020

Principle and Procedure of Flame Photometer

What is the flame photometer?

A flame photometer is a device that is used in inorganic chemical analysis for the determination of certain metal ions such as potassium, sodium, calcium, and lithium. The photoelectric flame photometry is based on determining the intensity of emitted light produced when a metal is introduced into a flame.


Working principle of flame photometer:

The working principle of the flame-photometer is simple. The liquid is sprayed with a non-shiny flame in the form of a fine mist that is colored according to the characteristic emissions of the elements, e.g. Sodium (Na), Potassium (K), Calcium (Ca), and Lithium (Li). The flame is detected by a photodetector that observes the flame through a very narrow band optical filter that only passes the wavelength (nm) based on the characteristic emission of the chosen element. 

The output of the photodetector is fed to electronic modules that provide digital readings of the desired element concentration. The method has to be calibrated with known concentrations of the solution, before testing the unknown liquid sample.

Compressed air is supplied to an atomizer in a measuring system with the aid of a compressor, leading to a draught of air at the tip of an atomizer, which is sucked in the mixing chamber sample fluid and into the mixing chamber. Liquid petroleum gas (LPG) is also used at a controlled pressure in the mixing chamber; here the atomized sample and gas are delivered to the burner and ignited. 

The light from the flame is emitted by the optical lenses and transmitted to the flame photometer detector through a selected filter. The detector data analyzed electronically and the sample solution results are shown correctly.


Procedure of flame photometer:

Before beginning a working procedure of flame photometry, we must recognize the various components essential to perform the process. The flame photometer consists of four major components such as a source of flame, a nebulizer and mixing chamber, an optical filter, and a photodetector.

1. Source of flame: A burner delivered flame and can be maintained in a stable form and at a constant temperature.
2. Nebulizer and mixing chamber: This helps to move the sample solution of the molecules into a flame at a constant rate.
3. Optical filter (optical system): The optical system consists of three elements such as lens, filter, and a convex mirror. This separates the wavelength of any other extraneous emissions to be measured.
4. Photodetector: Detect the emitted light and determine the radiation level produced by the flame.

Working procedure of flame Photometer:

  • Prepare standard and sample solutions as required
  • Ensure the proper connection of the air, gas, and drain tube.
  • Power the flame photometer and compressor according to the instruction manual
  • Adjust the output pressure and drown the capillary tube into distilled water
  • Turn on the gas supply and immediately ignite the flame.
  • Set the blank with the diluent used for standard and sample preparation.
  • Aspirate the standard samples sequentially.
  • Aspirate the sample solution and record the readings.
  • Do the device shutdown process.

Applications of flame photometer:

  • The flame photometer determines the concentration of potassium (K), sodium (Na), and calcium (Ca) ions that are extremely significant for the activity of different metabolic functions in the human body.
  • It is used for the qualitative and quantitative study of the components.
  • The amount of various alkali and alkaline earth metals in the soil can be determined with the help of a flame test.
  • It can be used for calcium determination from milk, drinks, beer, and other products.
  • Flame photometry is used to determine the concentration of different elements and metals are in fruit juices, alcoholic beverages, and soft drinks.
  • Flame photometry is used in pharmaceutical analysis.

Advantages of flame photometer:

  • Flame photometry is a precise, fast, and responsive technique.
  • This is a cost-effective technique compared with others.
  • Qualitative and quantitative analysis of metal ions is possible using flame photometry.
  • It does not require a specialist to handle it, as it is easy to operate
  • Very low concentrations (PPM/PPB) of metal ions can be determined by this method.


Disadvantages of the flame photometer:

  • The major disadvantage of flame photometry is it only analyzes the liquid sample.
  • This technique does not provide details about the metal ion's molecular structure.
  • This often requires calibration, in some cases, it takes longer to prepare the sample.
  • The metal ion concentration can't be accurately measured using this method.
  • Flame-photometry cannot analyze each metal atom.

Difference between a spectrophotometer and a flame photometer:

The major difference between the spectrophotometer and the flame photometer is that the spectrophotometer uses the absorption of light by the molecules in a sample., whereas the flame photometry makes use of a controlled flame test.


Frequently asked question (FAQ):


What is a flame photometer?
It is a spectrophotometer in which a mist of metal salts in solution is vaporized in a very hot flame and quantitatively evaluated by calculating the strength of the metal spectral lines.

What is the basic principle flame photometer?
The principle of flame photometry is based on determining the intensity of emitted light when a metal ion is introduced in a flame.

What type of spectroscopy is flame photometry?
It is a type of atomic spectroscopy used to determine the concentration of certain metal ions, for example, calcium, sodium, potassium, lithium, etc.

What is the major advantage of flame photometry?
Its major advantage is the analysis of alkali and alkaline earth metals by flame photometry.


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Wednesday, March 4, 2020

Principle and Procedure of Colorimeter

What is a Colorimeter?

A colorimeter is a light-sensitive tool used to measure the absorption and transmission of light that passes through a sample solution. A colorimetric device works based on Beer Lambert's law. The photoelectric colorimeter is a sensitive apparatus intended for use in various colorimetric analyses such as soil component analysis, building materials, water analysis, food ingredients, textile products, additives, and employ in different manufacturing processes.  

Colorimeter Principle:

Colorimetry is a sensitive tool used to determine the intensity and concentration of a sample at a particular wavelength. In general, two types of colorimeters are used which are spectrophotometers and tristimulus colorimeters. The colorimeter principle works based on Beer-Lambert's law. This rule states that the absorption of light when passing through a medium is directly proportional to the intermediate convergence. While using a colorimeter, there is a beam of light wherein a given wavelength is directed toward a liquid sample. By entering a sample solution, the light beam travels through a series of different lenses, and the microprocessor is used to determine the absorption or emission of light through the liquid sample. If the concentration of the sample is high, more light will be absorbed and if the sample has a low concentration, it will transmit more light.

You may determine colorimetric reactions on a colorimeter or a spectrophotometer. Both measure the intensity of light that passes through a liquid sample and convert the intensity of this light into a concentration based on a specific calibration curve.

The colorimetry follows the principles of the Beer-Lambert law is expressed as: 
A = Ɛ x b x c
A is the absorbance of the sample component
Ɛ is a wavelength-dependent absorptivity coefficient
b is the path length of the cell
c is the concentration of the analyte

Working principle of colorimeter:

The colorimeter's working principle depends on the Beer-Lambert law which states that the amount of light absorbed by a sample corresponds to the sample solution concentration and the length of a light path through the solution. A low voltage lamp that is turned on by a constant voltage renders the light source. This goes through a color filter and liquid sample on the detector. The current generated by the photocell is then converted into a voltage to display the result on the screen. The current that the photocell generates will then be converted to voltage to show the result of either an absorbance or a transmittance on the screen.

Types of Colorimeter:

The tristimulus colorimeter and spectrophotometer are the types of colorimeter used for color measurement.
Tristimulus colorimeter: Tristimulus colorimeter is usually used for quality control, and appropriate with color variations and resistance determination. The tristimulus method measures the light reflected from the object to have a similar sensitivity using three separate sensors.
Spectrophotometer: A spectrophotometer is a device that can determine light intensity as a function of color, or more precisely, the wavelength of light, and other liquid samples. This detects both the entire UV spectrum in the range of 200-400 nm and the visible range of 400-800 nm. It gives accurate data by providing the wavelength of the sample absorbance or transmittance properties by wavelength spectral analysis. A spectrophotometer is simple and fast to operate and is most commonly used for light absorption measurement.

Colorimeter Procedure:

Before beginning a colorimetry analysis, we must recognize the various parts essential to perform the process.
Light source: Generally a tungsten or xenon lamp is used to produce the light.
Filter: It is made of colored glass, and is used to choose the light of narrow wavelength.
Cuvette: It is used to hold the solution of the sample. The monochromatic light passes through the sample solution put in a cuvette. Cuvettes are made up of special quartz or glass.
Detector or Photocell: it is used to detect the light transmitted through the sample. It is the photosensitive component that transforms light energy into electric energy.

Experimental Procedure of Colorimeter:

  • A colorimeter requires the first calibration using standard solutions of the specified solute concentration to be measured in a test sample. 
  • Prepare samples according to the procedure. 
  • Turn the instrument ON and allow it to warm up for 10-15 minutes. 
  • Choose the correct filter. 
  • Select the appropriate mode, i.e. % transmittance or absorbance. 
  • Insert the test tube containing the “Blank” or “Reference” solution. 
  • Make auto-zero with the blank solution. 
  • Remove the test tube containing the blank solution and insert the sample solution. 
  • Note down the reading in %T mode or optical density.

Colorimeter Applications:

  • It uses to confirm the quality and consistency of fabric and paint colors 
  • Applications of colorimeter have the qualitative and quantitative analysis of the samples. 
  • The food industry uses this to ensure the quality of the product. 
  • It is used to determine the quality of the food, to ensure that it does not spoil by determining its particular color. 
  • It can also be used to calculate a reaction's path by evaluating the rate of formation and the disappearance of light-absorbing analytes within the range of the visible light spectrum. 
  • The quality of the water is measured using colorimetry. 
  • It is used by manufacturers of paints, pharmaceuticals, and textiles. 
  • Colorimetry is frequently used to determine the concentration of the sample by determining the transmittance, optical density, or absorption thereof. 
  • By determining the absorption spectrum in the visible range, the component can be identified. 

The Advantages of Colorimeter are as Follows.

  • It is cost-effective, rapid, and is easy to operate. 
  • Compared to the volumetric or gravimetric processes it is a fast and convenient technique. 
  • No expert is required to handle this. 
  • Using the colorimetry process the chemical substances can be identified in the water. 
  • It subjected colored compounds to quantitative analysis. 
  • It can be used in the quantitative analysis of colored compounds. 
  • Another advantage of colorimeter is that it's a portable system that convenient to carry  

The Disadvantages of Colorimeter are as Follows.

  • Not analyzing colorless compounds is the major disadvantage of colorimetry. 
  • It requires more sample amounts for analysis. 
  • It requires the standard solution to be prepared. 
  • It has a lower sensitivity than other techniques. 
  • Colorimetry is not functioning in UV and IR regions. 
  • The accurate bandwidth of wavelengths can be essential for a more precise analysis of the molecules.

Commonly asked questions on colorimeter are as follows.

What is a Colorimeter?
A colorimeter is a light-sensitive tool used to determine the emission and absorption of light that passes through a sample solution.

What are the types of colorimeters?
In colorimetry, two types of analysis are used: the spectrophotometer and the tristimulus colorimeter.

Which light source is used in the colorimeter?
Generally, a tungsten or xenon lamp is used as a light source in colorimetry.

What is the major advantage of colorimeter?
The major advantage of colorimeter such as economical, quick, and has simple operation.

What is the major difference between spectrophotometer and colorimeter?
The major difference between a spectrophotometer and a colorimeter is that the spectrophotometer determines the absorption, or transmittance as a function of wavelength, and can operate on a range of wavelengths, i.e. UV or VIS, while a colorimeter is a tool that determines the absorption of particular colors at a fixed wavelength.

What is the major difference between absorbance and transmittance?
The major difference between absorbance and transmittance is that absorbance is to determine the amount of light absorbed by the analyte when a beam of light travels through it. Whereas, transmittance is the amount of light transmits by the analyte when a beam of light travels through it.


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