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Tuesday, September 24, 2019

Factors affecting the resolution in gas chromatography



GC is a chromatographic technique of separation in which the gas (e.g. Nitrogen, Helium) used as a mobile phase. Gas chromatography is one of the most accepted techniques for separating and analyzing analytes, because of its high accuracy, reproducibility, resolution, speed, and low range of detection. GC can be useful for the separation of any volatile compound, hence it GC useful in the separation of many organic and inorganic compounds.
The factors that affect the resolution in the GC is mentioned here.
The temperature of the column:
The extremely high temperature of the column is the result in low RT and poor separation of the analytes, as all components are mainly in the gas phase. However, the analytes require interaction with the stationary phase to be separated.
Vapor pressure
The compounds boiling point is often associated with its polarity. If the boiling point of the compound is low, the higher the vapor pressure and the retention time are shorter since the compound will use more time in the gas phase.
The concentration and volume of the sample solution:
Generally, the peaks have an asymmetric shape. If the concentration and the volume of the sample solution are too high, there is a tailing in the peaks, which is the reason for poor separation. The detectors used in the GC are extremely sensitive and they don’t need much material to give a detectable signal. E.g. Flame Ionization Detector (FID), Mass Spectrometer (MS), Electrolytic Conductivity Detector (ELCD), Flame Photometric Detector (FPD), Photoionization Detector (PID) etc.
The flow rate of carrier gas:
A higher flow rate shortens the retention time, but a poor the separation will also be observed. Since the molecules have little or no time to interact with the stationary phase and are simply pushed through the column by the carrier gas.
The polarity of the stationary phase on the column and polarity of components:
If the polarity of the compound and the stationary phase are the same, the component's RT will increase since the strong interaction with the stationary phase. As a result, polar molecules have a longer retention time when using a polar stationary phase and shorter retention times when using non-polar polar stationary phase.
The length of the column used:
If you use a longer length of the column, then the retention time of the component will increase in proportion to the column length and a significant peak broadening will be seen. Generally, separation improves when long columns are used in the analysis.

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Effect of flow rate on column chromatography

Column chromatography is a widely used technique for separation and purification of compounds from complex mixtures. In which the compounds pass through the stationary phase with the help of the mobile phase and get separate on the basis on varying degrees of adhesion. There are different types of column chromatography are used for several applications and they work on the same principle of column chromatography i.e. adsorption.

There is an optimal solvent flow rate for each column, it relies on which type of mobile phase, analyte, and column dimension is being used. If the solvent flow rate is too fast, is not getting adequate time to equilibrium for the compounds and will be forced down the column with leaving a long tail. If the solvent flow rate is used very slowly in column chromatography, the diffusion processes will lead to band widening. For columns of smaller diameter, the optimal rate is lower than columns with a larger diameter. Therefore, compared with smaller columns the larger columns can be run with the higher flow rate.

We know that the size of the collected fractions relies on Rf value and size of the column and that the flow rate of the solvent can affect the separation process. The suitable flow rate in column chromatography is dependent on the dimensions of the column. The major effect of flow rate on column chromatography is that changing the flow rate of the analysis can change the separation quality of the component.


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HPLC Principle and Types of HPLC Chromatography
Applications of HPLC
HPLC column care and maintenance
Resolution in Chromatography
Factor Affecting Retention Time in HPLC
Advantages and disadvantages of Chromatography
Advantages and Disadvantages of Affinity Chromatography
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How does temperature affect paper chromatography?

Paper chromatography is a technique in chemistry that is used to separate a complex mixture of components or solutes with varying solubility and a degree of adsorption. In this technique applying a sample (dot) near one corner of the paper and the mobile phase runs throughout the paper and samples can be separated according to the affinity toward the stationary phase. Both thin layer chromatography (TLC) and paper chromatography work on the same principle.

Temperature can affect the separation of components in all chromatography types. If the temperature rises, the heat transfers further energy to the solvent-giving the molecule the power to escape from the surface of the liquid hence increases the transfer of liquid to the vapor phase. 

Paper chromatography and thin-layer chromatography (TLC) has two counteracting effects of temperature first is the changes in the retention time, that is, if the temperature increases, the retention decreases, and the second one are increased temperature will reduce the elution strength of the molecule and density of the mobile phase. The temperature also affects the solubility of substances, the more the temperature, the more the solubility of the substances.

 
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Factors affecting IR spectroscopy


This method offers a simple and quick technique for determining the presence of functional group species in an organic compound or molecule. In which the IR radiation passes through the sample and that spectrum is a plot of the percentage of IR radiation. Several functions of the wavelength of radiation associated with covalent bonding. Fourier transform infrared spectrometer (FTIR), Dispersive infrared spectrometers and Raman spectroscopy are various kinds of infrared spectroscopy available. The FTIR is a commonly used instrument in which the absorption spectrum is acquired by a Fourier transformation of an interferogram. The IR instrument uses a monochromatic light, while FTIR instrument uses a polychromatic light to identify the functional group.
There are mainly 4 factors affecting the vibrational frequencies in IR spectroscopy.
1. Coupled vibrations
2. Fermi resonance
3. Electronic effects
4. Hydrogen bonding
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Factors Affecting Separation in Column Chromatography
Factors Affecting Paper Chromatography
HPLC Principle and Types of HPLC Chromatography
Applications of HPLC
HPLC column care and maintenance
Resolution in Chromatography
Advantages and disadvantages of Chromatography
Advantage and disadvantage of IR spectroscopy
Advantages and Disadvantages of Affinity Chromatography
Advantages and disadvantages of FTIR spectroscopy 
Advantages and Disadvantages of Gas Chromatography 
Advantages and Disadvantages of HPLC 
Advantages and disadvantages of ion-pair chromatography 
Advantages and Disadvantages of Thin Layer Chromatography 
Advantages and Disadvantages of UV Visible Spectroscopy 
Advantages of FTIR Spectroscopy 

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Wednesday, September 11, 2019

Difference between fluorescence and absorbance

The major difference between fluorescence and absorbance is that the absorption is the process that devours a photon and places a molecule or atom in an excited state. Fluorescence is the process that first devours a photon and places a molecule or atom into an excited state and after that emits photons that have low energy that takes the molecule or an atom reverse to the ground state.
The amount of light absorbed by a substance is measured in absorption spectrometer, while fluorescence spectrometry measures the intensity or the amount of light emitted by a molecule.


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Advantages of fluorescence spectroscopy over UV

Absorption and fluorescence are different, but integral techniques of quantitation of molecules. Fluorescence spectroscopy has numerous advantages over ultraviolet-visible spectroscopy. It’s very low detection limit is a major advantage of fluorescence spectroscopy over ultraviolet-visible spectroscopy. It can be a highly sensitive than the absorption measurement in the ultraviolet spectrophotometer.
The advantages of fluorescence spectroscopy over UV are as follows.
High Sensitivity: Because of the high extinction coefficient of fluorophores, fluorescence assay is very sensitive and it allows the molecule detection at hundreds of times lower concentrations than those detectable by conventional absorption.
Specificity: The binding properties of fluorophores make this technique extremely selective for particular components.



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What are the advantages of colorimeter?
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Difference between fluorescence and absorption spectroscopy

Fluorescence and absorption spectroscopy both are the most important laboratory techniques in chemistry. Both tools are sensitive, simple to operate, and can give a broad range of information. Both these methods are determined over the same region of wavelengths but are caused by two various phenomena. UV-Vis measures the absorption of the light near-ultraviolet region (200 to 400 nm) and the visible region (400 to 800 nm), whereas fluorescence spectroscopy measures the light emitted by a sample component in this region after absorbing light at an energy higher than that emitted.
The main difference between fluorescence spectroscopy and absorption spectroscopy is that the fluorescence spectroscopy measures the intensity or the amount of light emitted by an analyte, whereas absorption spectroscopy measures the amount of light absorbed by a particular component.


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Advantages and disadvantages of fluorescence spectroscopy

The fluorescence spectroscopy is a type of spectroscopy and is also called fluorometry. It is used to identify and measure the concentrations of analytes in a sample. The processes involve the excitation of electrons in molecules of a particular analyte by a beam of light (ultraviolet) and encourage them to emit light (visible). It is a molecular absorption of light energy at a wavelength and its almost immediate re-emission at a different, longer wavelength. Some components are fluorescent naturally, and others should be modified for fluorescence.
The advantages of fluorescence spectroscopy are as follows.
  • Its high sensitivity is the main advantage of fluorometry.
  • Due to the unique optical properties of the component, it has high specificity.
  • It can determine fluorescence intensity, decay time, and the concentration of the component.
  • It may immune to the scattering of light.
  • The emitted light is read at the right angle to the exciting light, reducing the background signal
  • These types of methods have a large range of linearity.
The disadvantages of fluorescence spectroscopy are as follows.
  • The major disadvantage of fluorescence spectroscopy is that not all molecules are fluorescent.
  • It has limitations related to loss of recognition capability and photostability.
  • Susceptible to interference because of the changes in pH and oxygen levels of the sample.
  • It is susceptible to the auto-fluorescence of the sample.
  • Issue related to potential toxicity, due to the foreign material in the biological media.
  • The short lifespan of fluorophores is another disadvantage of fluorometry.


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What are the advantages of colorimeter?
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What are the advantages of colorimeter?

The colorimeter is one of the excellent measurement methods as other color measurement methods. A colorimeter is a light-sensitive device that determines the transmittance and absorption of light which is passed through a sample component.
Here are mentioned some advantages of a colorimeter.
  • It determines the concentration or intensity of color into a sample solution.
  • This is a more rapidly and suitable method than gravimetric or volumetric processes and they are simply optimized for automation.
  • They provide significant features at affordable prices.
  • The operation of the spectrometer is simple, which does not require a trained person
  • This method can be used to identify the chemical substances in water.
  • It is portable hence it not required much space.
  • It is commonly used as a quality control device in most applications where color sampling is used
  • The analysis of colorimetry is inexpensive and rapid.
  • Another advantage of colorimetry is that the quantitative analyses of colored compounds are possible.

 
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Advantages and disadvantages of colorimetry

Colorimetry is a tool that measures the color of a substance or sample and classifies it according to a color chart. The colorimeter is a light-sensitive instrument used to determine the absorption and transmittance of light passing through a sample solution. It calculates the concentration or intensity of the color which developed by introducing a particular reagent into a sample solution. 

Advantages of colorimetry:

  • It is economical, fast and has the simple operation of a spectrometer.
  • It is a fast and convenient method as compared with the volumetric or gravimetric processes and they are easily optimized for automation.
  • It does not require an experienced person to handle it.
  • The chemical substances in water can be identified by this method.
  • It applied to the quantitative analysis of colored compounds.
  • Another advantage of colorimetry is that it is a portable system you can easily carry and transport.

Disadvantages of colorimetry:

  • The major disadvantage of colorimetry is that colorless compounds cannot be analyzed.
  • It needs more amount of sample for analysis.
  • You require preparing a standard solution.
  • Its sensitivity is low.
  • The same colors from interfering material may create errors in results.
  • The precise wavelength bandwidth may be required for more accurate analysis.
  • The interference with the matrix can lead to poor results in uncontrolled conditions.


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What are the uses of colorimetry?


A colorimeter is any device which uses to determine the colors and its intensity from the sample solution based on the Beer-Lambert Law. It has a wide range of applications, including analysis of soil components, laboratory research, environmental analysis of water quality and analysis of chemicals used in various manufacturing settings.
The uses of colorimetry are as follows.
  • Colorimetry is commonly used to determine the concentration of a colored compound by determining transmittance, optical density or its absorption.
  • A colorimeter ensures the consistency and quality of the colors in the fabric and paint
  • It is used to determine the food quality, to confirm that they do not spoil by determining their specific color.
  • It is used in the quantitative analysis of colored samples.
  • The colorimetry is used to test water quality.
  • A component can be identified by measuring the absorption spectrum in the visible range of the light spectrum.
  • It is used by the paints and textile manufacturers.
  • The colorimeter used as qualitative and quantitative analysis of colored samples.
  • It is used by the food industry to ensure the quality of the product.
  • Applications of colorimeter have the measurement of color and it is temperature.
  • Colorimetry is also used by pharmaceutical manufacturers.
  • It can also be used to measure the course of a reaction by determining the formation rate and disappearance of light-absorbing analytes in the region of the visible spectrum of light.
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What are the applications of colorimetry?

The colorimeter is a device used to determine the colors based on the transmittance and absorption of light. A colorimeter can measure the concentration of a substance which is depending on the intensity of the color of the in the sample solution.
The colorimeter has a wide scope of applications is used for quantitative and qualitative analysis of colored compounds, including determining the concentration of a solution, laboratory research, the analysis of blood, environmental analysis of water quality, determining the rates of reaction, applications across the chemical and biological fields, analysis of soil components, nutrients in soil and foodstuffs, analysis of chemicals used indifferent industrial settings. Some other applications of colorimeter the analysis is used in the food ingredients, textile products, building materials, beverages, chemical solutions, and many others. 



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What are the types of colorimeter?

Colorimetry is a tool for the determination of intensity and the wavelength of electromagnetic radiation in the visible range of the spectrum. It is a color measurement tool used to communicate, capture, and evaluate the color, and also broadly used to identify and determine the concentrations of color substances which absorb light.

It compares the amount of light of the solution with the pure solvent. They can work quantitatively and qualitatively for the analysis of color analytes in various applications.

The spectrophotometer and tristimulus colorimeter are the two types of colorimeters are used for color measurement.

Tristimulus colorimeters:

Tristimulus colorimeters are typically used in quality control and are consistent with determination color differences and tolerance. The tristimulus technique estimates the light reflected from the item utilizing three sensors separated to have a parallel sensitivity. 

The colorimeter can be used broadly for different applications, today has several tristimulus colorimeters are available for the manufacturing of color samples, and examination of color quality control.

Spectrophotometer:

A spectrophotometer is a tool that can measure the intensity of light as a function of color or more particularly, the wavelength of light and other sample solutions. It detects the whole range of UV (200-400 nm) as well as the visible range (400-800 nm). 

By giving the wavelength by wavelength spectral analysis of the sample absorbance or transmittance properties it gives accurate data. A spectrophotometer is easy and rapid to operate and is most generally used for the measurement of light absorption.

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What is the principle of colorimeter?


The colorimeter is a very sensitive instrument used to determine the concentration and intensity of a particular color used in a sample solution. There are generally two types of colorimeters that are used in industries that are spectrophotometer and tristimulus colorimeter. The principle of colorimeter is working on the base of Beer-Lambert's law. This law expresses that the absorption of light while goes through a medium is straightforwardly proportional to the medium convergence. While a colorimeter is used, there is a ray of light in which a particular wavelength is directed towards a sample solution. The beam of light passes through a series of different lenses before reaching a sample solution and the microprocessor is utilized for the calculation of the absorbance or transmittance of the light through the sample solution. If the absorption of the solution is high, there will be the high-light absorbed by the compound and if the sample solution has low concentration then more light will be transmitted through it.
Colorimetric reactions can be determined on a colorimeter or a spectrophotometer.
Both determine the light intensity that passing through a color sample compound and convert this intensity of the light to a concentration on the base of the stored standard calibration curve.
The colorimetry follows the principles of the Beer-Lambert Law is written 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
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Working principle of colorimeter

A colorimeter is a device used in chemistry to determine the absorption or transmittance of a color sample solution and it is working on the base of Beer Lambert's law. The photoelectric colorimeter is a precision instrument intended to be used in the different colorimetric analysis, such as analysis of water, building materials, chemicals, food ingredients, analysis of soil components, textile products, and used in various manufacturing. It is easy to work, robust and precise.
The working principle of colorimeter:
A lamp of low voltage-activated by a constant voltage makes the light source. It passes through a designated color filter and sample solution on the detector (Photocell). The current produced by the photocell is then converted to voltage to display the result on the screen. The switch on the front panel allows being selected in either transmittance (% T) or absorbance (OD).
Simply, the working principle of the colorimeter depends on Beer-Lambert's law which expresses that the measure of light absorbed by a sample is corresponding to the concentration of the sample solution and the length of a light path through the solution.


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Advantages and disadvantages of mass spectrometry

Mass spectrometry is the technique in analytical chemistry for measuring molecules and atoms to determine their molecular weight. The information of such weight or mass data is once in a while adequate, much of the time essential, and constantly valuable in deciding the identity of a species. Mass spectrometry (MS) provides information regarding the molecular weight of compound or analyte and, when a combustion analysis, is performed in conjunction with relative percentages of hydrogen, oxygen, and carbon. This is relatively helpful in determining a molecular formula for the compound you are trying to identify.
The advantages of mass spectrometry:
  1. A major advantage of mass spectrometry than other technologies is that it is extremely sensitive.
  2. It is an exceptional technique to identify unknown components in a sample solution.
  3. It can work combining with other techniques, such as high-performance liquid chromatography (LC-MS) and gas chromatography (GC-MS).
  4. It is a very precise, rapid and sensitive method.
  5. It works with very small sample quantities which are in parts per million (PPM).
  6. This gives the relative molecular mass of every molecule.
The disadvantages of mass spectrometry:
  1. The main disadvantage of mass spectrometry is that it is costly, need a skilled technician, and it is not a portable system.
  2. We will unable to differentiate among isomers of the molecule with the same charge-to-mass ratio.
  3. Chiral columns may be required to separate enantiomers.
  4. The disadvantages of the mass spectrophotometer are that it is not nice to recognizing hydrocarbons that generate parallel ions and is unable to separate optical and geometric isomers.

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