Monday, September 23, 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.

Effect of flow rate on column chromatography


Column chromatography is a widely used technique for separation and purification of compounds from the complex mixtures. In which the compounds pass through the stationary phase with the help of mobile phase and get separate on the basis on varying degrees of adhesion.
There is an optimal solvent flow rate for each column, it relies on which type of mobile phase, analyte, and column dimension are 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.

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.

 

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




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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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 is the difference between colorimeter and spectrophotometer?


In chemistry, the colorimetry and spectrophotometry both techniques are used to determine the absorption and transmittance properties of molecules. Besides, it is a simple method to identify the concentration of a sample solution. The colorimeter and spectrophotometer both are working on the same principle i.e. Beer-Lambert Law.
The colorimeter is tools that perform "tristimulus" color measurements on the base of light passing through three primary filters. The color measurement gives information about how much amount of light absorbs or transmitted by an analyte. 
Ultraviolet-visible spectrophotometer refers to absorption spectroscopy in the region of ultraviolet-visible. A UV/VIS spectrophotometer has high accuracy and increased versatility. It is appropriate for more complex color analysis since it can measure the spectral reflectance at every wavelength. Though, the spectrometry can be more costly as compared to colorimeter
The main difference between a spectrometer and a spectrophotometer is that the spectrometer operates at fixed wavelengths in the visible range, while the UV/Vis spectrophotometer works on a wide range of wavelengths. 

Difference between spectrophotometry and colorimetry

Both spectrophotometers and colorimeters are used to measure the absorption and transmittance of components based on their wavelength. The Tristimulus colorimeter has color filters and a light source which works on color sample only, whereas the spectrophotometer work with tungsten lamp, deuterium lamp, and monochromators to select the fixed wavelength as well as the range of UV/VIS.
Here some differences between spectrophotometer and colorimeter are mentioned.
  • Spectrophotometer consists of a sensor, data processor, and a computer that gives data as per the given format, whereas a colorimeter typically consists of a sensor and a data processor.
  • Spectrophotometer has a more expensive device than colorimetry.
  • The spectrophotometer can serve for research, development, and quality control, while the colorimeter is typically used for inspection and quality control purposes.
  • Spectrophotometry uses a wide range of wavelengths i.e., Ultraviolet and visible regions, whereas colorimetry uses a fixed wavelength in the visible range.
  • The spectrophotometer can recognize the strength and metamerism of color, whereas the colorimeter cannot identify the strength and metamerism of color.
  • The spectrophotometer determines the amount of light, whereas the colorimeter determines the absorption of light that passes through a sample solution.


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Difference between colorimeter and spectrophotometer

The spectrophotometer and colorimeter are used to determine the concentration of analytes by measuring the absorption of the sample solution. The Tristimulus colorimeter is usually portable to use color filters and LED light sources. Consequently, they work in fixed wavelengths and can accommodate tests involving only those wavelengths. Spectrophotometers are commonly used instruments since they can work on a range of wavelengths. In spectrophotometers, a tungsten lamp works on the visible range and deuterium lamp to work over the UV range. Spectrophotometers also have monochromators to choose particular wavelengths. Consequently, spectrophotometers can be used for a wide range of tests.
In analytical chemistry, both tools work qualitatively and quantitatively to determine color absorption, particularly by sample solutions. The main difference between colorimeter and a spectrophotometer is that the colorimeter is a device that measures the absorption of specific colors at a fixed the wavelength, while a spectrometer measures absorbance, transmittance or reflection as a function of wavelength and they can work on a range of wavelengths (UV/VIS).


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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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