Saturday, March 2, 2019

The Principle of Affinity Chromatography

In the affinity chromatography, the proteins are separated as per the dissimilarity in their binding affinity. There is a high binding affinity for specific ligands in enzymes, antibodies, and receptors. While the protein mixture is passed by a column which is composed with a material of legend coupled matrix, just proteins that bind the ligand can be retained. The target protein is eluted from the column by a solution, which competes for the target of the protein binding, i.e. the ligand.
The principle of affinity chromatography is as follows:
1. Initially inject a sample into an equilibrated column of affinity chromatography.
2. The substance in which there is an affinity to the ligand, only that substance retained in the column.
3. The substance is eluted from the column in which there is no affinity to the ligand.
4. By modifying the salt or organic solvent concentrations or pH, retained substances can be eluted from the column.
Affinity chromatography is broadly used for the separation and purification with the specific properties.


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What is Affinity Chromatography?

Affinity chromatography is the method by which the interactions of reversible biospecific between molecular species, its legand is utilized in the separation of such molecular species from a biological environment. Affinity is used to tell how two molecules behave with one another. Polar substances have a preference for each other to the substance of non-polar and we generate antibodies with intimacy to antigen for help fight against the infections. Affinity chromatography depends on biological relationships between molecules such as substrates and enzymes there is an affinity for each other. As the method depends on this molecule interaction, it could be separate a molecule based on its individual chemical composition and it is the only method that can execute this.


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Advantages and disadvantages of Chromatography

Chromatography is a technique of physical separations that are applying for the separation and identification of different solutes or components, it consists of the mobile phase and stationary phase, the mobile phase can be a gas or a liquid (Water, Methanol, nitrogen, etc.) whereas the stationary phase is either a liquid or a solid (TLC plate of silica, HPLC column etc.).
Here are some advantages and disadvantages of chromatography.
The Advantages of Chromatography:
  • Chromatography is the simplest method for the separation of components.
  • Chromatography can be controlled by a single person.
  • Components from a complex mixture can separate using chromatography method.
  • The small amount of sample (Gram, PPM, and ng/ml) can be detected by this chromatography.
  • It is a rapid and precise method of separation.
  • Very few sample volume/quantity is required for analysis.
  • It works on a broad range of samples.
  • In some chromatography techniques, it is possible to separate different components of a complex mixture.
  • Continuous operation possible on a large scale
  • The separation of components can be achieved in different methods.
  • Different kinds of chromatography tools are available for the separation of the compounds. Some examples are liquid chromatography, Gas chromatography, High-performance liquid chromatography, Column Chromatography, Solid Chromatography, Liquid-liquid Chromatography, Solid-liquid Chromatography, Thin Layer Chromatography.

 The disadvantages of Chromatography:
  • The chromatography equipment can only be operated by a trained person.
  • Chromatography instruments are expensive.
  • An error occurs due to the overloading of the samples.
  • Chromatography equipment must be handled with care because of these parts are expensive and sensitive.
  • Some of the chromatography techniques require more solvent to separate the analytes.
  • Periodic maintenance and parts need to be changed
  • Some of the chromatography methods require high power consumption.
  • High operational pressure may be required to achieve efficient separation.

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Factors Affecting Retention Time in HPLC

Retention time is the time which is spent by a molecule on the stationary phase after injecting. Retention time is one of the important parameters in chromatography because it is used to describe the chromatographic behavior of a sample component in a column. Several factors can affect the retention time (Rt value) of the molecules and can impact the analysis.
Chromatography has the peaks of separated components and peaks have the particular retention time, area, height, HPLC works on the UV or PDA detectors, it’s also showing the results in peaks. The time required to travels the component to reach the detector through a column is known as its retention time. This time is calculated from the time at which the sample is injected into the time when the chromatogram shows a maximum peak height for that analyte. Different analytes have dissimilar retention times, for a specific compound, the RT will vary it depends on the different factors.
Here are some points of the factors that influence retention time in high-performance liquid chromatography.
  • Insufficient saturation of column
  • Mobile phase composition
  • The polarity of the mobile phase
  • The viscosity of the mobile phase
  • The polarity of the sample
  • Type of column used
  • Column length
  • Column degradation
  • The overloading of HPLC column
  • The dimension of the column
  • The flow rate of analysis
  • The pH of the mobile phase
  • The pH value of the sample
This means that if you are using retention time as a way of identifying compounds, then the chromatographic conditions need to be carefully controlled.


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Factors Affecting Separation in Column Chromatography
Factors Affecting Paper Chromatography
HPLC Principle and Types of HPLC Chromatography
Applications of HPLC
Principle and Procedure of HPLC
HPLC column care and maintenance
Resolution in Chromatography
Advantages and disadvantages of Chromatography
Advantages and Disadvantages of Affinity Chromatography
Factors Affecting Retention Time in Chromatography
Factors Affecting the Resolution in HPLC
Factor Affecting Retention Time in HPLC
Factors affecting column efficiency in column chromatography
Factors affecting column efficiency in HPLC
Factors affecting IR spectroscopy
Factors Affecting Paper Chromatography
Factors affecting resolution in chromatography
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Difference between C8 and C18 column in HPLC

High-performance liquid chromatography is a method used to separate, identify, and quantity of each analyte in the complex mixture using a mobile phase. Both C8 and C18 refer to the bonded face of the alkyl chain and both are used in HPLC separation. There are numerous different kinds of reverse phases there in the market, including C8 and C18 columns. C18 is the most popular one than a C8 column. C8 and C18 are both reversed-phase columns, both columns refer to the alkyl chain length of the bonded phase. The length of the chain affects the hydrophobicity of the sorbent phase and therefore increases the retention time of the component. C18 have the maximum amount of hydrophobicity, since the longer length of the carbon chain, C-18 is extra hydrophobic compared to the reverse phases.

C8 column is used while small RT is desired, if hydrophobicity is low, there is less retention time for non-polar analytes, therefore, the non-polar analytes or compounds separate out more quickly with C8 column. The C8 is select over the C18, in the reverse phase matrix where the degree of hydrophobicity is low. But, the C18 column is more accepted and broadly used because C18 silica gel interacts with the broad range of analytes, hence it used in the separation, qualitative and quantitative studies in the pharmaceutical industries, chemical analysis, and environmental science.

The key difference between the C8 and C18 column is that the C18 column consists of 18 carbons attached to the silica that is a longer carbon chain than C8. Because of the extra carbons chain, the C18 has a wide surface area which offers an additional time of reaction between the bonded phase and the compound. As a result, the compounds are eluted more gradually from the column, and there is more isolation.
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Difference between HPLC and TLC

Chromatography is a method of physical separations that is applying for separate and recognizes analytes from the sample of complex mixtures. HPLC and TLC are the chromatographic techniques; both are useful for the separation of less volatile and non-volatile analytes. The important difference between HPLC and TLC is the quantitative separation of a sample is possible for HPLC, whereas TLC in which there is no quantitative separation of the sample.
Here are some points of difference between HPLC and TLC.
  • HPLC is a high-performance liquid chromatography.
  • TLC is a thin layer chromatography.
  • In HPLC, columns are used in place of plates and partition coefficient or adsorption is the method of separation.
  • In TLC, the fine solid material is spread on a plate, and capillary action is permitted to migrate the mobile phase to the surface of the TLC plate.
  • The liquid mobile phase in the HPLC system pumped with pressure through the HPLC column, which is packed with a stationary phase.
  • The liquid mobile phase in TLC travels with capillary action through the TLC plate which is made of the silica or alumina.
  • HPLC is high efficient than thin layer chromatography.
  • TLC is less efficient than high-performance liquid chromatography.
  • HPLC column is reusable.
  • The TLC plate is not reusable.
  • Very low sample quantity can be applied to TLC compared to HPLC.
  • TLC takes less time for method development than HPLC.
  • HPLC can be connected to Mass spectrometer.
  • HPTLC is the automated form of TLC.
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Monday, February 25, 2019

Applications of IR Spectroscopy in Pharmaceutical Analysis

Infrared spectroscopy is a significant technique in organic chemistry. It is a simple method to identify the functional groups or the structure of the analytes.

Applications of IR spectroscopy in pharmaceutical analysis:

  • Identification of a functional group and structure elucidation:
The range of the group frequency is 4000-1500 cm-1 whereas the fingerprint area is 1500-400 cm-1. By using the fingerprint area in IR spectroscopy an organic compound can be identified. Each functional group absorbs a certain frequency of the radiation and shows the peaks. Hence, the position of peaks provides information regarding the functional group’s presence in the sample components or analytes.
  • Studying the progress of the reaction:
Advancement of the chemical reaction can be studied by looking at the small segment of the reaction mixture from time to time.
  • Identification of the structure of substances:
Infrared spectroscopy is applied to find the structure of the unknown sample compound. From the peak numbers or wave numbers provided by IR, the entire structure of the molecule can be predicted.
  • Quantitative analysis:
By measuring the intensity of each analyte, Quantitative analysis of an organic mixture can be done.
  • Detection of impurities:
Infrared spectroscopy is likewise valuable in the detection of the impurity in a compound by contrasting its spectrum and the spectrum of the standard sample of the compound.

Different applications of IR spectroscopy:

  • Environmental analysis:
In order to overcome health and environmental concerns, FTIR spectroscopy is an important technique for analyzing the quality of water, air, and pollution levels.
  • Food analysis:
Infrared fitted total reflection (ATR system) is used by the manufacturer of food products for the food analysis. This examination is necessities for compliance with food labeling requirements and maintains its quality.
  • Uses for various manufacturing products:
IR spectroscopy technology is used for product development, and quality control of the paint, coatings, plastic, resins, rubbers, fillers, and adhesive manufacturer.
  • Forensics applications:
These techniques can give rapid, simple and reliable analysis, hence it used by the forensic department for the evidence and identification of the substance.

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