Saturday, May 2, 2020

What are the major advantages of HPTLC over TLC?

High-performance thin-layer chromatography (HPTLC) and thin-layer chromatography (TLC) both are planar chromatography methods that are used to separate the analytes on a flat thin layer of stationary phase using mobile phase. The HPTLC is an automated form of TLC. Several enhancements can be made to the conventional method of thin-layer chromatography to computerize the various steps, to increase the resolution obtained and automation is useful to overcome the errors caused by the manual process.
The major advantages of HPTLC over TLC includes,
  • Autosampler makes better than the TLC since it overcomes the manual error
  • Provides high resolving power
  • It reduces solvent consumption
  • More rapidly development of chromatograms
  • HPTLC has a broad range of stationary phase
  • It utilizes UV/VIS or fluorescence detectors
  • Enables simultaneous analysis of multiple compounds
  • In HPTLC the sample spotting by the applicator
  • It is highly prĂ©cised than TLC


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    Why is water not suitable solvent in paper chromatography

    Paper chromatography is a type of chromatography process that used to separate the compounds. It is a technique of planar chromatography in which a special cellulose paper acts as a stationary phase in that the molecules are separated. The principle involved in paper chromatography is a partition in which the components are distributed or partitioned into liquid phases. The same basic principle (partition) involved in column chromatography, liquid chromatography, and thin-layer chromatography (TLC).
    The stationary phase of paper chromatography is cellulose paper which polar and water are also quite polar, therefore there is no distinction by which to separate the molecules. The water is not used in the paper chromatography, due to the water the paper swells up and ultimately dissolves.




    Factors affecting separation in paper chromatography

    Paper chromatography is a method of planar chromatography that uses specialized cellulose paper used as a stationary phase to operate chromatographic procedures. Paper chromatography is working similar to thin-layer chromatography (TLC), it is easy and most commonly used technique due to its applicability for the separation, detection, and quantitative determination of organic and inorganic molecules. The separation of analytes in each type of chromatographic technique is significant to qualitative and quantitative estimation, but the many factors can affect the separation process in paper chromatography.
    Some factors are mentioned here that affect the separation in paper chromatography.
    Mobile phase saturation: Rf values in paper chromatography and TLC mainly dependent on the mobile phase saturation of the atmosphere in the chamber.
    Effect of temperature: The variation of temperature between processes can affect the separation of components.
    Composition of the mobile phase: The appropriate combination of solvents are required to separate the analytes, this can mainly affect the separation
    Quality of paper used: The paper needs to better adsorb and retained the compounds, as the quality of the paper also affects the separation.
    Quality of solvent used: High-quality solvents need to be used each time because low-quality solvents affect the separation
    Sample concentration: A highly concentrated and highly diluted sample cannot separate appropriately.

    Commonly asked questions on paper chromatography are as follows.
    What are the major applications of paper chromatography?
    Paper chromatography is mainly used in analytical chemistry to identify and separate color mixtures and it is also used as a qualitative method.

    Why are two solvents used in paper chromatography?
    Two solvents are used in the mobile phase of paper chromatography because it helps separate complex mixtures of molecules having similar polarity.

    Why is water used as a solvent in chromatography?
    Water is a cost-effective, readily available, act as a polar solvent and is more efficient when used with a non-polar stationary phase.




    What are the types of paper chromatography?

    The principle involved in paper chromatography is partition chromatography. The paper chromatography has a stationary phase made of a special cellulose paper and is called chromatography paper. The mobile phase is either a solvent or combination of solvents. A sample mixture is spotted on a line approximately 2 cm over the base of the stationary phase and then suspended in a chromatography chamber that has the appropriate mobile phase. The mobile phase elevates the paper and flows at the spot through capillary action. The paper retains different molecules according to their affinity into two phases. After the development of chromatograms, the spots of the isolated compounds are apparent from the original location position on the chromatogram at varying heights. This can be seen either by the use of a suitable spray reagent or under ultraviolet light. The relative adsorption of each analyte of the sample is articulated as the retention factor (Rf value).
    There are five types of paper chromatography are available on the basis of how the chromatogram is developed.
    Ascending chromatography: In this type of paper chromatography, the chromatogram is ascending. The development of chromatogram takes place because of mobile phase upward moves on the paper. The mobile phase is at the bottom of the chamber. The tip of the paper with sample spots only dips to the bottom of the mobile phase thus the spots remain just above the mixture of solvents.
    Descending chromatography: In this type of paper chromatography, for the development of paper, the solvent travels to the downwards of the paper. The mobile phase is placed at the top of the system and it moves by gravity.
    Ascending- descending mode: In this hybrid version of paper chromatography, the mobile phase first flows up over a paper folded on rod and begins its travels to downward after crossing the rod. This process allows for long-term development for better resolution of complex sample mixtures.
    Radial or horizontal mode: In this mode of paper chromatography, the mobile phase moves from the center towards the periphery of the circular paper. This is usually done in a covered petri dish for the development of chromatograms. The wick in the middle point of the paper is dipped in the mobile phase, by which the solvent is drained on the paper and radially transfers the sample as concentric rings to form sample spots of various components.
    Two-dimensional chromatography: In this mode of paper chromatography, the chromatogram development arises at right angles to directions of the first run. The sample mixture is spotted in a corner of rectangular paper, followed by the paper for the second chromatogram is again immersed in the solvent at the right angle to the preceding development.

    Commonly asked questions on paper chromatography are as follows.
    What are the applications of paper chromatography?
    Paper chromatography is a type of chromatography that is used for the isolation and identification of colored sample mixtures including pigments.
     
    What are the steps in paper chromatography?
    Steps involved in paper chromatography are preparation of the sample, spotting sample on the paper, development of chromatogram, drying of paper, and detection of molecules.
     
    Which type of paper is used in paper chromatography?
    The selection of the correct type of paper used is significant. Generally cellulose or filter paper is one of the most used as a stationary phase in paper chromatography.
     
    What is the major difference between paper chromatography and TLC?
    The major difference between paper chromatography and TLC is that the paper chromatography uses a paper as its stationary phase and TLC uses a thin layer of silica gel or alumina as its stationary phase.




    Principle and applications of affinity chromatography

    Affinity chromatography is a kind of liquid chromatography that is used for separation and purification of molecules in a particular technique. It uses a reversible biological interaction called affinity which applies to the different degrees of forced attraction used by the atoms, which allows them to remain in combination. Contrasting to other chromatography-based purification techniques, which isolate the molecules based on the size (size-exclusion chromatography) or the ionic interactions strength with a stationary phase (ion-exchange chromatography), affinity purification workings by controlling of properties and particular binding interactions amongst molecules to purify the protein.
    Principle of affinity chromatography:
    Affinity chromatography is one of the most effective chromatographic processes for the separation of analytes from the sample mixture. It depends on particular biological interactions between two analytes, for example, antibodies and antigens, enzyme-substrate interactions, receptor, and ligand. The stationary phase in affinity chromatography consists of a cellulose beads support medium on that the substrate is covalently attached to expose the reactive groups required for enzyme bonding. When the sample protein mixture is passed into the column, the protein binds to the stationary phase with an immobilized substrate site to the stationary phase, while all other sample analytes are eluted at void volume in the column.
    Biomolecules are purified by a purification method that differs according to differences in precise properties as follows.
    Property:                                                         Method:
    Biorecognition (ligand specificity)-Affinity chromatography (AC)
    Charge -                                               Ion exchange chromatography (IEX)
    Size -                                                    Size exclusion chromatography (SEC) or gel filtration (GF)
    Hydrophobicity -                              Reversed-phase chromatography (RPC)
    The different components essential to perform the process are as follows.
    Matrix: It is passive support that can be directly or indirectly linked to a ligand.
    Spacer arm: It is used to increase the binding amongst the ligand and the target molecule by eliminating any satirical hindrance effect.
    Ligand: It refers to an analyte that binds in contrast to a specific target molecule.
    The processes involved in affinity chromatography are as follows. 
    1. Column Preparation:
    2. Equilibration:
    3. Sample Loading:
    4. Elution of Ligand-Molecule:
    5. Re-equilibration:
    Applications of affinity chromatography:
    The major application of affinity chromatography is that it is used for separation and purification of all biological macromolecule.
    Affinity chromatography is an adaptable and valuable separation technique for pharmaceutical and biomedical analysis.
    Affinity chromatography is used to isolate and purify enzymes, antibodies, and nucleic acids.
    It is used in several types of clinical applications.
    Affinity chromatography is used to purify and concentrate on an enzyme solution.
    Affinity chromatography used in analytical chemistry to studying the kinetics of biological interactions.
    This can be used to reduce the number of unnecessary substances in the sample mixture.
    It is often selected method for purification of biomolecules due to its ease of operation, specificity, yield, and throughput.
    It is used for purification and concentrates the substance from a sample mixture into a buffering solution.

    Commonly asked questions on affinity chromatography are as follows.

    What type of chromatography is an affinity chromatography?

    Affinity chromatography is a kind of liquid chromatography, which is used for the purification of particular biomolecules, including proteins.
     
    What is the basis for the separation of proteins by the technique affinity chromatography?
    Affinity chromatography isolates the proteins based on interactions between a specific ligand and a protein.
     
    What is the purpose of affinity chromatography?
    Affinity chromatography can be used to purify and concentrate an enzyme solution, reduce the number of unwanted components in a sample mixture, purify, and concentrate a component from a sample mixture into a buffering solution.




    Principle and applications of gas chromatography

    Gas chromatography is the most widely used analytical method for quality control as well as identification and quantification of molecules in sample mixtures in many research and industrial laboratories. GC is frequently used in many applications of forensic and environmental as it allows for very small amounts of detection. A large range of samples can be determined as long as the analytes are enough thermally stable and are relatively volatile. Depending on the stationary phase used there are two types of GC, such as gas-liquid chromatography (GLC) and gas-solid chromatography (GSC).
    Principle of gas chromatography (GC):
    Gas chromatography is a technique for separating and quantifying vaporized analytes using an inert carrier gas (Nitrogen or Helium). The GC works on the same principles as column permeation chromatography, in which analytes are dissolved in the mobile phase and moved through a porous stationary phase. The basic principle involved in gas chromatography is partitioning, in which the analytes are distributed or partitioned between the two-phase i.e. stationary phase and mobile phase. The analytes are separated based on their affinity, the more affinity will elute slowly while lower affinity toward the stationary phase will elute rapidly from the column.
    Gas-liquid chromatography (GLC) is the most regular form of GC, in that a stationary phase is a non-volatile liquid. The stationary phase can be coated on small inert solid particles packed in a coiled metal or glass column. Most analyzes of GC performed in the early years of GC using this kind of packed column. But, in recent years the capillary columns have been widely used since it has more separation efficiency and sensitivity. The carrier gas, sample injection port, column, column oven, and detector, etc. are the components essential to perform the process of GC.
    Applications of gas chromatography (GC):
    Pharmaceutical applications of GC: The gas chromatography is widely used for the qualitative and quantitative analysis of a sample of formulations and medicinal products in pharmaceutical production.
    Analysis of food, flavors, and fragrances: The quality and quantity of fragrances, flavors, and food products are required to be maintained; hence GC is used to analyze them. It is also used to determine the pesticides, and contaminants from foodstuff, vegetables, and fruits.
    Applications of GC in the analysis of pesticides: The column of GC provides high resolution, efficiency, and exceptional inertness towards neutral, basic, and acidic compounds, therefore, it is appropriate for the analysis of volatiles and pesticides.
    Applications of gas chromatography in academic research: GC-MS is the most powerful technique, it is used for the identification and characterization of molecules, which provide high resolution compared to the HPLC column.
    Environmental applications of gas chromatography: GC is the most generally used technique for the identification of organic molecules in environmental samples.
    Applications of GC in clinical and forensic purposes: In several fields of forensic science the gas chromatography is used, ranging from the analysis of crime investigations, alcohol, and drug abuse. It is also essential in clinical applications to the analysis of drugs in the blood.
    Applications of GC in a variety of industries: Gas chromatography has wide applications in many industries such as the petroleum industry, food industry, beverage industry, pharmaceutical industry, chemical industry, and gas industry, etc. 

    Commonly asked questions on gas chromatography are as follows.
     
    Which factors influence the separation of the components in gas chromatography?
    The separation of the components in GC is affected by the vapor pressure, length of the column, the polarity of analyte versus the polarity of stationary phase on the column, the temperature of column and oven, the flow rate of carrier gas and amount of sample injected.
     
    What is the most commonly used detector in gas chromatography?
    There are many types of detectors are used in gas chromatography such as flame ionization detector (FID), thermal conductivity detector (TCD), mass spectrometer (GC/MS), thermal conductivity detector (TCD)and electron capture detector (ECD), but out of these, the flame ionization detector is most commonly used detector in gas chromatography.
     
    What is the major difference between GC-MS and LC-MS?
    Both GC-MS and LC-MS are separation technique used to separate the compounds. The major difference between GC-MS and LC-MS is that the in GC-MS mobile phase uses as inert gases such as nitrogen or helium, whereas LC-MS uses a solvent or mixture of solvents as its mobile phase.