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?

    Learn about five types of paper chromatography which is according to how the chromatogram is developed.

    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 chromatography: 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 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 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 HPTLC

    HPTLC is an advanced and automated version of thin layer chromatography with superior and higher separation efficiency and detection limits. It is also known as planar chromatography or high-pressure thin layer chromatography. It is a potent analytical technique and suitable for quantitative and qualitative analysis of components. In this type of chromatography, separation can result from the partition or, adsorption, or both, it is based on the type of the adsorbents used for development.

    The basic principle of HPTLC:
    Similar to other chromatographic processes such as column chromatography, HPLC, gas chromatography (GC), and paper chromatography, thin-layer chromatography is also based on the principle of separation. The separation of analytes is based on its relative affinity towards the stationary phase and mobile phase. Under the influence of the mobile phase, analytes are moves (by capillary action) over the surface of the stationary phase. The analytes have a higher affinity toward the stationary phase, they travel slowly while the others elute rapidly. When separation happens, the individual molecules are visualized as spots and we can determine the Rf value and other required parameters.

    Applications of high-performance thin-layer chromatography (HPTLC):
    • Qualitative and quantitative analysis of compounds is the major application of high-performance thin-layer chromatography.
    • HPTLC used in the quality control of pharmaceuticals.
    • It is used to qualitative analysis of components in biological fluids and formulations.
    • For the detection of drugs and metabolites, it is used in pharmacokinetics.
    • The food industry uses HPTLC to estimate vitamins, nutrients, pesticides, and beverages from foodstuff, fruits, and vegetables.
    • High-performance thin-layer chromatography can use in many industrial applications
    • This method is significantly used in the forensic department for identification of substances, including drug abuse adulteration, and counterfeit drugs,
    • It can be used for the stability testing of substance.
    • High-performance thin-layer chromatography can use in environmental analysis.
    Commonly asked questions on chromatography are as follows.
      

    What is the major disadvantage between HPTLC

    The major disadvantage of HPTLC is that it has a higher analysis cost than TLC.


    What are the basic steps involved in the HPTLC procedure?

    The basic steps involved in HPTLC are the selection of the stationary phase, selection and optimization of the mobile phase, preparation of the sample, application of sample, chromatogram development, and detection of the separated sample.


    What is the principle of thin-layer chromatography (TLC)?

    TLC operates on the principle that different molecules have different solubility and adsorption between two phases that separate them.


    What is the major difference between HPTLC and TLC?

    The major difference between HPTLC and TLC is the particle and pore size of the sorbents.


    Principle and applications of HPLC

    High-performance liquid chromatography (HPLC) is a separation technique of analytical chemistry that is used for the isolation, identity, and quantification of compounds present in a sample mixture. It is the most essential technique used in laboratories worldwide.
    Principle of high-performance liquid chromatography (HPLC):
    All types of chromatography, for example, column chromatography, thin-layer chromatography (TLC), and gas chromatography, etc. work on the same principle. They have stationary phase and mobile phase, the solvent moves through the stationary phase and bringing the analytes with it.
    In the principle of HPLC, A sample is injected through the injector in a flow of mobile phase and travels through a stationary phase (column). The analytes in the sample mixture move with the flow of the mobile phase and interact with the solid support. The rate of movement relies on the affinity of the analytes toward the stationary phase. The strongly interacting analytes with the stationary phase travel gradually, while the less interacting compounds elute quickly.
    The separation in the HPLC relies on the interaction of molecules with the stationary phase and mobile phase. Below is a description of the isolation mechanisms which are commonly used in HPLC.
    Normal phase chromatography (NP-HPLC): In this technique the compounds are separates according to polarity, using the non-polar mobile phase and polar stationary phase.
    Reverse-phase chromatography (NP-HPLC): This type of chromatography works on the principle of hydrophobic interactions therefore the more non-polar compound has the longer be retained. In this technique the stationary phase is non-polar and the mobile phase is polar.
    Size-exclusion chromatography (SE-HPLC): In this technique, the separation of compounds depends on the size. The compounds are separated from the stationary phase based on their exclusion from pores.
    Ion-exchange chromatography (IE-HPLC): The ion exchange chromatography is a widely used technique for purification of proteins and some charged molecules. In cation exchange chromatography, positively charged molecules are attracted to a negatively charged stationary. Ion exchange chromatography works in contrast.

    The various applications of HPLC are as follows:
    Environmental applications: The phenolic compounds in water can be determined by HPLC, and also used to monitor the pollutants.
    Clinical applications: it is used for the clinical diagnosis of diseases, disorders, and analysis of antibiotics, bilirubin, and other drugs in the blood.
    Pharmaceutical applications: The high-performance liquid chromatography is used in the quality control, to observe the stability of molecules and analysis of pharmaceuticals.
    Food and beverage applications: The HPLC is used to determination of soft drinks and water quality, analysis of preservatives, sugars, and additives in foodstuffs
    Forensics applications: It is used to find out the steroids, cocaine, and some abused drugs from the urine, blood, etc.
    Some other applications of HPLC include:
    • HPLC is used for analysis in pharmaceutical laboratories as well as scientific research for discovery.
    • It is also helpful in the isolation and purification of chemicals.
    • It can use for the analysis of synthetic polymers.
    • It is used for standards control by the government.
    • It can use the manufacturing of highly pure goods.
    • It can use to control the microbiological process.
    Commonly asked questions on HPLC chromatography are as follows.  

    What are the types of HPLC chromatography?
    There are different types of HPLC chromatography used in the process of separation such as normal phase HPLC, reverse phase HPLC, size-exclusion HPLC, ion exchange HPLC.
     
    What is the principle of HPLC?
    The separation principle of high-performance liquid chromatography (HPLC) depends on the distribution of solute amongst the stationary phase (column) and the mobile phase (solvent mixture).
     
    How many types of detectors are available for HPLC?
    Liquid chromatography uses various detectors such as UV/VIS detector, photodiode array detector (PDA), refractive index detector, mass detector (LCMS), fluorescence detector, electrochemical detector, conductivity detector, light scattering detector, and an infrared detector.
     
    What is the difference between UV and PDA detector?
    The major difference between the UV and PDA detector is that the photodiode array detector can detect the in the different range of wavelengths 200 to 800 nm, while UV detector can detect only at a specific wavelength.

    Principle and applications of paper chromatography

    Paper chromatography is a separation technique in chemistry that is used to separate molecules or substances, particularly colored substances. It is mainly used as a teaching tool, replaced by other chromatography methods, for example, thin-layer chromatography (TLC). It is also used to testing the purity and identifying of molecules. The major advantage of paper chromatography is that it is relatively quick, simple, and requires a small amount of sample.
    The principle of paper chromatography:
    The basic principle involved in paper chromatography is partition chromatography in which the compounds are partitioned. The water that is held in the pores of the filter paper used is one phase and another is the mobile phase that passes over the stationary phase. Under the capillary action, the mobile phase is run over the stationary phase, the analytes are isolated due to differences in their affinity towards the stationary phase. The analytes with a low affinity toward the stationary phase are rapid travels than the analytes of higher affinity.
    The principle may also be adsorption chromatography of solid and liquid phases, in which the stationary phase is the solid paper surface and the mobile phase is the solvent or mixture of solvents. However, the principle of partition chromatography is the most used in paper chromatography. Once the molecules get separated, the Rf value can be calculated, which is used to determine the characteristic of the molecules and to identify the compounds.
    Various essential components involved in a paper chromatography process.
    Specialized cellulose paper, chromatographic chamber, solvent, and detecting or visualizing agents, etc.
    Major steps involved in the process of paper chromatography are.
    1. Choose the appropriate type of development
    2. Choose the appropriate filter paper
    3. Sample Preparation
    4. Sample spotting on the paper
    5. Development of chromatogram
    6. Drying of paper and detection of components

    The applications of paper chromatography are as follows.
    • To analyze the processes of fermentation and ripening it can be used.
    • Forensic and pathology science are also used in this technique, mainly in DNA and RNA fingerprinting.
    • The presence of chemicals or alcohol in the blood can be determined by paper chromatography
    • It is one of the techniques used to isolate amino acids.
    • It is used to detect the presence of impurities beverages and foodstuffs.
    • This technique is mainly useful in separating colored pigments from a complex mixture.
    • Paper chromatography is also used by manufacturers of cosmetics.
    • The reaction mixture can be determined using paper chromatography.
    Commonly asked questions on paper chromatography are as follows.  

    What are the different types of paper chromatography?
    Paper chromatography has 5 types such as ascending chromatography, descending chromatography, ascending-descending chromatography, radial or circular chromatography, and two-dimensional chromatography.

    What is the basic principle of paper chromatography?

    The basic principle of paper chromatography is partition chromatography.

    What does a high RF value mean in paper chromatography?
    A high Rf value in paper chromatography means that the component is very non-polar.

    Why do amino acids travel different distances in chromatography?

    Due to the variations in R groups of amino acids, the different amino acids travel at different rates on the paper.


    Principle and applications of column chromatography

    Column chromatography is a basic and simple type of chromatography technique used for separating and purifying the compounds. It is a kind of adsorption chromatography in which the compounds are separated based on their different affinity to the stationary phase. In column chromatography, generally, silica gel is used as a stationary phase (adsorbent), and solvent or a mixture of solvent used as a mobile phase. Column chromatography has a polar stationary phase. The separation of the compounds based on its polarity since the polar compounds elute slowly and nonpolar compounds travel rapidly through the column.
    The principle of column chromatography:
    The adsorption is the basic principle involved in the separation process of column chromatography. The liquid mobile phase is allowed to travel through a solid stationary phase with the sample mixture under the force of gravity. The analytes of the sample become adsorbed in the stationary phase throughout their travel and they get separated due to differences in affinity of individual analytes. The analytes have low affinity to the stationary phase they rapidly eluted, while those analytes have greater affinity they elute slowly from the column. The solute reversibly adsorbs to the stationary phase. Therefore, with a constant input of the solvent from above all the samples flow out.
    Various essential components involved in the column chromatography process.
    Tubular glass column, stationary phase, solvent, stand with clamp and funnel, etc.
    Major steps involved in the process of column chromatography.
    1. Preparing the column
    2. Selecting the suitable solvent to run the column
    3. Loading the column
    4. Fractions Collection
     
    The applications of column chromatography are as follows.
    • One of the major applications of column chromatography is that it can be used for isolation and purification of many classes of components.
    • Essential components can be separated from plant extracts, formulations, active components, from plant extracts.
    • It is very useful in the removal of impurities from the sample.
    • It can be used to isolate amino acids, glycosides, and alkaloids.
    • This method is used in the purification of reaction mixtures in chemical synthesis.
    • Metabolites from biological fluids such as blood, serum, etc. are isolated using column chromatography.
    • The impurities of the sample can be separated by using the appropriate mobile and stationary phases.
    • This is used for the determination of drugs from formulations.
    • Pharmaceutical manufacturers typically used to purify compounds.
    • It is used for the purification of proteins (biomolecules) for pharmaceutical research.
    • The column chromatography is used to isolate any type of sample mixture.
    • The column chromatography is used to analysis of environmental samples.
    Commonly asked questions on column chromatography are as follows.

    What are the types of column chromatography?
    Based on the stationary phase there are four types of column chromatography are adsorption chromatography, ion-exchange chromatography, partition chromatography and, gel chromatography
     
    What is the most common absorbent in column chromatography?
    The silica gel (SiO2) and alumina (Al2O3) are the most widely used adsorbents in column chromatography. The calcium phosphate, calcium carbonate, starch, and magnesia, are also used as stationary phase in column chromatography.

    What are the factors affecting column chromatography?
    The dimension of the column, the particle size of the adsorbent, the temperature of the column, flow rate of solvent, nature of the solvent used, and pressure on the system, etc.

    How do you pack columns for column chromatography?
    Usually, two methods are used to prepare a column i.e. dry method and wet method.

    Principle and applications of TLC

    TLC is one of the economical, simple, and widely used chromatographic methods in qualitative and quantitative analysis, and is used for separation and analyzes the purity of organic compounds and test the purity of compounds. Thin-layer chromatography is a type of liquid chromatography in which a stationary phase (a thin plate coated with a silica gel or alumina) and a mobile phase (a mixture of solvents) are used to analyze the molecule on a flat surface, and this process works under the capillary action, ambient temperature, and atmospheric pressure.

    The principle of thin-layer chromatography (TLC):
    Thin-layer chromatography also relies on the separation principle, like other chromatographic methods. The separation depends on the relative affinity of the analytes against the stationary phase and mobile phase. The analytes in the solvent mixture move over the surface of the stationary phase. The movement occurs in such a way that analytes that have a high affinity towards the stationary phase move slowly while other analytes move quickly that have less affinity. Consequently, separation of the complex mixture is achieved. When the separation process is complete, the individual analytes from the sample mixture visualized as spots at particular levels on the TLC plate.
    Various essential components involved in the TLC process.
    TLC plates, mobile phase, TLC chamber, filter paper, and TLC pipettes, etc.
    Major steps involved in TLC procedure:
    Saturation: It consists of equilibration the TLC chamber with solvent vapors.
    Spotting: It consists of transferring a small volume of sample to one end of the plate.
    Development:  It is a method of putting the bottom of the TLC plate in a solvent of development.
    Visualization: It is a technique for viewing the distance traveled by molecules along with the stationary phase.

    The various applications of TLC are as follows.
    To check the sample purity: The purity of the compound can be determined by comparing it with the standard. When the impurity is observed, it shows additional spots so these can be easily identified.
    Used in chemistry: Thin layer chromatography is widely used in chemistry to determine the retention factor (Rf value) by the separation and identification of molecules and also used in the determination of cations and anions in inorganic chemistry.
    To identification of analytes: Another major application of TLC is it can use to separate, purification, and identification of natural substances such as glycosides, essential oils, alkaloids, pure oils, steroids, volatile oil, waxes, etc.
    Used in food and cosmetic industries: The thin layer chromatography technique is used to isolate and identify the sweetening agents, colors, preservatives, foodstuffs, and some cosmetic products.
    To separation of biochemical metabolites: To isolate and evaluate the biochemical constituent from blood plasma, urine, and body fluids the thin layer chromatography is useful.
    Used in pharmaceuticals: Many pharmaceutical manufacturers use the TLC technique to identify impurities in a chemical substance as well as pharmaceutical formulations.
    Used to determine the mobile phase: It is quick, easy, requires a minimum amount of solvent to operate, hence it is used to determine a suitable solvent system for column chromatography.
    To analyze the reactions: TLC can be employed to analyze the reaction or rate of reaction and also used to check whether the reaction is completed or not.
    Used in forensic science: TLC is useful in forensics to identify chemicals, including adulteration, poisoning, illegal drugs, and drug abuse.

    Commonly asked questions on TLC chromatography are as follows.
     
    What is the basic principle of TLC?
    The TLC, paper chromatography, and high-performance thin-layer chromatography (HPTLC) are works on the same basic principle that is adsorption.

    What are the three major applications of thin-layer chromatography?
    The pharmaceutical, clinical, and food testing applications are 3 major application of TLC.

    Why is TLC better than paper chromatography?
    TLC provides better separations of analytes as compared to paper chromatography because as the stationary the TLC uses silica or alumina and paper chromatography uses cellulose paper.