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Thursday, April 23, 2020

What is the difference between LC and HPLC?

High-performance liquid chromatography (HPLC) is a widely used separation technique for the qualitative and quantitative analysis of compounds. HPLC, also known as high-pressure liquid chromatography, it is an advanced version of LC. In HPLC, we inject the sample solution through the syringe into the injector, the mobile phase carries it through a stationary phase (column) that isolates the molecule based on its affinity.HPLC has a wide range of applications, such as pharmaceuticals, forensics, clinical, and food analysis.
The major difference between traditional LC and HPLC is that the mobile phase of liquid chromatography moves by the gravity force while the mobile phase of HPLC flows through the high pressure that is created by the pump, which decreases the analysis time.

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Why silica gel is used as a stationary phase in column chromatography?
Advantages of TLC over column chromatography
What is the stationary phase in column chromatography?
What is the stationary phase in thin-layer chromatography?
What are the types of paper chromatography?
Factors affecting separation in paper chromatography
Why is water not suitable solvent in paper chromatography
What are the major advantages of HPTLC over TLC?
What is the principle of chromatography technique?
Classification of chromatography based on the mechanism of separation
What are the uses of chromatography?
Principle and applications of TLC
Principle and applications of column chromatography
Principle and applications of paper chromatography
Principle and applications of HPLC
Principle and applications of affinity chromatography

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Sunday, April 12, 2020

What is the stationary phase in thin-layer chromatography?

Thin Layer Chromatography is a type of chromatography that is used to separate the non-volatile molecules. The separation process of TLC is executed on the sheet of aluminum, plastic, or glass. This sheet is usually formed by a thin layer of silica gel, aluminum oxide, or cellulose and acts as a stationary phase. The analytes are move by the mobile phase in the capillary action and they separate based on their affinity towards the stationary phase.
The uncoated silica gel is one of the most commonly used stationary phases in thin layer chromatography (TLC). Silica gel is a kind of silicon dioxide. The silica gel remains the significant adsorbent for different types of chromatography, such as high-performance liquid chromatography (HPLC), column chromatography, high-performance thin-layer chromatography (HPTLC), and thin-layer chromatography (TLC). The significant benefit of silica is its incredible affinity for adsorption. Silica gel is a slightly acidic and polar adsorbent in nature that has a high ability to adsorb basic compounds. The alumina also used as a stationary phase in TLC since it has a high ability to retain acidic components because it is slightly acidic. This is ideal to isolate the weak or moderately polar components.


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What is the stationary phase in column chromatography?

Column chromatography is a type of chromatography used to separate and purification of sample components. It is a solid-liquid method where the solid stationary phase and mobile phase is a liquid that is used in the process of separation. The principle of column chromatography is based on the differential adsorption of the analytes by the adsorbent. 
The stationary phase of column chromatography is the adsorbent, generally it is a solid (adsorption) or a liquid (partition) material used in the stationary phase depends on the compounds want to separate. Generally, silica, alumina, calcium phosphate, calcium carbonate, starch, and magnesium are used. Solvent or mobile phase selection is dependent on both the solvent and the adsorbent nature. The rate of separation of the analytes of a sample mixture depends on the solvent polarity and activity of the adsorbent. If the adsorbent activity is high and the polarity of the solvent is low, the separation is very slow however providing a good separation.
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Advantages of TLC over column chromatography

The thin-layer chromatography and column chromatography both are the types of chromatography that are used to separates the compounds. The basic principle of TLC and column chromatography is the adsorption of the solutes. The column chromatography and TLC are one of the most commonly used techniques in analytical techniques since they have many advantages and applications. Both techniques have the same stationary and mobile phases hence the TLC is used to find an appropriate mobile phase or solvent system and Rf value.
Here are mentioned some advantages of TLC over column chromatography.
  • The Rf value in TLC can be easily calculated.
  • TLC has a broad range of solvents than column chromatography.
  • The results of thin-layer chromatography have more specific individual spots; therefore it becomes easier to differentiate them.
  • TLC required fewer amounts of solvents to separate the analytes than the column chromatography.
  • The molecules in TLC have to travel distance less as compared with column chromatography, hence it saves time.
  • The preparation of samples in thin layer chromatography is easy as compared to column chromatography, the column chromatography required a large amount of sample.
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Why silica gel is used as a stationary phase in column chromatography?

Column chromatography is the most efficient technique of separating and purifying components, in which the solid stationary phase and a liquid mobile phase used. In the process of separation, the column is filled with silica gel, a sample compounds placed on top of the column, and then the mobile phase moves from the solid support under the force of gravity. Depending on differing affinity of molecules to the stationary phase they are isolated and separated into fractions.
Silica gel as a stationary phase is largely accepted as one of the top adsorbents used in column chromatography as well as other separation techniques. One of the major advantages is its tremendous affinity for adsorption. Additionally, it is commercially very readily available in several different sizes and types and there is a huge of information and research that is given by producer on its many uses, particularly in chromatography. The major significant reason for silica gel used as a stationary phase in column chromatography is that it has feasible to obtain the extract essential size of the particle size for a particular method.
Silica gel is a polar adsorbent that is slightly acidic and has a strong capacity to adsorb the basic substance. Alumina is slightly basic and has a strong capacity to retain acidic molecules. This is better to separate the analytes that are moderately or weak polar. The silica gel is most widely used in the reversed-phase partition chromatography and it has broad applications that consist of separation of steroids, amino acids, lipids, alkaloids, and several pharmaceutical processes.
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Advantages and disadvantages of volumetric titration

Titration is a method used to determine the concentration of an unknown sample solution by a recognized concentration of solute. The titrimetric method includes three types such as volumetric method, gravimetric titrimetry, and coulometric titrimetry. The volumetric titration used to determine the volume of solution with the known concentration involving quantitative reaction with a substance solution to be analyzed.
The advantages of volumetric titration are as follows.
  • The major advantage of volumetric titration is that it is a simple and cost-effective method.
  • Volumetric titration is not sophisticated, so skill is not required to handle it
  • The volumetric titration is rapid and gives accurate results.
  • It is a simple method compared to other types of titration.
  • The reaction can identify visually at equilibrium or endpoint.
The disadvantages of volumetric titration are as follows.
  • Certain factors such as pH, temperature, and humidity may affect the titration results, as this is an open system.
  • Human error may occur during processing and may affect accuracy.
  • It requires an indicator for the reactions to occur.
  • The major disadvantage of volumetric titration is that this can produce large volumes of chemical waste and that needs to be disposed of it.
  • It requires reactions occurring in a liquid phase.
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What is the principle of volumetric analysis?

Volumetric analysis is a sub-branch of a quantitative analytical method that is broadly used. This process can precisely determine the quantity of a substance in terms of volume, thus the name volumetric analysis. For this type of analysis, a solution of a substance that is to be calculated is taken and a known quantity of another substance whose concentration is determined. When the chemical reaction is completed, the volume of the solution is measured.

The endpoint of this reaction is specifying by a change in precipitation or color etc. So in this measurement process, the estimated volume of two analytes is taken and the concentration of one of them is recognized then conveniently measured the concentration of the other.

The basic principle of volumetric analysis is as follows.
  • The sample solution to be analyzed have unknown amounts of the substance.
  • The solution that is of unknown concentration reacts with an unknown amount of compounds in the presence of an indicator to indicate the endpoint of the titration.
  • Volume is measured by titration that completes the reaction between the solution and the reagent.
  • The volumes of the solution are determined by titration that completes the solution and reagent reaction.
  • Reagents quantity and concentration used in the titration indicate the volume of solution and reagent.
  • The quantity of unknown substances in the specific solution volume is calculated by the equation mole fraction.
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Advantages and disadvantages of conductometric titration

Conductometric titration is used to determine the electrical conductivity of the solution. This process will calculate the concentration of the sample by adding titrant until reaching the endpoint. It is observed by measuring the conductance of the solution. Conductometric titrations are used in analytical chemistry to measure the progress of chemical reactions.
The advantages of conductometric titration are as follows.
  • As compared with other titration methods, the endpoint of the conductometric titration is much faster and more precise.
  • Very dilute solutions and weak acids can be used in this titration.
  • It is used with solutions (turbid/colored) that endpoint cannot be seen with the naked eye.
  • If there is no suitable indicator is available in titration, conductometric titration can be used.
  • Conductometric titration gives a more accurate end-point.
  • It is applied in the titration of the acid-base, precipitation, and redox.
The disadvantages of conductometric titration are as follows.
  • It is not possible to measure high concentrations by this method.
  • This is less accurate when total electrolyte concentrations are higher.
  • A sample solution must be diluted for measurement.
  • The salt levels can change the conductivity of the solution.


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Difference between conductometric and potentiometric titration
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Advantages and disadvantages of fluorometry

Fluorometry is an analytical device for fluorescence determination and measurement in compounds that use ultraviolet light in compounds. It is an analytical method for detecting and measuring fluorescence in molecules that use fluorescence light in a molecule. Fluorometry technique is mostly applicable to low concentration ranges and is consequently sensitive analytical methods compared to spectrophotometric determination. Typically, fluorometric methods have sensitivities that are two to four orders of magnitude and higher compared to spectrophotometric procedures.
The advantages of fluorometry are as follows.
  • The major advantage of fluorometry is its high sensitivity compared to other absorption methods.
  • Fluorometry has a high specificity due to unique optical properties compounds.
  • This will measure the intensity of fluorescence, and sample concentration.
  • It is more precise on the characteristics of wavelengths of excitation and emission.
  • It is more precise than the absorption process because both emission and excitation wavelength are characteristics.
  • Fluorometry has a wide range of concentrations.
  • It is a user-friendly and economical method.
The disadvantages of fluorometry are as follows.
  • The major disadvantage of the fluorescence method is that it only analyzed fluorescent molecules, not all.
  • Rapid scanning is not possible to obtain the excitation and emission spectrum of the analyte.
  • Reference and sample solution cannot be analyses at a time.
  • It is not useful in identification.
  • The dilute sample solutions of compounds are less stable.
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