Tuesday, October 16, 2018

Different types of Column Chromatography

Learn the four major types of column chromatography used to separate different types of samples.

The column chromatography is a type of chromatography it is one of the most effective separation techniques. Stationary phase (adsorbent) either silica gel or alumina is used and filled from the top of the column. The mobile phase is either solvent or a combination of solvents, passed along the sample through the stationary phase. The separation of the components depends on their polarity, as the polar compounds travel slowly through the column. This means that polar compounds absorb more strongly than non-polar.
Based on the stationary phase, there are four types of column chromatography available, and they are working on a different mechanism.
There are different types of column chromatography as follows.
1. Adsorption chromatography.
2. Ion-exchange chromatography
3. Partition chromatography
4. Gel chromatography

1. Adsorption chromatography:

Adsorption chromatography is based on the interaction on active sites and polarity of solid molecules and analytes at the stationary phase. Since the polarity of the stationary phase than the mobile phase is more polar, then the high polar compounds will remain firm until the stationary phase, while the low polar compounds will be tied less than the stationary phase. Compared with firmly bound compounds, the less-bound compounds will be eluted by the first mobile stage, so this technique proves that polar like polar.

2. Ion-exchange chromatography:

Ion-exchange chromatography is a method that permits ions and polar molecules to be separated on the basis of their charge. It can be used for about any type of charge molecule contains large amino acids and proteins. It is regularly used in quality control, water analysis, and protein purification.

3. Partition chromatography:

components are distinguished between the stationary and mobile phase, In the Partition chromatography, the stationary phase and the mobile phase both are liquid.

4. The gel chromatography:

The gel chromatography column is packed with gel in which the separation takes place. The stationary phase of gel chromatography is a solvent. 

Types of Column Chromatography

Commonly asked questions on column chromatography is as follows.

Which principle is involved in column chromatography?
The adsorption of analytes of a sample through a stationary phase and separating the mixture into individual components is the basic principle involved in column chromatography.

What is the main advantage of column chromatography?
All types of the complex sample mixture can be separated using column chromatography, which is the major advantage of column chromatography.

Which compound elutes first in column chromatography?
First, there is a non-polar molecule has eluted from the column, since the non-polar molecules travel rapidly through the stationary phase.

What are the 4 main types of chromatography?
The four primary types of chromatography are liquid chromatography (HPTLC), thin-layer chromatography (TLC), gas chromatography (GC), and paper chromatography.

What are the major applications of column chromatography?
The applications of column chromatography consist of isolation compound mixtures, active ingredients, identification of solutes, and used to remove impurities.


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What are the Advantages of HPLC over GC

High-performance liquid chromatography and Gas chromatography both are the separation techniques for the components in analytical chemistry and they are well established from overtime. Each technique has many advantages and some disadvantages over each other.
Here are mentioned some advantages of HPLC over GC.
Mobile Carrier Phase:
Gas chromatography evaporates the sample and it is taken with the system by an inert gas like nitrogen or helium. The use of hydrogen gas gives improved efficiency and separation. But, due to the flammable nature of gases, many laboratories prevent the use of these gases. While using liquid chromatography, samples are solid or liquid forms and travel through high pressure through solvents or mobile phases such as methanol, acetonitrile, and water, etc. through the column. The solvent composition can be modified as per the requirement of samples / complex mixture of components gives better separation, but in GC a gas passes through the column at constant proportion. 
Column Types:
The gas chromatography column has an extremely small internal diameter and 10 to 45 meters in length. These are silica-based columns and it can be heated up to 150 C. Columns of HPLC are also made up of silica-based covered with stainless steel outer surface and are up to 50 to 250 cm in length, These columns are used under ambient temperate on high pressure. 
Compound Stability:
In gas chromatography, the sample is introduced into the injector and vaporizes it at a specific temperature and travels through the column. Thus, the compound should be capable of withstanding the heat at high temperatures without degradation. On the contrary, in the high-performance liquid chromatography system, you allow the analysis of stable or less stable compounds because the heating to sample is not necessary. 
Sample Requirement:
Samples by GC required volatile compounds but in HPLC both volatile and nonvolatile samples are analyzed.


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Advantages and disadvantages of paper chromatography

Paper chromatography is the oldest technique of separation of analytes from the sample; it is applied particularly in the analysis of lipid samples and chemical compounds. In paper chromatography, a small dot of sample applied to the piece of paper using capillary, the edge of the paper is deep in a solvent and it runs the solvent up through the paper by the capillary action. Separation of sample compounds can depend on the structure, molecular size, weight, and polarity of the compound.
The advantages of paper chromatography are as follows:
  • It requires fever quantitative material. 
  • Separation of compounds in a short time. 
  • Analysis requires a low amount of sample. 
  • Compare to other chromatography methods paper chromatography is a cheap technique. 
  • Organic as well as inorganic compounds can be identified by paper chromatography method. 
  • The setup of paper chromatography occupies less space than the other chromatographic method. 
  • Easy to handle and setup. 
  • The less sample quantity required for the analysis. 
  • Cost-effective method. 
The disadvantages of paper chromatography are as follows:
  • Volatile substances cannot be separated using paper chromatography techniques. 
  • Paper chromatography cannot be compatible with large amounts of sample. 
  • Quantitative analysis is not useful in paper chromatography. 
  • Paper chromatography cannot be separated complex mixture. 
  • As compared to the HPLC, TLC or HPTLC, paper chromatography has less accuracy. 
  • Data cannot be saved for long periods 


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What are the advantages of HPLC over TLC

High-performance liquid chromatography is the heir of thin-layer chromatography method and has contributed significantly to the analytical science development and its different applications in various fields like pharmaceuticals, clinical analysis, environmental analysis, polymers, and plastics. Each technique has its importance and utilities. It fulfills each other and all rely upon your necessities. Both techniques are used in the separation of drug products or analyte.
Here are mentioned some special highlights of HPLC, which makes the HPLC better than TLC, Let’s check some Advantages of HPLC over TLC.
Speed of analysis:
Mobile phase progression on a plate in TLC separation is dependent on capillary action or gravitational force. However, the flow of the mobile phase through the column in HPLC is speedy because it is pushed with the help of a pump.
Resolution:
TLC resolution makes it difficult to distinguish between overlapping spots and bands. On the contrary, in HPLC, it can be separated by controlling operational parameters such as mobile phase pH, mobile phase concentration, column temperature, and flow rate.
Sensitivity:
HPLC has a variety of detectors such as UV, photodiode array, mass, electrochemical, fluorescence, conductivity, and refractive index detectors, so it is a highly sensitive method. Such types of detectors are not available in the TLC analysis.
Quantification of results:
The HPLC programming is able to show exact and precise results based on the area of the peak since it’s suitable for quantitative analysis. But in TLC, Quantitation depends on the intensity matching of the spot which is not giving accurate and precise results.
Choice of stationary phase and columns:
Stationary phases are accessible in a large number, according to different categories of applications. Likewise, varieties of columns with different specifications are available in HPLC chromatography.
Reuse of Columns:
New TLC plates are required every time of TLC analysis, but in HPLC, columns can be reused for various components or complex blends.
Software:
HPLC is a computerized system, so it’s run on software and depends on peak area; it gives accurate and precise reports. It produces and saves the record of the chromatogram and all parameters, like mobile phase composition, flow rate, temperature, and wavelength, etc. are controlled through the software which is not possible in TLC analysis.

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Various advantages of HPTLC over HPLC

Both HPTLC and HPLC are well-recognized for the separation of analytes and for qualitative and quantitative analysis and they have many advantages.
Here are some of the advantages of HPTLC on HPLC as follows: 

  • Preparation of the sample is simple.
  • Maintenance costs are low. 
  • The whole spectrum can be seen. 
  • Many analysts can work together on the same system. 
  • Consumption of solvents per sample is very low. 
  • Solvents do not need any prior treatment, such as filtration and degassing. 
  • There is no need to clean the sample. 
  • There is no effect of the turbid sample on analysis. 
  • Using the freshly prepared mobile phase, there is no chance of interference from the previous analysis.
  • Analytical grade solvents are suitable. There is no need for expensive solvents. 
  • UV absorption and Corrosive mobile phase can also be used. 
  • High flexibility in the different steps, such as the selection of mobile phase, stationary phase, and developing techniques. 
  • More basic pH can be used in a mobile phase, which is not suitable in HPLC. 
  • An analytical requirement is least. 
  • Technically simple, no skilled person is required to learn and operate. 
  • Precoated HPTLC plates are available in low cost. 
  • The solution of the solvent is not extremely significant for sample solubility because it is completely removed prior to developing chromatogram. 
  • Sampling and standard can be processed simultaneously under the same condition provide better analytical accuracy and precision.


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What are the advantages of HPTLC

HPTLC is a widely accepted analytical technique and applies to a broad variety of analytes in various types of matrix. High-performance thin layer chromatography is one of the most broadly used methods for the pharmaceutical analysis, food and drug analysis, environmental analysis, cosmetic products, forensic chemistry, biochemistry, clinical chemistry, and other fields. It is the only chromatographic method which gives the option to present the result as an image.
Advantages of HPTLC (High-performance thin layer chromatography) include the following.
  • Technically, learning and operating is easy.
  • Many analysts work together on the system.
  • Low Analysis Time and Low-cost Analysis.
  • Low maintenance cost compared to other instruments.
  • Visual recognition is possible because it is an open method.
  • Availability huge ranges of stationary phases.
  • UV-absorbing and corrosive mobile phases can be working on this system.
  • Filtration and degassing for solvents are not necessary. 
  • Due to the use of the freshly prepared mobile phase and stationary phase, this does not have any chance of interference or contamination.
  • Samples cleaning not often require. 
  • Sensitivity range of analysis usually occurs at the pictogram (pg) and Nanogram (ng).


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Applications of HPTLC in Pharmaceutical Analysis

High-performance thin layer chromatography is an advanced form of thin layer chromatography. Various improvements can be added in the basic method of thin layer chromatography to automate various stages, to raise the achieved resolution and allow for more precise quantitative analysis. The applications of HPTLC (high-performance thin layer chromatography) are very huge, since any compound, which can be analyzed by TLC analyze using HPTLC techniques. Also, because the technique is very sensitive and small sample volume is enough to detect and estimation of analytes, using HPTLC different applications are possible in many areas.

Here are mentioned some applications of HPTLC (high-performance thin layer chromatography).
  • Qualitative analysis of analytes in biological fluids and formulations.
  • Assay of active components and multi-components analysis.
  • Content uniformity in a dosage formulation.
  • Presence of impurities in the drug.
  • Stability testing and forced degradation studies.
  • Phytoconstituents in plant extract.
  • Quality control/authentication.
  • Phytochemical analysis of volatile oils.
  • Used in pharmacokinetics and metabolism to detect drugs and metabolites.
  • Forensic applications
  • Cosmetic analysis
  • Food analysis
  • Environmental analysis
  • Industrial application
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What is the basic principle of HPTLC

HPTLC is TLC based analytical technique of separation; hence, adsorption is a principle of HPTLC, Where the solid stationary phase and the liquid mobile phase are used. The stationary phase is either coated on glass plates or aluminum foil of different sizes. If the plates are not freshly opened, they need to be activated before the application of the sample, using an Autosampler, a small amount (μl) of the sample or samples are applied to the bottom edge of the plate. By placing in a development chamber of HPTLC, this plate is developed, while the mobile phase travels up against gravity of the stationary phase up, separation of compounds due to the difference in relation to different compounds towards the stationary phase. The required time for the separation of the compound is called as development time, for the better separation, normally a distance of approximately 5 cm-15 cm is used. Separating spot can be seen using the densitometry scanner UV or visible or fluorescence detectors, it can be based on the properties of the components. Both qualitatively and quantitatively analysis can be done by using this method.


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What is HPTLC Chromatography

High-performance thin layer chromatography (HPTLC) is a sophisticated, enhanced, and computerized form of thin layer chromatography. It is also known as flatbed chromatography, As compared to thin layer chromatography (TLC), it provides high-performance separations, there is different levels of operation at each step like Sample applications on HPTLC plates by auto applicants, selection of pre-coated stationary phases, Detecting compounds using different detection like UV, visible or fluorescence detectors and software used for data processing. All these benefits compared to TLC make HPTLC a better, high-performance and expensive technology.
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Advantages of TLC over Paper Chromatography

TLC is a chromatographic method of separation of the analytes or components in the mixture of samples. TLC consists of two phases, one mobile and one solid phase, in which the TCL plate acts as a stationary phase.
Paper chromatography is the types of chromatography, which are used qualitatively for the separation and identification of sample components, or analytes, that can be colored or especially pigments. It runs on a piece of paper that acts as a stationary phase on which the separation occurs. 
Thin-layer chromatography is parallel to paper chromatography, TLC and paper chromatography as compare with each other, The TLC has a wide range of adsorbent, depending on sample and development. After development sample can be easily separated from TLC. 
Thin-layer chromatography offers many advantages over paper chromatography, some of which have been mentioned here. 

  • Time-saving method.
  • The sensitivity of detection is more in TLC.
  • Rigid support.
  • The option of support.
  • Simple Setup.
  • The amount of the sample application is in minute quantity (in microlitre).
  • Strong reagents can be used for the purpose of identification.
  • Choice of spray reagents.
  • Higher sample throughput and lower analysis time.
  • If required for spot development, the thin-layer chromatography plates can be heated.

In short, the thin layer, chromatography provides several advantages for separation than the paper chromatography. 


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Advantages and Disadvantages of Thin Layer Chromatography

Thin Layer Chromatography (TLC) is an analytical technique of separation which is used to qualitative analysis and observes the reaction of complex mixtures of analytes and also for identifying the unknown compounds. It is also significant to determine the right solvent system with which to implement in the column chromatography.

Advantages of Thin Layer Chromatography:
  • This is a very easy way to separate the components.
  • TLC is a sensitive method.
  • In comparison to other separation techniques, very few types of equipment are used. The components are separated in a very short time because the components will elute rapidly.
  • It is feasible to visualize all components of UV light.
  • The non-volatile compounds are separated by the TCL method.
  • The only small sample size is required in TLC, and it can be in microlitre.
  • A comparison with standard material, tentative identification is possible.
  • The components there in the complex mixture of samples are able to easily separate and recovered by scratching the plate.

Disdvantages of Thin Layer Chromatography:

  • There is a no longer stationary are available in TCL plates Therefore, its separation length is insufficient in comparison to other chromatographic techniques.
  • Results obtained from TLC are difficult to reproduce.
  • Only soluble components of the mixtures are possible.
  • Only qualitative analysis is possible, not quantitative.
  • Usually, it is not automatic.
  • TCL works in the open system, therefore, due to temperature and humidity can affect the results.


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Applications of Thin Layer Chromatography in Pharmaceutical Analysis

Applications of thin layer chromatography in pharmaceutical analysis contain purity testing, identification, stability testing, assay, and content uniformity testing of intermediates, raw materials, and drug products, with the analysis of sample analytes.
TLC is dependent on its unique detection, the speed of analysis, flexibility, and simplicity methods on together quantitative and qualitative basis. Often degradation products, synthetic intermediates, and process related impurity they do not have chromophores which cannot be detected by the UV detector. Thus, these types of impurities are frequently specified by the TLC analysis. Sometimes impurities are eluted on the solvent front in the HPLC, and they may be complicated to quantities and monitor, and modify in the mobile phase or HPLC column could not be able to solve them adequately.
On the other hand, occasionally the impurities have taken more time to elute from the column, and they can’t be detected, but in TLC method is open in which whole samples are evaluated. Thin layer chromatography use in the early stage of drug development.
Here are some applications of thin layer chromatography mentioned. Let’s check it.
  • Separation of multi-component pharmaceutical formulations is one of the most significant applications of TLC. 
  • TLC is also used to identify the components of traditional medicines.
  • Thin-layer chromatography is applied to identify the nature of various plant compounds.
  • The reaction mixture in the synthesis of chemical compounds can be detected by this method.
  • TLC can use for the identification of cations and anions in inorganic chemistry.
  • Preservatives, sweetening agents, colors, and various cosmetic ingredients are separated and identified using this technique.
  • TLC is a method for separating, analyzing, and characterizing metabolites and compounds in blood, urine, serum, and body fluids.
  • It is used in the food and cosmetic industry to identify drug residues.
 
 
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Basic Principle of Thin Layer Chromatography

In Thin layer chromatography (TLC) the analytes or sample mixture is applied as a minute dot or spot at the bottom of the stationary phase it holds up on a glass, metal or plastic plate. A solvent of the sample is allowed to evaporate from the plate, which is after that, positioned in a closed chamber, in which at the bottom there is a shallow pool of the mobile phase. Mobile phase goes through capillary forces through the stationary phase. Components of the analyte mixture travel at the different rates throughout the movement through the stationary phase of the mobile phase While the mobile phase has taken a suitable distance, from the chamber plate is removed and the marked the solvent front and through drying at room temperature the mobile phase is evaporated from the plate or by placing an oven. If the mixture components are not colored or fluorescent naturally, an identification reagent is applied to see the bands. To ensure, the use of one or more identification techniques to detect all the components in the mixture is done. Rf value is a suitable method of expressing the position of an analyte on a establish chromatogram and it is calculated as, Rf ¼ Distance of the analyte from the origin/solvent distance from the origin.
Different kinds of solvents are used as stationary phases in TLC; consist of silica gel, chemically modified silica gel, ion exchangers, polyamides, alumina, and cellulose. Now, pre-coated high-performance TLC plates with fine particles are generally used in the pharmaceutical industry for reproducible, fast, and efficient separations. The options for the mobile phase are from a solitary component solvent system of many component solvent systems, and the multiple component solvent systems are the most common ones. The movement of every analyte in the mixture of mobile phase throughout TLC is the result of multi opposite forces, for example, the mobile phase capillary action and the stationary phase retardation action. Both forces give towards achieving the migration of each analyte.
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Introduction to Thin Layer Chromatography

Chromatography is intended to use to separate the analytes from the mixture of substances. Each type of chromatography works on the similar principle, and each all has mobile phase and stationary phase, Passes the mobile phase throughout the stationary phase and travel the sample components mixture with it.
The Thin layer chromatography (TLC) is a popular and broadly used technique of separation due to the cost efficiency, simple to use, rapidity of isolation, ability to analyze many samples concurrently as well as the high sensitivity. TLC is used for separation, quantification of components from the mixture, identification and the isolation of analytes and it can also be used to separate a particular component on a preparative scale, it has been used in chemistry, toxicology, biochemistry, food science, environmental science, and in pharmacology discipline, a large variety of TLC equipment is available commercially in the market.


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Monday, September 24, 2018

Definition of Buffer Capacity of Buffer

Buffers are compounds that resist the pH modify when a small number of acids or bases adding to the buffer. The buffer is typically prepared for a weak base or weak acid and its conjugate salt.
Buffer capacity has calculated the efficiency of a buffer in opposing changes in pH. Or Buffer capacity is the number of acids that are able to absorb buffer strong acid before breaking the ability to add strong acid.
The buffer capacity is articulated as the strong acid or base quantity in gram-equivalents to change pH by an entity that should be added to 1 liter of solution. When adding strong acids, there are buffer capacities in weak base solutions and a high amount of weak acid are high levels of buffer capacity when adding a strong base. The basic behind the buffer is to remain pH in a narrow range of the solution.


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