Advantages and Disadvantages of HPLC

High-performance liquid chromatography is one of the most popular and widely accepted separation techniques of chromatography, in which the mobile phase containing a sample of complex mixture pumped with a certain pressure through the column, which already filled with the adsorbent material, analytes interact with the adsorbent according to their properties and detector detects the retention time, area, and height of the peak. Compared to other technologies, HPLC has both advantages and disadvantages, let’s check it.
The advantage of HPLC:

• HPLC offers a rapid, automated and highly precise method to recognize certain chemical components in a sample.

• High-performance liquid chromatography offers a fast and precise quantitative analysis.

• A gradient solvent system can be applied in certain methods.

• It is highly reproducible.

• HPLC can be upgraded to mass spectroscopy (MS).

• The HPLC is very rapid, efficient, and delivers high resolution as compared to other chromatographic techniques, such as TLC, column chromatography, and paper chromatography.

• Manages all areas of analysis to increase productivity

The disadvantage of HPLC:

• HPLC can be an expensive method, it required a large number of expensive organics, needs a power supply, and regular maintenance is required.

• It can be complicated to troubleshoot problems or develop new methods.

• The lack of a universal detector for HPLC, however, the UV-Vis detector only detects chromophoric compounds.

• The separation in High-performance liquid chromatography has less efficiency than GC.

• It is more difficult for the beginner.

• HPLC pump process reliability relies on of cleanliness of the sample, mobile phase, and proper operation of the system.

 

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Applications of HPLC

HPLC is an analytical technique to separate, identify, and quantify components in a mixture. It is the single leading chromatography technique essential to most laboratories worldwide.
The use of HPLC can be done in both qualitative and quantitative applications, which are both for the identification and quantification of compounds. About all HPLC separation can be done in the reverse phase HPLC, Normal phase is not regularly used. The High-performance liquid chromatography can be applied in the pharmaceutical, drug science, molecular weight determination, environmental chemistry, in analytical chemistry forensic, food industries, and clinical tests.
Let’s check some applications of HPLC.
Pharmaceutical Applications of HPLC:

• The tablet dissolution study of the pharmaceutical dosage form
• To control the stability of the drug
• The HPLC is used for drug quality control
• The qualitative and quantitative study of analytes
• It can be used to the mass determination by coupling it with mass spectroscopy (MS)

Applications of HPLC in Food and Flavour:

• Analysis of preservatives
• Analysis of sugar
• Measuring soft drinks and water quality
• Analysis of polycyclic

 Forensic Applications of HPLC:

• It is used to determine the cocaine and other drugs of abuse
• The quantity of drug in the biological samples
• The HPLC is used for the identification of steroids in urine and blood

 Environmental Applications of HPLC:

• The HPLC is used to finding phenolic compounds in drinking water
• The HPLC is used for the identification and determination of compounds in drinking waters, air, waste, and the surface
• It used for the determination of Bio-monitoring of pollutants.

Clinical Applications of HPLC:

• Quantitative estimation hemoglobin
• Analysis of urine
• Analysis of antibiotics in blood
• Analysis of Bilirubin

 

 

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Applications of High Performance Liquid Chromatography

High-performance liquid chromatography (HPLC) is a chromatographic technique in analytical chemistry used to separate a complex mixture of analytes, in order to identify and quantify of each component in the sample. For the identification and quantification of compounds, the HPLC technology is used. There are many applications, both in normal and reverse phase HPLC, RP-HPLC is not useful for the separation of polysaccharides, inorganic ions, and polynucleotides. Inorganic ions separated by ion exchange chromatography, whereas polysaccharides are hydrophilic in nature, so they are rapidly absorbed in the solid phase. Reverse phase HPLC can be used for the separation of different compounds such as amino acids, aromatic hydrocarbons, peptides, proteins, lipids, and sugars. High-performance liquid chromatography is also used in the medical, analysis of pharmaceuticals, forensic, food industries, clinical tests, environment, and Pharma manufacturing units.

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Factors affecting buffer capacity

Buffer capacity is also known as buffer efficiency, buffer value, buffer coefficient, and buffer index. Buffer capacity of a solution is determined the magnitude of resistance to a pH in adding to an acid or base. Buffer capacity relies on the concentration of the buffer components. The highest concentration of the pair of conjugate acid-base is the higher the buffer capacity. If there is not an adequate amount buffer available as compared to the acid/base added, the buffer can be swamped, and the pH of the sample solution will significantly change. The concentrations buffer should be adequately large by the amount of acid or base. Buffer's capacity is measured by the definite quantity of acids and salt.

The buffer capacity basically relies on two factors:

• The proportion of the salt to the acid or base.
• Total buffer concentration.

Factors that affect buffer capacity:

• The relative concentrations of the weak acid and its conjugate.
• The concentrations of the weak acid and its conjugate base.

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Advantages and Disadvantages of Affinity Chromatography

Affinity chromatography in which a macromolecule, (for example, a protein) is separated and purified in a solution with a molecule containing a ligand for which the macromolecule has an affinity that reason it is retained on the column.

The Advantages of Affinity Chromatography are:

• High sensitivity compared to TCD & FID.
• Affinity chromatography is used in the production of vaccines.
• Affinity chromatography is used in the purification of protein and enzyme.
• Affinity chromatography gives High specificity.
• Enzymes and other proteins are studied by affinity chromatography.
• To maintain the quality of the product, this chromatography is used in the pharmaceutical manufacturer in the production of vaccines.
• Affinity chromatography doesn’t rely on ionic strength, pH, temperature, and composition of the buffer.
• The high degree of purity can be obtained by Affinity Chromatography.
• This is a very reproducible process.
• This is the simplification method.
• Used to increase the solubility.
• In genetic engineering, affinity chromatography is used.

The Disadvantages of Affinity Chromatography are:

• It takes a lot of skill to handle it.
• It interferes with the structure.
• Transfer and the leakage of metal ion lead to protein loss.
• Sometimes ligands leakage is observed.
• The volume of the sample is limited.
• The carrier gas used must be pure such as pure nitrogen.
• The ligands used in affinity chromatography are costly.
• Relatively low productivity.
• It has a non-specific adsorption.
• Degradation of the solid support.
• Metal-ion transfer and metal ion leakage lead to loss of protein.
• Affinity chromatography is non-specific for adsorption than other chromatography methods.

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