Tuesday, September 29, 2020

Advantages and disadvantages of LC-MS

Liquid chromatography-mass spectrometry (LC-MS) is one of the most reliable modern analytical technique offers several advantages.

LC-MS is a combination of liquid chromatography and mass spectrometry used to isolate the compounds of a sample mixture with the power of mass spectrometry as a detector. High-performance liquid chromatography (HPLC) separates molecules by sample mixture and mass spectrometry (MS) provides structural identification of individual compounds with high detection sensitivity and molecular specificity.
The sample is pumped through a stationary phase (LC column) by a mobile phase flowing at high pressure. As per the affinity the analytes move through the HPLC column at different migration rates. After elution from the LC column, the effluent is directed to the mass spectrometer. The mass determines the mass-to-charge ratio of ions, this data can be used to determine the exact molecular mass and structural information about the component of the sample.
Advantages of LC-MS:
  • The major advantage of LC-MS is that it is the most robust analytical technique that provides higher sensitivity and selectivity required to detect an exact molecular weight of a wide range of samples.
  • This can help in the manufacturing of pharmaceuticals to streamline their research or analysis processes and compliance with regulatory guidelines.
  • The combination of the two analytical techniques (MS and HPLC) reduces experimental error and improves accuracy.
  • It can separate and identify solutes in low concentrations (which are in parts per million- PPM) in a complex mixture.
  • LC-MS is widely applied as regulatory compliance in the pharmaceutical, bio-pharmaceutical, research, forensic, food, and environmental sectors.
  • The LC-MS offers high selectivity, resolution, precise mass, and specificity as compared with other chromatography techniques.
  • It is applied to a wide range of samples and able to identify the different types of impurities.
  • This is an incomparable method to identify unknown components of a sample solution.
  • Mass spectroscopy can also work with gas chromatography such as GC-MS.
Disadvantages of LC-MS:
  • The major disadvantage of LC-MS is that it only works with volatile buffers that are required to avoid fouling of the API interface.
  • LC-MS has another disadvantage is that the residual impurities being analyzed should be ionized.
  • Phosphate buffer is not compatible with the LC-MS analysis, which is the most commonly used buffer in HPLC method development.
  • LC-MS is an expensive technique both in terms of capital and analysis costs.
  • This is not a portable instrument; it requires special and more space.
  • To operate and data analysis of the liquid chromatography and mass spectrometry (LC-MS) requires a skilled and trained person.
  • The LC-MS has a high maintenance cost as compared with other analytical instruments.

Commonly asked questions on LC-MS are as follows.

What are the applications of LC MC?
LC-MS is an analytical tool that has many industrial and research applications in the fields of pharmaceuticals, environmental, bio-pharmaceutical, and forensics.
 
What is the advantage of LC-MS over HPLC?
The major advantage of LC-MS over HPLC is that LC-MS can achieve a complete elucidation of the chemical structure of the molecule.
 
What is the difference between LC-MS and HPLC?
The main difference between LC-MS and HPLC is that the HPLC is used to isolate, identify, and determine each component of the sample mixture, whereas LC-MS is a combination of liquid chromatography and mass spectrometry that is used to determine the structural identity of the compound.


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Saturday, September 19, 2020

Advantages of disintegration testing

Disintegration is a physical process related to the mechanical fragmentation of small particles of a tablet or granulate particle. It is used as a quality control test and has many advantages, let's check it out.

The disintegration test is performed to determine the disintegration time taken by the solid dosage form by which the quality of the tablet and capsule can measure. Disintegration is broadly applied in the pharmaceutical industry. It is an essential test performed to check the disintegration ability of tablets and capsules, as per pharmacopoeia standards such as IP, USP, and BP.
Advantages of the disintegration test:
  • It determines the disintegration time, which is significant for the development of the oral disintegrating tablet.
  • According to pharmacopoeia standards such as IP, USP, and BP, it checks the quality of pharmaceutical products.
  • Multi samples at a time can be tested using dissolution media.
  • This gives important safety data on the bioavailability of the drug (API) in the body without using in vivo methods.
  • No expert required, easy to operate, and robust.
  • The disintegration test takes less time and it is less complex than the dissolution test.
  • The pH of the buffer of the medium can be easily changed than the dissolution testing.
  • It can be easily automated.
The disintegration test apparatus has one or two baskets assemblies with separate timers and motors, a beaker, an external probe for beaker temperature, timer and temperature display, and one water bath connected to the programmable temperature controller.
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Friday, September 18, 2020

Factors affecting granulation process

Granulation is the process of particle enlargement by the agglomeration method. It is the most important unit operations in the manufacturing of pharmaceutical dosage forms, particularly for tablets and capsules. The granulation has two types such as wet granulation and dry granulation. In wet granulation that uses a granulation liquid in the process and dry granulation does not use the liquid. The type of process is selected based on the physicochemical properties of drugs, excipients, essential flow, and release properties.
The granulation method converts fine powder into free-flowing granules, dust-free granules which are easy to compress. However, due to the high-quality requirement of the formed granules, granulation has various challenges such as uniformity of material and physicochemical properties such as porosity, bulk density, size of granule, compressibility, moisture, and hardness, etc.
Here are mentioned some factors that affect the granulation process.
  • Compatibility between material and binding solvents such as particle size, hydrophobicity, compressibility, solubility in granulation liquid, and surface area
  • Packing properties of the solids
  • Selection of excipients, since the role of excipients, is important in the granulation process
  • The quality of the machine that is used in the process
  • Stirring speed for the method: it used according to the raw material characteristics and requirement of the finished product
  • Particle shape and surface morphology
  • The solubility of particles in the granulation liquid
  • Characteristics of the drug, excipients, and other material which is used in the process
  • The contact angle of the binder liquid to the solids
  • Mean particle size and size distribution of the solids

Commonly asked questions on granulation are as follows.

What is the advantage of granulation?
The major advantage of granulation includes it increases flowability, suitable for most compounds, and suitable for different types of dissolution rates.

What are the two types of granulation?
Wet granulation and dry granulation are types of granulation. 

What is the application of the granulation technique?
The granulation is commonly applied in pharmaceutical industries for the enlargement of granules by the agglomeration technique.

What is the difference between dry and wet granulation?
The major difference between dry and wet granulation is that wet granulation uses a granulation liquid in the process while dry granulation does not use the liquid.
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Thursday, September 17, 2020

Applications of granulation

Granulation is the process of making granules from a powder, which produces granular material. The granulation is applied in several technical processes in the chemical and pharmaceutical manufacturing.

Granulation is a technique where powder particles bind to each other, resulting in homogeneous and larger granules. It is a mechanism of bonding particles together by forming bonds, and this bonding is formed by compression or by using a binding agent.
Granules are prepared in two ways: dry granulation or wet granulation. Dry granulation involves a dry mixing, slug-de-slug, and roller compaction processes and wet granulation includes aqueous (using granulation liquid) and non-aqueous granulation.
  • The major application of granulation is that it is broadly used in the pharmaceutical industry to manufacture solid dosage forms such as tablets and capsule granules.
  • The major applications of wet granulation are that it is used to improve compressibility, electrostatic properties, flow, and the stability of solid dosage forms.
  • The major applications of dry granulation are that it is used when the drug does not compress well after wet granulation, and it is also suitable for moisture sensitive drugs (APIs).
  • Granulation is essential to enhance the powder flow and mechanical properties of the tablet.
  • Granulation is used to improve the appearance of the product.
  • It is also used to improve fluidity, the compressibility of the material, and decrease dusting.

Commonly asked questions o granulation are as follows.
What are the two types of granulation?
Granulation technique is generally classified into two types, namely dry granulation, and wet granulation.

What is the advantage of using dry granulation?
The advantage of dry granulation is that there is no moisture content in the formulation, so it is suitable for moisture-sensitive compounds.

What is the advantage of using wet granulation?
Wet granulation makes granules more spherical than powder, so it has better flow properties and is suitable for a wide variety of dissolution rates.
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Tuesday, September 15, 2020

Different types of the granulation process

The granulation process is broadly classified into two types: dry granulation and wet granulation.

Granulation is the method of making granules from a powder or solid material which is used to produce a granular material in pharmaceutical industries.

What is granulation?

Granulation is the agglomeration technique of particle enlargement it is one of the most important unit operations in the manufacturing of pharmaceutical dosage forms, particularly tablets and capsules. The granulation process turns the fine powder into free-flowing granules which are easy to compress.

However, granulation has various challenges in terms of content uniformity and physio-chemical properties such as moisture, bulk density, compressibility, granule size, hardness, porosity, etc., due to the high-quality requirement of the formed granules.

The granulation technique is divided into two types: dry granulation and wet granulation.

Wet granulation method:

The wet granulation method is one of the most widely used processes in the pharmaceutical industry to manufacture a solid dosage form as it has many advantages over other techniques. In this process a mixture of powder particles is made using a granular liquid, the liquid contains a volatile solvent that is removed by drying and they are non-toxic. There are three parts in the wet granulation process are wetting the powder, nucleation, and agglomeration.

The mixing of excipients, drug (API), mixing the powder with binder solution, sieving, drying, and shifting are the steps involved in wet granulation.

Dry granulation method:

The dry granulation method is a type of granulation it is widely used in conditions where the effective drug dose for direct compression is too high, and the drug is responsive to moisture, heat. In this method, granules are prepared without using the granulating liquid and there are two ways to achieve compact when using dry granulation: slugging and roller compaction. 

The dry granulation method can perform with a special instrument called a roller compactor, which can be screened in granulation and is appropriate for compression of the tablet.


Commonly asked questions on granulation are as follows.

What are the advantages of granulation?
The advantages of granulation include, improves the flowability, cohesiveness, and compressibility, of the powder, disintegration time, increase porosity to maximize dissolution rate, reduce dustiness, change bulk density, and change material’s shape, size, and surface smoothness for coatings, etc.

What is the difference between wet and dry granulation?
The major difference between wet and dry granulation is that dry granulation is not used granulation liquid and wet granulation uses liquid/solvent in the process.

What is the major application of granulation?
The granulation is used in the pharmaceutical and food processing processes. It is mainly used in the pharmaceutical industry in the process of manufacturing solid dosage forms such as tablets and capsules.



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Monday, September 14, 2020

Difference between dry granulation and wet granulation

The major difference between dry granulation and wet granulation is that in the process of dry granulation the granules are produced without using a granulating liquid, while in the wet granulation process the granules are produced by the addition of granulating liquid.

The granulation is the method in which primary powder particles are followed to form larger multi-entities called granules. Generally, it is used to prepare the tablet dosage form. The granulation techniques for tablet manufacture are broadly classified into two types, dry granulation, and wet granulation.
Dry granulation is a type of granulation process in which no granulating liquid is used in the formation of granules. This process is mainly suitable for the components that are extremely sensitive to heat and moisture. The primary powder particles in this process are aggregated under high pressure.
Wet granulation is a type of granulation process in which granulating liquid is used in the formation of granules. The liquid used in this process is volatile and non-toxic which can be easily removed by drying out the final product.
Difference between dry granulation and wet granulation:
  • In dry granulation, finely ground powder is directly compressed.
  • In wet granulation, firstly powered mixed with a granulating fluid by forcing through a sieve and then compressed to prepare a tablet.
  • If there is a delay in the case of dry granular batch manufacturing, there is no possibility of losing the batch.
  • The probability of losing the batch is high, for example, electrical failure, instrument failure, or manual delay.
  • In dry granulation there is no wetting of materials, hence there are fewer chances of influence of raw material.
  • In wet granulation there is the wetting of materials, hence there are more chances of influence of raw material, for example, density, and particle size distribution.
  • Dry granulation is used for compounds that are sensitive to moisture and heat.
  • Wet granulation is used to prevent the degradation of active ingredients in the powder.
  • The dry granulation process involves less equipment and space which are cost-effective.
  • The wet granulation process involves more equipment and space which are more expensive than the dry granulation process.
  • If there is a delay in the case of wet granulation batch manufacturing, the probability of losing the batch is high, for example, electrical failure, instrument failure, or manual delay.
  • The dry granulation requires less cleaning cycle compared with wet granulation.
  • The scale-up of dry granulation is less complex than wet granulation.


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Saturday, September 12, 2020

Advantages and disadvantages of dry granulation

The granulation is a method where powder particles are made to bind to one another, resulting in large, multi-particle bodies, called granules. If the process is done with the addition of granulating liquid, it is known as wet granulation and if the process is carried without liquid, it is known as dry granulation.
The dry granulation process is used to make granules without liquid, as the granulating material can be sensitive to heat and moisture. In this process, the powder mixture is compacted by exerting a force on the powder, which in general causes a considerable size increase. There are two ways to achieve compact when using dry granulation: slugging and roller compaction.
The steps involved in dry granulation are- weighing formulation ingredients, mixing of formulation ingredients, compression of mixed powders into slugs, milling, and sieving of slugs, mixing with disintegrants and lubricants, and compression of granules into tablets, etc.
Advantages of dry granulation:
  • The major advantage of dry granulation is that the formulation does not have moisture content, because granular liquid or solvent is not used in the process. It is suitable for pharmaceuticals (drugs or excipients) that are sensitive to moisture.
  • The dry granulation process requires less equipment, less space, more economic than wet granulation.
  • The drying step is not involved in this type of method, hence it is suitable for heat-sensitive materials.
  • Dry granulation does not require any special excipients; it uses conventional grades of material.
  • Tablet manufacturing by dry granulation improves the disintegration time.
  • The rate of dissolution of tablets produced by dry granulation does not decrease with aging.
Disadvantages of dry granulation:
  • The main disadvantage of dry granulation is that since it does not form granules, it may not have better flow properties.
  • The dry granulation method leads to low flowability, compressibility, and cohesiveness of the powder, causing the powder to not easily flowing and compressed.
  • The process produces a lot of dust which can lead to cross-contamination.
  • It requires a special heavy-duty apparatus for granulation.
  • Dry granulation tablets are softer than wet granulation tablets which make them difficult to tablet coating or film coating.
  • It extensively damages the sieving screening and other tools which are used in the process.
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Advantages and disadvantages of wet granulation

The wet granulation is the traditional process, in which the mixing of powder particles can be done using granulating liquid (Volatile solvent). The granulating liquid contains a volatile solvent that can be removed by drying and they are non-toxic. 
The wet granulation process has three parts such as powder wetting, nucleation, and further nucleation or agglomeration. This method is widely used in pharmaceutical manufacturing to manufacture a solid dosage form. 
The steps involved in the wet granulation are mingling of excipients and API (drug), mixing of powder with binder solution, granular sieving, drying, and sifting of granules. 
Advantages of wet granulation: 
  • The major advantage of wet granulation is that it forms granules and is more spherical than the powder, hence has better flow properties, and suitable for different types of dissolution rates.
  • The wet granulation process improves the flowability, compressibility, and cohesiveness of the powder, due to which the powder is easily flowing and compressed.
  • The method of wet granulation can be used for high-dose pharmaceuticals with weak compressibility that is not affected by moisture and heat.
  • The presence of water in the form of moisture can help to improve the dissolution rate of hydrophobic drugs.
  • Wet granulation reduces the level of dust present during the manufacturing process as compared with other methods.
  • It maintains the same density of all granules since good material uniformity is maintained due to particle segregation prevention.
  • Tablets made from a wet granulation process are ideal for tablet coating.
Disadvantages of wet granulation: 
  • The major disadvantage of wet granulation is that the presence of moisture can cause a reaction between the drug/drugs and the additive.
  • The process is not suitable for compounds and excipients that are moisture-sensitive.
  • The dissolution rate of tablets produced by wet granulation may decrease with aging.
  • It includes several processing steps compared with other methods.
  • It takes more time, energy, space hence it is a costly method as compared with conventional methods.
  • To conduct the granulation process, we need an experienced person.
  • Wet granulation process requires more equipment and less space than dry granulation.


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Thursday, September 10, 2020

Advantages and disadvantages of friability test

The friability testing is a method that is used to determine the physical strength of tablets (compressed/uncoated) exposed to mechanical shocks and attraction, let's check their some advantages and disadvantages.

The design, quality control, and post-formulation of tablet dosage form include comprehensive assessments and evaluations of the chemical, physical, and bioavailability properties of the tablet. The tablets are subject to different tests (post-production) such as friability, hardness, and dissolution as per the standard procedure of pharmacopoeias such as USP, BP, and IP. Friability testing is a method that is performed using the friability tester and that is employed to determine the physical strength of uncoated and compressed tablets when exposed to mechanical shock.
Advantages of friability testing:
  • The Friability test is commonly used in the pharmaceutical industries or research and it tells us how the tablets have durability during packing processes and transit.
  • It helps in the consistency and reproducibility of product in batch-to-batch production.
  • It determines the quality of pharmaceutical products.
  • As per the standards of pharmacopoeia such as USP, BP, and IP it is used in the research and development as a regulatory perspective.
  • There is no need for repetition; a sample of 10/20 tablets is checked at a time so that results are given at once.
  • It is an automated process and is easy to use so there is no need for an expert to handle it.
  • The most advanced apparatus provides automatic pre-set count stops, balance interface, and a printing facility.
Disadvantages of friability testing:
  • Friability can be affected by several factors such as moisture content of tablet granules, humidity, and hardness.
  • Acrylic drums have to be handled carefully due to their fragility.
  • It needs to be frequently calibrated such as drum alignment, counting, etc.
  • It works only for compressed or uncoated tablets.
  • The process is slow it takes time to complete.

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Wednesday, September 9, 2020

Advantages and disadvantages of dissolution testing

The official dissolution apparatus has its inherent advantages and disadvantages and is therefore ideal for dissolution testing of various pharmaceutical studies.

Dissolution testing of solid dosage forms has been well-known as a standard (official test by pharmacopeias) method for evaluating drug release from tablets as well as capsules. Presently, this is the most useful in vitro technique to verify batch uniformity and is an important quality control process for evaluating the release profiles of various finished product batches.
There are different types of dissolution apparatus are used in different studies, it ranges from apparatus I to VII according to USP, BP, and IP. Generally, they consist of vessels, paddles, basket, pump, water tank, rotating motor, and temperature controller, etc.
Advantages of dissolution testing:
  • The major advantage of dissolution testing is that the rate of release and the extent of absorption of a drug is determined by the dissolution of the dosage form.
  • The dissolution test apparatus is robust and simple to use, requiring no specialist to handle.
  • It is well standardized and sufficiently flexible to allow perform testing for a broad range of oral solid dosage forms.
  • Dissolution testing is used to determine the quality of pharmaceutical products (tablets/capsules) based on the characteristics of in vitro release of the products.
  • Dissolution testing is used to establish the quality of pharmaceutical products, mostly tablets and capsules, based on the in vitro drug release characteristics of these products.
  • Dissolution testing of solid dosage forms plays a significant role in the decision-making process from a regulatory perspective, mainly in the development and approval of generic medicines, where it is possible to avoid unnecessary human studies without compromising the quality of pharmaceutical products.
  • The pharmaceutical manufacturer takes advantage of dissolution testing by using it to assess product development, product stability, quality control, and quality assurance, and to establish in-vitro-in-vivo correlation.
  • The dissolution apparatus can be easily automated which is important for routine investigations. 
Disadvantages of dissolution testing:
  • The major disadvantage of the dissolution process is that the amount of media required to be maintained at each interval time, which feels the analyst uncomfortable.
  • The results depend on other instruments, the sample of dissolution needs to be analyzed in UV/VIS spectrophotometer or high-performance liquid chromatography (HPLC).
  • Dissolution requires large amounts of media and takes longer to prepare and adjust the pH of the buffer.
  • Liquid dosage forms are not tested using this process; it is suitable only for solid dosage forms.
  • Between the processes, it is often difficult to change the pH/media.
  • Each drug and product has different properties, so in some cases, there is a problem of sticking and floating.
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Monday, August 31, 2020

Selection of the column for RP-HPLC

Column selection is the first and most important step for the development of an analytical method, whether it is gas chromatography or high-performance liquid chromatography. It is not possible to develop a rugged, robust, and reproducible method without high-performance and stable HPLC columns. Significantly, columns are reproducible and stable to avoid errors with irrelevant sample retention time during the development of the HPLC method.
The RP-HPLC column is selected based on the nature of the molecule and the information about the solute. Reversed-phase chromatography is the types of HPLC which have broad columns range such as dimethyl silane (C2), butylsilane (C4), octylsalane (C8), octadecylsalen (C18), cyanopropyl, BDS phenyl, and nitro, etc. A C18 or C8 column is specially made with less acidic, high-quality pure silica and is particularly suitable for the isolation of basic analytes. They are normally appropriate for all the samples and are strongly recommended by the chromatographers.
The following few points give guidance in the selection of column for RP-HPLC:
  • Almost all HPLC columns are made of steel. The major advantage of steel is that it is relatively inert to corrosion and pressure resistance.
  • It is significant for a better separation of solutes that there is no scoring, roughness, inside a chromatographic column.
  • Silica gel is the most popular stationary phase in high-performance liquid chromatography, which consists of Si atoms and oxygen atoms and it carries OH groups (silanol groups). It is available in different particle sizes and is used within a pH range of 1.00 to 8.00.
  • Polymer-based columns are also used in reversed-phase HPLC, providing higher pH stability than silica gel columns. It can be used for the isolation of strongly acidic or basic components.
  • Column packing materials of various types and sizes are available according to the nature of the compound and HPLC method.
1. Fully porous particles are the most used shapes in the HPLC column, which are available in 3, 5,     or 10 μm.
2. Coreshell particles have a solid core, and the outer shell is porous, resulting in improved efficiency in the separation. One major disadvantage of coreshell particles is that it has low load-ability compared to porous materials.
3. The monolithic phase column consists of a piece of porous material, like a silica gel. A disadvantage of monolithic phases is that the availability of selectivity is limited in contrast to fully porous materials.
  • The longer columns generally give a better separation because of the higher theoretical plate numbers. If the particle size decreases, then surface area available for coating increases. 5-μm particle size HPLC columns offer the best solution in terms of reproducibility efficiency, and reliability.
The C18 is the most common reversed-phase column may be used in several analyses. To decide which stationary phase or column is suitable, the C18 column is considered the first choice at the beginning of method development. Since it has longer carbon chains (typically 18 carbons) than other column and provides a wide surface area which offers an additional time of reaction between the bonded phases. The very-polar molecule does not interact with the C18 material and is eluted at the dead time, in such cases you can use C4, CN, diol, or phenyl phases. Conversely, if the molecules are very un-polar you can use silica, CN, or NH2 phase.
While doing the HPLC method development, there are some aspects to be considered: separation scheme, mobile phase, type of HPLC column, the diameter of the column (such as particle size, pore size, internal diameter, diameter, and carbon content), analytes of interest, pKa value, pH of the solvent system, detector, and injector, etc. Method development has different conditions in each case; each chromatographer should consider these factors when performing the HPLC analysis.

Commonly asked questions on the HPLC column are as follows.

How many types of HPLC columns are there?
There are four main types of HPLC columns-reversed phase HPLC column, normal phase HPLC column, the size-exclusion HPLC column, and the ion-exchange HPLC column.

Is c18 column polar or non-polar?
The C18 column is used in the reversed-phase chromatography; it’s packed with non-polar material as compared to the polar stationary phase. 

What is the difference between HPLC and RP-HPLC?
HPLC is the type of chromatography in which RP-HPLC is the most commonly used modes of HPLC. HPLC stands for high-performance liquid chromatography and RP-HPLC stand for reversed-phase high-performance liquid chromatography.

What is the difference between c8 and c18 HPLC columns?
Both C8 and C18 are the types of columns used in HPLC, the major difference between C8 and C18 is that C18 has 18 carbon atoms while C8 has only 8 carbon atoms.


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Saturday, August 29, 2020

Advantages and disadvantages of buffer solution

Definition of the buffer: Buffer is a compound or mixtures of compounds that resist changes in pH by the addition of small amounts of acid or alkali by their presence in the solution.
A buffer solution is an aqueous solution that contains a weak base and its conjugate acid or a weak acid mixture and its conjugate base. It avoids the changes in pH or slight changes as applying small amounts of strong acid or base to it. Buffer solutions are widely used in a range of chemical applications as a way of maintaining pH at a nearly constant value. Several systems use buffering for pH regulation.
Advantages of buffer solution:
  • The major advantage of a buffer is that it is keeps the pH stable and increase the solubility of the substance or formulation.
  • Buffer solution plays a significant role in chromatography, as retention of ionizable molecules is extremely sensitive to the pH of the mobile phase in such cases the pH can be regulated by adding a buffer solution.
  • The buffer solution can remain stable for several weeks if it is stored in the fridge.
  • Different types of buffers such as acetate, ammonium, sodium, and phosphate, etc. are highly soluble in water.
  • The pKa of a buffer solution is the same as being body fluids; hence it is used in the dissolution testing to determine the extent and rate of solution formation from a dosage form.
  • It maintains the solubility of the therapeutic agent in the pharmaceutical product.
  • Buffer solution improves the stability of products where the active agent is pH-dependent.
  • It is non-toxic to cells and inhibits enzymatic reactions.
  • By using the buffer solution the onset of action of dosage form increases.
  • It is used to optimize biological activity.
  • Since the pH of the buffer solution is precise, standard buffers i.e. pH 07.00, 04.00, and 09.20 are used to calibrate the analytical instruments (pH meter).
Disadvantages of buffer solution:
  • The major disadvantage of the buffer solution is that it is an aqueous solution that can be contaminated with microorganisms and is not suitable for long-term storage, so you need to prepare a fresh buffer solution each time.
  • To adjust the pH of the solution, it is necessary to know the pKa value of the component. 
  • The drug dissolution may precipitate when the pH of the solution is not maintained properly.
  • Some types of the buffer are precipitated and tend to react.
  • Drug ingredients and some vitamins are dissolved only at a particular pH therefore, it is essential to maintain the correct pH of the solution.

Commonly asked questions about buffer solutions are as follows.

What are the types of buffer solutions?
There are two types of buffer solutions: acidic buffer solutions and alkaline buffers.

What is the range of pH scale?
The pH scale determines how acidic or basic a substance is in the range of 0.00 to 14.00 pH. A pH of 7.00 is neutral; a pH less than 7.00 is acidic and a pH greater than 7.00 is basic.

What are the applications of buffer solution in the pharmacy?
The buffer solutions are used in pharmaceutical industries for improving purity, increased stability, enhancing solubility, and optimizing the biological activity of the compounds or products.
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Wednesday, August 26, 2020

The separation mechanism of reversed-phase chromatography

Chromatography is a primary separation technique which is used in chemical analysis. It isolates the sample mixture by continuous partitioning between the mobile and stationary phases. The principle of chromatography involves the isolation obtained by dissolving compounds in a mobile phase and traveling over a stationary phase. Each compound of sample mixture has different retention time, as the compound with strongly interacting with the stationary phase appear to travel very slowly, while compounds that have less affinity tend to elute rapidly.
The high-performance liquid chromatography is one of the most powerful separation techniques since it can apply different types of separation techniques for different types of molecules. The reversed-phase chromatography is a type of HPLC that is currently the most broadly used method of isolation in HPLC since it is versatile, simple, separate the analytes of diverse polarity and molecular mass, and has more scope of the technique. 
The principle of reversed-phase chromatography depends on the interaction of the solutes with the hydrophobic group. It has analytical and preparative applications in the field of biochemical isolation and purification. It is typified by a mobile phase of polar properties and a stationary phase with non-polar properties. The reversed-phase chromatography does works in contrast to normal phase chromatography.
Separation mechanism of reversed-phase chromatography:
The separation mechanism in reversed-phase HPLC generally depends on the distribution of components between the mobile phase and the stationary phase. This process can also be interpreted as a type of component extraction between the stationary phase and the mobile phase. In the stationary phase, a non-polar substance dissolves preferentially, remains there preferentially, and is thus gradually eluted from the chromatographic column. Polar substances are easily eluted from the column, as they tend to remain in the solvent system. Subsequently, the retention time of the solutes varies as their distinct affinity for the stationary phase.
The stationary phase in reverse phase chromatography:
The stationary phase of reversed-phase HPLC is usually composed of hydrophobic alkyl chains that interact with the solutes. Octadecyl carbon chain (C18) bonded silica is the most popular column used in RP-HPLC as it has a longer carbon chain compared with the C4 and C8.
The mobile phase of reversed-phase chromatography:
The mobile phase used in reversed-phase HPLC has a certain amount of water or aqueous buffers and organic solvents are used to isolate as well as to obtain a symmetrical peak shape. The water-miscible solvents such as methanol, acetonitrile, and tetrahydrofuran are most commonly used organic solvents. The retention of ionizable compounds is very sensitive hence the pH of the mobile phase or buffer can play an important role.

Commonly asked questions on chromatography is as follows.

Why is reversed-phase chromatography useful?
The RP_HPLC is most widely used technique amongst the different types of HPLC chromatography since it has a hydrophobic stationary phase and can be used with the polar (hydrophilic), non-polar (hydrophobic), ionic and ionizable molecules to isolate their different molecules, which is depends on the procedure being used.

Which compound will elute first in a reversed-phase HPLC separation?
Polar compounds will elute first in a reversed-phase HPLC separation. The polar (aqueous) mobile phase is used in the reversed-phase HPLC As a consequence, in the polar solvent system hydrophobic solutes begin to adsorb into the hydrophobic stationary phase, and in the mobile phase, hydrophilic solutes pass through the column and are first eluted.

What is the difference between the normal phase and reversed-phase chromatography?
The major difference between reversed-phase chromatography and normal phase chromatography is that reverse phase chromatography has a non-polar stationary phase and a polar mobile phase while normal phase chromatography has a very polar stationary phase and a non-polar phase.

What is the major advantage of reversed-phase chromatography?
The major advantage of reversed-phase HPLC is that its hydrophobic stationary phase works well for the retention of most organic analytes. This allows the aqueous mobile phase to be used that includes water and the type of buffer solution such as phosphate, acetate, etc. RP-chromatography also has the advantage of being able to use pH according to the pKa of the molecule to improve the separation.

What are the major types of chromatography?
There are various types of chromatography has, such as high-performance liquid chromatography (HPLC), gas chromatography (GC), column chromatography, paper chromatography, thin-layer chromatography (TLC), and mass spectroscopy (LC-MS), etc.
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