A Brief Review on: Separation Techniques Chromatography

Gautam R. Ramraje*, Sumesh D. Patil*, Dr. P. H. Patil, Amol R. Pawar

KVPSS Institute of Pharmaceutical Education, Boradi, Tal-Shirpur, Dist-Dhule (M.S.) India 425405.

*Corresponding Author E-mail: amolpharma9@gmail.com

ABSTRACT:

Chromatography is an important biophysical process, allowing the components of a mixture to be isolated, classified and extracted for qualitative and quantitative analysis. Proteins can be purified based on features such as size and shape, total charge, surface hydrophobic groups and stationary phaset binding ability. Four separation strategies based on molecular characteristics and form of interaction Four separation techniques based on molecular characteristics and form of interaction use ion exchange mechanisms, surface adsorption, partition, and exclusion of the size. Column chromatography is one of the most common methods of protein purification Chromatography is based on the theory of separation of fluid stationary phase (stable phase) when traveling with the support of a mobile phase. The variables that are involved in this separation process include adsorption (liquid-solid), partition (liquid-solid), and affinity or variations between their molecular weights. Due of these variations, certain components of the mixture stay in the stationary phase longer, and travel gradually in the chromatography system, while others move rapidly through the mobile phase, leaving the system faster. Based on this method, three components form the basis of the chromatography technique. Stationary phase: is a phase consisting of a "solid" phase or a "solid surface adsorbed layer" Mobile phase: is a phase consisting of a "liquid" or a "gaseous part." Separate molecules which form the interaction between stationary phase, mobile phase and substances contained in the mixture are the basic component that is successful in separating molecules from the partition-based chromatography methods are very effective in separating small molecules and distinguishing them as amino acids, carbohydrates and fatty acids. In the separation of macromolecules as nucleic acids and proteins, however, affinity chromatography’s (i.e. ion-exchange chromatography) are more efficient. Paper chromatography is used in protein separation and protein synthesis studies; gas-liquid chromatography is used to isolate alcohol, ethanol, lipid, and amino groups, and to analyze enzymatic interactions, while molecular-sieve chromatography is used in particular to evaluate molecular protein weights. Agarose gel chromatography is used to purify RNA, DNA particles, and viruses. The chromatography stationary phase is a solid phase or liquid phase that is sprayed on a solid phase surface. A gaseous or liquid phase is a mobile phase which flows through the stationary phase. If the mobile phase is liquid, it is called liquid chromatography, and if it is gas, it is called gas chromatography. of gases and mixtures of volatile liquids, and solid material, gas chromatography is used. Liquid chromatography is used in particular for thermal unstable and non-volatile materials. The purpose of applying chromatography, which is used as a quantitative analysis tool apart from its separation, is to record a sufficient separation within an acceptable time interval. Various chromatography methods have been developed to that end. Some of these have chromatography on columns, thin layer chromatography (TLC), paper chromatography, gas chromatography, ion exchange chromatography, gel permeation chromatography, liquid high pressure chromatography, and affinity chromatography.

KEYWORDS: HPLC, Mobile Phase, Stationary Phase, Detector, Chromatography, Instrumentation.

INTRODUCTION:^(1,2,7,8)

High performance Liquid Chromatography (HPLC) is one of the most common and established analytical techniques, and by far the most commonly used technique of separation. This has been used for pharmaceutical research, medicinal chemistry, food and environmental studies, organic chemistry, etc. in laboratories around the world over the past 40-plus years. HPLC has gained popularity mainly because of its reliability (use of pressure-driven liquid support) and flexibility (possibility of changing mobile and stationary phase composition).

The chromatographic mode or process of separation depends on the overall interpersonal relations between the stationary phase, the mobile phase, and the analyte. Particle packed columns are used in traditional or miniaturized HPLC systems with either totally porous- or the newly formed core-shell particles and monolithic columns.

The aim of this analysis is to highlight fundamental aspects and practical considerations on: column flipping, traditional (heart-cut), and detailed two-dimensional LC and various aspects of micro column chromatography, nanoliquid and ultra-performance (UPLC). A summary of recent advances in column technology and in miniaturized LC instrumentation; chip-based nano-LC system.

Type of HPLC: ^{(3, 4, 5, 17, 18, 21)}

HPLC types typically depend on the phase system that is being used in the process. Following forms of HPLC widely used in analyzes

1. Normal phase chromatography

2. Reversed phase chromatography

3. Size exclusion chromatography

4. Ion exchange chromatography

5. Bio-affinity chromatography

1. Normal phase chromatography:

Also known as Normal phase HPLC (NP-HPLC), this method separates polarity-based analytes. The NP-HPLC uses a stationary polar phase and a mobile non-polar phase. The polar analyte interacted with and remains in the polar stationary phase. Adsorption forces increase with increased polarity of the analyte, and the interaction between the polar analyte and the polar stationary phase increases elution.

2. Reversed phase chromatography:

Reversed phase HPLC (RP- or RPC) has a stationary non- phase and a mobile phase which is aqueous, mildly polar. RPC works on the theory of hydrophobic interactions, resulting from repulsive forces between a polar eluent, the relatively non- analyte, and the stationary non- layer. The analyte binding to the stationary phase is proportional to the area of the contact surface

3. Size exclusion chromatography:

Size exclusion chromatography (SEC), also known as gel permeation chromatography, or gel filtration chromatography, separates primarily particles based on dimension. This is also useful for determining the tertiary structure of proteins and amino acids and their quaternary structures. This technique is commonly used for the determination of polysaccharides by molecular weight.

4. Ion exchange chromatography:

Retention is based in Ion-exchange chromatography on the attraction between solute ions and charged sites bound to the stationary phase. Same-charge ions are removed. This method of chromatography is commonly used in water purification, ligand-exchange chromatography, protein Ion-exchange chromatography, carbohydrate and oligosaccharide high-pH anion-exchange chromatography, etc.

5. Bio-affinity chromatography:

Separation based on complex reversible protein-to-ligand interaction. Ligands are covalently connected to strong support on a matrix of bio-affinity, maintaining proteins interacting with the ligands bound to the base.

a. Biospecific elution:

free ligand is included in the elution buffer which is competing with the bound ligand board.

b. Relevant elution:

pH transition, salt transition, etc., which weakens protein interaction with column-bound substrate. Because of the interaction specificity, chromatography with bioaffinity can result in very high purification in one single step (10-1000-fold)

· PARAMETERS: ^(44,45,46)

There are several parameters which are used as a norm for a particular compound for the accurate analysis of a compound. If a shift occurs in the parameters the result can be greatly affected. The parameters commonly used are internal diameter, particle size, pore size, pump pressure. The parameters can be modified for various compounds according to their existence and chemical prop.

1. Internal diameter:

An HPLC column's internal diameter (ID) is a crucial aspect that defines the amount of analyte that can be loaded onto the column, and also affects sensitivity. Larger columns are typically used for later use in industrial applications such as purifying a drug product. Low ID columns at the cost of loading efficiency have increased sensitivity and lower solvent usage.

2. Particle size:

Most typical HPLC is done with the stationary step of small spherical silica particles (very small beads) attached to the exterior. Generally, smaller particles have more surface area and better separations, but the pressure needed for optimum linear velocity increases by the reverse of the squared particle diameter. Size of the pores: Several stationary phases are porous to give more surface.

Small pores have greater surface area while larger pores provide stronger kinetics for larger analytes in particular. Pore size determines the analyte molecules 'ability to penetrate the particle's interior and interact with its inner surface. This is particularly important since the ratio of the surface of the outer particle to its inner one is about 1:1000. Molecular surface contact occurs mainly.

3. Pump pressure:

The pressure capacity of pumps varies, but their efficiency is determined by their ability to yield consistent and reproducible flow. Current HPLC devices have been developed to operate at much higher pressures and can also use much smaller particle sizes (< 2 micrometers) in the columns.

· Solvents System:^{(25,26,22,23,24)}

It depends on column form and analyte form of HPLC. Commonly:

1) Normal phase or reverse phase: water/buffer and organic solvents: ^(42,43)

Normal Phase Chromatography:

Separation is achieved by using analytes of varying affinity for the polar stationary phase and using non-polar solvents such as chloroform, hexane, cyclohexane, etc. (this type of chromatography may be useful for separating stereo-isomers). I have never had to perform this type of chromatography in all my experience.

Reverse Phase Chromatography:

Separation is accomplished by using analytes with varying affinity for the polar stationary phase and by using non-polar solvents such as chloroform, hexane, cyclohexane, etc. (This method with chromatography can be useful for stereo-isomer separation). I have never had this sort of chromatography in all my experience.

2) Size exclusion: whatever solvent which can dissolve the analyte.

The methods that I was introduced to using an isocratic buffer with a column unique to the SEC. Separation are accomplished by taking advantage of various size proteins which move at different rates through the column. In the stationary phase, lower molecular weight proteins can travel easily in and out of the small pores while the higher molecular weight proteins cannot. Separation occurs with the elution.

Ideal characteristics of solvent:

· Product Grade HPLC can be used

· Mobile process, free from dust and impurities sample to be analyzed is soluble in the mobile phase

· The stationary phase should not respond to mobile phase

· It is important to track the mobile phase level and ensure that it is continuously updated and that the device is never allowed to run dry

INSTRUMENTATION

1. PUMPS:^{(30, 31, 35, 41)}

HPLC Devices like the pump come in various sizes and for different uses. Nevertheless, a strong pump has the capacity to generate a pressure of up to 6,000 psi, or pounds per square inch, applied after injection of the sample. What happens is that it helps the sample to move through the column faster and more effectively than if only the force of gravity were to flow through. You can look at various parameters such as flow rate, pressure generation, suitability to different solvents, ease of use, etc. before buying HPLC Equipment and specifically pump. Some key types of HPLC pumps available on the market today are listed below

a. Reciprocating Pump:

It is one of the most popular pumps used in HPLC. The mobile phase is pushed back and forth action of piston action present about a cylindrical chamber. It is highly sought after because of its constant flow rate, low space occupancy, excellent pressure generation and it is useful for both isocratic and gradient runs.

b. Syringe Type HPLC Pump:

These pumps generally consist of a cylinder that holds the mobile phase which is expelled by a piston. Now, the piston is advanced by a motor connected through worm gears, to provide smooth pulse less flow. These types of HPLC pumps have their own advantages. Their pressure capability is usually quite high (up to 78,000 psi) and maintenance is infrequent since there are no fluctuating check valves; gears are simple and strong.

c. Pneumatic Pump:

One of the simplest of pumps, the Pneumatic Pump as the name goes uses gas to pressurize the mobile phase that is present in a collapsible solvent container. These are considered to be some of the most cost-effective of all HPLC accessories.

2. INJECTORS:^(32,33,34)

1) Syringe injection method.

2) Sampling loops method.

3) Automatic injection method.

1) Syringe injection method:

In this method the sample is introduced through a self sealing elastomeric septum, for this purpose micro syringe designed to withstand pressure up to 1500 psi are used. In stop flow injections, the flow of solvent is stopped momentarily and a fitting at the column head is removed. Then the sample is injected directly onto the head of column packing. After replacing the fitting, the system is again pressurized. This method is simple but the reproducibility of the result can’t be obtained.

2) Sampling loop method:

These loop valve devices are the integral part of high pressure liquid chromatography (HPLC) equipment and have interchangeable loops providing a choice of sample sizes from 5 to 500ul. Sampling loops of this type permit the introduction of samples at pressure up to 7000psi. Micro sample injection valves with sampling loops having volumes of 0.5 to 5 ul are also available.

3. COLUMNS:^{(35,36,37,40)}

Columns are the main component in HPLC because the column is responsible for the separation of the sample components. The sample passes through the column with the mobile phase and separates in its components when it comes out from the column. Generally, silica gel is filled in the high-performance liquid chromatography columns because of its particle size and porosity that helps in separation of components and silica gel is also an inert material that does not react with mobile phases. There fore silica columns can be used to analyze the compounds of different chemical natures. The material filled in the HPLC columns is known as a stationary phase. There are different types of chromatography columns on the basis of their composition and method of separation. Some of them are described here.

1) Normal Phase Columns

2) Reverse Phase Columns

3) lon Exchange Columns

4) Size Exclusion Columns

1) Normal Phase HPLC Columns:

This type of columns has more polar stationary phase than the mobile phase. The packing material of the column should be more polar than the mobile phase and this condition is fulfilled by the silica that is polar material. But water is more polar than the silica, therefore, water is not used and methylene chloride, hexane and chloroform or a mixture of these with diethyl ether is used as mobile phase. Separation ofthe sample components occur on the basis of the polarity of the sample components. Sample components having more polarity interact more with polar stationary phase resulting in separation from the less polar component that interacts with less polar mobile phase. Silica columns are widely used in the pharmaceutical analysis. The chromatography column packing in which normal phase columns are used is known as Normal Phase Chromatography.

2) Reverse Phase HPLC Columns:

In reverse phase columns as its name states, it is reverse of the normal phase columns. It has a non-polar or less polar stationary phase than the more polar mobile phase. Bonded hydrocarbons like C8 and C18 and other non-polar hydrocarbons are used as stationary phase in reverse phase columns while aqueous organic solution like water-methanol or water-acetonitrile mixture is used as mobile phase. Separation of sample components in reverse phase columns also occurs on the basis on the polarity of the sample components but it happens just opposite of the normal phase HPLC columns, therefore, this type of chromatography is known as Reverse Phase Chromatography.

3) lon Exchange HPLC Columns:

The compounds those can easily ionize are analyzed using these columns. Stationary phase in these columns remains acidic or basic having negative or positive charge while mobile phase is a polar liquid as the salt solution in water. Separation of molecules occurs on the basis of the attractive ionic force between molecules and the charged stationary phase. Due to the exchange of ions during the separation of sample components, it is known as lon Exchange Chromatography.

4) Size Exclusion HPLC Columns:

Porous stationary phase in these columns allows the separation of the components according to their size. Combination of polymers like polysaccharides and silica is used as stationary phase in these columns. Small sample molecules penetrate in the pores of stationary phase while the large molecules penetrate partially into the pores. There fore the large molecules of the sample elute first than the small molecules and this chromatography is called Size Exclusion Chromatography. These columns are generally not used in the analysis of pharmaceutical compounds.

HPLC columns have a different length varying from 30 mm to 250mm and their particle size or porosity from 3µ to 5p. These factors affect the analysis of sample, therefore, these are considered important during the HPLC analytical method development Columns are selected according to the nature of the compound to be analyzed and the mobile phase. Column performance should also be evaluated time to time generally after 1000 runs or as required.

4. TYPES OF DETECTOS USED IN HPLC:^{(19,20,27-29)}

Detectors:

HPLC detectors fall into two main categories: universal or selective. Universal detectors typically measure a bulk property (e.g., refractive index) by measuring a difference of a physical property between the mobile phase and mobile phase with solute while selective detectors measure a solute property (e.g., UV-Vis absorbance) by simply responding to the physical or chemical property of the solute. HPLC most commonly uses a UV-Vis absorbance detector, however, a wide range of other chromatography detectors can be used. A universal detector that complements UV-Vis absorbance detection is the charged aerosol detector (CAD). A kind of commonly utilized detector includes refractive index detectors, which provide readings by measuring the changes in the refractive index of the effluent as it moves through the flow cell. In certain cases, it is possible to use multiple detectors, for example LCMS normally combines UV-Vis with a mass spectrometer.

a) UV - Visible detector:

The SPD-20A and SPD-20AV are general-purpose UV-Vis detectors offering an exceptional level of sensitivity and stability. With improved light-source compensation and stray light correction, high sensitivity is achieved across an extremely broad linear range (2.5AU). A Temperature controlled flow cell assists in reducing noise and allows for baseline stability.

b) Photodiode Array Detectors (M30A, M20A, 30AM):

The SPD-M20A is a photodiode array detector (PDA) that achieves high sensitivity and superior linearity (2.0AU) due to improved light-source compensation and stray light correction. Two slit widths are available, 1.2nm for high-resolution work and 8nm for quantitative runs. Temperature controlled flow cells are standard to reduce noise and assist in baseline stability.

The SPD-M30A is a photodiode array detector (PDA) that uses a newly designed capillary cell to achieve ultra-low dispersion in UHPLC separations. This detector also supports high-sensitivity analysis due to the improved signal level and reduced noise level. Stray light compensation technology and temperature control functions allow for reliable analysis.

The SPD-30AM is a UHPLC multi-wavelength photodiode detector for high-speed multi-wavelength (up to 4 discrete channels) sampling. This detector was designed to satisfy demands for greater accuracy and sensitivity than a typical UV-Vis detector but with a flexible multi-wavelength design.

c) Refractive Index Detector:

The RID-20A is a high-performance, easy-to-use refractive index detector that offers excellent stability. A dual temperature control structure and an improved thermal design is adopted for the optical system to provide better baseline stability and a shorter initial stabilization time.

d) Fluorescence Detectors:

The RF-20A and RF-20Axs are fluorescence detectors for UHPLC and HPLC separations with industry-leading sensitivity and fast sampling. These detectors offer superb ease-of-maintenance, thanks to cell and lamp replacements from the front panel with no additional position adjustment. The RF-20Axs also offers a Temperature controlled flow cell with cooling functions allowing excellent peak area reproducibility with respect to room temperature fluctuations.

e) Evaporative Light Scattering Detector:

The ELSD-LT II is an evaporative light scattering detector using a unique nebulizer and evaporation tube to allow low-temperature operation. This universal detector is a powerful tool for the gradient analysis of compounds that cannot be analyzed using an absorbance detector.

Fig. 1: Evaporative light scattering detector (ELSD)

f) Conductivity Detector:

The CDD-10AVP is a conductivity detector applicable for ion chromatography or organic acid analyses. This detector offers low noise and low drifts assurance with a wide dynamic range. High sensitivity is achieved even as a non-suppressed system which also allows for a high degree of versatility.

5. RECORDER:^(38,39)

Once the detection is completed the detected signals is converted into electronic signals and then amplified by means of amplifier and recorded as a chromatogram in the form of data points. Then these are manually or by aide of software it’s interpreted as per requirement to convert as a presentable format.

· Applications of HPLC:^(8-17)

a) Pharmaceutical applications:

· Tablet dissolution study of pharmaceutical dosages form.

· Shelf-life determinations of pharmaceutical products

· Identification of active ingredients of dosage forms

· Pharmaceutical quality control

b) Environmental applications:

· Detection of phenolic compounds in Drinking Water

· Identification of diphenhydramine in sedimented samples

· Bio-monitoring of pollutant

c) Forensics:

· Quantification of the drug in biological samples.

· Identification of anabolic steroids in serum, urine, sweat, and hair

· Forensic analysis of textile dyes.

· Determination of cocaine and metabolites in blood

d) Clinical:

· Quantification of ions in human urine Analysis of antibiotics in blood plasma.

· Estimation of bilirubin and bilivirdin in blood plasma in case of hepatic disorders.

· Detection of endogenous neuropeptides in extracellular fluids of brain.

e) Food and Flavor:

· Ensuring the quality of soft drink and drinking water.

· Analysis of beer.

· Sugar analysis in fruit juices.

· Analysis of polycyclic compounds in vegetables.

· Trace analysis of military high explosives in agricultural crops.

CONCLUSION:

It can be concluded from the entire review that HPLC is a versatile, reproducible chromatographic technique for the estimation of drug products. It has wide applications in different fields in term of quantitative and qualitative estimation of active molecules.

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