INTRODUCTION:

Chromatography is basically a modern tool for separation of a mixture into its basic constituents as per need. Separation of the mixture refers to separation of the main potent moiety from its additives, separation of impurities and their determination, or separation of various in-process related substances that come with the dosage form while it is manufacturing.

The basic principle of any type of chromatography depends on the two basic and most important parts comprising of the stationary phase and the mobile phase¹. The key to appropriate separation and detection does not lie on the mixture to be analyzed but on the supporting mobile phase compositions and stationary phase characteristics. The mixture or the drug to be analyzed maybe categorized into various headings which describes its nature. Based on the nature of the analyte, various factors play role in the proper selection of the mobile phase and the stationary phase². Majorly in case of mobile phase, the pH, ionization degree, temperature of mobile phase, ratio of organic and aqueous solvents etc are the parameters that create an impression on the efficacies of separation procedure. Likewise the stability of stationary phases over various pH ranges, the composition of stationary phases, stability, polarity and other factors, as discussed in the following, play a very important role in the selection of the stationary phase³. Not only the identification and the separation matters, but the R_t of the analyzing component also depends on the selection of mobile phase and stationary phase. Effective selection of the chromatographic components can be established when it allows for a lesser retention time of the analyzing component.

Effective selection of mobile phase and stationary phase also refers to the fact that how economical is our process of analysis⁴. A process becomes economical when in a minimum amount of solvent utilization and minimum amount of drug concentration we obtain our analyte peak, in the minimum possible time. That can be only achieved if we can study the properties of our analyte of interest and use compatible stationary phase and mobile phase for our analysis. Sometimes, apart from selection of the compatible solvent and column, we observe a non-refined peak. This refers to a peak which is broad, or split, fronting, tailing, and even overlapped peaks. Hence, not only selection, but optimization of the column parameters like particle size of the silica, its bonded phase, column length, column temperature, and parameters of mobile phase including the ratio of organic and aqueous solvent, pH etc should be taken into consideration.

Stationary Phase:

Ideal characteristics of stationary phases used in HPLC are retentivity and sample holding capacity, Chemical stability at varied range of pH and mechanical stability at varied range of pressure, the particle size distribution should be less in order to get maximum surface area for absorption, the pore size should be such, to pass the analyte (in terms of diameter) for optimum mass transfer, the stationary phase should possess minimum swelling properties, the support material should be homogenous as coated on the stationary phase but should be chemically modifiable when required⁵. Stationary phases are selected on the basis of: Stability over pH, Chemical stability, Chiral separation of enantiomeric analyte, Surface chemistry of stationary phase, Effect of temperature, Lipophilicity of analyte, Ion exchange separation

Stability over pH:

Silica based stationary phases are used widely. Their stability is checked both at very low pH as well as very high pH. At very low pH (acidic), the stability of the stationary phases are increased in the following ways. End-capping of the silica based stationary phases is not done by any methyl group so that it remains stable at acidic environment and does not dissolve. Moreover increasing the amount of covalent bond at the silica surface, resist the attacks of acid on the stationary phases. It is noted that more the hydrophobicity of the column material increases, more acid resistant it becomes. Hence, C₁₈ columns are more stable at lower pH compared to C₁, C₄, C₈ columns. A multi-dentate synthetic approach of C₁₈-C₁₈ bonded silica phases show very high resistivity against low pH ranges. Bi-functional or tri-functional substitution of the silica bonds also provide acid resistance but are not reproducible in their retention. pH resistivity can also be performed by end-capping the silica bond with a halogen group, an aromatic bulky group or even clubbing C₁₈ with C₁, C₄, C₈ stationary phase. In order to allow the silica based stationary phases survive high pH limits, they are mostly exposed to end-capping reactions. The disadvantage of these reactions is that they provide steric hindrances between bulky groups hence leaving much of the silica unexposed. Often double end capping is performed. This provides better pH resistance but often results in steric hindrance. Double end-capping can be done for example with C₁₈ with hexamethylcyclotrisiloxane or hexamethyldisilazane^6,7.

Chemical Stability:

The widely used silica stationary phases are soluble in an organic solvent or aqueous solvent, in even slightly alkaline media and even at moderate temperature elevations. Siloxane bonds are also unstable at acidic pH. To overcome this problem, nowadays metal oxide phases e.g. Zirconia, Titania, Alumina is being widely used. These metal oxide columns are able to withstand pH of very low to high as well as broad spectrum of pressure, while provides a longer column life. They provide a faster analysis also which in turn, makes the process cost-effective⁸.

Chiral separation of Enantiomeric analyte:

Chiral drugs e.g. Ketoprofen, Ibuprofen, Betaxolol etc has a chiral carbon in their chemical structure. Hence they are able to synthesize a racemic mixture of 2 of their enantiomers e.g R & S. The best chiral stationary phases used are cellulose or amylase based phases. The use of stationary phase which has a cellulose base in enantiomeric separation compensates the association power of mobile phase used. The chiral stationary phases fall under ‘polysaccharide sorbents’ which eventually result into the formation of diastereoisomeric complexes involving H-bond, π-π bond and dipole-dipole interaction. Basically cellulose or amylose are the polysaccharides that are used for this purpose, having carbamates added on them. The basic difference between cellulose and amylose phase is the spatial arrangement of the two. For cellulose, it is inflexible and linear. On the other hand for amylose, it is spiral. Carbamate groups are responsible for chiral separation. The difference in spacial arrangement, leads to the difference in arrangement of the carbamate groups. Studies show, amylose performs better chiral separation than cellulose^9,10,11,12.

Surface chemistry of stationary phase:

Surface chemistry defines the surface nature of the stationary phase used: crystalline or amorphous. Usually, silica is used extensively as a reverse-phase stationary material since it has a number of advantages in retention of analyte and proving itself inert to chemical reactions. But this polymeric base is thermally unstable. Even at highly alkaline medium, the silica stationary phase tends to wash away from the column and tend to elute. In order to overcome this problem, nowadays metal oxides replace silica as a stationary phase material. Metal oxides are crystalline in nature hence has minimum possibility to be washed out under extreme pH conditions. Metal oxides are able to withstand extreme thermal conditions. Silica has a low pH_pzc value, hence only considers cationic exchange^13,14. Whereas, the metal oxides e.g. zirconiam, titanium, alumina, are amphoteric in nature, hence provide exchange of both cationic and anionic entities. The silanols have a low pH_pzc value, hence it dissociates at neutral pH and the surface acquires a negative charge. The metal oxides have Lewis Acid sites on the surface which are responsible for ligand exchange ability of those metal oxides. Sometimes covalently bonded water molecules enables in the exchange of a Lewis Base. Harder the base, easier is the exchange.

Effect of temperature:

Selection of stationary phase, greatly rely on the degrees of temperature to be applied on them during the process of separation. As the temperature change provide a change in the chemical reaction in terms of analyte retention, we consider the van’t Hoff equation which relates to the change in equilibrium constant K, of a chemical reaction to the change in temperature T¹⁵. It has been found that increased temperature is advantageous for the peak shapes of bases however degradation of the stationary phase is rapid hence specially designed stationary phases for elevated temperatures should be used.

Lipophilicity of analyte:

Lipophilicity is basically explained as the partition coefficients in immiscible organic or aqueous systems which tend to be biphasic such as octanol/water, alkane/water, chloroform/water. In order to find the extent of Lipophilicity of the analyte, and its compatibility with the stationary phase, structure analysis of the analyte is performed with the help of linear salvation-free relationships (LSER’s) based on solvometric parameters. Solvometric parameters are effects of the medium on the UV/visible spectra of substances that are sensitive to the properties of medium e.g. polarity, Lewis acidity, basicity, dipolarity and polarizability.

Ion exchange separation:

Analyte which are ionic in nature, e.g. proteins, requires stationary phase which has capacity of ion exchange. Such ionic analyte are separated in the reverse phase mode^16,17.

Evolution of Stationary Phases:

Organic polymer monoliths are extensively used as stationary phases¹⁸. They act as a continuous polymer bed as well as a continuous column support. Monolithic material comprises of cellulose, silica, synthetic polymer of various shapes, molded to confine it in capillaries. The polymer monoliths allow macromolecular particles to pass through and provide a rapid flow. They also improve separation and increases mass transfer. The monomers used are hydrophilic, hydrophobic, ionisable and tailor-made. The polymers maintain the hydrodynamic properties of monolithic columns. Monolithic columns have inter-particular voids; porous polymer is used for ion exchangeable resin in order to provide better osmotic shock resistance and faster kinetics. The latest advancement among HPLC columns are supercritically porous silica columns¹⁹. This modern day invention provides faster analysis, good efficiency, adequate sample-loading capacity, reduced plate height and reduced back pressure. These columns absorb less organic solvents and hence can be termed as economical and environmentally friendly both. The various kinds of columns of this kind are Halo columns, Kinetex columns, Ascentis Express column, Poroshell columns, Accucore columns, Sunniest columns etc.

Halo Columns²⁰: Modern day Halo columns have an overall diameter of 2.7µ with a porous layer of 0.5µm along with a pore size of 90Å. These columns have the capability to separate larger molecules in a faster pace of mobile phase flow. It comprises of type B silica end-capping which is a densely bonded phase in the C₁₈and C₈ columns which enables it to sustain a pH range of 2.0 to 9.0 even at elevated pressures.

Kinetex Columns²¹: These columns emerged three years after the invention of Halo columns. Hence it carries a lot of modification that makes it even more effective than the previous one. The particle size was reduced from 2.7µ to 2.5µ, and the pore size was increased from 90Å to 96Å. Previously the surface area of Halo columns were 127m²/g, providing excellent mass transfer but later on with Kinetex columns. The surface area for mass transfer was further increased to 200m²/g. The total carbon content in the column was also increased from 7.5 to 12. Overall this column helped the separation and analysis to a much greater extent.

Ascentis Express columns²²: Very limited information has been obtained regarding this column type. But it is evident from the research that this column provides a rapid tool for lipophilicity determination. Along with silica gel B it consists of phenylhexyl. Poroshell columns^.Basically modern day columns share the same internal chemistry with slight modifications on pore sizes, lengths and coating layers. This differs from company to company.

Mobile Phase:

Isocratic or gradient:

Mobile phase preparation, in any method development or for any analysis, can be broadly classified into the techniques; whether to prepare an isocratic system or a gradient one²³. For isocratic mode of elution, the mobile phase comprises of a mixture of organic and aqueous phase. The contents of mobile and aqueous phases are in specific proportions. For example: the aqueous phase maybe a buffer, mixed with an organic solvent. Hence this mixture is used as the mobile phase for isocratic elution. For gradient elution, the mobile phase is separately prepared consisting of one aqueous mobile phase and another organic mobile phase.

Selection of mobile phase:

Mobile phase selection is basically dependent on the kind of drug we want to analyze. The nature of the drug implies to the polarity of the drug. Other than polarity, pH and pKa are amongst the most important criteria to look into when selecting a mobile phase, or developing a mobile phase.

Buffers as mobile phase:

Buffers are often used as the aqueous part of the mobile phase for separation of analyte with acid-base properties. As we know there is a very interlinked relation between pH²⁹ and pKa³⁰. pka is basically the pH value at which a chemical entity will either accept or donate a proton. So, if the pKa of an acid is very low, it will have a greater ability to donate a proton in an aqueous solution²⁴.

pH of the solvent:

Talking of measuring the pH of solvent includes electrode²⁵. pH has many scales on which it could be quantified. In pharmaceutical practice, pH is measured on molarity scale and requires a glass electrode and a reference electrode. Gradient elution technique provides a deciding factor whether the sample is suitable for isocratic elution or not. In case it is, it establishes the required ratio of the solvents to be used for effective separation²⁶. In RPHPLC, the main factor which decides the retention time and resolution is the composition of the mobile phase. Initially, in case of every unknown development method, a gradient run is performed. This initial gradient run decides whether an isocratic run can be performed or not depending on peak and retention times.

Selection of isocratic condition for HPLC:

Initially gradient elution is considered for any process that is yet to be developed. In this first attempt if any peak is obtained then it is seen whether isocratic elution is possible or not. If no peak is obtained, we have to change the chromatographic system. If isocratic elution is possible, the conditions for obtaining that elution are to be selected and run should be made. If the obtained peaks are distinct and are well resolved from each other, then it can be reported. Peak resolution can be well achieved by addition of modifiers, when required²⁷.

After initial run (gradient) we have to check if the sample is suitable with the stationary phase and solvent used and if isocratic is possible or not. If it is possible, organic modifiers e.g.: Methanol, Acetonitrile, Tetrahydrofuran can be used for better separation. If good chromatograms are obtained, then the approach is successful. And if not, the scheme shall be abandoned.

Isocratic Behavior:

Retention behavior of solute in RP shows a linear relationship. S = p + qlnko; where p and q are constants for a mobile phase system having two types of solvents. Only S or k_o is required to describe the behavior of an isocratic system.

Gradient Behavior:

Retention behavior is determined by: Isocratic retention characteristics of the solute, Characteristics of gradient program. Alternative approach of reversed phase liquid chromatography is to use water instead of any organic modifier as a mobile phase. But the water used should be of very high temperature. This water is termed as pressurized hot water^28,29. Instead of changing the solvent composition, we can increase the temperature of the mobile phase in order to get increased elution strength. There are a lot of advantages in using pressurized hot water over organic modifiers e.g.: ACN/water, MeOH/water, THF/water. The advantages are: Pressurized hot water is environment friendly. This can also be termed as “green mobile phase”, Provides higher separation speed, hence lower back pressure, Improved column efficiency, Extended detector selection.

Instead of pressurized hot water, D₂O can also be used at elevated temperature^30,31. When such elevated temperature is used to prepare the mobile phase, normal silica based columns cannot be used. Temperature resistant columns e.g. polysterene-divinylbenzene or zirconia particles with polybutadiene, polysterene or a carbon coating are used instead. Carbon coated zirconia resists till 370^oC and polybutadiene zirconia can withstand temperatures of 260^oC and 300^oC. At high temperature, thin walled capillary columns are advantageous due to faster rate of heat transfer and temperature equilibrium. The silica based columns with can withstand elevated temperatures are: Zorbax RX-C columns.

The pressurized hot water when used should be treated with inert gas. The pressure needed to retain the pressurized hot water in a liquid state is less e.g.: 40 bar at 250^oC. At 300^oC the pressure is 86 bar. When pressurized hot water is used as the solvent, instead of using syringe pump, reciprocating pump is used, as corrosion is less. Application area where pressurized hot water is used as solvent in separation procedure of active chemicals is natural product chemistry. PHW-LC finds its application in the analysis of thermolabile vitamins as well. It can also be applied for the separation of carbohydrates.

There is a scope of using multi-solvent mobile phase also. Multi-solvent mobile consist of both aqueous as well as organic solvent.

Solvents for Reversed Phase HPLC:

1. Acetonitrile: It has low viscosity and lessens back pressure. It also ends up providing better peak shape. It provides an advantage for UV detection as can help detect samples at lower λ’s. Forms binary mixtures with water.

2. Methanol: It is less expensive and less harmful. It is more polar, hence lessens the probability of precipitation of the drug in the buffer of the mobile phase. Methanol can undergo polar-polar or ionic interactions with solutes. Usually, it results in better selectivity for more polar compounds as it provides longer run times.

Degree of analyte ionization: This change with solvent pH. Ionised form has a greater polarity. Due to this, it gets eluted quickly with the polar mobile phase, in reversed phase conditions. Non-ionised form, on the other hand, is less polar in nature. Therefore, it retains longer in reversed phase as the stationary phase is also non-polar in nature.

General conditions to be followed during selection of buffer:

1. When Phosphate buffer solubility is checked, it is seen that it tends to dissolve more in methanol/water than in acetonitrile/water or tetrahydrofuran/water.

2. Hygroscopic buffers i.e. moisture absorbing buffers are mostly buffers of certain metallic salts e.g. Sodium. These buffers result into tailing associated with peaks of drugs which are chemically basic in nature.

3. Ammonia salts show solubility more in organic mobile phases compared to aqueous mobile phases.

4. Trifluroacetic acid, when used as a mobile phase constituent, should be taken care of the fact that it evaporates very quickly as it is volatile in nature. It also gets absorbed at very low UV frequencies.

5. Microbial contamination occur mostly in places comprising of water. Hence mobile phase compositions which are devoid of any organic modifiers and are fully aqueous have a chance of faster microbial contamination which can get deposited in the inlet tubes, mobile phase bottles, pipes etc and damage the system as well as give rise to inaccurate peaks.

6. Mobile phase should be degassed.

Retention time:

C₈ and C₁₈ columns consist of an agglomeration of bonded hydrocarbons in chain like fashion. There is a loss of retention noticed in these C8 and C18 columns when highly aqueous mobile phase is used. C8 and C18 columns are mostly used during reversed phase separations. The various theories that have been projected, over the years, as an explanation to retention loss are^32,33: Drying out of sorbent bed after conditioning, Loss due to folding of alkyl chains of the stationary phase, Formation of internal structures due to entrapment of solvent in the stationary phase, Pore size of the bonded phase particles. Retention in pore size causes partial filling of the pore network with the bonded groups, Low surface concentration of the stationary phase material. Hence, in order to increase retention in such columns, increment of the organic solvent content should be done in the mobile phase. Apart from that the retention can be increased by increasing the back pressure by gradual increment in the flow rate.

Dependence on temperature:

Often the mobile phase used is given a certain higher temperature due to many advantages^34,35.36: These are acceleration of rate of separation, efficiency and resolution of peak responses, temperature is one of the easiest way to tune chromatographic selectivity, especially in case of polar and ionizable compounds, if the temperature is elevated, the concentration of organic modifier, used in the mobile phase composition, can be lowered, increased temperature increases the interaction of analyte with the stationary phase, increasing retention or allowing proper retention. Hence peak shape improves. But temperature optimization should be taken into consideration because at unnecessary elevated temperatures the concerned column might suffer damage. It also alters the stability of the analyte.

Eco-friendly mobile phase:

Eco-friendly reagents refer to those which produce less waste in the form of fumes or residues which cannot be recycled in any other form. Such an example is Ionic Liquids^37,38. Ionic liquids belongs to a category of solvents used for the selective extraction, separation, and detection of proteins, peptides, nucleic acids, and other analyte from complex biological samples. With the help of ionic liquids, it has been possible to analyze samples directly in the instrument even when it has a lower concentration or lower abundance. Ionic liquids are also called “molten salt” which has their melting point similar to the ambient temperature. Ionic liquids consist of equal number of positive and negative charges which makes its electrically neutral. Some of the commonly used ionic liquids are Imidazolium, Alkylpyridinium, N-ethylpyridinium etc. Ionic liquids are not only present in the mobile phase as modifiers but sometimes coated as a thin coating on the stationary phase also. The silanol groups of the stationary phase interact with the ionic liquids. Generous addition of ionic liquid to the mobile phase allows an interaction between the ions of the ionic liquids with the silanol groups on the alkyl silica surface of the stationary phase, hence reducing the retention time^39,40.

When ionic liquid modifiers are added in the mobile phase, there arises a competition among the polar groups of the analyte and the positively charged particles of ionic liquids, in order to attach to the silanol groups on the alkylsilica surface of the stationary phase. As the concentration of the ionic liquids start to increase, the positively charged particles interact more with the silanol groups. The interaction is basically electrostatic in nature. This reaction gives rise to a bi-layer electronic structure that is chemically weak. This bi-layer structure in return reacts with alkyl groups via hydrophobic interactions and decreases the retention of analyte. If the concentrations of the ionic liquids are slightly increased further, their cation interaction with the silanol groups, on the alkyl silica surface gradually strengthens. This results in an increase in the carbon content of the stationary phase. Thus the retention times of substances are increased^41,42.

Lipophilicity of the analyte:

It is one of the most distinguished parameter in judging a molecule on the attributes of its chemical nature. It plays important role in both pharmacokinetics and pharmaco-dynamic determination.

Reverse phase HPLC is a strong tool to determine lipophilicity. Retention factor (log k) is used to estimate the partition coefficient. Determination of Lipophilicity of an analyte is advantageous in many ways; requires less amount of the analyte, consumes less time, and provides with highly reproducible results. Many trials have been carried out in the past to reach down to an ideal mobile phase composition for such lipophilicity determination. Out of all the trials performed, octanol-water system has been observed to be the most suitable. To add to mobile phase system, addition of methanol as an organic modifier further helps in the analysis. Methanol is added in various percentages, depending on the analyte to be separated^43,44. n-octanol has a hydrophobic chain with polar head. The hydroxyl group in the molecule has hydrogen bonding capacity. A very high degree of water saturation produces practical difficulties in analysis. For n-octanol it is only 0.25 mol%. The water molecules in octanol are variedly dispersed into clusters of four molecules. These clusters of water molecules are further surrounded by 16 molecules of octanol molecules. The hydroxyl groups of octanol direct themselves towards the water molecule creating a hydrophilic network. On the other hand, the hydrocarbon part of the n-octanol creates a hydrophobic zone and this total arrangement replicates the inner structure of a lipid bilayer. Hence partitioning of an analyte in terms of its lipophilicity becomes easy with n-octanol and water system. Furthermore, methanol is chosen as the organic modifier in lipophilicity determination because it does not interferes or disrupts the hydrogen bonding of water. Methanol bonds with stationary phase forming a monolayer. This monolayer formation provides hydrogen bonding with n-octanol^45,46,47.

CONCLUSION:

HPLC analysis of analyte will be helpful in getting more accurate results in precise time and in an economical and eco-friendly way if the above mentioned parameters are studied prior to their analysis. The major factors on which any HPLC analysis depends are the pH-pKa stability, polarity of stationary and mobile phase, temperature stability of the analyzing sample and the column, hydrophobicity/hydrophilicity, chemical stability, time consumption for analysis and sample load. Considering these parameters as per literature review provides a faster and economical yet effective analysis.

CONFLICTS OF INTERESTS:

The authors claims that there is no conflict of interest.

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Received on 31.12.2020 Modified on 20.06.2021

Research J. Pharm. and Tech 2022; 15(9):4325-4332.

DOI: 10.52711/0974-360X.2022.00726