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Phani. R. S. Ch et al.

, IJSID, 2012, 2 (6), 218-228

ISSN:2249-5347

IJSID

International Journal of Science Innovations and Discoveries


Review Article
1Department

An International peer Review Journal for Science

Available online through www.ijsidonline.info


SCIENTIFIC APPROACH FOR RP-HPLC METHOD DEVELOPMENT: COMPLETE REVIEW of Chemistry, K.L. University, Vijayawada, Andhra Pradesh, India; 2RV Labs, Guntur, AP, India Phani.R.S.Ch*1, K.R.S. Prasad1 and Useni Reddy Mallu2

Received: 14-11-2012 Accepted: 20-12-2012


*Corresponding Author

components in a mixture is based on their interaction between a stationary and a mobile phase. These interaction differences are achieved based on the properties such as polarity, polarity, pH and pKa values, molecules spectral absorbance and functional groups activity understanding of the chemical structure of the molecules, synthetic route, solubility,

electric charge (for ionic compounds), pH, functional groups and size of the molecule. HPLC method development depends on the how much the researcher have the method development. For adjucent and late elution case gradient program is best to elution and this will give longer column usage for regular analysis. spectrum and Synthetic Route. KEY WORDS: etc. Nowadays in the market so many type of columns are available for the unique HPLC

HPLC is the key instrument for separating the chemical substances. Separation of ABSTRACT

Address: Name: Phani.R. S. Ch. Place: Guntur, AP, India E-mail: phani.r.s.ch@gmail.com

reduce the analysis time with accurate quantitation. Isocratic elution is suitable for wide RP-HPLC, Method development, Gradient program, Isocratic elution, UV

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Phani. R. S. Ch et al., IJSID, 2012, 2 (6), 218-228 an indication of the identity of the chemical species in the sample. Quantitative analysis estimates, how much quantity is present in a mixture. Modern analytical chemistry is functioning by instrumental analysis. Separation of components in a mixture is based on their interaction between a stationary and a mobile phase. These interaction differences are achieved and Column chromatography, UV/Visible spectroscopy, FT-IR, LC-MS, GC-MS, MASS and NMR. based on the properties such as polarity, electric charge (for ionic compounds), pH, functional groups and size of the molecule. Analysis can be divided in to two classes, i.e. Qualitative analysis and Quantitative analysis. Qualitative analysis gives INTRODUCTION

Different types of quantitative analytical techniques are there, for ex: HPLC, Gas chromatography, TLC, Ion chromatography

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY: stationary phase used.

mobile phase. Separations are achieved by partition, adsorption, or ion-exchange processes, depending upon the type of

High-performance liquid chromatography is a separation technique based on a solid stationary phase and a liquid

Figure-1: Qualitative and quantitative analysis

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Figure-2. High-performance liquid chromatography instrument and flow diagram

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Phani. R. S. Ch et al., IJSID, 2012, 2 (6), 218-228 Instrumentation recorder. Figure-2 represents the shematic diagram of HPLC instrument. Figure-3 represents the HPLC individual parts. General HPLC instrument have reservoir, degasser, pump, column, column oven, injector, auto sampler, detector and

Reservoir: Degasser: Pump: Column:

Isocratic Mixture: Column oven: Injector: Detectors:

Gradient mixture:

Solvent reservoirs should hold at least 500 ml of mobile phase. Single solvent contains both buffer and organic solvent. Contains two solvents (Sol-A and Sol-B). Packed with very fine particles Used for the sample injection Used for mobile phase pumping (up to 10,000psi) Facilitate for 100-200vials in to the system.

Degassing is used to eliminate dissolved gases in the mobile phase.

Figure-3: HPLC instrument parts

Auto sampler:

Facilitate the temperature to HPLC column from 25C to 60C UV, RID, ELSD, Fluorescence and chemical detector.

Chromatographic Calculation: Area %, RRT, RRF etc. 1. 2. 3. 4.

Chromatographic calculations are, Resolution, Theoretical Plates, System suitability, S/N Ratio, Void volume, Area, HPLC method development requires the scientific approach for developing the method for the chemical substances Literature survey, Buffer selection, Column selection, Chemical properties of the molecules,

HPLC METHOD DEVELOPMENT:

and drug products. Method development will involves the following steps like

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5. 6. 7. 8. 9.

Mobile phase selection, Detector selection,

Phani. R. S. Ch et al., IJSID, 2012, 2 (6), 218-228

Isocratic and gradient elution, Pre-validation of the method

Chromatographic parameters selection and

STAGE NUMBER-1: 1. Literature Survey:

Literature sources are Pharmacopeial monographs (USP, EP, JP, IP and etc.), online published journals, books and manufacturer catalogues and patents. 2. Synthetic route: the product. Draw the chemical structure of the each stage reactants, products, bi-products, solvents, catalysts and carry over products. The typical synthetic route represented in the below figure. Synthesis of the product information is mandatory to develop the method for impurity profile and quantification of

Literature survey is important for method development and it will give better idea for method development.

Figure-4: HPLC method development approach

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Phani. R. S. Ch et al., IJSID, 2012, 2 (6), 218-228

development and toluene also. If any bi-products in the target synthetic route it is also important. 3. Chemical properties:

As per the above model synthetic route reseacher need to consider the reactant compound-1 and 2 for the method

Figure-5: Typical synthetic route

development and it will give scientific approach for the method development. Draw the comparative difference b/w the impurities, starting materials, byproducts, intermediates and degradation products with final product. 4. pH and pKa value of compound diluent for all molecules. Polarity of the molecules information is important for selecting the polar /non-polar HPLC columns. Outside this range the compound is either ionized or non-ionized, and its retention does not change much with pH. 5. Spectral data: IR spectral data is the source for understanding the functional groups activity. substances by HPLC. Solubility study at different pH values information for all targeted molecules is best input for selecting the common Based on pH or pKa values the nature of the compound and polarity of the compound can be assumed. When pH is

Chemical structure of the known and expected product chemical structures are good inputs for initiating the method

equivalent to pKa, the compound is half ionized. Almost all the pH related change occurs within 1.5 units of the pKa value.

development the reseacher need to know the molecule spectral absorbance. FT-IR spectrum will give the information which

All listed molecules UV/VISIBLE and FT-IR spectrums are required to select the UV detector nm for all molecules. FTFrom the above UV spectrum the suitable nanometer for analysis is 260nm so before initiating the method

functional group have the high active nature in the molecule, this will give better support for separating the related chemical

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Phani. R. S. Ch et al., IJSID, 2012, 2 (6), 218-228

Figure-6: Typical UV spectrum

STAGE NUMBER-2:

1. Selection of initial chromatographic conditions: the below sections.

chromatographic conditions were represented in the below table. The selection of each parameter has discussed in-detail in 2. Selection of diluent and concentration and water-organic mixtures. Mobile phase is best choice for sample diluent, as it eliminates ghost peaks, base line noise and negative peaks. All the components should be completely soluble to obtain clear solution. All the components of the drug substance to be checked in solutions like mobile phase, mobile phase organic mixtures

This step requires the basic properties of the targeted molecules (above all information).

Figure-7: Typical FT-IR spectrum

The selection of

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need to consider the quality of the column by verifying batch to batch and lot to lot reproducibility. Column parameters like analysis the mobile phase is polar and column is nonpolar. C8, C18, cyano, phenyl and amino columns can be used against a Cyano, phenyl, silica and chiral columns are used in normal phase mode. 4. Selection of column oven temperature: 5. Selection of injection volume: Column oven temperature can be selected from the elution of the analytes and peak shapes.

Chromatographic condition HPLC column Flow rate Detector Sample concentration Diluent Mobile phase buffer Organic modifier (solvent) Mobile phase composition (buffer: solvent) Runtime (min) 3. Selection of column:

Table-1: Selection of chromatographic conditions

Phani. R. S. Ch et al., IJSID, 2012, 2 (6), 218-228 Required information for selection Polarity, pH and PKa values Selected column details Molecule chemical structure and UV spectrum UV spectrums Solubility over the pH range Polarity, solubility, pH and pKa values Solubility and polarity Elution of the molecules Elution of the samples

internal diameter, particle size, surface area, carbon load to be checked to verify system suitability criteria. In reverse phase

In the commercial market, there are so many types of columns are available. Before choosing the column researcher

more polar mobile phase. Similar way in normal phase analysis the column is more polar when compared to mobile phase.

have high absorbance at the selected detector conditions then researcher can use the less microliter also. 6. Selection of runtime: 7. Selection of organic modifier:

General case 20L is used and if the samples have less absorbance then it may change to 100L also and if the samples Samples analysis time can be fixed from the late elution of the peak. General case after elution of the late elution

elution of the molecules and peak shapes and mobile phase buffers. 8. Selection of isocratic and gradient:

The most useful solvents are Acetonitrile, Methanol and THF. The selection of organic modifier is depending on the Isocratic elution can be selected for wide elution peaks and this can give better baseline than gradient elution.

Isocratic is the best for regular quality control case but if any adjacent elution peaks and late elution peaks gradient elution is the best method. By using the Gradient elution researcher can reduce the analysis time and peak shapes. Gradient elution will be useful for impurity profiling. 9. Selection of mobile phase pH value: stability, column stationary phase, elution of the analytes and peak shapes. 10. Selection of Detector In the development mobile phase pH selection is critical and it can be selected based on the solubility, solution A detector is an important part of the liquid chromatography, it should be chosen very carefully for selective

separation and accurate determination. The single most crucial factor is continuous detection based on the progress of International Journal of Science Innovations and Discoveries, Volume 2, Issue 6, November-December 2012

separation of a component which may be immediately displayed and then recorded. However, a good detector should have

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linear response to solutes that extends over several orders of magnitude. It must have good stability, reproducibility and reliability. It should have low dead volume to minimize extra-column band broadening. basic types, bulk property detectors and solute property detectors. Type of Detector UV-Detectors Fluorescence detectors Electrochemical detectors: Refractive Index detectors: Evaporative Light detectors(ELSD): Table 2: Detector selection based on application of compound

Phani. R. S. Ch et al., IJSID, 2012, 2 (6), 218-228

Based on the structure and nature of the molecule suitable detection technique is to be selected. Detectors are of two Applications Used for compound having chromophore. Used for compounds exhibits fluorescence properties. For easily oxidizable compounds. These are universal, but cannot be used with Gradient elution. Used for the compounds which are not having chromophores and where high sensitivity is not required. These are superior to RI detectors can be used for higher sensitivity with gradient elution.

other detectors like RI, ELSD or gas chromatography.

the components of molecule. Select absorption maximum for product by considering all components in the product. pure are not.

Types of detector suitable for analysis are shown in Table 1. If the compounds are not having chromophores choose If selected detection technique is UV-Visible spectrophotometer or photo diode array detector record spectrum of all

Scattering

Components to be considered are degradants, intermediates, process related impurities and raw materials. Also check the data at different scanning range if any component behaves differently, analyze in dual wavelength. Ensure peak purity of all the 11. Selection of Buffer

components, purity angle should be less than purity threshold. Peak purity test measures whether the compound is spectrally analyte, the longer it is retained. When an analyte is ionized, it becomes less hydrophobic and, therefore, its retention decreases. Acids lose a proton and become ionized when pH increases and bases gain a proton and become ionized when pH control the pH of the mobile phase using an appropriate buffer in order to achieve reproducible results. Phosphate Citrate Buffer Table.3: commonly used hplc buffers for reverse phase HPLC pKa Buffer Range 2.1 1.1-3.1 7.2 6.2-8.2 12.3 11.3-13.3 3.8 2.8-4.8 4.8 3.8-5.8 3.1 2.1-4.1 4.7 3.7-5.7 5.4 4.4-6.4 8.3 7.3-9.3 11.0 10.0-12.0 11.3 10.3-12.3 * Volatile buffers UV Cut off (nm) 200 210 210 230 205 200 200 decreases. Therefore, when separating mixtures containing acids and/or bases by reversed phase HPLC, it is necessary to

In reversed phase HPLC, the retention of analytes is related to their hydrophobicity. The more hydrophobic the

Formic acid* Acetic acid* Tris Triethylamine* Pyrrolidine

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tetrahydrofuran. Ammonium acetate is the most soluble buffer salt, ammonium phosphate is less soluble, and potassium

phosphate is the least soluble buffer salt in each aqueous-organic mixture. Phosphate buffers at pH 3.0 are more soluble than 25 to 50 mM is adequate for most reversed phase applications.

phosphate buffers at pH 7.0. Commonly used hplc buffers are showed in Table 2. Optimum buffering capacity occurs at a pH

Ideally the buffer should transmit light below 220nm. All buffers are most soluble in methanol and least soluble in

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equal to the pKa of the buffer. In general, most buffers may provide adequate buffering capacity for controlling mobile phase HPLC is the key technique in the pharmaceutical industry and by using HPLC researcher can estimate the impurities, CONCLUSION AND RECOMMENDATIONS

pH only within 1 unit of their pKa, beyond that, buffering capacity will be inadequate. A buffer concentration in the range of content, drug release etc. For best method development, the researcher should proceed with scientific manner i.e. understand the chemical structure, solubility, polarity, spectral data, pH and pKa values. Literature survey will provide the basic either isocratic or gradient. After finalizing the method, researcher should perform the method evaluation so that if any method errors all can be resolved before proceeding for the complete method validation. 1. 2. 3. 4. 5. 6. 7. 8. 9. REFERENCES chromatography experiment, Journal of Chemical Education, 50 (5) (1973) 369. Analytical Chemistry, 72 (21) (2000) 54505458. (247) (1997) 67377. information for initiating the method development trials. Based on the peaks elution and peak shape researcher can select

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