INTRODUCTION:

As the solutes pass down a chromatographic column, they are eluted at varying rates, which is how the HPLC analytical procedure separates the solutes. Distribution between the mobile phase and stationary phase controls how this instrument separates material. The proper balancing of many operating conditions, including the kind of column packing and mobile phase, column length and diameter, mobile phase flow rate, column temperature, and sample size, is necessary for the successful application of HPLC to a potential issue¹. Due to its wide range of applications, reversed-phase chromatography is now the most widely used separation method in HPLC. The two primary stationary phase types are reverse phase and normal phase. Whether a column can be used for normal phase or reverse phase chromatography depends on the characteristics of the stationary phase.

Table 1: Normal Vs Reversed Phase Chromatography²

	Normal Phase	Reversed Phase
Stationary Phase	Polar - Cyano - Amino - Diol - Silica	Non-Polar - C-18 - Cyano - C-8 - Phenyl - C-3 - C-12
Mobile Phase	Non-polar (Organic solvent) - Acetone - Toluene - Ether - Hexane - Cyclohexane - Isopropyl alcohol	Polar - Methanol - Water - Acetonitrile
Sample Movement	Non-polar fastest	Polar fastest
Separation Based On	Different Polarities	Different Hydrocarbon content

Reversed-phase chromatography is generally more use than Normal phase Liquid chromatography nowadays. Due to following reason.³

Large proportion of analytes are water soluble in nature. So, it will easy to dissolve in the mobile phase because in RP-HPLC polar mobile phase used.

¨ Wide range of stable stationary phases are available.

¨ Simple mobile phases work for many applications. (i.e water: acetonitrile)

¨ Retention and selectivity are alter by changing:

• Stationary Phase: chain length and chemistry, polar end capping reagents etc.

• Mobile Phase: Organic solvent type, pH, buffers and other additives

• Temperature: specially with ionizable analytes

NEED OF METHOD DEVELOPMENT:⁴

§ New molecules method development

§ Stability indicating method development

§ Herbal Products

§ New Process and Reaction

§ Impurity Profiling

§ Active ingredients (Macro-analysis)

§ Residues (Micro-analysis)

STEP INVOLVE IN METHOD DEVELOPMENT:

The method development strategy can be in the following sequence,⁵

1. Analysis of Physicochemical properties of a compound

2. Set up HPLC condition

i. Separation modes

ii. Selection of detector

iii. Selection of Stationary phase

iv. Selection of Mobile Phase

v. Selection of pH/buffer/organic modifier

vi. Washing of column

3. Sample pre-treatment

4. Method validation

1. Analysis Of Physicochemical properties of a Compound:

Before beginning method development, we need to review what is known about the sample. The kind of sample-related information that can be summarized in table 2.

Table 2: Important information concerning sample³

Important information concerning sample composition and properties

Chemical structure of compounds

Molecular weight of compounds

Polarity of a compound

Solubility of compound in solvent

Ionization constant of a molecule

Log P value

A. Chemical structure of compound^3,7:

· It provides best clue for the selection of initial HPLC conditions.

§ Chiral molecule: chiral chromatography

§ Ionic compound : ion exchange chromatography

B. Molecular weight of a compound⁸:

§ Molecular weight of a compound can be useful in the selection of chromatographic separation principle.

§ High molecular weight compound: (macromolecules, proteins, biomolecules) size exclusion chromatography

§ Low molecular weight compound: Normal phase chromatography, RP-HPLC

C. Polarity of a compound:⁸

§ Polar molecule: RP-HPLC

§ Non polar molecule: normal phase chromatography

D. Solubility of compound in solvent:⁸

§ The solubility of sample in commonly used is important otherwise it may lead to incompatibility.

§ Solubility of a particular drug is prerequisite for any salt selection program.

§ The free acid/free base and their corresponding salt will all have different solubilities in the diluents.

§ The analytes and its reactivity with diluent.

E. Ionization constant of a molecule:⁹

§ It is called pKa value of a molecule.

pH=pK_a+log([salt]/[acid])

§ if the pH of the mobile phase is 2 pH unit less than pKa value, the molecule remains in unionized form.

§ if the pH of the mobile phase is 2 pH unit higher than pKa value, the molecule remains in ionized form.

§ So, the selection of the mobile phase pH is important parameter in the separation of a compound.

Figure 1: Effect of mobile phase pH

F. Log P value⁹ :

§ It is the partition coefficient of a compound between octanal and water.

§ The log P value indicate that the molecule is hydrophilic or hydrophobic.

§ If log P value is higher which indicate that the molecule is hydrophobic in nature. It affect the solubility of a compound.

i. Separation Modes^11,12:

Figure 3: Isocratic and Gradient separation

¨ Traditionally, isocratic separation are deemed as more reproducible than gradient separation.

Disadvantages of gradient elution¹³:

¨ Longer run time

¨ Column re-equilibration required after every analysis

¨ Require a pump with at least two-solvent capability

¨ Not compatible with some forms of detection. (RI, EC)

¨ More variable to control for reproducibility

¨ Delay volume (dwell volume) becomes important

¨ Delay volume: volume of mobile phase contained in the HPLC sustem between pump and column.

· Tips for a successful gradient run¹⁴:

¨ Keep it as simple as possible

¨ Be aware that delay volumn will vary from instrument to instrument

¨ Make sure post run equilibration time is adequate to return column to initial conditions.

¨ Pre-mix mobile phase modifiers use.

ii. Selection of Detectors^15-18:

Following types of detectors generally use in HPLC.²⁹

· UV Visible detectors

· Photo diode array detector

· Fluorescent detector

· Refractive index detectors

· Electrochemical detectors

· Mass detectors

· Detector selection: UV/Vis detector:

· Requirement: analyte must be UV active

· Choose detection wavelength that maximize sensitivity and specificity.

· Solvent used may cause slight shifts in UV_nmfrom published values (2-5nm)

¨ Check absorbance of analyte in mobile phase

· Mobile phase solvent have UV Cutoff points.

· Operating below cut-off point will reduse sensitivity and add to baseline noice.

· Diode array detector (DAD) can monitor multiple wave length simultaneously.

· Detector selection: Mass spectrometer:

· Requirement: analyte must be ionizable

· Can discriminate between co-eluting peaks in selected ion mode.

· Reduces resolution required

· For best sensitivity, work at pH where analytes are ionized.

· Neutral to basic pH (7-9) for acids.

· Acidic pH (3-4) for bases.

· Detector selection: Refractive Index:

· It is a Universal detector

· Monitors difference in the refractive index of the sample cell vs. the reference cell.

· Non- selective

· Concentration dependent

· Sensitivity is typically 100 x -1000x less than a UV/Vis detector.

· It cannot be used with gradients.

ii. Selection of Stationary phases^19-21:

· Stationary phases are packed in column and column are made up of stainless steel.

· Most liquid-chromatographic column range from 5 to 25 cm long. Straight column are used.

· The inside diameter of analytical column is often 3 to 5 mm. the common particle size of packaging is 3 to 5 micrometer.

· Column Dimensions

¨ Dimensions of column effect on chromatography.

Table 4: Column dimensions and its effect on chromatography

Short (30-50 mm)	Short run times, low backpressure
Long (250-300 mm)	Higher resolution, long run times
Narrow (≤ 2.1 mm)	Higher detector sensitivity
Wide (10-2- mm)	High sample loading

iii. Selection of Mobile Phase^22-24:

Figure 4: Commonly used mobile phases

· In partition chromatography, the mobile phase should be a moderate to poor solvent for samples.

· Produce a capacity factor of 1 to 10 (Preferably 2 to 5)

· For ion exchange and size Exclusion the Mobile Phase should be a strong solvent for the sample.

· The use of additives or Modifiers can

¨ Enhance separation

¨ Improve peak shape

¨ Alter selectivity

· Viscosity

¨ Low viscosity solvents are required

¨ As viscosity increases the efficiency of the system decreases.

· UV cut off:

¨ Wavelength below which solvent will absorb more than 1 unit in a 1 cm cell.

Table 6: Solvent and its UV-cutoff

Solvent	UV cutoff (nm)
Acetonitrile	190
Isopropyl alcohol	205
Methanol	205
Ethanol	205
Tetrahydrofuran (THF)	215
Ethyl acetate	256

· Compressibility

¨ Solvent with high compressibility show greater pulsation during pumping.

· Polarity

¨ It is a measure of solvent strength or ability to elute a particular type of compound

· Vapor pressure

¨ Due to evaporation of solvent composition of mobile phase may change.

iv. Selection of Buffer^25-26:

· Buffer as a part a mobile phase.

· In order to develop rugged HPLC method, knowledge of choosing the right buffer is very important.

· Buffer that are selected should have a good buffering capacity at the specified mobile-phase pH.

· The concentration of the buffer should be at least 10 mM.

· Optimum buffering capacity occurs at a pH=pKa of the buffer.

· In general, most buffers provide adequate buffering capacity for controlling mobile-phase pH only within ±2 unit of their respective pKa.

· Also, buffer are great media for growing bacteria. It is recommended to have at least 10 % V/V of organic in the aqueous phase to prevent bacterial growth.

Table 7: HPL CB uffers, pKaValues and Usefulp HRange

Buffer	pKa	Useful pH Range
Ammonium acetate	4.8 9.2	3.8-5.8 8.2-10.2
Ammonium formate	3.8 9.2	2.8-4.8 8.2-10.2
KH2PO4/ phosphoric acid	2.1	1.1-3.1
KH2PO4/K2PO4	7.2	6.2-8.2
Potassium Acetate/ acetic acid	4.8	3.8-5.8
Borate (H3BO3/Na2B4O7 10H2O)	9.2	8.2-10.2
Ammonium hydroxide/ amm	9.2	8.2-10.2

¨ Choice of Organic Modifier²⁷:

· Selection of the organic modifier type could be viewed as relatively simple.

· The usual choice between acetonitrile and methanol (Rarely THF)

· In short, methanol shows more predictable influence on the analyte elution.

· The viscosity of water/organic mixture should be considered as an additional parameter in the selection of organic modifier.

· The stability of the mobile phase should also be considered. THF is known to form peroxides.

Figure 5: Different Peak modifiers

v. Washing of the column²⁸:

· If no buffer is used in mobile phase than

¨ Once analysis is comleted, HPLC instrument should be run for 30min using methanol: water (70:30% v/v) or Acetonitrile: water (70:30% v/v)

· If buffer is used in the mobile phase than

¨ Continue HPLC instrument with warm HPLC water for 30min and then use Methanol: water (70:30 %v/v) or Acetonitrile: water (70:30 %v/v) for further washing.

vi. Sample Pretreatment^29-30:

· Sample are pretreated to achieve

§ Improving Detectability of compound

§ Minimizing matrix effect

§ Stabilize an unstable analyte

1. Regular sample/normal sample/synthetic sample:

¨ Neutral: if the sample dissolve in solvent than RP-HPLC is the preferred choice.

¨ Ionic: ion pair chromatography or ion exchange chromatography is the preferred choice.

2. Special samples:

¨ Biomolecules, proteins: size exclusion chromatography

¨ Chiral molecules: chiral chromatography

¨ Enzyme, Antibody: affinity chromatography

· Sample Preparation³⁰:

· Weighing and Volumetric Dilution

¨ Sonication

¨ Homogenization

¨ Dissolution

· Sample Extraction (For solid and liquid sample)

· Filtration

· Centrifugation

· Solid Phase Extraction (SPE)

· Derivatization

vii. VALIDATION OF DEVELOPED METHOD:^31-33

The following are typical analytical performance characteristics which may be tested during methods validation:

1. Specificity

2. Linearity

3. Range

4. Accuracy

5. Precision

6. Detection limit

7. Quantitation limit

8. Robustness

1. Specificity:

· Specificity is the ability to assess unequivocally the analyte in the presence of components which may be expected to be present. Typically these might include impurities, degradants, matrix, etc.

· Specificity for an assay ensures that the signal measured comes from the substance of interest, and that there is no interference from excipient and/or degradation products and/or impurities.

2. Linearity:

· The linearity of an analytical procedure is its ability (within a given range) to obtain test results which are directly proportional to the concentration (amount) of analyte in the sample.

· For the establishment of linearity, a minimum of 5 concentrations is recommended. Other approaches should be justified.

3. Range:

· The range of an analytical procedure is the interval between the upper and lower concentration (amounts) of analyte in the sample (including these concentrations) for which it has been demonstrated that the analytical procedure has a suitable level of precision, accuracy and linearity.

· The range is normally expressed in the same units as the test results. For assay tests, ICH requires the minimum specified range to be 80to120 percent of the test concentration.

4. Accuracy:

· The accuracy of an analytical procedure expresses the closeness of agreement between the value which is accepted either as a conventional true value or an accepted reference value and the value found.

· Accuracy should be assessed using a minimum of 9 determinations over a minimum of 3 concentration levels covering the specified range (e.g., 3 concentrations/3 replicates each of the total analytical procedure).

· Acceptance limit for % recovery is 98-100%

5. Precision:

· The precision of an analytical procedure expresses the closeness of agreement (degree of scatter) between a series of measurements obtained from multiple sampling of the same homogeneous sample under the prescribed conditions. Precision may be considered at three levels: repeatability, intermediate precision and reproducibility.

6. Detection Limit:

· The detection limit of an individual analytical procedure is the lowest amount of analyte in a sample which can be detected but not necessarily quantitated as an exact value.

· Limit of detection (LOD) was calculated by using the formula

LOD=3.3x σ/S

Where, σ=Standard deviation of response; S = Slope of regression equation

7. Quantitation Limit:

· The quantitation limit of an individual analytical procedure is the lowest amount of analyte in a sample which can be quantitatively determined with suitable precision and accuracy.

LOQ=10xσ/S

Where, σ=Standard deviation of response, S=Slope of regression equation.

8. Robustness:

· The robustness of an analytical procedure is a measure of its capacity to remain unaffected by small, but deliberate variations in method parameters and provides an indication of its reliability during normal usage.

9. System Suitability:

System suitability test parameters are:

Table 8: System Suitability Test Parameters and Recommendations:

Parameters	Recommendation
Theoretical Plates(N)	should be >2000
CapacityFactor(k‟)	The peak should be well-resolved from other peaks and the void Volume, generally k‟>2.0
Relativeretention	Not essential as long as the resolution is stated.
Resolution (Rs)	Rsof > 2 between the peak of interest and the closest eluting potential interferent (impurity, excipient, degradation product, internal standard) etc.
TailingFactor(T)	T of</= 2

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Received on 18.12.2022 Modified on 11.08.2023

Asian J. Res. Pharm. Sci. 2024; 14(1):71-76.

DOI: 10.52711/2231-5659.2024.00011