INTRODUCTION:

High Performance Liquid Chromatography (HPLC) has emerged as a cornerstone in analytical chemistry, offering unparalleled precision in the identification, separation, and quantification of diverse substances. As technology continues to advance and analytical challenges become more complex, the need for innovative methodologies in HPLC has become increasingly imperative.

This review article aims to provide a comprehensive exploration of the recent advancements in developing techniques for HPLC and their extensive applications across various scientific domains.

HPLC's pivotal role in analytical sciences lies in its ability to navigate the intricacies of molecular composition, making it an indispensable tool in fields such as pharmaceuticals, environmental monitoring, clinical diagnostics, and biotechnology.^1,2 The continuous evolution of HPLC methodologies has been marked by significant breakthroughs in sample pretreatment, mobile phase selection, column optimization, and detector technologies. These advancements collectively contribute to enhancing the precision, sensitivity, and versatility of HPLC, thereby ensuring its adaptability to an ever-expanding range of analytical scenarios.

The complexities inherent in the chemical structures of substances under analysis necessitate a nuanced approach to method development in HPLC. This review will delve into the impact of various chemical attributes such as molecule structure, synthetic pathways, solubility, polarity, pH, and functional group activity on the creation of tailored HPLC techniques. Understanding these chemical intricacies is fundamental to optimizing HPLC methods for specific applications, and recent developments in this realm will be thoroughly examined.

Moreover, this review will explore the diverse applications of HPLC, showcasing its effectiveness in addressing the unique analytical demands of different scientific disciplines.^3-6

As we relve into the intricacies of HPLC advancements, it is crucial to consider the validation protocols that underpin the reliability and reproducibility of HPLC methods. Adherence to international guidelines, particularly those established by the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH), ensures the credibility and acceptance of HPLC techniques in the scientific community.⁷

HPLC Classification:

It can be classified based on various criteria, considering the diverse methodologies and applications within the field. Here's a classification based on different aspects:

1. Based on Stationary Phase:

a. Normal Phase HPLC (NP-HPLC): In NP-HPLC, the stationary phase is polar, and the mobile phase is nonpolar. Separation is achieved based on the polarity differences between the components.

b. Reverse Phase HPLC (RP-HPLC): RP-HPLC is characterized by a nonpolar stationary phase and a polar mobile phase. It is the most common mode of HPLC and is widely used for separating nonpolar and moderately polar compounds.^8-11

When silica was first employed, hydrophobic ligands were attached to it as a stationary phase. However, the unreliability of this assistance with sharp pH shifts is a drawback. When used as a base matrix for a cation exchanger to separate amino acids, polystyrene performs far better than silica support. Being chemically stable in both basic and acidic environments is polystyrene's biggest benefit.^12,13

c. Ion Exchange Chromatography: In this method, separation is based on the charge of the analyte. The stationary phase contains charged groups that attract ions with the opposite charge.¹⁴

d. Size Exclusion Chromatography (SEC) or Gel Filtration Chromatography: Separation is achieved based on the size of the molecules. Larger molecules elute first, while smaller molecules are retained longer.^15,16

2. Based on Mobile Phase:

a. Normal Phase Chromatography: Non-polar solvents are used as the mobile phase.

b. Reversed Phase Chromatography: Polar solvents are used as the mobile phase.^17-19

3. Based on Analyte Interaction:

a. Adsorption Chromatography: Separation is achieved based on the adsorption of analytes onto the surface of the stationary phase.

b. Partition Chromatography: Separation is based on the distribution of analytes between the mobile and stationary phases.

c. Ion Exchange Chromatography: Separation is achieved by the exchange of ions between the analyte and the stationary phase.^20-24

4. Based on Detection Method:

a. UV-Visible Detection: The most common detection method, based on the absorption of ultraviolet or visible light by the analyte.

b. Fluorescence Detection: Some compounds exhibit fluorescence, and this property is utilized for detection.

c. Mass Spectrometry (MS) Detection: Coupling HPLC with mass spectrometry allows for highly sensitive and specific detection of analytes.^25-27

5. Based on Mode of Operation:

a. Isocratic HPLC: A constant mobile phase composition is maintained throughout the analysis.

b. Gradient HPLC: The composition of the mobile phase is changed during the analysis, providing enhanced separation capabilities.^28-31

6. Based on Instrumentation:

a. Conventional HPLC: Traditional HPLC systems with moderate pressure capabilities.

b. UHPLC (Ultra High-Performance Liquid Chromatography): Uses columns with smaller particle sizes, enabling higher resolution and faster separations.^32-34

7. Based on Application:

a. Pharmaceutical HPLC: Focuses on drug analysis, pharmacokinetics, and formulation studies.

b. Environmental HPLC: Used for analyzing environmental samples for pollutants and contaminants.

c. Biomedical HPLC: Applied in the analysis of biomolecules, peptides, and proteins.

This classification provides an overview of the diverse approaches and applications within the realm of HPLC, reflecting its versatility and widespread use in analytical sciences.^35-40

HPLC Instrumentation:

High Performance Liquid Chromatography (HPLC) instrumentation is a complex system designed to separate, detect, and quantify components of a liquid sample. Modern HPLC systems are sophisticated and consist of several key components, each playing a crucial role in the analytical process.⁴¹ Here's a detailed description of the main components of HPLC instrumentation:

Pump:

· Function: The pump is responsible for delivering the mobile phase at a constant flow rate to the chromatographic system.⁴²

· Types: There are different types of pumps, such as reciprocating, syringe, and gradient pumps.

· Features: Precise and stable flow rates are crucial for reproducible and accurate separations.^43-45

Injector:

· Function: The injector introduces the sample into the chromatographic system by injecting it onto the column.

· Types: Common injectors include manual injectors and automated injectors with sample loops.^46,47

· Features: Injectors should provide precise and reproducible sample introduction.⁴⁸

Column:

· Function: The column is the heart of the chromatographic system, where separation of analytes occurs based on their interaction with the stationary phase.⁴⁹

· Types: Different columns are available, such as reverse phase, normal phase, ion exchange, and size exclusion columns.

· Features: Column dimensions, particle size, and stationary phase chemistry affect separation efficiency as shown in (figure 1).^50-52

Figure 1. Selection of column: pore size v/s particle size.

Detector:

· Function: The detector monitors the eluent leaving the column and produces a signal that corresponds to the concentration of analytes.

· Types: Common detectors include UV-Visible detectors, fluorescence detectors, refractive index detectors, and mass spectrometers.^53-56

· Features: Sensitivity, linearity, and selectivity are critical aspects of detector performance.⁵⁷

Data System:

· Function: The data system acquires, processes, and analyzes the signals generated by the detector.

· Features: It includes software for instrument control, data acquisition, and data analysis. Modern systems often offer user-friendly interfaces and data storage capabilities.^58-60

Column Oven/Heater:

· Function: The column oven or heater maintains a constant temperature for the column, improving separation reproducibility.

· Features: Temperature control is crucial for achieving consistent results, especially for thermally labile compounds.^61-63

Auto Sampler:

· Function: An auto sampler automates the injection of samples, improving precision and sample throughput.

· Features: It can accommodate multiple samples in a tray, and some advanced models offer temperature control for sample storage.^64-67

Mobile Phase Reservoir:

· Function: The mobile phase reservoir stores the solvent or mixture of solvents used as the mobile phase.

· Features: It should provide a continuous and consistent supply of mobile phase to the pump.⁶⁸

Gradient Controller (for Gradient HPLC):

· Function: In gradient HPLC, a gradient controller manages the changing composition of the mobile phase during the analysis.

· Features: Precise control of the gradient profile is essential for optimizing separations.^69-71

Waste Collector:

· Function: The waste collector collects the effluent from the column after separation.

· Features: Efficient waste collection is crucial for maintaining a clean and safe working environment.⁷²

Pressure Regulator:

· Function: The pressure regulator ensures that the system operates at the desired pressure.

· Features: It helps prevent damage to the system components and maintains consistent flow rates.^73-75

Tubing and Fittings:

· Function: Tubing and fittings connect various components of the HPLC system, ensuring a leak-free flow of liquids.

· Features: Inert materials are often used to prevent interactions with the analytes.^76-78

Development of method in HPLC:

Method development in High Performance Liquid Chromatography (HPLC) involves optimizing various parameters to achieve accurate, precise, and efficient separation of analytes. Here are the key parameters considered during HPLC method development:⁷⁹

Stationary Phase Selection:

· Choose an appropriate stationary phase based on the nature of the analytes (polar, nonpolar, acidic, and basic).

· Consider factors such as column chemistry, particle size, and pore size.⁸⁰

Mobile Phase Composition:

· Select a suitable combination of solvents for the mobile phase (binary or ternary).

· Optimize the ratio of polar and nonpolar solvents for reverse phase HPLC or select appropriate solvents for normal phase HPLC.

· Consider the addition of modifiers or ion-pairing agents to enhance separation.

· Tetrahydrofuran (THF), acetonitrile (ACN), and methanol (MeOH) are commonly employed as solvents in reverse phase high-performance liquid chromatography (RP-HPLC), each possessing low UV cut-off wavelengths at 190, 205, and 212 nm, respectively.

· Various types of Chromatography’s, with their different mobile phases and applications are given below in (Table 1).^81-83

Table 1. Various types of Chromatography’s, their mobile phases with applications.

Type of Chromatography	Mobile Phase used	Applications
Normal Phase	Organic solvents	Sample insoluble in organic solvents
Reverse Phase	Water, Buffer, Methanol, ACN	Un-ionisable samples soluble in organic solvents
Ion Pair	Water, Buffer, Methanol, ACN	Ionisable samples
Ion Exchange	Water, Buffer	Proteins, Nucleic acids, Inorganic ions and organic samples
Size Exclusion	Water, THF, Chloroform	Compounds with high molecular masses

Column Selection:

· Choose a column with the appropriate dimensions (length, diameter) for the desired separation.

· Consider the particle size of the packing material, which influences resolution and efficiency.

· Particle size less than 2.5μ is desirable.

Mobile Phase pH:

· Adjust the pH of the mobile phase to optimize separation, especially for ionizable compounds.

· pH can impact the ionization state of analytes and affect their retention time.

Column Temperature:

· Control the column temperature to optimize the separation.

· Elevated temperatures can improve efficiency but may affect analyte stability.^84,85

Flow Rate:

· Optimize the flow rate to balance separation efficiency and analysis time.

· Higher flow rates can reduce analysis time but may compromise resolution.

Injection Volume:

· Determine the appropriate sample injection volume to achieve detectable signals without overloading the column.

· Consider the concentration of analytes in the sample.

Detector Wavelength (for UV-Visible Detection):

· Select the appropriate detection wavelength based on the absorbance maxima of the analytes.

· Optimize detector settings for maximum sensitivity.

Detector Sensitivity and Response Time:

· Adjust detector sensitivity to ensure accurate detection of low-concentration analytes.

· Optimize response time to capture peaks accurately.

Gradient Program (for Gradient HPLC):

· Develop a gradient program for elution to enhance separation.

· Optimize gradient slope and duration for improved resolution.

Sample Pretreatment:

· Evaluate and optimize sample preparation techniques such as extraction, filtration, or derivatization.

· Ensure that sample pretreatment is compatible with the chosen HPLC method.

Standard Solution Preparation:

· Prepare standard solutions of analytes at various concentrations to construct calibration curves.

· Use these solutions to assess linearity, accuracy, and precision.

System Suitability Testing:

· Conduct system suitability tests to ensure that the HPLC system is performing adequately.

· Assess parameters such as resolution, peak symmetry, and column efficiency.

Method Validation:

· Validate the developed method according to regulatory guidelines (ICH, FDA).

· Evaluate parameters like accuracy, precision, specificity, limit of detection (LOD), and limit of quantification (LOQ).^84,85

Parameters for Chromatography:

Resolution (Rs):

In HPLC, getting the best resolution in the shortest amount of time is crucial. In order to reliably quantify the area or height of each peak, the sample components must be well (baseline) separated, with a resolution value of 1.5 or higher between two peaks.

The resolution is determined by dividing the difference between two peaks by their average. peak width.at the. base (tR2 > tR1). The width of the second peak can be used in place of the average value when there are two nearby peaks since it can be assumed. that the peak. width at the base.wb1 ≈ wb2. Refer (figure 2).

Figure 2. Resolution (Rs) calculation

· Retention Factor (k):

It is also called as retention capacity. Analyte retention on a chromatographic column is measured using the retention (or capacity) factor (k). A high retention rate and prolonged interaction between the sample and the stationary phase are indicated by a high k value. Refer (figure 3).

Figure 3. Retention factor (k) determination

· Separation Factor (α)-

It is also called as selectivity factor. It is the chromatographic system's capacity to "chemically" discriminate between the various components of the sample. It can be represented as the separation between the apices of the two peaks and is commonly expressed as a ratio. of the retention factor (k) of two peaks.in determination.

· Efficiency –

The distribution of the analyte band as it passes through the HPLC system and column is measured by the efficiency. of a chromatographic peak. Chromatographic peaks should, in theory, be pencil-thin lines; but dispersion effects cause the peaks to assume their well-known, "Guassian" shape.

The HPLC column's peak dispersion, which indicates the column's performance, is measured by the plate number (N). Refer (figure 4).⁸⁶

Figure 4. Efficiency (N) determination

Method Validation Parameters according to ICH guidelines:

The validation of a High-Performance Liquid Chromatography (HPLC) method is a critical step to ensure its reliability and suitability for its intended purpose. The International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) provides guidelines that outline the key parameters for method validation. These parameters ensure that the HPLC method meets predefined criteria for accuracy, precision, specificity, robustness, and other relevant characteristics. Here's an in-depth discussion of the key validation parameters according to ICH guidelines:

Accuracy:

· Definition: Accuracy refers to the closeness of measured values to the true or accepted reference values.

· Validation Approach: Assess accuracy by comparing the measured values obtained from the HPLC method with known reference values.

· Acceptance Criteria: Typically expressed as a percentage recovery, with results within an acceptable range, often set at ±2% of the true value (called as Relative Standard Deviation i.e., RSD).

Standard deviation

% RSD= --------------------------- x 100

Mean

Precision:

· Definition: Precision is the degree of agreement between individual measurements when the procedure is applied repeatedly under the same conditions.

· Validation Approach: Evaluate precision by performing repeatability (intraday precision) and intermediate precision (interday precision) studies.

· Acceptance Criteria: Usually expressed as the relative standard deviation (RSD), with RSD values below a predefined limit, often set at 2% or 5%.

Specificity:

· Definition: Specificity is the ability of the method to measure the analyte accurately in the presence of potential interfering substances.

· Validation Approach: Assess specificity by analyzing samples containing the analyte of interest along with potential impurities or matrix components.

· Acceptance Criteria: Demonstrate that the method can selectively identify and quantify the analyte in the presence of potential interferences.

Robustness:

· Definition: Robustness is the ability of the method to remain unaffected by small, deliberate variations in method parameters.

· Validation Approach: Introduce small changes to critical method parameters (e.g., flow rate, column temperature) and evaluate the impact on method performance.

· Acceptance Criteria: The method should remain within predefined limits for critical parameters, demonstrating its robustness.

Limit of Detection (LOD) and Limit of Quantification (LOQ):

· Definition: LOD is the lowest concentration of an analyte that can be reliably detected but not necessarily quantified. LOQ is the lowest concentration at which the analyte can be quantified with acceptable precision and accuracy.

· Validation Approach: Determine LOD and LOQ using statistical methods, such as signal-to-noise ratio or standard deviation of the response.

A concentration at a given signal-to-noise ratio—typically 3:1—is used to express LOD.

The ICH has suggested a signal-to-noise ratio of 10:1 for LOQ.

· Acceptance Criteria: LOD and LOQ should be experimentally determined and meet the desired sensitivity requirements.

The following formulas can also be used to compute LOD and LOQ according to the standard deviation (SD) of response and the slope of the calibration curve (S).

LOD = 3.3 × S /SD

LOQ = 10 × S /SD

Linearity/ Range:

· Definition: Linearity is the ability of the method to provide test results that are directly proportional to the concentration of the analyte.

· Validation Approach: Construct a calibration curve by analyzing samples at different concentrations and plotting the response against concentration.

· Acceptance Criteria: The calibration curve should be linear, and correlation coefficient (r) should be close to 1.

System Suitability:

· Definition: System suitability tests ensure that the HPLC system is adequate for the intended analysis.

· Validation Approach: Conduct system suitability tests before each batch of sample analysis, assessing parameters like resolution, tailing factor, and capacity factor.

· Acceptance Criteria: Set predefined limits for system suitability parameters based on method requirements.

Stability:

· Definition: Stability assesses the ability of the sample to remain stable during the analytical process.

· Validation Approach: Evaluate the stability of the sample solution over time, under different storage conditions (e.g., ambient, refrigerated).

· Acceptance Criteria: The sample should remain stable within specified limits for the duration of the analysis.

Ruggedness:

· Definition: Ruggedness is the ability of the method to produce consistent results under various conditions and by different analysts.

· Validation Approach: Conduct the method under different laboratory conditions, using different instruments or analysts.

· Acceptance Criteria: Results should be comparable, demonstrating that the method is rugged.

Forced Degradation Studies (for Stability-Indicating Methods):

· Definition: Forced degradation studies assess the method's ability to detect and separate impurities or degradation products.

· Validation Approach: Subject the analyte to various stress conditions (heat, light, acid/base hydrolysis) and analyze the resulting degradation products.

· Acceptance Criteria: The method should successfully separate and quantify degradation products.^87-91

Technological Leap:

· Miniaturization: Microfluidic chips and nanoscale columns have significantly reduced sample sizes and analysis times, paving the way for high-throughput analysis and minimal sample consumption.

· Advanced column materials: Core-shell and monolithic columns offer superior efficiency and peak resolution, improving sensitivity and detection limits.

· Coupling with other techniques: Hyphenation with mass spectrometry (MS) and nuclear magnetic resonance (NMR) provides valuable structural information alongside precise separation, unlocking deeper insights into complex samples.

· Automation and artificial intelligence (AI): Automated sample preparation, injection, and data analysis significantly improve workflow efficiency and minimize human error. AI-driven method development and optimization further streamline the process.⁸⁶

Expanding Applications:

· Emerging analytes: From complex biomolecules like proteins and peptides to environmental pollutants and food contaminants, the range of analytes amenable to HPLC analysis is constantly expanding.

· Multidimensional separations: Combining different separation mechanisms in a single run facilitates the analysis of highly intricate mixtures, even within small sample volumes.

· Biopharmaceutical analysis: HPLC plays a crucial role in drug discovery and development, from purity assays to metabolite identification and pharmacokinetic studies.

· Food safety and quality control: Detecting food contaminants, assessing nutritional components, and ensuring food authenticity are areas where HPLC shines.

· Environmental monitoring: Tracking pollutants in air, water, and soil, and identifying sources of contamination are crucial applications of advanced HPLC techniques.⁸⁷

CONCLUSION:

HPLC has undergone a remarkable transformation, transcending its traditional boundaries through continuous advancements in technique development and application. The evolution of smaller particle columns, increased flow rates, and novel stationary phases has given rise to powerful variants like UPLC and Nano LC, offering unparalleled speed, resolution, and sensitivity. These advancements have revolutionized method development and validation, providing faster, more reliable, and cost-effective analyses for a wider range of analytes.

In conclusion, advancements in HPLC techniques have not only refined existing procedures but also opened doors to previously unexplored analytical domains. As research continues to push the boundaries of separation science, HPLC remains a cornerstone of analytical instrumentation, poised to reshape the future of diverse fields from pharmaceutical and environmental monitoring to clinical diagnostics and nanomaterial characterization. The future of HPLC is a symphony of cutting-edge technology, robust applications, and boundless possibilities, waiting to be unlocked.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

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Received on 18.01.2024 Revised on 11.05.2024

Accepted on 19.08.2024 Published on 28.02.2025

Available online from March 04, 2025

Asian Journal of Pharmaceutical Analysis. 2025;15(1):57-65.

DOI: 10.52711/2231-5675.2025.00010

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