Abstract

Chromatography is a widely used analytical technique for separating and analyzing compounds present in complex mixtures. It operates on the principle of differential distribution of components between a stationary phase and a mobile phase, enabling the separation of substances based on their varying affinities for these phases. Over the years, chromatography has evolved into a versatile and indispensable tool in various fields, including chemistry, biochemistry, pharmacology, food science, environmental analysis, and clinical diagnostics. The primary types of chromatography include gas chromatography (GC), liquid chromatography (LC), ion-exchange chromatography (IEC), affinity chromatography, and size exclusion chromatography (SEC). Each type is suited to different applications, based on factors like the nature of the analytes, separation efficiency, and detection sensitivity. High-performance liquid chromatography (HPLC) and ultra-high-performance liquid chromatography (UHPLC) are particularly renowned for their resolution and speed, making them indispensable in pharmaceutical and clinical settings. This review provides an overview of the principles, types, applications, and recent advancements in chromatography. It highlights the integration of chromatography with other techniques, such as mass spectrometry (MS) and spectroscopy, to enhance its sensitivity and precision. Additionally, it discusses emerging trends, such as miniaturization and automation that are shaping the future of chromatographic analysis in research, quality control, and diagnostics.

Introduction

I. INTRODUCTION

Chromatography is a powerful analytical technique widely used for the separation, identification, and quantification of components in complex mixtures. It was first developed by Russian botanist Mikhail Tsvet in the early 20th century as a method for separating plant pigments, but it has since evolved into a cornerstone technique in numerous scientific disciplines, including chemistry, biochemistry, environmental science, and clinical diagnostics. The term "chromatography" derives from the Greek words chroma (color) and grapho (to write), initially referring to its use in separating colored compounds, though the method now extends to a vast range of analytes, many of which are colorless (1).

Chromatography works on the principle of differential partitioning between a stationary phase and a mobile phase. The sample mixture is introduced to a column or surface, where it interacts with the stationary phase. The mobile phase, typically a liquid or gas, moves the components through the system, with different substances in the mixture migrating at different rates depending on their chemical properties (such as polarity, size, or affinity for the stationary phase). This differential movement leads to the separation of the components as they travel through the chromatographic system (2).

The primary types of chromatography include:

1. Gas Chromatography (GC): Utilizes a gaseous mobile phase and is typically used for the separation of volatile organic compounds. GC is commonly employed in environmental analysis, forensic science, and the pharmaceutical industry for purity testing (3).
2.  Liquid Chromatography (LC): Involves a liquid mobile phase and is versatile for separating a broad range of substances, including drugs, peptides, and nucleic acids. High-performance liquid chromatography (HPLC), a subtype of LC, is known for its high resolution and is widely applied in both research and clinical settings for drug analysis and environmental monitoring (4).
3.  Ion-Exchange Chromatography (IEC): This method is based on the exchange of ions between the stationary phase and the mobile phase. IEC is often used for the separation of charged molecules, such as proteins, nucleic acids, and small ions (5).
4. Affinity Chromatography: Based on specific interactions between a target molecule (such as an enzyme, antibody, or receptor) and its ligand attached to the stationary phase. This technique is highly selective and is commonly used in biochemistry and biotechnology for protein purification (6)
5. Size upgrade Chromatography: Recent advances in chromatography have focused on increasing separation efficiency, speed, and sensitivity. Additionally, chromatography has become an integral part of two-dimensional chromatography, which combines multiple separation mechanisms to handle more complex mixtures, such as in proteomics and metabolomics research.

The growing demand for green and sustainable methods in scientific analysis has led to the emergence of green chromatography, which emphasizes the reduction of harmful solvents and minimizes waste production. Such advancements not only improve environmental sustainability but also align with modern trends in sustainable analytical chemistry (7).

II. PRINCIPLES OF CHROMATOGRAPHY

 This interaction is influenced by the size, polarity, and charge of the components, leading to the successful separation of mixtures. Through this mechanism, chromatography serves as a critical tool for purifying compounds and analyzing complex mixtures in diverse scientific fields (8,9).

A. Types of Chromatography

Chromatography techniques can be categorized in several ways, including by the type of stationary phase used, the method of separation and the types of instruments involved. Below are some of the most common chromatography methods:

1.  Gas Chromatography (GC): Gas chromatography is used for separating and analyzing volatile compounds. The stationary phase is typically a solid or liquid coating inside a column, and the mobile phase is an inert gas like helium or nitrogen (10). GC is widely used in environmental science for pollutant detection and in the petrochemical industry (11).
2.  Liquid Chromatography (LC): like drugs, peptides, and organic compounds (12). HPLC and UHPLC are especially useful in pharmaceutical analysis for ensuring drug purity and quality control (13).
3.  Thin-Layer Chromatography (TLC): TLC is commonly used for qualitative analysis and rapid screening of samples in the food, pharmaceutical, and forensic industries (14).
4.  Paper Chromatography: It is widely used in educational settings and for basic research (15).
5.  Ion-Exchange Chromatography:It is widely used for purifying proteins, nucleic acids, and separating metal ions (16). This technique plays a critical role in the biotechnology and pharmaceutical industries (17).
6.  Size Exclusion Chromatography (SEC): This technique is particularly useful for analyzing polymers and proteins (18).
7.  Affinity Chromatography: Affinity chromatography is based on the specific interactions between the stationary phase (often functionalized with a ligand) and the target molecule. This method is commonly used for the purification of biomolecules, such as antibodies and enzymes, and in clinical diagnostics (19).

B. Recent Advancements in Chromatography           

1.  Ultra-High-Performance Liquid Chromatography (UHPLC): One of the most notable advancements in chromatography is the development of UHPLC, which enables faster and more efficient separations. Using smaller particle sizes for the stationary phase, UHPLC offers increased resolution and sensitivity, making it ideal for complex analyses in the pharmaceutical and environmental industries (20).
2.  Two-Dimensional Chromatography: This advanced method combines two different chromatographic techniques (e.g., GC and HPLC) to enhance separation efficiency. It is particularly useful in proteomics for separating complex protein mixtures (21).
3. Green Chromatography: With the growing emphasis on sustainability, green chromatography aims to minimize the use of harmful solvents and reduce waste. Techniques like reversed-phase HPLC and the use of ionic liquids in mobile phases are paving the way for more eco-friendly methods (22).
4. Automated Chromatography: Advances in automation and integration with robotics have significantly improved chromatography processes. Automated sample preparation, method optimization, and data analysis are becoming the norm in many laboratories, increasing throughput and reproducibility (23).
5. Miniaturization and Microfluidic Systems: It has allowed chromatography to be performed on a much smaller scale. These systems offer high sensitivity, reduced sample requirements, and faster analysis times, which are crucial for clinical diagnostics and point-of-care applications (24).

C. Applications of Chromatography

Chromatography finds applications in a wide range of industries and research areas:

1. Pharmaceutical Industry: It is also used in pharmacokinetics to monitor the concentration of drugs in biological samples (25).  HPLC is a gold standard for analyzing complex drug formulations (26).
2. Environmental Science: The ability to detect trace contaminants is essential for public health and safety (27).
3. Food Industry: In food analysis, chromatography is used to detect contaminants such as preservatives, pesticides, and food additives. It is also used for determining nutritional components like vitamins and fatty acids (28). TLC and HPLC are frequently used for these applications (29).
4. Clinical Diagnostics: Chromatography is integral to clinical diagnostics for separating and analyzing biomarkers in blood, urine, and other bodily fluids. It is widely used in monitoring diseases like diabetes, cancer, and cardiovascular diseases by analyzing specific proteins, hormones, and metabolites (30).
5. Biotechnology and Proteomics: Affinity chromatography, in particular, is crucial for isolating high-value biomolecules .Two-dimensional chromatography and HPLC are extensively used in proteomics (31).
6. Forensic Science: Chromatography is an invaluable tool in forensic science for detecting illicit drugs, explosives, and toxins in biological samples and crime scene evidence. GC and HPLC are often employed for these analyses .

D. Objective and Purpose of Chromatography

The purpose of chromatography extends across a broad spectrum of scientific disciplines, from basic research to industrial applications, owing to its high sensitivity, accuracy, and adaptability to various types of samples. The specific objectives include:

1. Separation of Components: The most fundamental objective is to separate the various components of a mixture. This is crucial for identifying the individual components, especially when dealing with complex biological, chemical, or environmental samples.
2. Pharmaceutical Applications: It is an essential tool in drug formulation, stability testing, and quality control. Techniques like HPLC are used for the determination of the purity of drug substances and formulations, ensuring they meet regulatory standards for safety and efficacy.
3. Status Monitoring: Implies to health safety.
4. Food and Beverage Quality Control: This includes the detection of contaminants like food preservatives, additives, and pesticides, as well as the analysis of flavor compounds, vitamins, and other nutritional components .
5. Clinical Diagnostics: It is an important tool in diagnosing diseases, monitoring treatment efficacy, and performing metabolic profiling.
6. Proteomics and Genomics: Chromatography, particularly in combination with mass spectrometry, has become indispensable in the fields of proteomics and genomics. It enables the separation of complex protein mixtures, identification of protein biomarkers, and the study of gene expression. Techniques like two-dimensional chromatography are pivotal in these areas, allowing for a more detailed analysis of biological systems.
7. Research and Development:  In scientific research, chromatography serves as a crucial tool for the isolation and characterization of novel compounds, whether they are natural products, synthetic chemicals, or metabolites. This is particularly relevant in fields like biochemistry, molecular biology, and environmental science .
8. Sustainability and Green Chemistry: The increasing emphasis on sustainability has led to the development of greener chromatography methods that use less toxic solvents, minimize waste, and reduce environmental impact. Green chromatography is essential for reducing the ecological footprint of analytical processes while maintaining analytical performance (32).

III. TECHNOLOGICAL AND METHODOLOGICAL ADVANCEMENTS

1. Automation and Miniaturization: Modern chromatographic systems are increasingly automated and miniaturized. Automation improves throughput, accuracy, and reproducibility, while miniaturization enables the analysis of smaller sample volumes with enhanced sensitivity. These advancements are particularly valuable in high-throughput screening, clinical diagnostics, and pharmaceutical testing.
2. Integration with Other Techniques: These hybrid methods are widely used in complex analyses like the identification of metabolites, the characterization of synthetic compounds, and environmental monitoring (33).

IV. FUTURE PROSPECTS OF CHROMATOGRAPHY

Chromatography, a foundational technique in analytical chemistry, continues to evolve with advancements in technology, methodology, and applications. Several trends are shaping the future of chromatography, including miniaturization, automation, and integration with other analytical techniques, sustainability, and the development of advanced materials.

1.  Miniaturization and Microfluidics: Miniaturization, particularly in the context of chromatography, is one of the most promising trends for the future. Advances in microfluidics allow for the development of portable, cost-effective, and faster chromatographic systems. The development of portable chromatographic devices, combined with sensors, could revolutionize fields such as field-based testing and rapid diagnostics. Furthermore, microfluidic technology is being leveraged to enhance the sensitivity and resolution of chromatographic separations, enabling highly efficient analyses in smaller sample sizes .
2.  (UHPLC): By using smaller particles for stationary phases, UHPLC significantly improves the speed and efficiency of separations, allowing for high-throughput analyses in pharmaceutical, environmental, and clinical applications. The continuous development of more robust and efficient columns, as well as better detection technologies, will further push the limits of UHPLC.  UHPLC's potential for separating complex mixtures with increased resolution has opened new doors in proteomics and metabolomics, offering unparalleled insights into complex biological systems.
3.  Integration with Mass Spectrometry (MS): Further advancements in ionization techniques and mass spectrometric instrumentation will allow for the precise detection of complex compounds, even in trace amounts, broadening the scope of chromatography-based MS applications (35).
4.  Automation and High-Throughput Systems: Automation in chromatography is advancing rapidly, enabling high-throughput and reproducible analyses. Robotic sample handling, automated injection systems, and integrated data analysis platforms allow for the faster processing of samples while reducing human error. In industries like pharmaceuticals, where large-scale testing and quality control are critical, automated chromatography systems are becoming standard practice. The future will see the further development of fully automated chromatography systems, offering even more efficient workflows in research and industrial applications. The integration of AI and machine learning algorithms to predict and optimize chromatographic conditions will likely revolutionize the automation of these systems. Additionally, fully integrated chromatographic systems that incorporate sample preparation, separation, and detection in a single platform will facilitate seamless, high-throughput operations (36).
5.  Green Chromatography and Sustainable Practices: As sustainability becomes increasingly important across scientific disciplines, green chromatography is emerging as a key trend. Green chromatography focuses on minimizing the use of hazardous solvents, reducing waste, and increasing the energy efficiency of chromatographic systems. The development of eco-friendly stationary phases, as well as alternative solvents like supercritical CO2, ionic liquids, and bio-based solvents, will lead to more sustainable chromatographic methods. This trend is particularly relevant in pharmaceutical and environmental analysis, where stringent environmental regulations are driving the adoption of more sustainable practices (37).
6. Two-Dimensional Chromatography (2D Chromatography): To achieve enhanced separation efficiency. This approach has gained considerable attention in complex analyses, particularly in proteomics and metabolomics, where the separation of thousands of biomolecules is required. Future advancements in 2D chromatography will likely focus on improving automation, reducing analysis times, and enhancing the resolution of complex biological samples. Recent innovations in two-dimensional liquid chromatography (2D-LC) have made it possible to separate proteins and metabolites with unprecedented resolution, aiding in the exploration of disease mechanisms and drug development .The increased coupling of 2D chromatography with high-resolution mass spectrometry (MS) further enhances its potential in complex proteomic and metabolomic analyses (38).
7.  Chromatography in Personalized Medicine: As personalized medicine continues to grow, chromatography will play an increasingly vital role in the characterization of individual patient profiles. Chromatographic techniques, combined with mass spectrometry and genomic technologies .The future of chromatography in personalized medicine lies in its ability to integrate with high-throughput technologies for the comprehensive profiling of human samples. Chromatography-based biomarkers can help physicians determine which treatment protocols are most effective for individual patients, especially in oncology, cardiovascular diseases, and rare genetic disorders . Moreover, the application of chromatography in pharmacogenomics will allow for the better understanding of drug metabolism and drug interactions, leading to more precise dosing regimens (39).
8.  Advancements in Stationary Phases and Materials: The development of novel stationary phases, such as those with higher surface areas, increased stability, and better selectivity, will continue to drive innovation in chromatography. New materials, including porous organic polymers, metal-organic frameworks (MOFs), and bio-inspired materials, promise to improve separation efficiency and sensitivity. These materials are particularly relevant in the separation of biomolecules, environmental contaminants, and complex mixtures. The future will likely see the use of nanomaterials and functionalized surfaces to enhance selectivity and improve separation performance. For example, the development of monolithic columns, which provide faster separations with less backpressure, is expected to revolutionize the speed and efficiency of chromatography.  Research into advanced materials such as nanostructured coatings and silica-based hybrids will continue to improve the stability and selectivity of stationary phases (40).
9.  Applications in Emerging Fields: Chromatography’s versatility allows it to be applied in a range of emerging fields, including nanotechnology, biotechnology, and renewable energy. In nanotechnology, chromatography will aid in the analysis of nanomaterials, ensuring the purity and quality of nanoparticles used in drug delivery systems or electronic devices.The renewable energy sector will also benefit from chromatography, particularly in the analysis of biofuels, which require detailed characterization of their chemical composition. Additionally, chromatography will continue to be pivotal in the development of new biocatalysts and in the production of bio sustainable chemicals (41).
10.  Point-of-Care Applications: The advent of portable chromatographic systems promises to revolutionize diagnostics by enabling point-of-care applications. Portable chromatography devices could be used for on-site analysis in clinical, environmental, and food safety settings. These devices could be integrated with smartphone technology for real-time data analysis and reporting, thus providing immediate results for clinical decisions, disaster management, and environmental monitoring (42). Advances in miniaturized chromatographic sensors and lab-on-a-chip technology will further enhance the feasibility of portable systems, offering quicker, more affordable testing in remote or low-resource settings (43).

Conclusion

Chromatography remains an essential and versatile analytical technique, with its applications spanning across numerous industries and research fields. As technologies advance, including miniaturization, automation, and sustainability efforts, chromatography continues to evolve, providing even more efficient, precise, and environmentally friendly solutions for scientific analysis. Furthermore, the increasing demand for sustainability and eco-friendly practices in analytical chemistry will likely drive the adoption of greener chromatographic methods, using alternative solvents and recyclable materials. Ultimately, chromatography will continue to evolve as a dynamic and indispensable tool, contributing to advancements in scientific research, environmental protection, and healthcare. As new applications emerge and technology progresses, the role of chromatography in both research and industry will remain vital in solving complex analytical challenges and providing critical insights into the composition of various substances.

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Copyright © 2025 Mohammad Khalid, Abubakar , Mohd Faizan, Lavkush Kumar. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.