HPLC – Expanding horizons

The future promises even more interesting discoveries as researchers continue to push the boundaries of what is possible, ensuring that HPLC remains at the forefront of analytical chemistry for years to come.

In the annals of analytical chemistry, chromatography stands as a testament to human ingenuity and scientific progress. From its inception in the early 20th century, when Mikhail Tswett first introduced the concept of liquid-phase chromatography, to the sophisticated techniques of today, such as high-performance liquid chromatography (HPLC) and gas chromatography (GC), the evolution of chromatography has been nothing short of remarkable.

This journey of innovation has been driven by a relentless pursuit of precision, reliability, and versatility in molecular separation and analysis. At the heart of this evolution lies HPLC, a cornerstone of modern analytical chemistry, offering insights into the intricate world of molecular composition.

The ideal HPLC column epitomizes the quest for perfection, characterized by reproducibility, stability across diverse mobile phases and temperatures, and the ability to generate narrow, symmetrical peaks for all analytes of interest. Over the past half-century, scientists have dedicated themselves to realizing these ideals, spurred by continuous advancements in stationary phase synthesis, column hardware fabrication, and packing methodologies.

But HPLC’s significance transcends its origins; it now stands as a versatile tool capable of analyzing a vast array of biological compounds, from small molecules to peptide therapeutics and proteins. Its applications span across research laboratories and pharmaceutical industries, playing an indispensable role in unraveling the mysteries of molecular composition.

As automation and robotics revolutionize laboratory workflows, the horizon for HPLC continues to expand. With promises of greater precision, throughput, and efficiency, HPLC remains at the forefront of analytical chemistry, propelling us toward new frontiers of scientific discovery.

Indian market dynamics
The Indian HPLC market is estimated at ₹1562 crore in 2023 by value, and 7260 units by numbers. The market has seen a 13.8 percent increase in quantity and a 12.8 percent increase in value over 2022. Waters and Shimadzu are neck-to-neck, and Thermo Fisher Scientific is a close second. Agilent has been losing market share in some segments.

The global movement to de-risk from China has presented opportunities to India. The international drug industry is weaning itself off China, particularly for ingredients for patented medicines that bring in most of the industry’s profits. The Russia-Ukraine war, too, has unsettled the decision makers, who wonder what would happen to supply chains if China took similar action against Taiwan. India, too, has been gradually de-linking from China for its over-dependence on key starting materials (KSM), intermediaries, and APIs. That, in turn, has provided the requisite impetus to the HPLC industry.

The pharmaceutical and biotechnology industry continues to be the main focus of the HPLC industry. Major customers in 2023 were Hector Pharmaceuticals, Lupin, Aurobindo Pharma, Mankind Pharma, ECHEMI, Cipla, Reliance Life Sciences, Natco Pharma, Kaplan Pharma, Strides Pharma Science Limited, Biocon, Amneal Pharmaceuticals, Torrent Pharma, Cadila Healthcare, and Radius. The replacement market is huge, and vendors have lately introduced buyback schemes. Since the installed base is large, the increase does not reflect huge growth.

Other end users include diagnostic laboratories, food and beverage testing, and the academic and research industry. When it comes to the environment, HPLC is helpful in the bio-monitoring of pollutants and pollution levels and the detection of phenolic compounds in drinking water.

In 2023, the Government of India launched the National Sickle Cell Anemia Elimination Mission to eliminate SCD by 2047. The best way to check for sickle cell trait or sickle cell disease is to look at the blood, using HPLC. This test identifies which type of hemoglobin is present.

At present, however, treatment and care for SCD remains grossly inadequate and inaccessible. The poor availability of the drug hydroxyurea – an important therapy for SCD blood transfusion – is limited to district-level facilities, and bone marrow transplantation (BMT), another cure for SCD, is out of reach due to the difficulty in finding matched donors, the high cost of the treatment at private facilities, and long waiting times in public hospitals are making treatment and care for SCD grossly inadequate and inaccessible. In light of this, applying the CRISPR gene-editing technology (short for clustered regularly interspaced short palindromic repeats) to treat SCD gains importance. CRISPR’s treatment cost of USD 2–3 million keeps it out of reach of most.

India has approved a five-year project to develop CRISPR for sickle cell anemia. Under its Sickle Cell Anemia Mission, the Council of Scientific and Industrial Research is developing gene-editing therapies for SCD. Around ₹34 crore has been allocated for this mission over 2020–2023. It is reportedly in the pre-clinical stage, with clinical trials awaited. Adopting and promoting CRISPR in India requires a comprehensive approach that accounts for inequities and disparities in the country’s overall healthcare access framework.

The sickle gene is widespread among three socio-economically disadvantaged ethnic groups, the scheduled tribes, scheduled castes, and other backward classes in India. New-born screening programs for SCD have recently been initiated in Maharashtra, Gujarat, Odisha, and Chhattisgarh, and monitoring these birth cohorts will help to understand the natural history of SCD in India. The ICMR and the NRHM in different states are undertaking outreach programs to manage better and control the disease. The states of Madhya Pradesh, Maharashtra, Odisha, Gujarat, Rajasthan, Jharkhand, Chhattisgarh, Andhra Pradesh, West Bengal, and Karnataka account for around 83 percent of the total scheduled tribe population in the country, and the majority of these tribal groups live in rural areas.

Global market dynamics
The global HPLC market is estimated to reach USD 5.60 billion by 2029 from USD 4.24 billion in 2024 at a CAGR of 5.7 percent. Over the past few years, the HPLC market has grown rapidly due to factors, such as technological advancements in HPLC that have improved the dependability and robustness of liquid chromatography operations; the latest HPLC systems, for instance, provide multidimensional capabilities, dual-injection systems, and intermediate pressure alternatives that increase the technique’s usefulness, for several research applications are anticipated to drive market growth in the years to come.

Furthermore, the pharmaceutical and biotechnology industries are major users of HPLC for drug discovery, development, and quality control likely to augment the growth of the HPLC market.

Major HPLC manufacturers who are extending their product lines for niche markets, such as preparative purification, two-dimensional liquid chromatography, amino acid analysis, method development, and clinical diagnostics, are also driving the market growth.

However, the high price of HPLC systems and the scarcity of trained workers are two factors that can slow the market’s expansion during the projection period. Moreover, there is a growing need for HPLC due to rising environmental concerns, and the necessity of monitoring toxins is boosting market growth.

Segment insights. In 2023, by product, the instruments segment held the largest revenue share of around 46 percent, driven by integrated HPLC systems and advanced detectors like the Solvere carbon selective detector. Manufacturers focus on developing systems for increased productivity and application specificity. Additionally, the expanding healthcare sector influences market growth positively.

By application, the clinical research segment held the largest market share of over 36 percent in 2023. Clinical laboratories use HPLC to examine intricate biological fluids including plasma, blood, and serum, assisting in the creation of precise, targeted medications. This usage aids in the development of precise medications, making it a key driver of segmental growth.

Moreover, the forensic segment is growing at a moderate CAGR as HPLC analysis is used by forensic and toxicology labs to screen, quantify, and confirm compounds used in drug tests and criminal investigations.

Regional insights. North America accounts for the largest revenue share of over 31.12 percent in 2023 and this is because of growing demand for high-quality and reliable analytical techniques in drug discovery and development, stringent regulatory requirements for quality control in pharmaceutical manufacturing, and a strong focus on precision medicine and personalized healthcare. Moreover, the US Food and Drug Administration’s recommendations on chromatographic techniques to ensure proper identity, purity, and concentration of pharmaceuticals increase the use of HPLC techniques.

The European market is projected to have the second-largest share in the global market in 2023. The growing investments by the key market players in the pharmaceutical and biotechnology industry and increasing demand for the LC-MS technique drive the European market growth.

The Asia-Pacific region is projected to have the fastest CAGR in the HPLC market due to the rising demand for advanced healthcare services, increasing focus on quality control and regulatory compliance in pharmaceutical manufacturing, and the presence of a large patient pool requiring advanced diagnostic and therapeutic solutions further fuel the market.

Some of the prominent players operating in global HPLC market are Waters Corporation, Agilent Technologies, Shimadzu Corporation, Thermo Fisher Scientific Inc., Sartorius AG, Merck KGaA, YOUNGIN Chromass, Phenomenex, Inc., PerkinElmer Inc., Tosoh Bioscience, Bio-Rad Laboratories Inc., and Gilson Inc.

The HPLC market has seen an influx of new products. In the past year, Knauer introduced a new Azura UHPLC 1240 bar system that targets high-throughput UHPLC and MS/MS applications. Agilent released the new 1290 Infinity II Bio Online LC system that blends high-performance and biocompatible features for the analysis and real-time monitoring in pharmaceutical processing. Waters also released the Alliance iS HPLC system, which includes a quaternary pump and UV absorbance detector that is meant to reduce common laboratory errors, operator training requirements, and method transfer time.

Significant advancements
Innovation opens doors for further exploration and optimization, highlighting the continuous evolution and advancement of HPLC technology.

RP HPLC. HPLC, particularly reversed-phase (RP) HPLC, has become essential in modern analytical chemistry due to its effectiveness in separating and analyzing complex mixtures. Recent advances in RP HPLC method development, including new stationary phases, improved column technology, and novel optimization strategies, have enhanced speed, resolution, sensitivity, selectivity, and reduced non-specific adsorption.

RP HPLC finds applications across various industries, such as pharmaceuticals, food and beverage, environmental analysis, forensic science, and biotechnology. Despite its benefits, challenges like column stability, sample preparation, and optimization persist.

However, optimization of key factors like stationary phase, mobile phase composition, and column temperature, along with the adoption of novel technologies like UHPLC and 2D-LC, addresses these challenges. The implications of RP HPLC findings for analytical chemistry are significant, enabling more accurate, sensitive, and selective analyses, thereby improving product quality, safety, and environmental protection.

Additionally, combining RP HPLC with other analytical techniques like mass spectrometry and NMR spectroscopy offers greater insight into complex mixtures. Future research in RP HPLC method development involves exploring new stationary phases, column technologies, sample preparation techniques, and optimization strategies.

New chromatographic material derived from diatoms shows potential in HPLC. Researchers from Nicolaus Copernicus University explored the potential of biosilica derived from diatoms, and microalgae, as a novel stationary phase in HPLC. Biosilica-filled columns showed improved efficiency, though less retention of polar compounds, compared to hydrophilic interaction chromatography (HILIC). Successful separation of nucleosides and nucleobases was demonstrated. Scanning electron microscopy (SEM) highlighted biosilica’s porous structure and its limited availability from laboratory-scale cultivation tanks was noted. Despite differences from commercial phases, biosilica offers promise for enhancing chromatographic techniques, emphasizing the need for further research and development.

The development of the CC19-R column represents a significant advancement in enantioselective HPLC, offering improved capabilities for chiral separations across various industries and scientific disciplines. This novel chiral stationary phase (CSP) based on a [4+6]-type homochiral porous organic cage (POC) demonstrates enhanced enantioselectivity and a significantly expanded separable range, compared to previous POC-based columns and commercial alternatives. By achieving exceptional resolution values and successfully separating challenging racemates, the CC19-R column offers promising prospects for improved chiral separations in HPLC, highlighting its potential to advance the field of chromatography. Additionally, the stability and reproducibility demonstrated by the CC19-R column underscore its reliability for practical applications, further enhancing its appeal for use in HPLC racemic separation.

HPLC-MS. HPLC has long been a preferred analytical tool in the pharmaceutical industry. With the rising use of mass spectrometry (MS), it continues to dominate analysis. High-performance liquid chromatography-mass spectrometry (HPLC-MS) merges LC’s separation power with MS’s detection specificity, becoming indispensable in pharmaceuticals for its throughput, sensitivity, and selectivity.

The ongoing evolution of HPLC-MS interface technologies marks a significant advancement in analytical chemistry, particularly in the realms of efficiency, throughput, and sensitivity. Automated sample preparation techniques, including solid-phase extraction, liquid-liquid extraction, and supported liquid extraction, are revolutionizing bioanalysis and pharmaceutical analysis by alleviating the labor-intensive nature of sample preparation and meeting the demand for rapid, high-throughput analysis.

Additionally, the adoption of parallel LC separation strategies enables simultaneous analysis of multiple samples by dividing a single injection into different columns, thereby significantly increasing throughput while maintaining separation efficiency.

Concurrently, continuous efforts to enhance the sensitivity of MS detection involve refining pretreatment procedures, ionization techniques, and mass analyzers, enabling the detection of analytes at lower concentrations and enhancing the overall performance of HPLC-MS systems.

Moreover, the development of small, portable MS systems addresses the need for quicker testing, higher throughput, and smaller footprints, providing flexibility and convenience for on-site analysis in various settings.

Together, these advancements underscore the dynamic and evolving landscape of HPLC-MS technology, paving the way for enhanced analytical capabilities and broader applications in research, pharmaceuticals, and beyond.

Integration of rapid HPLC techniques with the advancements in HPLC-MS interface technologies further accelerates analytical workflows, offering enhanced efficiency and throughput. Rapid HPLC methods are characterized by shortened analysis times and increased sample throughput.

A study published by Research Square outlines a rapid, accurate, and precise high-performance liquid chromatography with diode-array detection (HPLC-DAD) method for determining levofloxacin concentration in liquid pharmaceutical formulations. Levofloxacin’s effectiveness can be influenced by its stability in formulations, emphasizing the need for precise quantitative determination. While various analytical methods for levofloxacin quantification exist, challenges persist, including lengthy separation times and the limited availability of specific detectors.

The novel HPLC method utilizes a mixture of acetonitrile and trifluoroacetic acid as the mobile phase, achieving a short analysis time and good peak resolution. Validation according to international guidelines confirms the method’s accuracy, linearity, and precision. Therefore, the HPLC-DAD method is suitable for routine analysis in pharmaceutical quality control and bioequivalence studies, highlighting its relevance to the field of HPLC and pharmaceutical analysis.

Hplc-py software. The hplc-py software package is a powerful tool designed to address the challenges associated with analyzing chromatographic data obtained from HPLC. HPLC and GC are widely used analytical techniques for the quantitative characterization of chemical components in mixtures. However, the increasing throughput of HPLC experiments has posed new challenges for reproducible and rapid analysis of resulting chromatograms.

hplc-py offers a solution by providing a python package that enables rapid and reliable identification and quantification of component signals within chromatograms. It employs a sophisticated signal detection and quantitation algorithm capable of identifying peaks, even in cases of significant signal overlap. This approach, based on inferring parameters from a mixture of distributions, allows for precise absolute quantitation of chemical constituents with similar chromatographic retention times.

The software’s methodology involves multiple steps, including baseline correction, peak identification, and fitting of models to each peak. It offers a programmatic interface that allows users to process chromatograms efficiently, quantify components accurately, and visualize results effectively. Notably, hplc-py can separate highly overlapping signals, enabling the accurate quantitation of mixtures that would otherwise require extensive experimental optimization. hplc-py serves as a valuable tool for enhancing the analytical capabilities of HPLC by providing automated and reliable data processing methods.

Clinical applications of HPLC
The HPLC-UV platform is a significant advancement in the clinical applications of HPLC. Designed with practicality and economy in mind, it can be effectively utilized in various hospitals, even by operators unfamiliar with HPLC analysis. The platform demonstrated reliable and reproducible results in quantifying drug concentrations in patient serum samples, making it suitable for daily therapeutic drug monitoring (TDM) operations. By optimizing pre-treatment conditions, HPLC-UV conditions, and employing the absolute calibration curve method, the platform proved its feasibility for clinical application.

Furthermore, the platform’s potential to expand TDM operations in hospitals could lead to improved quality of medical care and cost-effective interventions in drug treatments. Its applicability to a wide range of drugs, including antimicrobial agents like vancomycin (VCM) and voriconazole (VRCZ), demonstrates its versatility and suitability for addressing diverse clinical needs. As efforts continue to further develop and refine the platform, it is poised to make significant contributions to therapeutic drug monitoring and enhance patient care in medical settings.

Challenges in the HPLC system
HPLC comes with its set of challenges that researchers and analysts must overcome to ensure accurate and reliable results.

Column degradation and performance issues, chromatographic columns are prone to degradation over time due to factors, such as chemical interactions, fouling, and mechanical wear. This can lead to poor chromatographic performance, decreased resolution, and irreproducible results.

Sample matrix effect. Samples, such as biological fluids or environmental samples, can contain interfering compounds that affect analyte detection and quantification. Matrix effects may result in signal suppression or enhancement, leading to inaccurate results.

Impurity identification and quantification, in pharmaceuticals and complex mixtures pose significant challenges in HPLC method development. Co-elution of impurities with analytes of interest or poor chromatographic resolution can hinder accurate identification and quantification.

Instrumental and operational challenges, such as detector drift, pump instability, or injector variability, can impact the accuracy and reproducibility of HPLC measurements. Moreover, operator errors or suboptimal operational conditions can introduce variability and uncertainty into analytical results.

Data analysis and interpretation. Analyzing complex chromatographic data, and interpreting results accurately can be challenging, particularly when dealing with large datasets or overlapping peaks. Manual data analysis may be time-consuming and subjective, leading to potential errors.

While HPLC offers unparalleled capabilities for analyzing diverse compounds, it is essential to address the challenges associated with this technique to obtain reliable and accurate results. By implementing appropriate strategies, including proper maintenance, optimized chromatographic conditions, and advanced data analysis techniques, researchers and analysts can overcome obstacles, and harness the full potential of HPLC in various applications.

Integration of AI in HPLC. The incorporation of AI into HPLC represents a ground-breaking advancement in data analysis. Through AI algorithms, HPLC systems streamline the interpretation of complex chromatographic data, enabling real-time analysis, precise peak detection, and accurate compound identification. For instance, a recent biotechnology and bioengineering study showcased an AI-driven solution for automating chromatographic peak integration in the pharmaceutical sector, promising enhanced accuracy and efficiency. This innovation, executed under a digital innovation management framework, holds potential benefits for the biopharmaceutical industry, including improved patient safety and cost reduction.

Moreover, AI and ML have witnessed tremendous growth and are rapidly gaining popularity in various prediction fields due to their potential abilities, accuracy, and speed. In chromatography, AI and ML are particularly attractive options for method development, offering faster, more accurate, and efficient results.

Various AI and ML models have been employed in determining chromatographic characteristics, with artificial neural network (ANN)-associated techniques demonstrating better accuracy and significant possibilities for prediction in liquid chromatography, compared to classical linear models. The integration of a fuzzy system with an ANN has been shown to provide more efficient and accurate method in chromatographic prediction.

Additionally, combining AI or ML algorithms with quantitative structure-retention relationship (QSRR) methodology has proven to yield more accurate retention predictions, highlighting the potential of artificial intelligence and machine learning to overcome challenges in analytical chemistry.

Outlook
The influx of ongoing innovation in HPLC technology, combined with increased investment in research and development initiatives, reinforces the technique, as well as the equipment to successfully perform it, as necessary assets in both industrial and academic investigation. This rising need from biotechnological and pharmaceutical sectors will result in manufacturers catering to the dynamic demands and standards prevalent within these flourishing industries.

Through this continuous innovation and integration with complementary technologies, HPLC will continue to drive advancements in medical research, supporting early disease diagnosis and treatment. HPLC stands poised to make significant contributions to scientific discovery and innovation, shaping the future of analytical chemistry.

Second Opinion:-

Overview of the HPLC industry.

Advancements in HPLC revolutionize drug development and clinical diagnostics.