High-performance liquid chromatography (HPLC) has proven its potential in separation science as a reliable and heavily used technique, with superior performance and wide applicability in many different fields. Over the years, it has become a well-established technology that is continuously evolving to face the challenges posed by research and industry, as well as regulatory agencies, and to meet the even more stringent requirements for qualitative and quantitative analysis, in terms of speed, accuracy, and sensitivity.

Currently, HPLC is being used in research and routine analysis across more than 30 fields, from environmental analysis and food and drink, through to pharmaceutical development, diagnostic research, and biomarker discovery. Efforts to extend its analytical detection limits and its capacity to separate and quantify analytes in complex samples has driven new developments and innovation at every level of this technique, from the column technologies and separation modes that are the heart of HPLC separations, to the instrumentation and data-handling systems.

Further development of this technique continues today, and these include multidimensional LC, mixed-mode HPLC, nanoflow and microflow HPLC, ultra-performance LC (UPLC), fast HPLC, LC integrated with MS, and chip-based LC. These advances have been accompanied by the scaling-down and miniaturization of HPLC systems, which have taken analytical speeds, separation efficiencies and detection limits to new levels, opening up new and valuable applications in this field.

To further achieve the dramatic increase in resolution, speed and sensitivity, a significant advancement in the instrumentation and column technology (column particle size and column dimension) are also being made. As this rapid advancement of technology continues, new approaches are emerging in the development of therapeutic, diagnostic, and treatment that are continually changing and adapting to the technological advancements of the modern era.

Nowadays, sample handling equipment has replaced the need for manual manipulation and has helped in streamlining the experiments. Automation of sample- handling processes not only reduces the chances of error but also allows greater control and precision of the quantity of the sample or solution to be used. Moreover, the integration of automated systems in the sample handling, separation, and purification processes in chromatography has led to the introduction of several new instruments and accessories that can work in tandem with chromatography instruments.

Indian Market Dynamics

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The Indian HPLC market in 2016 is estimated at 1160 crore, with 5650 units. Modular systems, seeing a 22 percent per annum increase in demand, are increasingly being preferred over integrated ones. All other segments are seeing an average growth of about 10 percent per annum in volume terms.

In value terms, the growth is on a similar trend, albeit with the currency depreciation, the prices have increased by about 15–20 percent in rupee terms. The import duty continues to be 10 percent, with an additional 1 percent landing charges, 12.5 percent countervailing duty, 3 percent CESS, and additional countervailing duty of 4 percent

Specialty systems, comprising gel permeation chromatography instruments, supercritical fluid chromatography instruments, bio-LC, and ion exchange chromatography instruments cater to niche demand.

The pharmaceutical industry continues to drive this segment, and constitutes 80 percent market share. Food, specialty chemicals, and agro led the non-pharma sector over the last couple of years.

Vendor Update

Waters' Acquity Arc System that bridges the gap between HPLC and UPLC performance, recently received the Frost & Sullivan 2016 New Product Innovation Award.

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In March 2017, Agilent expanded its portfolio of scientific instruments with a new 6495B Triple Quadrupole LC/MS System that provides even greater sensitivity and accuracy for applications, such as peptide quantitation, food safety, environmental testing, clinical research and forensic toxicology. The LC/MS System combines HPLC and triple quadrupole MS in an integrated system. In May 2016, the vendor had launched InfinityLab, a product family of liquid chromatography instruments, columns, and supplies.

Shimadzu Analytical had a series of launches last year. In September 2016, the vendor released nine new analytical balance models in three series, namely, the AP-W, AP-X, and AP-Y. These models offer enhanced functionality that is especially useful for HPLC and other analytical instrument users. This enhanced the August 2016 release of two i-Series HPLC analyzers for food safety requirements – a mycotoxin screening system and a synthetic antimicrobial screening system.

In May 2016, Thermo Scientific launched UHPLC-MS workflow solutions for biopharmaceutical characterization, which include solutions for everything from sample prep to data analysis, and feature various new instruments, consumables, and software such as the Thermo Scientific DNAPac RP columns that use reversed-phase HPLC for DNA/RNA oligonucleotides separation, the Thermo Scientific Vanquish Flex UHPLC System that delivers separations in high-throughput analyses, the Thermo Scientific Dionex Chromeleon 7.2 Chromatography Data System that allows scientists to answer questions that frequently arise during method development, the Thermo Scientific MAbPac RP column for high-resolution separation of intact proteins, and the Thermo Scientific BioPharma Finder software to provide intelligent batch/lot comparisons to promote consistency and quality throughout.

Global Market

The global HPLC systems market is projected to reach USD 4.13 billion by 2021 from an estimated USD 3.23 billion in 2016, growing at a CAGR of 5.1 percent during 2016 to 2021, estimates MarketsandMarkets. High sensitivity and accuracy of HPLC techniques, growing popularity of LC-MS technique, increasing importance of HPLC tests in drug approvals, and growth in life science R&D spending are major factors driving the market.

The increase in demand for HPLC systems is also attributed to their growing requirement in the life sciences, pharmaceutical, and diagnostic industries. Moreover, some instruments for liquid chromatography are readily available in laboratories which are further providing boost to the growth of the market. Due to their rising demand, manufacturers are focusing on developing better technologies that help researchers with high-quality analysis. However, the high cost of HPLC systems, limited sensitivity, and specificity of detectors in applications involving complex samples are expected to restrain the growth of the market to a certain extent.

On the basis of product, the consumables segment is projected to grow at the highest CAGR between 2016 and 2021, primarily due to recurring requirement of consumables. In 2016, the systems segment commanded the largest share and the highest growth in the instruments market, attributed by the increasing use of HPLC for analysis across various application areas. The columns segment is estimated to show tremendous growth over the next 5 years attributing to technological advancements in the columns for HPLC.

In the coming 5 years, the HPLC market is expected to witness the highest growth rate in the Asia-Pacific region, attributing to the growth in biomedical and medical research in Japan, strategic expansions by key players in China, increasing government initiatives and growing pharmaceutical industry in India, and the favorable regulatory scenario in New Zealand and Australia.

In 2016, North America accounted for the largest share of the global HPLC market owing to government initiatives to promote R&D. Furthermore, the flourishing pharmaceutical industry in Canada, the increase in funding for R&D, preclinical activities by contract research organizations (CROs) and pharmaceutical companies, and the growing food industry in Ontario are further positively impacting the market.

The key players are expected to tap into the market opportunities to penetrate the market. Furthermore, the untapped opportunities in emerging economies will provide a considerable impetus to the small, medium, and large companies operating in the global market. Players in the market are focusing on innovation and broader range of products, which has resulted in a lot of mergers, acquisitions, collaborations, and partnerships. The major competition among the players is based on the basis of the cost of the products in the market.

Technology Trends

New systems contain extremely small particles and higher pressures (15,000–20,000 psi). Faster analysis has long been a primary objective for HPLC development. Downscaling or miniaturization of analytical platforms is a major trend. The advantage of this is that it reduces solvent and sample consumption and, eventually, costs, while at the same time reducing the environmental impact.

Multidimensional chromatography. Multi-D chromatography is a powerful technique for separating complex and difficult substances for qualitative and quantitative analysis. This enables researchers to separate compounds using multiple properties and potentially generating greater selected end-products.

Many laboratories today are faced with producing timely and accurate results at reduced cost. The pace of life science R&D and manufacturing is being accelerated but the hours in a single day have not changed. The need for companies to shorten their development or manufacturing time means that process development must be fast and inexpensive without compromising the quality of the end product. To address this challenge, many companies are moving toward automation. This trend is definitely being seen in the chromatography space with the advent of new multi-D chromatography instruments. With multi-D workflow templates and automation, there is no need for direct and constant manual interaction. The hands-on time drops dramatically while purification levels and reproducibility escalates.

Multi-D chromatography-mass spectrometry. While the major developments and emphasis in multidimensional techniques have involved conventional 2DLC and when coupled with mass spectrometry, with TOFMS as one of the prime instruments, a further variation has come through the interfacing of travelling wave ion mobility (TWIM) separation component to the mass spectrometer of the combined instrumentation. This also enables an increase in peak capacity over 1D chromatography akin to the front end 2D mode. TWIM allows access to this additional dimension of separation based on molecular size and shape through the acquisition and computation of molecular collision cross-sectional data.

Chip-based microflow LC-tandem mass spectrometry. Microflow LC-tandem MS (LC–MS-MS) has seen a surge of attention, development, and popularity among research scientists and bioanalysts over the last few years. The potential of this technology to provide better sensitivity, less solvent waste, near-zero dead volume, and high throughput are a big part of this renewed interest. LC–MS has the potential to address and improve each of these areas, along with the ability to analyze precious samples of limited quantity, reduce source contamination, reduce matrix effects, and produce a higher ESI response.

There has been a recent surge of interest in the development of LC–MS as hardware and technological capabilities have improved to meet the demands of ever-smaller sample volumes and the limited supply of precious materials. These technological developments are largely the result of the availability of better hardware, the ability to micro manufacture the tiny components necessary, and improved software interfaces. Now, instead of 1-m column lengths, vendors have produced column-on-a-chip devices. These devices are often self-contained, and fully integrated with the source of an MS detector.

Mixed-mode HPLC. MHPLC, which can achieve more than one type of separation modes in a single column, is now a hot-spot in chromatographic science. It has extraordinary separation properties, such as adjustable selectivity, high resolution, and high sample-loading capacity. As the name suggests, MHPLC utilizes more than one form of interactions between the stationary phases and the solutes for multi-mode chromatographic retention mechanisms, and thus one MHPLC column may replace two or more traditional single-mode columns. The physiochemical properties of mixed-mode stationary phase materials are the foundation of the development of MHPLC.

Column technologies. HPLC has evolved dramatically in the recent years, especially due to the development of new column technologies. In fact, in order to maximize HPLC performances in separation efficiency and speed, the size of the particles packed into the columns has been considerably decreased. The increased separation efficiency provided by the new technology of column packed with core-shell particles in HPLC has resulted in their widespread diffusion in several analytical fields such as pharmaceutical, biological, environmental, food analysis, and toxicological.

The current sub-2-m particle columns require a dedicated instrumentation that can work at very high pressure (up to 1200–1300 bars). As a main drawback, the cost of such apparatus is still prohibitive for an average laboratory, or it is difficult to switch from known procedures. The modern sub-3-m core-shell particles have a 1.7-m solid core wrapped in a porous layer or shell of a 0.5-m silica adsorbent, with a final particle size of 2.6-m. This combination of materials provides columns with speed and efficiency similar to columns packed with sub-2-m totally porous particles, while maintaining low back pressure, and thus can be used on conventional HPLC instrument, with a maximum pressure of 300 bars.

Currently, sub-3-m and sub-2-m superficially porous particles are available from several manufacturers with different length, diameter, and stationary phases.

Automation. Despite remarkable improvements in the speed of HPLC over the last decade, due to the development of columns containing sub-2-m particles and UHPLC instrumentation, chromatographic method development remains a significant bottleneck in many analytical laboratory workflows. Developing suitable chromatographic conditions can take weeks or even months, especially if extensive column and eluent optimization are required. This can limit laboratory productivity and increase operational budgets through the extensive use of costly resources such as solvents, even when systematic approaches are employed.

The latest automated method scouting techniques offer a solution to these problems, and can be used to truly leverage the ultra-fast analytical speeds of UHPLC. These systems combine automated multi-column and solvent screening and intelligent run analysis software, which allow chromatographers to develop effective (U) HPLC methods more rapidly, saving valuable time and resources.

Research Update

Professor Milton Lee of Brigham Young University, Utah, has recently reported on the development of a compact nanoflow UHPLC. The system comprises a small dual-pump gradient system with a maximum pressure of 16,000 psi and a maximum flow of 500 nL/min, capillary columns packed with sub-2-m, and even sub-micron column packings. With this low flow rate, the nanoflow LC is compatible with MS detection. Its small size facilitates placement close to the MS inlet, which improves detection and throughput. Alternatively, it also uses a small light-emitting diode (LED) UV detector. It is surprising how well the UV works, given the small capillary column diameter.

The nanoLC may be an interesting and potentially timely development since it enables the sub-micron column packing technology. It is not clear if nanoflow UHPLC technology will fill a significant application need. In any case, it is something to watch.

The Institute of Energy and Environmental Technology (IUTA) in Duisburg, Germany, along with the University Duisburg-Essen, Germany, is currently involved in a research project aiming to build a 4D separation and detection system, combining LCLC, ion mobility, and mass spectrometry.

Moving Forward

Although it has often been claimed that we cannot expect any further improvements related to HPLC, time has shown again and again that there is always progress. Research associates at Institute of Energy and Environmental Technology (IUTA), Duisburg, Germany, with colleagues from the University Duisburg-Essen, Germany, are currently involved in a research project aiming to build a four-dimensional separation and detection system, combining LCLC, ion mobility, and mass spectrometry.

It is foreseeable that both instrumental and column technology will continue to develop into the micro- and nanoscale region, spurred on by the requirements of life science applications in lipidomics or proteomics. Column manufacturers will respond with efforts directed toward improved batch-to-batch and column-to-column reproducibility, and extended column lifetime for general purposes, as well as biocompatible columns for enhanced selectivity and higher recovery for bioanalytical separations.

The HPLC resolution is dominated by the selectivity of the separation materials; aside from efficiency, a key factor will be the design and synthesis of tailored stationary phases, through the choice of adequate porous materials, improved bonding, and surface chemistry. However, the biggest area for improvement will not so much be on the analytical side, but more on the sample preparation side. Improvement of auto samplers will be a big push. Autosamplers will start to be manufactured larger and to be run faster, eliminating potential bottle necks of sample introduction to the LC.

Since liquid chromatography coupled to mass spectrometry is not regularly applied in the quality control/GMP environment for product release, the need for additional features/ technologies within the next five years may not be there. However, data integrity and compliance with CGMP would be essential as LCMS instruments continue their steady march from early discovery and R&D to the more regulated QC environment. With the increasing numbers of large molecule drugs in the pharma pipelines, LCMS systems will see deeper integration with complex sample preparation, typical of biologic samples. LCMS opens the possibilities for multi-attribute analysis. The data complexity for these types of analyses will mandate intuitive and easy-to-use software that is more application specific, and will drive the analyzer concept that will integrate sample prep, data acquisition, analysis, and reporting in a walkup manner.

Shailesh Damale,Asst. Manager LC/LCMS,Shimadzu Analytical India Pvt. Ltd.
Industry Speak
Newer Trends in Diagnostics

Medical diagnosis is one of the most challenging areas of healthcare industry as precise and careful diagnosis of disease biomarkers and giving treatment to the patient are the deciding factors while prescribing medicines. It should be fast, reliable, specific, accurate, and should minimize possibility of false positives. Diagnostics tools and methods with high degree of sensitivity and specificity aid in early detection of diseases and disorders; and hence can provide better prognosis. Cutting edge technology for medical diagnosis is very vital as it directly affects healthcare of the general population.

Nevertheless, rather than simply being a way to identify which patients have a specific disease, diagnostics are now used to support clinical development of drugs, predict disease before symptoms begin, forecast the progress of a disorder, and identify patients who are most likely to respond/not respond to specific treatment.

Many forms of chromatography have been used over the years in the clinical laboratory for the separation and quantification of a variety of clinically relevant analytes. The main advantages of high-performance liquid chromatography (HPLC) method over other techniques are their high selectivity, sensitivity, reliability, versatility with low cost of operations. Its use in the clinical laboratory has steadily increased over the past decades as its unmatched analytical performance and versatility allows for testing of many different types of clinically relevant analytes.

HPLC being a versatile tool can also be used for the separation and analysis of biological and pharmaceutical compounds. Trace level analysis even in complex matrix can be carried out with the effective utilization of HPLC and proper selection of a highly sensitive detection technique. It has been used for low level quantitation of vitamins, nucleic acids, biogenic amines, TDM like, immunosuppressant, and so many. HPLC-based methods would remain the gold standard in clinical testing for many of the current but also future biomarkers and therapeutic drugs.

With increasing health awareness amongst Indian population, government initiatives, and strict regulations, testing labs are forced to go far more selective, precise and automated diagnostic tools like HPLC that would find central place in any diagnostic lab.

Shailesh Damale,
Asst. Manager LC/LCMS,
Shimadzu Analytical India Pvt. Ltd.

Dr Anjali Tewari,Director, Lab Sciences,Regency HealthCare, Kanpur
Second Opinion
Utility of HPLC in Clinical Practice

HPLC (high performance/pressure liquid chromatography) has emerged as one of the most important chromatography techniques in last 5 decades. It is a highly sophisticated separation technique in which the mobile phase is an aqueous solution or an organic solvent that is miscible with water, and the compounds of interest are dissolved in water or an organic solvent such as methanol which passes under high pressure through a column and extracts analytes.

HPLC is given prominent importance over other methods due to its attributes like high sensitivity; high selectivity; versatility; precision; highest accuracy and reliability; low cost of operation; and rapid results.

HPLC is used in clinical laboratories for analysis of aminoacids, peptides, proteins, carbohydrates, lipids, nucleic acids, vitamins, hormones, metabolites, therapeutic drug levels, and presence of substance of drug abuse.It is most commonly used worldwide for monitoring of diabetes (HbA1c), screening of thalassemia, and analysis of other Hb variants.

The HPLC apparatus includes a solvent reservoir for the mobile phase, a pump, an automatic sampler, an injector, an analytical column, a detector and a computer for controlling and processing. Techniques used in various machines are based on normal phase, reverse phase, and ion exchange chromatography coupled with various detectors such as UV, fluorescence, or mass spectrometry. Total automation has minimized time spent on sample preparation, extraction, and clean up procedures.

For hemoglobin variants, most commonly used technology is ion exchange chromatography. Various models are available in market with loader capacity of 10 to 100 samples capable of delivering fast results ranging from 3 to 10 min/test.Depending on need, sample load and investment estimate laboratories have a choice out of various brands available.

While selecting the instrument one must consider robust post-sale support, reagent availability, ease of operation and interpretation, traceability of calibrators, and controls.

Dr Anjali Tewari,
Director, Lab Sciences,
Regency HealthCare, Kanpur


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