Improving HPLC for Better Separation in Shorter Time

SPP columns with greatly reduced backpressures are now being offered by all major column vendors and provide high performance to users who have not or cannot step up to true UHPLC.

Classic column chromatography has evolved over the years, with chromatographic innovations introduced at intervals of roughly decades offering major improvements in speed, resolving power, detection, quantification, convenience, and applicability to new sample types. The most notable of these modifications is high performance liquid chromatography (HPLC). It is one of the most popular and mature analytical techniques and by far the most widely used separation technique. HPLC has been used in laboratories worldwide over the past 40-plus years for pharmaceutical sciences, clinical chemistry, food and environmental analyses, synthetic chemistry, etc., and has gained its popularity mainly due to its reliability and versatility. Since its inception, HPLC has undergone technological enhancements in all aspects, whether that is improvements in detectors, better accuracy and precision, or new column chemistries.

HPLC began an explosive growth and its present popularity results from its convenient separation of a wide range of sample types, exceptional resolving power, speed, and nanomolar detection levels. However, at a time when many scientists reached separation barriers with conventional HPLC, UPLC (ultra-high-performance liquid chromatography) seems to be the possibility to extend and expand the utility of chromatography. Since the millennium, most of the advances have been centered on performing faster separations through the advent of UHPLC complemented by smaller column particles and diameters. That is not to say that other enhancements have not been made in the areas of robustness, reliability, and detection sensitivity. All of these developments have given rise to increases in productivity such as higher throughput, reduced cost per sample, greater return-on-investment (ROI) for high-value instrument purchases, and basically a faster route from sample to knowledge.

Recently, significant efforts have been made to increase the efficiency of LC supports. Conventional 4.6×250 mm columns, packed with 5 µm fully porous particles (FPPs) have steadily been replaced by short (5–10 cm), narrow bore (1.0–2.1 mm) columns packed with sub-2 µm fully and even sub-2 µm superficially porous particles (SPPs). The increased column back pressure that is inevitably encountered in columns packed with small particles has largely been overcome by the introduction of instrumentation that can operate at pressures well above 400 bar (currently up to 1500 bar). These new separation supports, in combination with high operating pressures, have made it possible to obtain the same separation quality in much shorter analysis times, or increase the separation quality in the same analysis time, compared to conventional columns. Current method development software can also be used to optimize mobile phase conditions, such as gradient time and composition, pH, temperature, and ternary composition, both for small molecules and large biomolecules, and for different retention mechanisms.

Pressure sensors are employed in advanced HPLC instruments which enhance the ability to measure high and ultra-high pressure, maintain zero internal dead volume, and provide fast frequency reports and ability to compensate for effects due to variation in ambient temperatures. In HPLC, membrane filters play a major role to determine which liquid or gas need to be filtered, check which membranes are chemically compatible with the sample, and determine which pore size will obtain the best results. In-line filters and guard columns are being used tremendously as it prevents particles under investigation from system wear and chemical contamination ultimately extending the life of HPLC components. Manufacturers are constantly striving to achieve value addition to the HPLC components such as technical modification in the sample injection port, chemical modification in the extraction columns, and sample detectors.

Manufacturers are also increasingly focusing on developing improved technologies that can help researchers with high-quality and fast analysis. There has been recent improvement in column selectivity that will be particularly valuable in characterizing biopharmaceuticals. Ongoing research and development is contributing to expanding application of HPLC in medical, research, and manufacturing sectors. These increasing research activities, rising government spending to harness the performance of R&D activities, and competitive market due to presence of existing and budding manufacturers involved in the production of HPLC will lead to the growth of the market in the coming years. Moreover, rising public health awareness and business expansion activities by major players in the untapped markets of the country are going to fuel the rapid growth of the market.

Indian Market Dynamics

The Indian HPLC market in 2017 is estimated at `1241 crore, with 6045 units. Across all segments, there was a uniform 7 percent increase over 2016, in value and quantity terms, since prices remained constant over the year. This is very different from 2016, when prices had increased by about 15–20 percent in rupee terms over 2015.

Basic HPLC systems (5000-8000 psi; 350-600 bar) continue to hold sway with a 75 percent share in quantity and a 66 percent share in value terms in 2017. Modular systems are increasingly being preferred over their integrated counterparts. Complex application demand is increasing, especially in the pharma and biopharma industries, traditional HPLC is not adequate to fulfil the requirement, and UHPLC with specialized detection system are being explored. Fast HPLC (9800-20000 psi; 900-1400 bar) is where the same (or better) separation is obtained, but with a shorter runtime. This is obtained simply by exploiting certain parameters (the column particle size, internal diameter, length, and particle morphology) to increase the efficiency of the separation. If the column and conditions are more efficient the same job is done in less time. Specialty systems, comprising gel permeation chromatography instruments, supercritical fluid chromatography instruments, bio-LC, and ion exchange chromatography instruments cater to niche demand.

Customers are looking for single vendor support for multi-vendor products. The pharmaceutical industry continues to drive this segment. Food, specialty chemicals, and agro led the non-pharma sector over the last couple of years. The Indian buyer is continuously giving weightage to operating cost, and the growth and increased spend will be determined by how much the vendors are able to decrease the operating cost.

Leading pharma companies continue to face serious compliance issues with regulatory authorities, particularly for FDA approvals, albeit today India has the 2nd largest number of USFDA-approved manufacturing plants outside the US. They are continually investing in remedial measures, making regular procurement of equipment a low priority.   The smaller companies, which export to non-US markets as Middle East, Russia and other developing nations, are better placed.

Global Market

The global HPLC market is projected to grow from USD 4.462 billion in 2017 to USD 5.672 billion in 2022, reflecting a CAGR of 4.91 percent, projects Research and Markets. Ongoing R&D is contributing to expanding application of HPLC applications in medical, research, and manufacturing sector. Moreover, the high sensitivity and accuracy of HPLC endorses its increasing adoption by law enforcement agencies for detecting performance enhancement drugs in urine, thereby augmenting the market growth.

However, high cost instruments will be key challenge to market growth over the forecast period. The developing economies like China and India are expected to create numerous opportunities for HPLC vendors over the projected period.

In the current scenario instruments holds the largest market share owing to increasing use of HPLC for analysis across various sectors. However, the consumables segment is expected to grow at a high rate in the coming years. The growth is majorly driven by technological advancements in the HPLC for consumables. The growing investments in the healthcare sector and expanding importance of HPLC in drug approval are significantly contributing to the segment’s growth.

The global HPLC market is dominated by few players owing to their capability to invest in continuous R&D. Moreover, the need for skilled labor and knowledge coupled with high initial investment restricts the entry of new entrants which further enhance the position of existing players. Some of the major players in the market include Shimadzu Corporation, Agilent Technologies, Phenomenex, GE Healthcare, Thermo Fisher Scientific, Waters Corporation, Jasco, PerkinElmer, Novasep, Bio-Rad Laboratories, and Gilson.

Vendor’s Update

Players in the market are focusing on innovation and broader range of products. New systems contain extremely small particles and higher pressures. Faster analysis has always been a primary objective for HPLC development.

The analytical complexity of new and emerging pharmaceuticals and biopharmaceuticals can now be met with advanced UHPLC systems designed to streamline workflows and help reduce cost per sample. The next-generation Vanquish Duo UHPLC systems from Thermo Scientific introduced in February 2018 are designed to maximize throughput and ROI, while providing scientists with a robust and comprehensive understanding of their samples

In January 2018, Waters Corporation has introduced the Acquity Arc Bio System, a versatile, iron-free, bio-inert, quaternary LC specifically engineered to enable the efficient transfer and improvement of bioseparation analytical methods. The Arc Multi-flow path technology delivers plug-and-play compatibility with HPLC or UHPLC methods through a selectable dwell volume, emulating the dwell volume of the original instrument. The company has also introduced the BioResolve RP mAb Polyphenyl 450 Å 2.7 µm solid core columns in January 2018, intended for the reversed-phase analysis of intact or sub-unit-digested monoclonal antibodies (mAbs) and antibody-drug conjugates (ADCs) using LC-UV and LC-MS. This newly introduced wider pore column provides superior recovery, separation, and resolution of therapeutic mAb subunits and domains, as compared to other RP C4 columns in the market

Shimadzu Corporation released the Nexera FV UHPLC in September 2017 to improve the operational efficiency of tests, to evaluate the dissolution of contained components in the development, and quality control of pharmaceutical dosage forms (e.g., tablets and capsules). The company has also launched the i-Series Plus integrated HPLC. With i-Series Plus, pretreatment operations, such as the processes to dilute samples and add reagents, have been automated, resulting in less mistakes and measurement errors and ensuring highly reproducible and reliable data in the analysis

Agilent Technologies has expanded its portfolio of scientific instruments with the launch of the 6495B triple quadrupole LC/MS system in April 2017 that provides even greater sensitivity and accuracy for peptide quantitation, food safety, environmental testing, clinical research, and forensic toxicology. The new LC/MS system combines HPLC and triple quadrupole MS in an integrated system to deliver outstanding operational efficiencies

Technological Advances

With many second-generation instruments debuted by all major manufacturers in prior years, now the market is seeing mostly line extensions, application-specific systems, modules (particularly new MS or MS add-ons), and software products.

Monolithic columns. Since late 1970s, particulate columns (5 µm) have been widely used in the field of chromatography. The decrease in the particle size gives better column efficiency but results in a higher back pressure. With the advent of monolithic columns, higher column efficiency can be provided with minimum back pressure. The recent invention and development of monolithic columns is a major technological change in column technology; indeed the first original breakthrough to have occurred in this area since Tswett invented chromatography, a century ago. The monolithic column is made up of continuous porous material, sealed against the wall of a tube, instead of beads. The decrease in chemicals and samples usage, improved sensitivity, reusable frit-less columns, and lower back pressure make them more efficient and user-friendly columns in the present world. Current they are available in three different types, that is, silica-based, polymer-based, and hybrid monolithic columns.

SPP columns. SPP columns that are now being offered by all major column vendors, provide high performance to users who have not or cannot step up to true UHPLC. Rugged, long-lasting SPP columns with greatly reduced backpressures provide efficiencies approaching those of UHPLC on conventional HPLC instruments. A popular particle size, 2.7 µm, allows users who are unprepared for UHPLC to experience shorter run times, better-resolved peaks, and reduced runtimes without investing in new hardware or specifying ultra-high purity solvents. Regardless, the need to transfer methods among various LC platforms is real. Marketing has made user’s desire fast chromatography – the ability to separate the maximum number of peaks in the shortest time.

Micro-pillar array column. Another new column format that has been introduced to increase the separation performance is the micro-pillar array column. The perfect order of these pillars significantly reduces the band broadening as a result of heterogeneous flow paths in the column, and hence the overall peak dispersion, while the inter-pillar distance can be tuned to decrease the column back pressure, allowing the use of very long columns. The distinctive properties of these columns can be used to address challenging separation problems, as encountered in lipidomics.

Core–shell technology. In contrast to the typical silica-based LC particles, core–shell particles consist of a solid, impermeable inner silica core that is surrounded by an outer shell of conventional fully porous silica. Due to this unique core–shell morphology, columns packed with core–shell particles deliver significantly greater efficiency than columns packed with fully porous media of equivalent particle size. Greater efficiency will translate into improved resolution and increased peak height (sensitivity). With that improved resolution, one can afford to go to shorter column lengths and/or increased flow rates (within allowable adjustment ranges) to dramatically reduce analysis times compared to your original method while still meeting initial system suitability requirements.

Fast HPLC. HPLC using short columns (3–10 cm) packed with small particles (<3 µm) and high flow rates has recently become a good strategy to save time and solvent consumption in HPLC. Many laboratory budgets do not allow the purchase of new UHPLC systems, and workload is constantly growing. Fast LC incorporates the use of faster mobile phase flow rates and smaller particles to obtain compound separations in a lower time and with an equivalent resolution to a traditional HPLC system. Fast HPLC allows to run more samples in the same time frame by developing fast methods with the existing HPLC systems in the laboratory.

Variable-wavelength detectors. Today, optical detectors are used most frequently in HPLC systems. The simplest detectors available in the market are of the fixed-wavelength type and usually contain low–pressure mercury lamps that have an intense emission line at 254 nm. Some instruments, however, offer conversion kits that allow the energy at 254 nm to excite a suitable phosphor to give a new detection wavelength (e.g., 280 nm). Variable-wavelength detectors offer a deuterium lamp with a continuous emission from 180 to 400 nm and use a manually operated diffraction grating to select the required wavelength. Tungsten lamps (400–700 nm) are used for the visible region. A variable-wavelength detector can be invaluable to increase the sensitivity of detection by using the wavelength of maximum absorption. This is particularly useful when analyzing proteins that absorb at 280 nm, or peptides that are detected commonly at 215 nm. Using a variable-wavelength detector can also increase the selectivity of detection by enhancing the peak of interest relative to interfering peaks.

Integrated solution. Manufactures are now launching HPLC systems build off by incorporating new modules (i.e., dual gradient pump and a unique dual auto-sampler) to offer three workflows that increase productivity in a single system – dual LC, tandem LC or LC-MS, and inverse gradients. Dual LC allows to run two samples in parallel or two different applications in parallel, doubling throughput or obtaining more information from a single sample respectively. Tandem LC or LC-MS uses two individual pumps, for example, a dual gradient pump and two columns. Whilst one is performing an analytical run on one column using one pump, the other column can be re-conditioned on the second pump. One can then simply switch the column used for the next sample. In this way, the detector does not sit idle waiting for the column to be re-conditioned before the next run begins. This not only saves time, but also maximizes the utilization and ROI of detector. Inverse gradient maximizes productivity. By using a dual-gradient pump it is possible to perform an inverse gradient on the second pump to match the analytical gradient. The two gradients then combine before the CAD so that it receives a constant solvent composition which greatly improves uniformity of response for quantitation.

Way Forward

The HPLC is constantly evolving, driven by the needs of the many areas in life sciences and technology wherein it plays a key role. The future for HPLC is focused around productivity. Technology enhancements, whether that is offering more pump pressure, reducing column particle sizes and diameters, or simply offering better system performance and robustness, all increase productivity. They will increase throughput and drive down cost per sample. Productivity is king and there are new solutions out there that can really enhance productivity. Recent introduction of integrated solution demonstrates that one can increase productivity in three different ways. Over the next decade HPLC will continue to evolve, with productivity being at the center of all developments and enhancements, whether that is in hardware, software or columns, and chemistries.

Industry Speak

Changing Trends in Clinical Diagnostics

HPLC has been widely used for years as an analytical method for sample separation in pharmaceutical quality control and life science research. Due to the power and flexibility of this technique, numerous applications have been adopted for routine use in the clinical laboratory. 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 last decade has seen a series of advances in the field of liquid chromatography that have resulted in improvements for many clinical diagnostic services. As a result, diagnostic services are able to offer faster turnaround times and measure analytes in patient types and disease states that were previously problematic.

The main advantages of high-performance liquid chromatography (HPLC) method over other techniques are its 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 a 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 and also future biomarkers and therapeutic drugs.

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

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

Second Opinion

HPLC – A Multifaceted Diagnostic Technique

Chromatography is a method of separating the components of a mixture by passing it through a porous medium based on their differential distribution and migration rates in stationary and mobile phases used in the process. High performance liquid chromatography (HPLC) is the most popular and versatile analytical method of chromatography technique. The characteristic feature and key point of HPLC is the high resolution that this method is able to provide while analyzing the parameters. It is a highly sensitive method and helps to detect even small amounts of the compound with accuracy.

HPLC allows efficient separation of molecules which can be analyzed with the help of various detectors. This system is able to measure multiple molecules in a single run. In the medical field it can be used to detect various substances like amino acids, proteins, lipids, vitamins, hormones. It is helpful in diagnosis of metabolic disorders such as pheochromocytoma, neuroblastoma, carcinoid syndrome, muscular dystrophy, Parkinson’s disease. It can also be used to detect glycosylated hemoglobin (HbA1c) with better precision, various hemoglobin variants to detect any hemoglobinopathies, C-DT (carbohydrate-deficient transferrin) in cases of alcohol abuse and therapeutic drug levels.

Apart from the medical diagnostic uses, HPLC is also used in pharmaceutical laboratories, food industry, and research fields. Though HPLC allows for detailed testing and it can be beneficial to provide appropriate treatment of patients, still it does not find widespread use in diagnostic labs due to its complex working pattern and requirement of expertise to run the system. However, with increasing automation in the overall processing of HPLC with minimal human intervention, integration of HPLC with mass spectrometry (MS) and possibility of sequential testing it has become more user-friendly and allows the routine laboratories to provide better diagnostic services with a shorter turnaround time.

Dr Gagandeep Kaur Sidhu
Associate Professor,
MP Shah Medical College

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