HPLC 2022: Instrument Advances and Fresh Applications at 50th International Symposium

The International Symposium on High Performance Liquid Phase Separations and Related Techniques—or HPLC 2022—enjoyed its 50^th edition earlier this summer. As a technology, HPLC is a giant in the laboratory market with forecast global sales of $10.5 billion.

The data in Table 1 shows that analytical HPLC, which includes UHPLC, dominates the LC market. This validates the prediction in the 1960s that HPLC would own the laboratory chromatography market since it was compatible for about 80% of the analyte candidates. Preparative LC has developed more slowly since the products, such as biotherapeutics, have to be developed in the laboratory before entering scale up and ultimately production and use.

*TDA estimates that global sales of chromatography products in calendar year 2021 were US$10.5 billion.

Table 1: Global 2021 Market Fraction, 2022 Growth Forecast and Shipments for HPLC (adapted from “Instrument Industry Outlook, Jan. 2022,” Top-Down Analytics, TDAresearch.com)

Multicolumn technology is being explored to improve throughput in analytical, as well as preparative, applications. Waters and Thermo Fisher Scientific are promoting multi-instrument analytical systems feeding high-speed, high-resolution and costly analytical MS/MS. These will be attractive for in-vitro diagnostics (IVD) where throughput and productivity are motivating concerns.

Bioprocessing is also coming up to speed in multicolumn technology to improve production economics. The endpoint will probably be processes using simulated moving bed (SMB) technology, which is difficult to understand and engineer but can reduce purification cost by 50% or more.

Supercritical fluid chromatography (SFC) fills applications niches, such as chiral separations. It has been a second choice to LC since there are more variables to control. However, CO2 and methanol are the most economical mobile phases, next to water.

MS has replaced UV absorbance to become the most important detector type for analytical HPLC. The single mass analyzer, usually a quadrupole, is a frequent choice. Even when UV and MS are used in tandem, users are often selecting the MS as the primary detector. This evolution has taken 20 years. Single quadrupole MS detectors are also suitable for targeted assays and routine work. However, tandem MS detectors offer superior detection sensitivity. Plus, MRM fragmentation provides useful confirmation of peak purity and identity, which can be valuable for detection of co-elution or when the unexpected happens.

Advances in HPLC Instrumentation

HPLC qualifies as a mature technology, especially if we focus on legacy instruments, which were designed for packed columns with 4 mm or larger ID. Today, the vast majority of applications of HPLC use such columns. However, column technology is not stagnant. Narrow capillaries are attracting attention due to improved chromatographic resolution and compatibility with MS detection. Also, the pillar and post columns on a chip are showing promise for high durability and efficiency.

As usual, column technology leads instrument development. Thermo Fisher Scientific (TFS) introduced the Vanquish product platform that features four analytical systems plus nine special purpose instruments designed for nano niche instruments, such as inverse gradient and heart cutting.

The Vanquish Core has specifications that are competitive with legacy analytical technology (~4 mm ID columns packed with 3, 5 or 10 um packing). The specifications for the Horizon and Flex instruments are compatible with UHPLC column technology. The Neo is designed for the emerging nano flow column technology.

Microflow LC is also attracting attention for at-site applications. AXcend Corp introduced upgrades to their very compact capillary LC/MS. It is designed for Process Analytical Technology (PAT) in pharmaceuticals to BTEX in various aqueous matrices. One huge advantage of capillary LC instruments is the dramatic reduction of mobile phase that is required for analysis and also post-run disposal.

Bridging the Gap from HPLC to UHPLC

The life cycle of analytical instruments is generally less than 20 years. The service life of HPLC is only about 10 years. Meanwhile, the life cycle of drug products is generally several decades longer. This has created a problem when new instrumentation replaces old.

Regulators often ask, “How does your current product compare with the product you licensed?” Twenty-five years later, this is not a nice question.

Waters ran into this when introducing the UPLC technology several years ago. This led the company to introduce the ARC product line, which is designed for bridging studies between legacy HPLC methods and UPLC. More recently, Waters and Eurofins developed a risk-based approach to method migration. This is based upon the growing acceptance that method migration need not present a serious risk. However, migration needs to be considered for each validated method.

Their Quality by Design (QBD) approach creates an Ishikawa map comparing different parameters such as flow rate, dead and dwell volume, detector noise, etc. The impact is predicted with a multi-element QBD approach. This model can be verified for items that have high risk. With the basic instrument parameters developed, it is easier to predict behavior of a new legacy validated method on a new instrument. One can then select and specify critical settings and more quickly and confidently demonstrate instrument and method suitability.

Improved Recovery

Non-specific adsorption is like a successful sneak thief. You do not know you’ve been robbed until after the loot is missing. Materials of construction can absorb or change certain analytes through chemical reactions, such as catalysis. Clues include low mass balance and distorted peak shape of certain analytes or trending response with successive runs. Troublesome compound classes include peptides, free glycans, oligonucleotides, and reactive chemicals such as linkers.

Waters is doing something about it with their MaxPeak HPS (High Performance Surfaces) technology available in ACQUITY Premier Columns and non-protected instrument systems.

To illustrate the point, they studied 2D chromatography of some phosphonothioate oligonucleotides that are encountered during synthesis of antisense DNA. The compounds in low oxidation state are potentially reactive with metals, even some stainless-steel alloys. By comparing systems with and without HPS technology, they found that well behaved analytes were recovered without change from both instruments but the recovery of the phosphonothioate compounds was lower for the non-HPS instrument.

Preparative and Process LC

Preparative gas chromatography is seldom practiced on any scale. However, preparative LC is very attractive at the industrial scale, particularly in bioprocessing. Fructose corn syrup is one example of a large-scale preparative LC process essential to the soft drink industry.

The main purpose of preparative LC is to purify material for further use. The scale and purpose are an important segmentation. Usually the steps involve column conditioning, sample loading, washing slightly retained materials, elution of the product, washing out the strongly retained contaminants, reconditioning the column for the injection of the next aliquot, then repeating the cycle. There is an opportunity to save time and reagents by coordinating these steps. Simulated moving bed chromatography (SMB) is an extreme example of an intensely engineered unit process.

According to J. K. O’Donnell of Tosoh Bioscience, multi-column continuous chromatography (MCC) is a more flexible technology that coordinates several flow paths to perform the individual steps simultaneously. This increases throughput and can also reduce reagent consumption. For example, a case study compared automated MMC with single batch purification of antibodies. The MCC reduced buffer volume by 40%.

New Liquid Separation Technology

For decades, the HPLC Meeting has carried the tag line of ”… and other liquid phase separations.” As such, the meeting series helped development of field flow fractionation (FFF), and HPCE. FFF uses a force field, such as thermal or sedimentation with a centrifuge, for orthogonal separation modes to HPLC.

Recently, Horiba introduced a centrifugal sedimentation instrument called “Partica CENTRIFUGE” for rapid assay of particles 10 nm to 40 um. The separation mechanism is governed by Stokes Law, as is FFF. The slurry is in a cuvette that can be spun at 18,000 RPM (~ 30,000g) or less. Run times are a few minutes, generally less than 5. While the technique is not destructive, the spun down pellet from a run is no longer representative of the original sample. Thus, repeat runs with the same identical sample is not recommended.

LC Applications

The fragmented meeting program of HPLC 2022 illustrates the breadth of applications. Environmental, drugs and therapeutics life science research, material science, clinical diagnostics, forensics—the list grows almost daily as new methods are developed and published. Let’s take a look at a few highlighted at HPLC to wrap up this article.

Smoke Taint Markers in Wine

Grapes are California’s second largest crop after marijuana. Recent wildfires in California and Australia have adversely affected wine quality due to “smoke taint.” Shimadzu recently published an assay for gualacol rutinoside and 4-methyguaiacol rutinoside, which are smoke taint markers, using their LCMS-8050 over a concentration range of 0.5 to 100 ng/mL.

Baby Formula

One of the timeliest applications reports was Shimadzu’s protocol for assay of 20 proteinogenic amino acids plus taurine in infant formula. The shortage of baby formula in the US majorly disrupted the global supply chain. Plane loads of formula were landing in the US from vendors with no experience in the American market. Package labels, therefore, were not in English, which leads to confusion.

Shimazu’s method uses an amino acid column (Intrada Amino Acid 100 X 3 mm, Imtak USA) eluted with an ammonium acetate gradient. Shimadzu’s LCMS-8045 triple quadrupole with ESI provided MRM peak identification and quantitation.

Pesticide Residues

YMC is a leading developer of columns for analytical and preparative HPLC. Responding to the global need for rapid tracking of glyphosate and AMPA, YMC developed a method derivatizing the analytes with FMOC-CL (fluorenylmethyloxycarbonyl chloride) to comply with the requirements of DIN ISO 16308. YMC recommends their YMC-Triart C-18 UHPLC column, which is packed with hybrid silica particles as is compatible with basic eluents, such as pH 9.5 in their method. Detection limits with MS were so low that direct injection of the sample was possible. The method covers the range of 30 to 800 ng/L. Stable isotopes were used as internal standards.

Gene Therapy

Virus-like particles (VLP) are attractive therapeutic agents since they can use their infection mechanism to enter the cell. Once inside, they may be directed to key locations to deliver their payload. As a therapeutic agent, a new set of key quality attributes have emerged. These include content of the VLP, such as number of payloads inside, size, surface chemistry, etc.

Tosoh Life Sciences has over 50 years’ experience with developing systems for steric exclusion chromatography (SEC). These include the PW 5000 and the recently introduced LenS3 MALS, which provides the molecular weight and radius of gyration (Rg) of the particle. The LenS3 measures scattered light at 10, 90 and 170 degrees. The low angle is ideal for characterizing large particles since the deflected light is near the axis of the incident beam. Larger angles are useful for characterizing smaller particles including determination of molecular weight.

The performance of the detector for Adeno Associated Virus (AAV) capsid showed the output of the LenS3 is linear from 4 X 10⁹ to 1 X 10¹² p/mL. The output also provided amounts of full, empty, partial filled and miss filled AAV capsids. The detection limit for empty is about 2% of the total. The latter includes capsids containing host cell DNA. These measurements are useful in process monitoring and quality control. Another study reported on the SEC –MALS of Parvovirus VLPS using a TSKgel GMPWXL SEC column, which has a very large pore diameter. The results showed that the diameter was 25.6 nm and ~4 MDa mole weight. The authors also reported that Tosoh’s deconvolution software improved determination of higher order structures.