HPLC, UPLC, & HIGH-PERFORMANCE FLASH
High-performance liquid chromatography (HPLC) and flash liquid chromatography (LC) remain among the most established and effective techniques for compound purification. Continuous advancements have ensured that preparative HPLC and flash LC remain essential tools, with ultra-high-performance liquid chromatography (UPLC) representing a significant leap forward—offering faster analyses and enhanced resolution.
Improvements in solid-phase technology have further enhanced resolution, selectivity, and reliability. Advancements in the physical properties of silica matrices—such as reduced particle sizes and increased superficial porosity—have contributed to superior separation performance. Meanwhile, chemical innovations, including novel functionalizations and enhanced cross-linking, have expanded the range of possible applications.
Hardware developments have also played a crucial role in refining preparative HPLC and flash LC, streamlining workflows and improving efficiency from benchtop research to large-scale manufacturing. Features such as intuitive system controls have simplified method programming, while enhanced hardware designs provide greater precision and operational reliability.
Benefits of Preparative HPLC & Flash LC
Resolution
HPLC columns offer the highest number of theoretical plates among chromatographic technologies, resulting in unparalleled resolution for compound separation. At the analytical scale, 1.6-micron solid phases provide exceptional resolving power, allowing even the most closely eluting components to be separated with precision. For preparative HPLC applications, columns with particle sizes below 5 microns enable efficient isolation of high-purity compounds, optimizing both yield and accuracy in purification processes
Selectivity
The diversity of chromatographic phases offers an extensive range of selectivities tailored for specific separation challenges. From HILIC and normal phase (NP) chromatography to C4, C8, C18, and more specialized options like biphenyl, hexyl-phenyl, and diol, the choices feel limitless. Each of these resins can be customized with various properties—cross-linking, particle size, pore size, and end-capping—allowing for fine-tuned optimization. Whether targeting hydrophilic interactions, reversed-phase retention, or mixed-mode selectivity, advancements in phase technology continue to expand chromatographic capabilities.
Scalability
Chromatographic technology offers seamless scalability, spanning from rapid analytical separations on <2 mm I.D. columns to large-scale manufacturing on columns exceeding a meter in diameter. Advancements in flow distribution plates have significantly improved flow linearity and band profile, even in the largest column formats. These innovations ensure that scaling up from bench-top methods to industrial-scale purification remains predictable and efficient, maintaining resolution and separation integrity across different column sizes.
Solid Phase Advancement Highlights
Solid-phase technology has seen countless advancements, but several key improvements stand out for their significant impact on chromatographic performance:
Particle Sizes
1.7 micron particle sizes mean reduced column dimensions and increased resolution. When paired with UPLCs, you get rapid high-resolution analysis.
Physical Strength
Improved manufacturing of solid phases and better column packing technologies result in columns that stand up to higher pressures and produce more consistent results.
Chemical Inertness
With improved end-capping reducing silanol interactions, possible catalytic interactions are reduced, which increases yields while minimizing non-targeted binding.
Semiporous Surfaces
Also referred to as "core shell", semiporous phases offer increased resolution at lower pressures, mimicking the benefits of < 2 micron particles with less drawbacks.
Technology Overview
Detectors
Advancements in preparative HPLC and flash LC detection technology have significantly enhanced analytical precision and efficiency. Modern UV detectors now offer higher sensitivities and faster refresh rates, enabling more detailed and accurate compound detection.
Secondary detection methods, such as mass spectrometry (MS) and evaporative light scattering detection (ELSD), have also seen notable improvements, expanding detection capabilities across a wider range of analytes. However, the most impactful progress lies in the enhanced ease-of-use, improved reliability, and seamless integration with sophisticated software systems—streamlining workflows and increasing operational efficiency.
Pumps
Advancements in pump design have led to the emergence of a new class of HPLC technology—ultra-high-performance liquid chromatography (UPLC). UPLC systems operate at pressures exceeding 800 bar and can reach up to 1,300 bar, enabling the use of smaller particle sizes. This innovation enhances resolution and analysis speed while simultaneously reducing solvent consumption.
These technological improvements have also benefited conventional HPLC systems, contributing to greater precision, faster processing times, and enhanced overall reliability.
Computing & the Cloud
The increasing speed and resolution of modern chromatography necessitate robust data processing capabilities. Laboratory analytics software suites, such as Agilent’s OpenLab CDS, have advanced significantly, enhancing data integrity, streamlining workflows, and optimizing systems management.
Implementing a centralized chromatography data system (CDS) allows for comprehensive oversight, ensuring seamless compliance with GMP requirements while improving operational efficiency.
Prep HPLC Technology Supplement
ABSTRACT: Echimidine is the main pyrrolizidine alkaloid of Echium plantagineum L, (Jane’s Salvation, Patersons Curse), endemic to Australia. The plant is a great attractant for bees and as a result echimidine finds its way into honey. Because of its hepatotoxicity, echimidine became a target of new EU regulation requiring testing of imported honey. Under normal RP HPLC conditions echimidine produces always a sharp peak. Only recently, we discovered that this single peak represents actually a mixture of three alkaloids.
Using relatively new “core-shell” RP HPLC column (Kinetex EVO C18, Phenomenex) in a buffer system we resolved “echimidine” into two well separated peaks. An NMR analysis proved that later eluting peak belonged indeed to echimidine, while the earlier peak contained two, largely unresolved alkaloids echihumiline (major) and hydroxymyoscorpine (minor). Each of them has been isolated before from other plants. All these alkaloids are isomeric C20H31NO7 and produce in MS a single MH+, signal at m/e 398, in and their NMR spectra are similar, which explains why they have not been detected before.