HPLC Basics: What You Should Know Vol. 2

By Chris Tuczemskyi

Types of liquid chromatography techniques

Given the vast number of compounds and structural diversity of potential analytes, HPLC is rarely a one-size-fits-all approach. From nano to preparative scale separations, here is a list of the most common types of HPLC techniques and when to apply each.

LC technique	How it works	Applications
High-performance liquid chromatography	HPLC is run at pressures below 700 bar with flow rates between 1 – 2 mL/min	Quality assurance/quality control of small and large molecules in pharmaceuticals, industrial chemicals, and food safety
Ultra-high-performance liquid chromatography	UHPLC uses pressures beyond 1000 bar and flow rates from 0.2 – 0.7 mL/min. Higher pressure ranges give better resolution and sensitivity, higher throughput, and less solvent usage than standard HPLC systems	Commonly applied in research and development labs and pharma and biopharma fields for the development and characterization of small molecule drugs, peptides, and antibodies
Liquid chromatography-mass spectrometry	Liquid chromatography-mass spectrometry (LC-MS) utilizes a mass spectrometer instead of the traditional optical detector like a UV-Vis detector	Mostly used for peptide and protein analysis
Low-flow liquid chromatography	Low-flow liquid chromatography encompasses nano-, micro-, and capillary-flow ranges spanning from low nL/min to about 50 µL/min. This technique increases sensitivity due to the associated decrease in column inner diameter, leading to less dilution of analyte bands	Ideal for high-sensitivity measurements of molecules in complex biological matrices where analyte concentrations can span several orders of magnitude
Preparative liquid chromatography	Preparative LC techniques involve collecting fractionated eluent into discrete sample containers to isolate one or more analytes in order to purify main components or segregate impurities for further investigations	Various applications like large scale purifications of drugs or smaller-scale for improving product yields or isolating pure compounds

Multichannel LC Systems

Multichannel LC systems use multiple flow paths to help chromatographers perform complex and/or parallel separation processes. The benefits of multichannel systems can include higher sample throughput, better resolution of complex samples, and enhanced analyte quantitation.

Multichannel LC technique	How it works	Applications
Two-dimensional liquid chromatography	2D-LC is an advanced separation technique using wo complementary column chemistries in series for a multi-dimensional separation instead of running the sample through one column	Applications of 2D-LC can apply to complex chemical mixtures like vaccines and foods with interfering sample matrices
Dual liquid chromatography	Dual LC is a multichannel HPLC method using two separate flow paths in a single system to run two analyses simultaneously	Helpful for situations when you need to increase sample throughput, like analyzing a sample for pesticides and phenol in a single run or performing replicate analyses simultaneously
Tandem liquid chromatography	Tandem LC techniques use a second pump and intelligent column switching to maximize detector usage by minimizing downtime from column reconditioning	Best used in lead selection for drug discovery labs to increase sample throughput and maximize detector utilization

Want to learn more about HPLC analysis?

It’s been incredible to see the evolution of liquid chromatography in real time. From the beginning, separating pigments, to basic liquid chromatography, it was a very manual process that took hours to separate compounds of interest. Today, with modern HPLC and UHPLC instruments, separations are done within minutes, even seconds in some cases.

Additionally, utilizing various detectors such as UV, mass spectrometry and charged aerosol detecting, identifying and quantifying your compounds is immensely more effortless than ever before.