High-performance liquid chromatography (HPLC) has become a widely used analytical technique in today’s laboratories – a go-to technology for separation, identification, and quantification of components in the most complex organic samples for a wide range of applications, from food safety to pharma QA/QC.
Based on the groundbreaking chromatographic work of J.P. Martin and R.L. Millington Synge1, which earned them the Nobel Prize in chemistry in 1952, HPLC came into its own in the 1960s and ‘70s, when scientists began relying less on simple gravity to move the mobile phase and began using a pump instead. The rest, as they say, is history.
From that point, HPLC (and ultrahigh-performance liquid chromatography, or UHPLC) has continued to evolve, incorporating innovations and different components to make it the widely used technique it is today. But it is important to understand the functions of the key components of HPLC, to better understand its potential.
We have identified six major components of an HPLC system:
- Mobile Phase
A liquid is used to carry a sample through the chromatographic system in HPLC, and this liquid is called the mobile phase. The composition of the mobile phase is exceptionally important because it can dramatically affect how different compounds partition between the mobile phase and the packing material inside the chromatographic column. The purity of the solvent used as the mobile phase, as well as the type and concentration of any buffers or additives, can dramatically affect the retention and subsequent separation of different compounds by the chromatograph.
It is not uncommon for HPLC analyses to use more than one composition of mobile phase, requiring a means of changing and mixing different solvents during a chromatographic separation.
- Stationary Phase
An HPLC or UHPLC column is most likely packed with tiny, porous silica particles that often (but not always!) have a surface that has been modified with some form of bonded chemistry that affects how the sample interacts with the silica particles. These coated silica particles ideally do not move during the chromatographic separation, which is why they are called the stationary phase. It is the relative interactions a particular chemical has with the mobile and stationary phases that define how well retained that chemical is, and varying degrees of interactions help define how well separated two or more chemicals can become as they pass through the chromatograph. The stationary phase is contained inside the chromatographic column.
Also known as the mobile phase (or solvent) delivery system, the pump in a HPLC system maintains a constant flow of mobile phase through the instrument, regardless of the back pressure caused by the flow resistance of the HPLC column. A standard HPLC pump can work against back pressures up to 6,000 psi (≈ 400 bar), whereas a UHPLC pump typically exceeds 8,500 psi (≈ 600 bar) and can go as high as 18,000 psi (≈1,200 bar).
After determining what type of solvents and mixtures you need, you must select a pump that is compatible to avoid damaging the system.2 The PerkinElmer LC 300™ HPLC and UHPLC Systems can come with two types of mobile phase pumps:
- Binary pumps, or high-pressure mixing pumps, use two independent pumps to deliver different solvents into a mixer
- Quaternary pumps, or low-pressure mixing pumps, consist of one pump that draws in a specific proportion of each solvent through a valve system before mixing
The column represents the heart of the separation process in chromatography. When choosing an LC column, deciding its stationary phase will affect the separation. In broad terms, LC columns are primarily divided into normal-phase columns (retaining polar compounds) and reversed-phase columns (retaining nonpolar compounds), with other types like ion exchange or hydrophilic interaction (HILIC) providing even more separating power. The dimension of the column and the size and type of particle also play big parts in defining the separation.
In general, smaller sizes lead to greater efficiencies, but they come at the cost of higher back pressures (perhaps requiring a UHPLC instrument for the smallest silica particles), lower sample loading capacities, and, unless all the column dimensions are scaled appropriately, lower resolving capacity. Picking the correct LC column is about finding the right balance between these aspects.
Detectors for HPLC/UHPLC determine the concentration of eluting compounds in the mobile phase, and they can be categorized into two types: specific detectors and bulk property detectors.3
- Specific detectors: As the name suggests, these detectors respond to specific compounds, and their response is not dependent on the composition of the mobile phase. UV/Vis detectors are the most common examples of specific detectors, as they respond to compounds that absorb UV or visible light at particular wavelengths.4
- Bulk property detectors evaluate properties common to all analytes by measuring differences in the mobile phase with and without the sample. The universal nature of bulk property detectors places an increased emphasis on the selectivity of the chromatographic column, and these detectors are limited in their sensitivity.4,5 The refractive index detector (RI) is the most common example of a bulk property detector.
Each type of detector differs with regards to its sensitivity, specificity, selectivity, and linear dynamic range, so the choice of detector is driven by method goals for the application.5 To understand the variety of detectors available read more in this guide.
- Chromatography Data System (CDS) Software
As with other chromatographic analytical techniques, HPLC requires use of a chromatography data system (CDS) software to support data acquisition, integration, quantitation, and reporting.
Over the decades, CDS software has undergone improvements aimed at making its functions more reliable, powerful, and easy to use. Innovations to enhance automation and productivity have made modern CDS software a key component of many labs’ chromatographic workflow.6
In this context, user interface (UI) and user experience (UX) aspects have become a key focus, as users demands intuitiveness, automation, and shorter learning curves. UI and UX are now shaping the analytical laboratory’s digital experience by:
- Advancing performance and productivity
- Balancing workflow and specification needs
- Cohesively integrating compliance solutions
Want to learn more about PerkinElmer solutions for HPLC analysis?
- PerkinElmer HOW TO GUIDE “High Performance Liquid Chromatography: Choosing the right Detector”
- HPLC DETECTORS: A BRIEF REVIEW – Instituto de …. Pdf4pro.com. https://pdf4pro.com/view/hplc-detectors-a-brief-review-instituto-de- 325ed.html Published August 8, 2018. Accessed May 11, 2021.
- Choudhary A. Different types of HPLC detectors. Pharmaguideline. com. https://www.pharmaguideline.com/2016/01/different-types-of-hplc-detectors.html Accessed May 11, 2021
- The A, B and Cs of a New Era in Chromatography CDS, https://www.perkinelmer.com/library/the-abcs-of-a-new-era-in-chromatography-cds.html