20 HPLC and UPLC*

High-performance liquid chromatography (HPLC) and its emerging successor, ultra-performance liquid chromatography (UPLC), are instruments that effect liquid chromatographic separations at high pressure. With flow from gravity in a vertically-oriented glass column, mobile phase pressures are small (< 16 psi). Pressures are much higher in HPLC (10³ psi) and UPLC (10⁴ psi).

High pressures are required because the columns are filled with very small stationary phase particles with high uniformity in size and shape, resulting in very little void space through which mobile phase can travel. Particle sizes are 3–5 µm for HPLC, < 2 µm for UPLC, and spherical in both cases. The result is a large increase in separation efficiency from reduction of the A and C terms in the van Deemter equation. Fast separations with high resolution are thus possible—especially with UPLC.

Instrument Design

The main components of an HPLC or UPLC instrument are the solvent proportioning valve, pump, injection system, column, and detection system.

Most HPLCs have valve and pump systems that push mobile phase solvent through the column at desired flow rates (0.05–1 mL/min). These systems also allow multiple solvents to be mixed, in real-time and at defined proportions (v/v), for gradient elution.

Injection systems can be manual or robotic and are designed to introduce a narrow plug of sample into the flowing mobile phase for delivery to the column. To withstand the applied pressure, columns are steel tubes packed with the stationary phase particles. Columns are usually housed in temperature-controlled ovens. Mobile phase and dissolved analytes eluting from the column are delivered to the detection system.

Absorbance detectors (HPLC-DAD) and mass spectrometers (HPLC-MS; discussed further in Ch. 25) are the two most common detector types for HPLC. Absorbance detectors are based on a diode-array design. A chromatogram is obtained for each wavelength and this capability supports application with a wide range of analytes, sometimes provides a mechanism of detection selectivity, and additional data that may help with peak identification. Mass spectrometric detection is much more expensive, but provides better detection limits, a much higher level of detection selectivity, and the most informative data for peak identification.

Other examples of detectors include those based on refractive index (RI), fluorescence detection, and electrochemical detection (e.g. voltammetric methods). Whereas fluorescence and electrochemical detection are more specialized, the main advantage of RI detection is its universal applicability to all analytes, albeit at the expense of poor sensitivity. Evaporative light scattering detectors (ELSD) also have near-universal applicability with better detection limits that RI detectors. A conductivity detector is used for IEC, as many ions are not readily detected by other means.

Some combinations of detector types can be used in sequence and eluent flows from one to the next. The requirement is that all but the last detector in line are non-destructive to the analytes.

Compatible Separations

Liquid chromatography (LC) instruments (inclusive of UPLC, HPLC, and lower-pressure instruments) support multiple separation types: NP and RP chromatography, IEC, SEC, HILIC, HIC, and affinity chromatography. LC methods are applicable to any soluble analyte, including highly-polar, high-molecular-weight, temperature-sensitive, and other non-volatile species. Non-destructive detectors also allow the separated species to be recovered afterwards.

LC is perhaps the most widely used analytical technique in the world.

Connections

• All of the general separation concepts described in Ch. 19 are able to be implemented with HPLC/UPLC.
• The designs of absorbance detectors are very similar to those discussed in Ch. 7, with the exception that the cuvette is replaced with a quartz flow cell.
• The design and operation of mass spectrometers, which are common LC detectors, are discussed in Ch. 25, Ch. 26, and Ch. 27.

Post-Reading Questions

1. Match the stationary phase particle sizes (2 µm, 4 µm, 10 µm) with the corresponding pressures (1000 psi, 6000 psi, 12000 psi).
2. Rank the following HPLC detectors in order of decreasing detection limit: absorbance, refractive index, mass spectrometry. (Assume that the analyte has at least one chromophore in its structure.)
3. Based on the content in this chapter, and your knowledge from prior chapters, categorize the following types of detectors as universal or selective: RI, ELSD, DAD, MS, fluorescence, electrochemistry. 

Topic Learning Objectives

The chapter is a primer for the following learning objectives, which will be covered in lecture and/or with additional assigned reading:

• Distinguish between high-pressure, high-performance, and ultra-performance LC.
• Know the parameters specified for LC columns.
• Understand how methods of improving separation efficiency affect the H,  A and C terms in the van Deemter equation (Ch. 19).
• Know the functional role for each major component of an HPLC/UPLC instrument.
• Draw a block diagram of an HPLC instrument.
• Know the operating principles of different types of detectors.
• Understand if and how detectors are able to be selective in what they detect.