High-performance liquid chromatography (HPLC), also known as high-pressure liquid chromatography, is an efficient, versatile and reliable analytical method for separating, identifying and quantifying chemical compounds.
While there are many different separation techniques available for HPLC, the most common include reverse phase, normal phase, size exclusion and ion exchange. But, the method doesn’t stand alone—it relies on other components to get the job done. In HPLC, a high-pressure pump forces the liquid mobile phase and sample through a column packed with solid stationary phase, which separates the sample based on polarity, size, charge or other qualities depending on the column type. A detector measures retention time for each compound, producing peaks that can be compared with standards to identify compounds and determine their concentrations.
Let’s take a look at some of these vital components.
Pumps
There are two common types of HPLC pumps: reciprocating pumps and syringe pumps. Reciprocating pumps are by far the most common pump used and can have single piston or dual pistons. Dual pistons are typically preferred, as the need for one piston to periodically refill results in pulses in pressure and flow, although dual piston pumps do require more maintenance. Syringe pumps are a type of pump that deliver solvent through a plunger, producing a constant flow rate through a motorized screw mechanism.
Three factors you’ll want to consider when choosing a high-pressure pump are flexibility in mixing solvents for gradient elution, compatibility with different solvents and the ability to pump against back pressure. Binary pumps, or high-pressure mixing pumps, use two independent pumps to deliver different solvents into a mixer, while 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. Determine what type of solvents and mixtures you will be using most often, and select a pump that is compatible to avoid damaging the system. Also, consider the range of backpressure the pump can withstand; the amount of backpressure you can expect will vary with the type of column you use.
Products to consider:
- LC-40B X3 HPLC/UHPLC Pump from Shimadzu
- 1260 Infinity II Quaternary Pump from Agilent Technologies
Columns
HPLC columns vary in sizes and materials and there are a wide range of different stationary phases available on the market. When it comes to packing material, particle size, pore size, particle shape and chemical nature are factors you’ll want to consider.
The most popular stationary phase for reverse-phase HPLC is C18-bonded silica, which provides reliable retention of a wide variety of hydrophobic molecules for reversed-phase HPLC. C18 silica gel comes in a variety of particle sizes and pore sizes, spherical or irregular shapes and can be porous throughout or have a solid core and porous surface, which is known as core-shell.
Smaller particle sizes will improve resolution but require more pressure and can lead to backpressure, but core-shell particles can help achieve higher resolution at lower pressures. Smaller pore sizes will also provide more surface area and better resolution, but are not suitable for larger molecules such as proteins. If you require a faster retention time, you could choose a silica gel with a shorter carbon chain than C18, such as C8, and there are other types of modified silica available that offer alternate selectivity for specific applications, such as cyanopropyl silane-bonded (cyano) and aminopropyl-bonded (amino) columns, which can also be used for normal-phase HPLC. Pure silica is a preferred stationary phase for normal-phase HPLC, and SEC columns contain a matrix of particles with different-sized pores. IEC columns contain resins of different positive or negative charges for stronger or weaker anion or cation exchange. Polymer-based stationary phases are one alternative to silica, as they can withstand greater variations in pH and temperature than silica.
The length, diameter and hardware material of a column will also have an impact on the analytical process. Using shorter columns will reduce run time, but will provide lower resolution than longer columns. Columns of a diameter smaller than 4.6 mm will provide higher sensitivity and are best paired with mass spectrometry (MS), while columns larger than 4.6 mm, up to the hundreds of millimeters, are typically only used for preparative HPLC. Most columns are made from stainless steel to tolerate high pressures, but other materials may be needed when the chemical properties of steel would interfere with the analysis, such as in IEC, which involves more extreme pH levels. Alternatives include glass and polyether ether ketone (PEEK).
Products to consider:
- Accucore™ XL 4µm C18 HPLC Columns from Thermo Fisher Scientific
- BIOshell™ UHPLC and HPLC Columns for Biomolecules from MilliporeSigma
Detectors
Ultra-violet/visible (UV/Vis) detectors are the most widely used detector for HPLC and provide sensitive, precise and repeatable data for analytes that absorb light in the visible and ultraviolet range, which includes most organic compounds. Standard UV/Vis detectors use one wavelength, usually 254 nm, for detection, while photo diode array (PDA) detectors can measure a range of wavelengths simultaneous and offer high speed and sensitivity. Fluorescence (FL) detectors are an alternative to UV/Vis detectors that can provide higher sensitivity and specificity, but fluorescence derivatives are needed to analyze compounds without natural fluorescence.
Evaporative light scattering detectors (ELSD) are used to measure analytes that cannot be detected through UV/Vis absorption methods, and are often used to test non-volatile and semi-volatile molecules and compounds such as sugars and fatty acids. Refractive index (RI) detectors can also detect analytes that UV/Vis detectors cannot, and are considered to be “universal” detectors because they can detect any component of a sample that differs from the mobile phase used; however, they offer lower sensitivity than UV/Vis or ELSD detectors.
MS can also be used with HPLC (LC-MS). While more expensive and not as easy-to-use as LC-UV systems, LC-MS systems offer very high sensitivity and selectivity, low detection limits and fast run times. Electrochemical (EC) detectors also offer very low detection limits and high sensitivity for trace analysis, and are often applied for testing of phenols and amines.
For size exclusion, light scattering detectors are typically used, such as multi-angle light scattering detectors (MLSDs) or ELSDs, which may also be paired with viscometers and UV detectors. Conductivity detectors are typically used in ion exchange chromatography, and can also be paired with others detector, including UV.
Here are some questions to ask yourself when deciding on a detector to use for your analysis:
- Are my samples chromophoric or non-chromophoric? Fluorescent or non-fluorescent?
- What sensitivity levels, selectivity levels and detection limits do I require?
- How time-consuming is this method and how much time am I willing to invest (including training and maintenance)?
- How much will this detector cost (including consumables and maintenance)?
Products to consider:
- LC-4000 Series UV/Visible Detectors from JASCO
- ACQUITY UPLC ELS Detector from Waters Corporation