HPLC, or High Performance Liquid Chromatography, is one of the fundamental techniques in analytical chemistry. The basic principle of liquid chromatography is to exploit differences in solubility or affinity between a solvent and a stationary material, between which substances can partition, in order to separate different compounds. The simplest forms use either a polar solvent and a less polar or non-polar stationary phase (reversed phase), or a non-polar solvent and a polar stationary phase (normal phase). Starting with a polar solvent is useful when analysing samples originating from water, whereas a non-polar solvent may be needed when the sample is, for example, a fat or tar. As the names suggest, normal phase came first, but reversed phase is probably the most widely used mode today. HPLC is commonly used for non-volatile organic compounds or polar compounds, for example in water.
The “High Performance” in HPLC refers to the use of columns packed with very small particles coated with the stationary phase. This increases the surface area available for compounds to interact with and partition into, thereby improving the efficiency of the separation. To force the solvent through the column, powerful pumps are required, since smaller particle sizes result in a substantial increase in the back pressure that the pump must overcome. A typical HPLC separation is often performed at pressures of around 100–200 bar, although 400 bar is not uncommon. Higher pressures and smaller particles generally make it possible to achieve better separation, higher sensitivity and shorter analysis times.
The technique has been further developed into what is known as UPLC, where even smaller particles and significantly higher pressures are used, up to around 1200 bar. Unfortunately, higher pressures also mean more expensive and less robust equipment, as well as increased wear on pumps, columns and other components. For many applications, HPLC therefore remains the more cost-effective choice.
Liquid chromatography (HPLC) and gas chromatography (GC) are complementary techniques, where polar and less volatile compounds are more easily analysed using HPLC, while non-polar and volatile compounds are analysed using gas chromatography. There is a significant overlap between the techniques, and many compounds and sample types can be analysed using both HPLC and GC.
HPLC can be coupled to several different detectors depending on the properties of the compounds, but the most common is a UV-VIS or absorbance detector, which today most often means a so-called diode array detector (DAD) that can acquire a full spectrum from the UV to the visible range. When higher sensitivity and better selectivity are required, i.e. the ability to identify or distinguish between different substances, HPLC is most often coupled to a mass spectrometer (MS). The development of mass spectrometers has progressed rapidly, and today there are mainly two types in use: either relatively low-resolution instruments, but with the ability to fragment molecules for increased selectivity, or high-resolution MS (HRMS), which can identify molecules because their mass can be determined with such high accuracy that even similar compounds can be identified. High-resolution MS can also be combined with fragmentation. The combination with HRMS enables non-target screening, i.e. the identification of unknown substances and substances that are not known or expected in advance, a capability that is becoming increasingly important. Low-resolution MS, such as triple quadrupoles (QQQ), is used more for routine applications because these instruments are less expensive and often better suited for quantification.
