High-Performance Liquid Chromatography
High-performance liquid chromatography (HPLC) also know as high-pressure liquid chromatography is an instrumental system based on chromatography that is widely used in forensic science. The “HP” portion of the acronym is sometimes assigned to the words high pressure (versus high performance), but it refers to the same analytical system. HPLC is used in drug analysis, toxicology, explosives analysis, ink analysis, fibers, and plastics to name a few forensic applications.
Like all chromatography, HPLC is based on selective partitioning of the molecules of interest between two different phases. Here, the mobile phase is a solvent or solvent mix that flows under high pressure over beads coated with the solid stationary phase. While traveling through the column, molecules in the sample partition selectively between the mobile phase and the stationary phase. Those that interact more with the stationary phase will lag behind those molecules that partition preferentially with the mobile phase. As a result, the sample introduced at the front of the column will emerge in separate bands (called peaks), with the bands emerging first being the components that interacted least with the stationary phase and as a result moved quicker through the column. The components that emerge last will be the ones that interacted most with the stationary phase and thus moved the slowest through the column. A detector is placed at the end of the column to identify the components that elute. Occasionally, the eluting solvent is collected at specific times correlating to specific components. This provides a pure or nearly pure sample of the component of interest. This technique is sometimes referred to as preparative chromatography.
Many different types of detectors are available for HPLC. The simplest and least expensive is the refractive index detector (RI). Although this detector is a universal detector, meaning it will respond to any compound that elutes, it does not respond well to very low concentrations and as a result is not widely used. On the other hand, detectors based on the absorption of light in the ultraviolet and visible ranges (UV/VIS detectors and UV/VIS spectrophotometers) are the most commonly used, responding to a wide variety of compounds of forensic interest with good to excellent sensitivity. The photodiode array detector (PDA) is especially useful since it can produce not only a peak-based output (a chromatogram) but also a UV/VIS scan of every component. In many ways, the ideal detector for HPLC is a mass spectrometer (MS), which provides both quantitative information and in most cases a definitive identification of each component (qualitative information). However, HPLC-MS systems are relatively complex and expensive and are not readily available in all labs. Other detectors that are sometimes used include fluorescence detectors (which are very sensitive) and electrochemical detectors.
Unlike in gas chromatography (GC) in which the mobile phase is an inert gas, the mobile phase in HPLC can be one of many different solvents or combinations of solvents. This imparts to HPLC a greater flexibility and range of application than has GC. Because the sample does not have to be converted to the gas phase, compounds such as explosives that break down at high temperatures are much more amenable to HPLC than GC. For HPLC, all that is required is that the sample be soluble in the solvents selected for the analysis. In addition, there are several types of HPLC defined by the type of mobile phase and stationary phase that is used. For forensic applications, one of the most commonly used types of HPLC is referred to as “reversed phase.” In this type of HPLC, the mobile phase is a solvent or mix of solvents that are “polar,” meaning that different parts of the individual solvent molecules carry a partial positive or negative charge. Water, methanol (methyl alcohol), ethanol (ethyl alcohol), and acetone are examples of polar solvents. The stationary phase in reverse phase HPLC is a nonpolar material such as a long chain hydrocarbon molecule. In this type of HPLC, components in the sample will partition and separate based on their degree of interaction with the stationary phase relative to the mobile phase. In other words, the separation is based primarily on the relative polarity of the sample molecules. Reverse phase HPLC is used in drug analysis (LSD for example), analysis of cutting agents such as sugars, explosives, and gunshot residue (GSR), and forensic toxicology.
Normal phase HPLC uses a polar stationary phase and a nonpolar mobile phase, but this is not widely used in forensic applications. Size exclusion chromatography (SEC) is more common and separates compounds based on relative sizes. The stationary phase in SEC is composed of a gel with different sizes of microscopic pores through it. The larger the molecule, the longer it takes for it to navigate through the pores and reach the detector. SEC is useful for the analysis of large molecules that come in a range of sizes such as plastic polymers, proteins, and nitrocellulose, a component of GSR. Chiral chromatography, a relatively recent development, is making inroads into forensic science since it is capable of separating enantiomers, molecules that are mirror images of each other. This capability is particularly valuable in forensic toxicology and drug analysis. Finally, ion exchange chromatography is available for detection species such as nitrate (NO,3-) and other ions.