High-performance liquid chromatography
High-performance liquid chromatography, formerly referred to as high-pressure liquid chromatography, is a technique in analytical chemistry used to separate, identify, and quantify specific components in mixtures. The mixtures can originate from food, chemicals, pharmaceuticals, biological, environmental and agriculture, etc., which is a liquid or has been dissolved into a liquid.
It relies on high pressure pumps, which deliver mixtures of various solvents, called the mobile phase, which flows through the system, collecting the sample mixture on the way, delivering it into a cylinder, called the column, filled with solid particles, made of adsorbent material, called the stationary phase.
Each component in the sample interacts differently with the adsorbent material, causing different elution rates for each component. These different rates lead to separation as the species flow out of the column into a specific detector such as UV detectors. The output of the detector is a graph, called a chromatogram. Chromatograms are graphical representations of the signal intensity versus time or volume, showing peaks, which represent components of the sample. Each sample appears in its respective time, called its retention time, having area proportional to its amount.
HPLC is widely used for manufacturing, legal, research, and medical purposes.
Chromatography can be described as a mass transfer process involving adsorption and/or partition. As mentioned, HPLC relies on pumps to pass a pressurized liquid and a sample mixture through a column filled with adsorbent, leading to the separation of the sample components. The active component of the column, the adsorbent, is typically a granular material made of solid particles, 1.5–50 μm in size, on which various reagents can be bonded. The components of the sample mixture are separated from each other due to their different degrees of interaction with the adsorbent particles. The pressurized liquid is typically a mixture of solvents and is referred to as a "mobile phase". Its composition and temperature play a major role in the separation process by influencing the interactions taking place between sample components and adsorbent. These interactions are physical in nature, such as hydrophobic, dipole–dipole and ionic, most often a combination.
Operation
The liquid chromatograph is complex and has sophisticated and delicate technology. In order to properly operate the system, there should be a minimum basis for understanding of how the device performs the data processing to avoid incorrect data and distorted results.HPLC is distinguished from traditional liquid chromatography because operational pressures are significantly higher, while ordinary liquid chromatography typically relies on the force of gravity to pass the mobile phase through the packed column. Due to the small sample amount separated in analytical HPLC, typical column dimensions are 2.1–4.6 mm diameter, and 30–250 mm length. Also HPLC columns are made with smaller adsorbent particles. This gives HPLC superior resolving power when separating mixtures in a shorter period of time, which makes it a popular chromatographic technique.
The schematic of an HPLC instrument typically includes solvents' reservoirs, one or more pumps, a solvent-degasser, a sampler, a column, and a detector. The solvents are prepared in advance according to the needs of the separation, they pass through the degasser to remove dissolved gasses, mixed to become the mobile phase, then flow through the sampler, which brings the sample mixture into the mobile phase stream, which then carries it into the column. The pumps deliver the desired flow and composition of the mobile phase through the stationary phase inside the column, then directly into a flow-cell inside the detector. The detector generates a signal proportional to the amount of sample component emerging from the column, hence allowing for quantitative analysis of the sample components. The detector also marks the time of emergence, the retention time, which serves for initial identification of the component. More advanced detectors, provide also additional information, specific to the analyte's characteristics, such as UV-VIS spectrum or mass spectrum, which can provide insight on its structural features. These detectors are in common use, such as UV/Vis, photodiode array / diode array detector and mass spectrometry detector.
A digital microprocessor and user software control the HPLC instrument and provide data analysis. Some models of mechanical pumps in an HPLC instrument can mix multiple solvents together at a ratios changing in time, generating a composition gradient in the mobile phase. Newer HPLC instruments have a column oven that allows for adjusting the temperature at which the separation is performed.
The sample mixture to be separated and analyzed is introduced, in a discrete small volume, into the stream of mobile phase percolating through the column. The components of the sample move through the column, each at a different velocity, which are a function of specific physical interactions with the adsorbent, the stationary phase. The velocity of each component depends on its chemical nature, on the nature of the stationary phase and on the composition of the mobile phase. The time at which a specific analyte elutes is called its retention time. The retention time, measured under particular conditions, is an identifying characteristic of a given analyte.
Many different types of columns are available, filled with adsorbents varying in particle size, porosity, and surface chemistry. The use of smaller particle size packing materials requires the use of higher operational pressure and typically improves chromatographic resolution. Sorbent particles may be ionic, hydrophobic or polar in nature.
The most common mode of liquid chromatography is reversed phase, whereby the mobile phases used, include any miscible combination of water or buffers with various organic solvents. Some HPLC techniques use water-free mobile phases. The aqueous component of the mobile phase may contain acids or salts to assist in the separation of the sample components. The composition of the mobile phase may be kept constant or varied during the chromatographic analysis. Isocratic elution is typically effective in the separation of simple mixtures. Gradient elution is required for complex mixtures, with varying interactions with the stationary and mobile phases. This is the reason why in gradient elution the composition of the mobile phase is varied typically from low to high eluting strength. The eluting strength of the mobile phase is reflected by analyte retention times, as the high eluting strength speeds up the elution. For example, a typical gradient profile in reversed phase chromatography for might start at 5% acetonitrile and progress linearly to 95% acetonitrile over 5–25 minutes. Periods of constant mobile phase composition may be also part of a gradient profile. For example, the mobile phase composition may be kept constant at 5% acetonitrile for 1–3 min, followed by a linear change up to 95% acetonitrile.
The chosen composition of the mobile phase depends on the intensity of interactions between various sample components and stationary phase. Depending on their affinity for the stationary and mobile phases, analytes partition between the two during the separation process taking place in the column. This partitioning process is similar to that which occurs during a liquid–liquid extraction but is continuous, not step-wise.
In the example using a water/acetonitrile gradient, the more hydrophobic components will elute later, then, once the mobile phase gets richer in acetonitrile, their elution speeds up.
The choice of mobile phase components, additives and gradient conditions depends on the nature of the column and sample components. Often a series of trial runs is performed with the sample in order to find the HPLC method which gives adequate separation.
History and development
Prior to HPLC, scientists used benchtop column liquid chromatographic techniques. Liquid chromatographic systems were largely inefficient due to the flow rate of solvents being dependent on gravity. Separations took many hours, and sometimes days to complete. Gas chromatography at the time was more powerful than liquid chromatography, however, it was obvious that gas phase separation and analysis of very polar high molecular weight biopolymers was impossible. GC was ineffective for many life science and health applications for biomolecules, because they are mostly non-volatile and thermally unstable at the high temperatures of GC. As a result, alternative methods were hypothesized which would soon result in the development of HPLC.Following on the seminal work of Martin and Synge in 1941, it was predicted by Calvin Giddings, Josef Huber, and others in the 1960s that LC could be operated in the high-efficiency mode by reducing the packing-particle diameter substantially below the typical LC level of 150 μm and using pressure to increase the mobile phase velocity. These predictions underwent extensive experimentation and refinement throughout the 60s into the 70s until these very days. Early developmental research began to improve LC particles, for example the historic Zipax, a superficially porous particle.
The 1970s brought about many developments in hardware and instrumentation. Researchers began using pumps and injectors to make a rudimentary design of an HPLC system. Gas amplifier pumps were ideal because they operated at constant pressure and did not require leak-free seals or check valves for steady flow and good quantitation. Hardware milestones were made at Dupont IPD such as a low-dwell-volume gradient device being utilized as well as replacing the septum injector with a loop injection valve.
While instrumentation developments were important, the history of HPLC is primarily about the history and evolution of particle technology. After the introduction of porous layer particles, there has been a steady trend to reduced particle size to improve efficiency. However, by decreasing particle size, new problems arose. The practical disadvantages stem from the excessive pressure drop needed to force mobile fluid through the column and the difficulty of preparing a uniform packing of extremely fine materials. Every time particle size is reduced significantly, another round of instrument development usually must occur to handle the pressure.