High Performance Liquid Chromatography(HPLC)

high-pressure liquid chromatography, is a technique in analytical chemistry used to separate, identify, and quantify each component in a mixture.

Principle

 The basic principle of separation by high performance liquid chromatography is similar to classical liquid or column chromatography (LC) though it differs with  regard to the size of the column and the sample. It differs from LC in terms of speed, automation, elution time and individual manual assays of collected fractions. In case of HPLC, microgram amounts of the sample is allowed to pass through a column containing stationary solid inert phase coated with non-volatile liquid phase by means of pressurized flow of a liquid mobile phase where components migrate at different rates due to different relative affinities. Comparison of column size, characteristics of packing material and pressure requirements to force the mobility of mobile phase in classical column chromatography. According to another version, HPLC may be considered as partition chromatography where stationary phase is a second liquid coated on an inert surface and it is immiscible with the liquid mobile phase. According to the stationary liquid phase, the technique may be subdivided into two types; liquid-liquid and liquid-bonded phase chromatography. These differ from each other in the way stationary phase is held on to the support particles of the packing. In LLC, the polar liquid is physically adsorbed on to an inert surface where it competes with the mobile phase. However, in case of bonded phase chromatography, liquid is chemically bonded making it more stable.

Types of HPLC

• Adsorption chromatography
• Ion-exchange chromatography
• Size exclusion chromatography

Adsorption chromatography

Adsorption Chromatography involves the analytical separation of a chemical mixture based on the interaction of the adsorbate with the adsorbent. The mixture of gas or liquid gets separated when it passes over the adsorbent bed that adsorbs different compounds at different rates.

the stationary phase is an adsorbent (like silica gel or any other silica based packing) and the separation is based on repeated adsorption-desorption steps.

Ion-exchange chromatography

Ion chromatography separates ions and polar molecules based on their affinity to the ion exchanger. It works on almost any kind of charged molecule including large proteins, small nucleotides, and amino acids. 

The stationary bed has an ionically charged surface of opposite charge to the sample ions. This technique is used almost exclusively with ionic or ionisable samples. The stronger the charge on the sample, the stronger it will be attracted to the ionic surface and thus, the longer it will take to elute. The mobile phase is an aqueous buffer, where both pH and ionic strength are used to control elution time. 

Size exclusion chromatography

Size-exclusion chromatography, also known as molecular sieve chromatography, is a chromatographic method in which molecules in solution are separated by their size, and in some cases molecular weight. 

The column is filled with material having precisely controlled pore sizes, and the sample is simply screened or filtered according to its solvated molecular size. Larger molecules are rapidly washed through the column; smaller molecules penetrate inside the porous of the packing particles and elute later. This technique is also called gel filtration or gel permeation chromatography. Concerning the first type, two modes are defined depending on the relative polarity of the two phases: normal and reversed-phase chromatography. In normal phase chromatography, the stationary bed is strongly polar in nature (e.g. silica gel), and the mobile phase is nonpolar (such as n-hexane). Polar samples are thus retained on the polar surface of the column packing for longer than less polar materials. Reversed-phase chromatography is the inverse of this. The stationary bed is (nonpolar) in nature, while the mobile phase is a polar liquid, such as mixtures of water and methanol or acetonitrile. Here the more nonpolar the material is, the longer it will be retained. Reverse phase chromatography is used for almost 90% of all chromatographic applications. Eluent polarity plays the major role in all types of HPLC. There are two elution types: isocratic and gradient. In the first type, constant eluent composition is pumped through the column during the whole analysis. In the second type, eluent composition (and strength) is steadily changed during the run.

HPLC as compared with the classical LC technique is characterized by

• High resolution
• Small diameter (4.6 mm), stainless steel, glass or titanium columns
• Column packing with very small (3, 5 and 10 µm) particles
• Relatively high inlet pressures and controlled flow of the mobile phase
• Continuous flow detectors capable of handling small flow rates and detecting very small amounts
• Rapid analysis

Initially, pressure was selected as the principal criterion of modern liquid chromatography and thus the name was "high pressure liquid chromatography" or HPLC. This was, however, an unfortunate Term because it seems to indicate that the improved performance is primarily due to the high pressure. This is, however, not true. In fact, high performance is the result of many factors: very small particles of narrow distribution range and uniform pore size and distribution, high pressure column slurry packing techniques, accurate low volume sample injectors, sensitive low volume detectors and, of course, good pumping systems. Naturally, pressure is needed to permit a given flow rate of the mobile phase.

Stationary Phases (Adsorbents)

HPLC separations are based on the surface interactions, and depend on the types of the adsorption sites. Modern HPLC adsorbents are the small rigid porous particles with high surface area.

Main adsorbent parameters are

• Particle size: 3 to 10 µm
• Particle size distribution: as narrow as possible, usually within 10% of the mean

Instrumentation for HPLC has following components.

• One or more solvent reservoirs for the mobile phase.
• A pump to deliver the mobile phase with varying range of pressures up to  several hundred atmospheres to achieve reasonable flow rates. 
• Sampling valves or loops where the sample may be injected into the flowing mobile phase. Sample may be dissolved in mobile phase.
• A guard column or an on-line filter to prevent contamination of the main column.
• A pressure gauge, inserted in front of the separation column, to measure column inlet pressure.
• Separation column containing packing to accomplish desired separation. These  may be modified silica gel, ion-exchange resin, gel or some other unique packing.
• A detector capable enough of measuring the solute concentrations.
• Display and recording device for plotting time vs. peak intensity.

Components of HPLC

Pump

In the earlier state of HPLC development, the pump was the most important part of the system. The development of HPLC can be said that it was a development of pump system. Pump is positioned in the most upper stream of the LC system and generates a flow of eluent from the solvent reservoir to the system. In the earlier stage of LC development, to be able to generate the high pressure was the one of the most important system requirements. However, nowadays, the high pressure generation is a “standard” requirement and what is more concerned nowadays is to be able to provide a consistent pressure at any condition, to provide a controllable and reproducible flow rate. Since a change in the flow rate can influence the analysis largely.

Most pumps used in current LC systems generate the flow by back-and-forth motion of a motor-driven piston (reciprocating pumps). Because of this piston motion, it produces “pulses”. There have been large system improvements to reduce this pulsation and the recent pumps create much less pulse compared to the older ones. However, recent analysis requires very high sensitivity to quantify a small amount of analytes, and thus even a minor change in the flow rate can influence the analysis. Therefore, the pumps required for the high sensitivity analysis needs to be highly precise.

Injector

An injector is placed next to the pump. The simplest method is to use a syringe, and the sample is introduced to the flow of eluent. Since the precision of LC measurement is largely affected by the reproducibility of sample injection, the design of injector is an important factor. The most widely used injection method is based on sampling loops. The use of autosampler (auto-injector) system is also widely used that allows repeated injections in a set scheduled-timing.

Column

The separation is performed inside the column; therefore, it can be said that the column is the heart of an LC system. The theory of chromatography column has not changed since Tswett’s time; however there has been continuous improvement in column development. The recent columns are often prepared in stainless steel housing, instead of glass columns used in Tswett’s experiment. The packing material generally used is silica or polymer gels compared to calcium carbonate used by Tswett.

The eluent used for LC varies from acidic to basic solvents. Most column housing is made of stainless steel, since stainless is tolerant towards a large variety of solvents. However, for the analysis of some analytes such as biomolecules and ionic compounds, contact with metal is not desired, thus polyether ether ketone (PEEK) column housing is used instead.

Detector

Separation of analytes is performed inside the column, whereas a detector is used to observe the obtained separation. The composition of the eluent is consistent when no analyte is present. While the presence of analyte changes the composition of the eluent. What detector does is to measure these differences. This difference is monitored as a form of electronic signal. There are different types of detectors available. 

Recorder

The change in eluent detected by a detector is in the form of electronic signal, and thus it is still not visible to our eyes. In older days, pen (paper)-chart recorder was popularly used. Nowadays, computer based data processor (integrator) is more common. There are various types of data processors; examples include a simple system consisting of in-built printer and word processor, and a personal computer type consisting of display monitor, keyboard, and printer. Also there are software that are specifically designed for LC system. It provides not only data acquisition, but features like peak-fitting, base line correction, automatic concentration calculation, molecular weight determination, etc…

 Degasser

The eluent used for LC analysis may contain gases such as oxygen that are non-visible to our eyes. When gas is present in the eluent, this is detected as a noise and causes unstable baseline. Generally used method includes sparging (bubbling of inert gas), use of aspirator, distillation system, and/or heating and stirring. However, the method is not convenient and also when the solvent is left for a certain time period (e.g., during the long analysis), gas will dissolve back gradually. Degasser uses special polymer membrane tubing to remove gases. The numerous very small pores on the surface of the polymer tube allow the air to go through while preventing any liquid to go through the pore. By placing this tubing under low pressure container, it created pressure differences inside and outside the tubing (higher inside the tubing). This difference let the dissolved gas to move through the pores and remove the gas. Compared to classical batch type degassing, the degasser can be used on-line, it is more convenient and efficient. Many of new HPLC unit system contain a degasser.

Column heater

The LC separation is often largely influenced by the column temperature. In order to obtain repeatable results, it is important to keep the consistent temperature conditions. Also for some analysis, such as sugar and organic acid, better resolutions can be obtained at elevated temperature (50~80 oC). It is also important to keep stable temperature to obtain repeatable results even it is analyzed at around room temperature. There are possibilities that small different of temperature causes different separation results.  Thus columns are generally kept inside the column oven (column heater).

How Does HPLC Work?

In column chromatography a solvent drips through a column filled with an adsorbent under gravity. HPLC is a highly improved form of column chromatography. A pump forces a solvent through a column under high pressures of up to 400 atmospheres. The column packing material or adsorbent or stationary phase is typically a granular material made of solid particles such as silica or polymers.

The pressure makes the technique much faster compared to column chromatography. This allows using much smaller particles for the column packing material. The smaller particles have a much greater surface area for interactions between the stationary phase and the molecules flowing past it. This results in a much better separation of the components of the mixture.

The pressurized liquid is typically a mixture of solvents such as water, acetonitrile and/or methanol and is referred to as the mobile phase.

The components of a mixture are separated from each other due to their different degrees of interaction with the absorbent particles. This causes different elution rates for the different components and leads to the separation of the components as they flow out the column. Compared to column chromatography, HPLC is highly automated and extremely sensitive.

Benefits of HPLC

• Controls and automates chromatography instrumentation
• Provides data management, security features, and reporting and instrument validation.
• Powerful and adaptable
• Increases productivity by managing all the areas of analysis - from sample to instrument, and from separation to reporting results.
• Affordable

Applications of HPLC

• Water purification
• Detection of impurities in pharmaceutical industries
• Pre-concentration of trace components
• Ligand-exchange chromatography
• Ion-exchange chromatography of proteins
• High-pH anion-exchange chromatography of carbohydrates and oligosaccharides