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 the pump system. The 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 high pressure was 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 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 number 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.
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 liquid chromatography measurement is largely affected by the reproducibility of sample injection, the design of the injector is an important factor. The most widely used injection method is based on sampling loops. The use of the autosampler (auto-injector) system is also widely used that allows repeated injections in a set scheduled-timing.
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 a 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.
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 the electronic signal. There are different types of detectors available.
The change in eluent detected by a detector is in the form of the electronic signal, and thus it is still not visible to our eyes. In older days, the pen (paper)-chart recorder was popularly used. Nowadays, a computer-based data processor (integrator) is more common. There are various types of data processors; examples include a simple system consisting of the in-built printer and a word processor, and a personal computer type consisting of a display monitor, keyboard, and printer. Also, there is software that is specifically designed for liquid chromatography systems. It provides not only data acquisition but features like peak-fitting, baseline correction, automatic concentration calculation, molecular weight determination, etc…
The components introduced so far are the basics of the liquid chromatography system. Below are some optional equipment used with the basic LC system.
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 noise and causes an 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 the 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 the new HPLC unit system contains a degasser.
The liquid chromatography separation is often largely influenced by the column temperature. In order to obtain repeatable results, it is important to keep consistent temperature conditions. Also for some analysis, such as sugar and organic acid, better resolutions can be obtained at elevated temperatures (50~80℃). It is also important to keep a stable temperature to obtain repeatable results even it is analyzed at around room temperature. There are possibilities that a small difference in temperature causes different separation results. Thus columns are generally kept inside the column oven (column heater).