2. What is liquid chromatography?
The history of Liquid Chromatography (LC) began at early 20th century. In 1906, a Russian botanist Mikhail Tswett invented LC in order to separate various plant pigments. He injected the plant extract and petroleum ether through a glass column packed with calcium carbonate.
Since this was done on a glass column, he was able to observe the changes inside the column. At the beginning, there is only one layer of pigment on the top of the column (Figure 1.a). But as time passes by, the pigment is separated into four different colored-layers (Figure 1.b). The later research discovered that those four-layers were (i) bluish green; chlorophyll a, (ii) yellowish green; chlorophyll b, (iii) yellow; xanthine, and (iv) orange; carotene.
The whole process of separation took several hours and thus it was not a very practical method. This long analysis time was a part of the reason that LC did not become a popular analytical tool until 1970s, a half century after Mikhail Tswett’s invention.
Below is some terms commonly used in chromatography analysis.
Chromatography is a separation technique and the word chromatography originated from chroma meaning “color” and graphein meaning “write”. Chromatograph is the separation equipment and chromatogram is an out-put chart obtained from the analysis.
Figure 2. Images of chromatography, chromatograph, and chromatogram.
3. What is HPLC?
HPLC stands for High Performance Liquid Chromatography. Before HPLC was available, LC analysis was carried by gravitational flow of the eluent (the solvent used for LC analysis) thus required several hours for the analysis to be completed. Even the improvements added in later time were able to shorten the analysis time slightly. Those classical/initial LC systems are called “low pressure chromatography” or “column chromatography”.
In 1970s in the US, Jim Waters founded Waters Corporation and started to sell HPLC instruments. This promoted the use of HPLC in practical analysis areas. The LC systems that Waters Corporation developed used high-pressure pump that generates rapid-flow of eluent, and thus resulted in dramatic improvement in the analysis time. Compared to the “low pressure chromatography” the newer types were called “high pressure liquid chromatography”. Therefore it was used to be thought that HPLC stands for High Pressure Liquid Chromatography, however nowadays it is a common agreement that HPLC stands for High Performance Liquid Chromatography. Another big change from Tswett’s date was the data acquisition methods. Instead of observing the changes of layers by eyes, detector system was coupled to the LC and out-put was recorded on paper chart. If we were to demonstrate Tswett’s analysis result on a chart (chromatogram), it will be like figure 3.
3. Yellowish Green
4. Bluish Green
Figure 3. Representation of Tswett’s LC analysis.
Initially, HPLC system was referred to Waters Corporation’s system. Still now, Waters Corporation is the HPLC pioneer, but there are several other companies that manufacture and sell HPLC systems.
Technically speaking, the word LC represents all the Liquid Chromatography, including low pressure LC, however most LC systems used these days are HPLC thus often the word LC is used as comparable as HPLC.
4. Components of HPLC
Details of each are explained below. Figure 4. Components of HPLC system
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.
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…
4.7 Column heater
5. Other chromatography
Another member of chromatography, as often used as LC, is the Gas Chromatography (GC). While LC uses eluent (flow generated by a pump system), GC uses gas (carrier gas) provided from a gas-tank, thus it does not require pump system. Therefore, GC system is simpler than LC system and for that reason; it was widely available before LC system become popular. At that time, the word chromatography was referred to GC. GC’s simple system was an advantage but also a disadvantage. The samples to be analyzed need to be in the gaseous form before introduced to the flow of carrier gas. Thus, if the original sample was in gaseous form, GC is a convenient tool. However most sample is in the form of liquid or solid and requires heating at high temperature to make it into a gas. Some analytes are alternated by heat, so the GC method is not an ideal for that case. Related problem is that GC has an upper limit for the analysis of large molecular weight compounds.
In comparison, liquid samples can be analyzed directly by LC. Also if solid sample can be dissolved in a solvent, it can be analyzed by LC. Some samples are insoluble in water, but most of those are soluble in organic solvent, there is a high chance that we can find a solvent that dissolves the sample. Therefore, LC can be used at ambient temperature (i.e., without causing alternation of analyte) for a wide range of analysis. This made the LC method become more popular than GC method.
There are other types of chromatography such as thin layer chromatography, super-critical fluid chromatography, paper chromatography etc… but their use is even less popular than GC.