WHAT IS HPLC AND HOW DOES IT WORK?
The term chromatography comes from the Greek word chroma- which means color and -graphein which means to write. The first recorded use of column chromatography is given to Russian scientist Mikhail Tsvet who crushed calcium carbonate in a tube and subsequently added homogenized green plant leaves, followed by an organic solvent. Tsvet observed separate colored bands as the solvent passed through the tube. This is how practical chromatography started at the beginning, successfully separating various pigments from the leaves. In today’s world, there are many analytes that are colorless and separated by chromatographic techniques, it is still known by the same name.
High Performance Liquid Chromatography (HPLC)
It is a type of column chromatography in which, by the action of a pump, a mixture of compounds or analytes is passed through a solvent system commonly known as the mobile phase. The mobile phase passes through a chromatographic column, which contains the stationary phase at a specified flow. The separation of the compounds occurs based on their interaction with the mobile phase and the stationary phase.
Ultra-High Performance Liquid Chromatography (UHPLC)
It is a technology based on the principle that a smaller particle size leads to higher efficiency, faster separations with higher resolution and sensitivity. However, to withstand the extreme pressure of particles smaller than 2 µm, the equipment needs to be able to work at high back pressure. The efficiency of these columns should not be lost in the rest of the equipment, due to the dead volume, for this reason equipment capable of working at high pressures was designed.
Because UHPLC instruments are expensive, in late 2000, Phenomenex released 2.6 μm Kinetex columns with Core-Shell technology, which provides UHPLC performance on traditional HPLC equipment. In this way you can use the traditional HPLC instrument you have in the laboratory and achieve an equivalent performance of UHPLC.
There are many modes of separation in chromatography and each has its own principles.
Here is an HPLC column selection guide to help readers choose the correct analysis mode. Although there are many separation modes available to resolve mixtures on a chromatograph, Reverse Phase (RP) separation is the most common mode in liquid chromatography.
“Why is the reverse phase called the reverse phase?”
The answer is simple:
Chromatography evolved from the use of the polar stationary phase and a non-polar eluent as the main component of the mobile phase, which is why it was considered normal practice, hence the name normal phase. While this model separated components based on their polar nature, there were a large number of analyte mixtures that were nonpolar and had hydrophobic characteristics that required separation.
The use of a non-polar stationary phase, with a polar mobile phase helped to separate these hydrophobic analytes. Since this practice is reverse to normal phase, the term reverse phase is used. This is similar to calling a right handed ping pong player as normal and a left handed ping pong player as the original.
Reverse Phase separation process
Now that we know that the most popular mode of liquid chromatography is reverse phase, let’s explore how it works.
Below is a schematic of the separation process. The analyte mixture is represented by blue, purple, and red dots, which are fed together into the column containing a non-polar reversed phase stationary phase. The red arrows represent the flow direction of the mobile phase. When the mixed analyte mix enters the column, the mobile phase pushes the analytes down the column. As they advance they come into contact with the stationary phase. Analytes that have a higher affinity for the stationary phase (blue dots) will be more strongly retained and will elute later in the run. Therefore, you can separate the analytes based on how strongly they interact with the stationary phase.
The following example represents a stationary, hydrophobic, non-polar phase, more specifically a C18. The mobile phase consists of a hydrophilic, polar aqueous component, usually water, and acetonitrile or methanol. The analytes will separate based on their relative affinity for these two phases. Hydrophobic compounds, such as benzopyrene, will have a strong affinity for the hydrophobic stationary phase, and will be tightly bound. Hydrophilic compounds such as ethyl sulfate will have low affinity for the stationary phase and will remain primarily in the mobile phase and transport rapidly through the column.
Although reversed phase separation occurs by hydrophobic interaction, there are three primary mechanisms of interaction that dictate overall chromatographic behavior.
- Hydrophobic interactions
- Polar interactions
- Ionic interactions
Apart from these three interactions, steric selectivity and the shape of the molecule can sometimes also contribute to the interaction. Using the example of tapentadol, a typical small pharmaceutical molecule, we can better show these three types of interaction, since it has polar, hydrophobic and also ionic components.
It is the primary mechanism in RP-HPLC and it determines retention behavior. That is, the hydrophobic interaction between the nonpolar stationary phase ligand (eg C18) and the hydrophobic nature of the sample molecule (eg the carbon skeleton).
This is a weak and transient interaction between a nonpolar stationary phase and molecules, which includes hydrophobic and van Der Waals interactions. A reasonable estimate of retention can be predicted based on the Log P value, or octanol-water partition coefficient coefficient. Log P is the ratio of octanol to water in a liquid-liquid extraction. In other words, the more hydrophobic a molecule is, the higher the Log P value it has, which translates into higher retention in RP-HPLC.
These are interactions that occur between the polar functional groups of the analytes. They also occur between residual silanols, embedded polar groups, surface polar groups, or polar end groups in the stationary phase. They interact with the analyte through hydrogen and dipole-dipole bonds. These interactions are relatively weak and transient compared to the ion exchange interaction.
Ion Exchange Interactions
Most of the stationary phases in RP are based on a silica support to which a non-polar phase such as C18 is anchored. Chromatographic column manufacturers such as Phenomenex, try to achieve complete inactivation of all silanol groups from silica, however this process cannot remove 100% of the groups, resulting in residual silanol groups on the surface (Si-OH) that are hidden. These silanols can deprotonate and acquire a negative charge, and ionically interact with positively charged basic analyte molecules. These ion exchange interactions are very strong and slow, in contrast to hydrophobic and polar interactions. Therefore, when ion exchange occurs, the analytes experience different rates of interaction (slow vs fast), and this can lead to peak distortion. This is a classic example of basic analytes that interact with residual silanols, which can be controlled by neutralizing the silanol or by neutralizing the analyte by testing at high pH.
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