What is HPLC and How Does it Work?

Guest author: Dr. Ramkumar Dhandapani

Chromatography is coined from the Greek word chroma- which means color and -graphein which means to write. First recorded use of column chromatography can be traced back to Russian scientist Mikhail Tsvet who crushed calcium carbonate into a tube and added homogenized green plant leaves followed by organic solvent. He saw colored bands separate as solvent passed through the tube. That’s how chromatography started in practice at first by successfully separating various pigments from the leaves. In today’s world, there are many analytes that are colorless and are separated by chromatographic techniques, like HPLC, that are still coined under the same name.

High Performance Liquid Chromatography (HPLC)

Is a form of column chromatography that pumps a sample mixture or analyte in a solvent system commonly known as the mobile phase at specified flow through a column which contains stationary phase. Separation of analyte happens based on the analyte’s interaction with the mobile phase and stationary phase.

Ultra-High Performance Liquid Chromatography (UHPLC)

It is technology based on the principle that smaller size particles lead to higher efficiency, faster separations with superior resolution, and sensitivity. However, to tolerate extreme pressure from particles smaller than 2 µm, the system needs to be able to handle high backpressure. The efficiency these columns produce should not be lost elsewhere in the instrument’s dwell volume. For these reasons, UHPLC instruments were designed. While UHPLC instruments cost a fortune, there is always a desire to use existing HPLC instrument and achieve UHPLC equivalent performance. In the late 2000’s, Phenomenex released 2.6 µm Kinetex Core-Shell Technology columns, which provides UHPLC performance on a traditional HPLC.

Modes of Separation
There are lots of chromatographic modes of separation and each has its own merits. Provided below is a HPLC column selection tree to guide readers to choose the correct mode of analysis. Although there are many separation modes available to resolve mixtures chromatographically, reversed phase (RP) separation is quite popular and the most common mode of liquid chromatography.

“Why is reversed phase called reversed phase?”

The answer is simple. Chromatography evolved from the use of polar stationary phase and non-polar mobile phase as the major mobile phase component and was considered as normal practice. Hence the name normal phase. While this mode separated analytes based on an analyte’s polar nature, there were a lot of analyte mixtures that were not polar and had hydrophobic characteristic that needed separation. The use of non-polar stationary phase and polar mobile phase helped to separate these hydrophobic analytes. Since this practice is reverse of normal phase, the term reversed phase is used. This is similar to calling a right-handed ping pong player as normal and a left-handed ping pong player as reverse of original.


Reversed Phase Separation Process
Now that we know the most popular mode of liquid chromatography being reversed phase, let us explore how it works. Presented below is a generic schematic representation of the separation process. Mixture of analytes represented by blue, purple, and red dots, are introduced as a band to the column, which contains a non-polar reversed phase stationary phase. The red arrows represent the mobile phase flow direction. As the mixed analytes band is applied to the column, the mobile phase pushes the analytes down the column. As they move down the column, they come into contact with the stationary phase. Analytes that have a higher affinity for the stationary phase (blue dots) will be retained more strongly and elute later in the run. Thus, you can separate the analytes based upon how strongly they interact with the stationary phase.

HPLC Separation Process

In this next example, the stationary phase consists of hydrophobic, non-polar and most often a C18. The mobile phase consists of a polar, hydrophilic, aqueous component, usually water and acetonitrile or methanol. Analytes will be separated based upon 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 strongly bound. Hydrophilic compounds such as ethyl sulfate will have little affinity for the stationary phase and will stay primarily in the mobile phase and be rapidly carried through the column.

Seperation Process HPLC particle view

Although reversed phase separation is contributed by hydrophobic interaction, there are three primary mechanisms of interaction that dictate overall chromatographic behavior. This includes:

  1. Hydrophobic interactions
  2. Polar interactions
  3. Ionic interactions

Aside from these three, stearic selectivity, or shape selectivity, can sometimes play a role. Using the example of tapentadol as a typical small-molecule pharmaceutical compound, let us explore the 3 major mechanisms of interaction. This molecule has polar, hydrophobic, and ionic components.

Hydrophobic Interactions
In RP-HPLC, the primary mechanism dictating retention behavior is hydrophobic interaction between the non-polar stationary phase ligand (e.g. C18) and the hydrophobic nature of the sample molecule (e.g. the carbon backbone). This is a weak, transient interaction between a non-polar stationary phase and the molecules, which includes hydrophobic & van Der Waals interactions. A fair estimate of retention can be predicted based on Log P value, which is the octanol; a water partition coefficient distribution ratio between octanol and water in a liquid- liquid extraction. In other words, the more hydrophobic a molecule is, the larger the Log P value it has, which translates to more retention in RP-HPLC.

hydrophobic interaction in HPLC

Polar Interactions
These are interactions that occur between polar function groups of analytes, residual silanols, embedded polar groups, surface polar groups, or polar end-capping groups in the stationary phase. They interact with the analyte through hydrogen bonding and dipole-dipole interactions. These interactions are relatively weak and transient compared to ion-exchange interaction.

Pola interactions in HPLC

Ion-Exchange interactions
Most RP media is based upon silica bonded with a non-polar stationary phase such as C18. While chromatographic manufacturers like Phenomenex try to achieve complete end-capping of all silanol groups, it cannot reach 100% complete. Resulting in residual surface silanol groups (Si-OH) that are hidden. These silanols can become deprotonated and acquire a negative charge, then can interact ionically 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 occurring (slow versus fast), and this can lead to peak distortion. This is a classic example of basic analytes interacting with residual silanols, which can be controlled by either neutralizing the silanol or by neutralizing the analyte by running them at high pH.

ion exchange in HPLC

For more information on HPLC/UHPLC and the different columns that can be used, please visit our site here.

How do HPLC Columns work
How do HPLC Columns work

HPLC Stands for High Pressure Chromatography. Here is a video that explains the basic concepts of HPLC columns.

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