In the early 1900s, Russian botanist Mikhail S Tswett began to develop his technique of separation, that he called “chromatography”.
Since those early days, his techniques have been experimented with and built upon, evolving into the modernized version of HPLC we know today.
High-performance liquid chromatography, or HPLC, is the scientific method used in the separation, quantification and the identification of the various components that form a particular mixture. HPLC columns specifically help in the separation of these components. However, it is important to note that, depending on the purpose of use, these HPLC columns come in various types.
So, let’s have a better look at what these types are, and the purpose they serve.
The Rationale Behind Differentiation
So, what is the reason for the differentiation between these columns? Well, chromatography columns are generally differentiated based on two factors. Those are:
- The nature of the composition
- The methods used for separation of components
Now, that the rationale is clear, we can better understand the four major types of HPLC columns there are.
1. The Normal Phase Columns
Normal phase HPLC columns use the power of polarity to help separate components in a mixture. It is based on an analytes ability to engage with polar surfaces. Here, the stationary phase within the column is polar, however, the mobile phase is non-polar. How it works is, the compounds with higher polarity will find their way towards the stationary phase, whereas the non-polar compounds will find their way towards the mobile phase of the column.
Now, when the sample is within the column, the least polar (or the non-polar) compound will be the first to separate from the mixture. The polar compounds will be the last to separate, within the column, making it easier to record and test for the compounds.
The normal-phase columns were one of the first of the HPLC columns to be developed. These kinds of columns are generally used with organic acid testing, drugs, medication, biomolecules and other neutral and ionisable compounds.
2. The Reverse Phase Columns
The reverse-phase column works in ways very similar to the normal phase HPLC columns. However, there is one major difference. In a reverse-phase column, the stationary phase is the non-polar one, and the mobile phase is the one that’s polar.
The time for retention in the stationary phase can be much longer, particularly for molecules that happen to be less polar than the others. In reverse-phase columns, the polar molecules are likely to elute at a much higher rate.
However, keep in mind that the lab assistant or investigator has the power to control the retention time by adding more water to the mobile phase, or adding more of the organic solvent to decrease the time of retention.
To truly understand what kind of column is right for your needs, it might be essential to understand the finer points of linear alkylsilane as a guide for HPLC columns.
3. Ion Exchange Columns
Ion exchange columns use positive or negatively charged ions. Cationic consist of a net charge of positive ions, whereas anionic has a net charge of negative ions. Generally, the stationary phase is either acidic or basic, with either kind of charge.
When the mobile charge enters the column, the compounds begin to separate as their response to the charged ions within the column. Depending on the kinds of compounds they are, their rate of separation may vary.
This ion effect can be manipulated by the lab assist by a variety of ways including increasing temperatures, increasing the concentration of salt within the solution, changing the pH levels of the solvent involved, and so on. These kind of columns are particularly useful in water purification, separation of carbohydrates, proteins, amino acids and more.
This is one of the reasons that ion-exchange columns are extremely useful for those practising within the food or beverage industries.
4. Size Exclusion Columns
The size exclusion column is perhaps, the most unique of the four HPLC columns on this list. That is because these columns do not rely on the interaction of the sample with the stationary or mobile phases for the separation of the compounds. What happens instead is, the sample is run through a filter that is designed to have a varying number of differently sized pores.
These pores come in the form of mesopores (2 – 50 nanometers) and micropores (>2 nanometers). Now as the sample passes through the filter, each compound is likely to pass through a different rate, and separate from the solution at a different rate as well.
Generally, the larger compounds are seen to avoid the holes altogether, allowing for quicker and smoother movement than their smaller counterparts. In a nutshell, size exclusion columns separate compounds based on the size of their molecules, and in some cases their weight.
Which of These HPLC Columns Are Right for You?
Now that you’re familiar with the major types of HPLC columns there are, it is important to know which of these are right for your needs. To make that decision you should have a decent understanding of the kind of compounds you are dealing with and their reactions to the various separation methods in question. Be sure to do a lot of research into your work, and talk to fellow professionals within your field to find out what’s best for you.
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