When you’re trying to analyze what’s in a certain mixture, high-performance liquid chromatography is one of the best tools at your disposal. This method runs your mixture through a sort of filter to pull out different molecules that may be in your sample. From there you can look at ratios and determine exactly concentration of your target molecules.
But not all HPLC columns work the same way. Some are based off of molecule polarization, while others have to do with size. Read on to learn about the different HPLC and UHPLC columns on the market and what each is used for.
What Are HPLC and UHPLC Columns?
High-performance liquid chromatography is a method for figuring out which substances are in certain mixtures. HPLC columns act somewhat like a filter to pull out each separate component as it passes through. Different layers in the column have different properties that will attract various substances, so by the time your mixture has flowed all the way through, you’ll know what substances were in it and how much of each there was.
Ultra-high-performance liquid chromatography provides even more resolution in their results than traditional HPLC. They have shorter column lengths and smaller sub-2-micron particles in the columns. They use less solvent and produce less waste, but they are more expensive on the front end.
Ion exchange HPLC columns use particles’ ionic charge to separate out the different substances in a mix. The packing in these columns is charged in order to separate out polar molecules. An ion exchange column may be either cationic or anionic, and the molecules are suspended in an aqueous buffer.
Ion exchange HPLC columns are useful in separating out amino acids, carbohydrates, and proteins. Amino acids are zwitterions with both carboxyl and amino groups in their structure, so cation exchange works for them. Carbohydrates can be separated with an anionic column, and proteins will use either cationic or anionic, depending on their net charge.
Ligand exchange HPLC columns target compounds that are able to form labile connections with transition metal cations. The stationary phase is an ion exchanger that carries those transition metal cations. You’ll often see these columns combined with ion exchange HPLC columns; they both contain polarized packing.
This type of HPLC columns can separate monosaccharides, which are smaller units of carbohydrates. They can also separate peptides and amines from amino acids and proteins.
Ligand exchange HPLC columns can separate enantiomers, among other things. This is useful for things such as developing new medications, since you can determine if there will be enough active components to carry out the desired function.
Reversed-phase HPLC columns are the most common in use today. They do not have polarized packing, unlike the ion or ligand exchange columns. The retention times and selectivity of these columns are dependent on a number of other factors, including the pH of the mobile solvent.
The mobile phases for reversed-phase HPLC columns are aqueous and water-miscible organic solvents. The most common solvents you’ll see for these columns are acetonitrile, methanol, and tetrahydrofuran. You’ll see both gradient and isocratic elution used with reversed-phase columns.
Normal-phase HPLC columns do have polar packing, unlike their reversed-phase cousins. These columns use a kind of partition chromatography that uses hydrophilic (water-loving) interaction liquid chromatography (HILC). The mobile phases in HILC contain a low amount of water.
The retention time and polarity of analytes in normal-phase HPLC columns can change with the addition of ionic compounds. These columns use organic solvents since normal-phase HPLC columns do not have polarized packing. They are most commonly used for organic acids, biomolecules, drugs, and other small molecules.
Size exclusion HPLC works much more like a traditional filter than the other column types we’ve discussed. They filter out different compounds based on molecular size. The packing contains both mesopores and micropores that are distributed in different ways to determine the size of the molecules that can diffuse into the pores.
The retention time and elution profile of size exclusion HPLC columns depend on the number of molecules that can diffuse into those pores. Larger molecules move through the column more quickly, eluting as a single peak after the void volume. So these filters are used primarily for larger molecules such as proteins and carbohydrates.
In addition to these larger categories of HPLC columns, there are also some application-specific columns that may have customized packing. For instance, aminoglycoside separation can create very harsh conditions that cause columns to break down and need replacing more quickly. Certain columns are designed to withstand these conditions, keeping your columns in good order for longer (which, as anyone with a research budget will be able to attest, is crucial to successful outcomes).
Some application-specific columns can separate pharmaceutical drug substances and their counter ions simultaneously. Others can separate all classes of surfactants out for you. Still others will separate herbicides like paraquat and diquat simultaneously, and some can perform rapid analysis of polyaromatic hydrocarbons.
Find the Right HPLC Columns for You
Every type of HPLC column functions differently and has variable applications. Knowing which HPLC columns are on the market is crucial when designing you analysis and generate more accurate results.
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Which Hplc column is best suitable for Insect pheromone separation?
Thank you Asif. Could you tell us which type of pheromones you are studying? We will be happy to recommend a column.