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What Is an HPLC Column and When Would You Need One?

    In 2018, there were over 331,000 clinical laboratory technologist and technician jobs available in the United States. The pay is very decent with an average of $25.16 per hour, which can make it an attractive career to switch to.

    But before you decide to drop this job for a completely different one, perhaps you should get a taste for the world of science. For some, it may be a highly fascinating field, while for others, it’ll bore them to tears.

    In this article, we’ll discuss what an HPLC column is and when you would need one.

    What Is an HPLC Column?

    “HPLC” stands for “high-performance liquid chromatography.” Basically, it is a lab technique used to separate, quantify, and identify everything that makes up a mixture.

    If you’ve taken biology in high school, then you may have done a lab technique called gel electrophoresis. With this, you made a gel plate and used two electrodes (an anode and cathode) to get an electrical charge and separate macromolecules in the sample you’ve loaded into the gel.

    With the result, you can then get a DNA fingerprint to compare and use in a variety of scenarios. For instance, you can determine the paternity of a baby or link a piece of bloody evidence to a perpetrator.

    An HPLC column works in a similar fashion, but instead of separating macromolecules, you’re separating components in a mixture.

    Types of HPLC Columns

    An HPLC column works by way of gravity. You drip a solvent through the column, which is filled with an adsorbent. It has a pump that forces the solvent through. By using high pressure, HPLC is a lot more efficient than regular column chromatography.

    In the HPLC, there are two main types of materials: stationary (the packing) and mobile. You’ll typically find silica gel in these columns since its characteristics facilitate the separation of components. Plus, it’s inert, which means it won’t react with materials in the mobile phase.

    Now that you know the basics of how an HPLC column works, we can move onto the types of HPLC columns used.

    Normal Phase

    In a normal phase HPLC column, you’ll find more polar stationary phase than the mobile phase. These HPLC columns are filled with silica gel and the solvent used will be non-polar; popularly used ones include hexane and methylene chloride.

    When you pass a mixture through, the non-polar compounds will be separated out quicker than the polar compounds. This is because the polar compounds will stick to the silica gel, which is polar as well.

    A normal phase HPLC column usually has a diameter of 4.6 mm or smaller. The length will be anywhere from 150 to 250 mm. This type of HPLC column is usually used for biomolecules, some drugs, and organic acids.

    Reverse Phase

    This is the most commonly used type of HPLC. As you’d expect, it’s the opposite of a normal phase HPLC column. Instead of polar packing, it has nonpolar packing. Because of this, it has less polar stationary phase than the mobile phase.

    In the stationary phase, you’ll find bonded and non-polar hydrocarbons. In the mobile phase, you’ll find aqueous organic solutions, such as water-acetonitrile or water-methanol mixtures.

    Silica gel particles are still used to pack the column, but they’re modified so they’re non-polar instead of polar. To make it a reverse phase HPLC column, you’d use a polar solvent. This means non-polar molecules will stick to the silica gel, while polar molecules are filtered out quicker.

    Ion Exchange

    In an ion exchange HPLC column, the packing in it is charged. This means it can either be cationic or anionic. Because this type of HPLC column depends on ion exchanges, this means instead of being separated by online casino go sizes, components of a mixture are separated by their charge.

    This type of HPLC column uses an aqueous buffer for its mobile phase. Do note that you can use this in conjunction with ligand exchange chromatography.

    You’d typically use an ion exchange HPLC column to analyze amino acids, carbohydrates, and proteins. If combined with ligand exchange chromatography, you can use it for analyzation of monosaccharides.

    Size Exclusion

    Size exclusion HPLC columns are a bit unique. While a normal HPLC column would separate compounds based on interactions with the column packing, this type does so by using a sieving effect, which happens through the difference in molecular weights.

    Because of this, instead of silica gel, you’d use mesopores and micropores for the packing. When the mixture passes through these pores, they’ll separate at different rates because they’re sized differently.

    Size exclusion HPLC columns are usually used for analyzing carbohydrates and proteins.

    When Would You Need an HPLC Column?

    In the above sections, we’ve listed what each type of HPLC column would typically be used for.

    But in general, HPLC columns are used for pharmaceutical compounds. This allows the user to determine what exactly is in the compound, as the individual components will be separated based on its particle size.

    However, you can use an HPLC column in a number of scenarios, especially if you need to separate, quantify, and/or identify anything in a chemical compound. For instance, HPLC columns are used in forensic labs to determine what’s in an unknown substance and to see if it matches up to any other evidence they have.

    Is a Lab Job Right for You?

    Now you know what an HPLC column is and what it’s used for. Do you still have the same viewpoint toward a lab job?

    After reading this article, you certainly know more about what the scientific field entails. Hopefully, this should give you a good idea of whether or not you want to pursue a career as a clinical laboratory technologist or technician.

    Want to learn more about science? Then check out our other blog articles for more interesting facts.