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Understanding HPLC: 7 Things You Need to Know

Have you ever wondered just how high-performance liquid chromatography works?

HPLC plays a key part in scientific fields from the food industry to pharmaceuticals. That’s great, but if you’re reading this you’re wondering just how it works!

Don’t worry! Read on to know the seven basics to understand how HPLC works.

1. What is HPLC?

HPLC or high-performance liquid chromatography is a technique of identifying different compounds.

HPLC will purify and separate compounds based on how well they mix with water. This is known as its polarity. An apolar liquid like oil doesn’t dissolve in water. Something like ethanol, though, mixes with water as all you spirit lovers will know!

2. What Components Do You Need?

There are a few different components that you will need to get a correct reading. Here they are:

The Column

Also called the stationary phase, it’s the engine of the HPLC process. You can make the column from a few different substances but most often it’s silica. This is because of its compact nature.

Long carbon particles cause the silica to functionalize. These carbon chains are apolar, and the longer the chain the more apolar the column becomes. It’s common to use a C18 column, named for its use of 18-carbon chains.

The Sample

Samples will vary as it depends on the field and what the compound is. You can analyze compounds in:

  • Chemical samples: drugs
  • Biological samples: urine, saliva, muscle, blood
  • Microbiological samples: toxins in bacteria and fungi

Injection of Sample

You then inject the sample into the HPLC column. Once upon a time, this was a manual process. Yep, you guessed it! Some poor intern had to sit there manually injecting each sample with a syringe for hours!

Luckily for all you interns, the newer machine models have an automatic injector. So no more sleepless nights! It also allows for the processing of a higher number of samples.

These machines have software that let you input a list of samples and set their order. They will then inject the samples in the order you set with the amounts you set too.

The Mobile Phase (Solvent)

This is a mix of solvent with water. Usually, it’s methanol or acetonitrile. It’s called the mobile phase because it moves through the column. At the time, it flushes out the compounds inside.

The compounds often get flushed out in a concentration gradient. In other words, the amount of water in the mobile phase is reduced over time, but the amount of solvent increases. As such, the mobile phase gets more and more apolar.

The HPLC Run

You can perform HPLC in a variety of modes. The most common method though is the reversed-phase or RP-HPLC. This phase separates compounds starting with the most polar. It ends with the most apolar compounds.

Whatever method you use, a high-powered pump will move the sample and the mobile phase along the column. This can take anything between 10-60 minutes.

3. Pay Attention to Buffers

HPLC will rely on a differential equilibria of the substance you are analyzing, the column, and the solvent. One common solvent is 50% acetonitrile and 50% of 10 mM potassium set to a pH level of 2.5.

To mix this, you want to mix phosphoric acid and potassium dihydrogen phosphate until you get to pH 2.5. Then mix this with an equal amount of acetonitrile.

Always set standard procedures that state buffer formulas and expiry dates. For more tips on how to improve your separations, check out this article.

4. A Close Look at the Principle Behind HPLC

Now you know the basics involved, here is a little more detail. As we’ve seen, HPLC separates compounds based on their polarity. But how does this work?

As the concentration gradient sets in, the solvent concentration level increase. At the same time, the water concentration decreases. The mobile phase will become more apolar as this goes on.

Compounds within the sample will then stick to the column’s carbon chains. The most apolar ones will stick the most while the more polar compounds will have a weak link.

Depending on whether they are more likely to stick to the column, the compounds will flush out during the mobile phase at different times. You call the time each compound flushes out the compound’s retention time (Rf).

5. How Do You Understand the Output

You will usually see the results of an HPLC run in the form of a chromatogram. This shows horizontal peaks which represent the compounds flushed out of the column and their Rf. Most modern HPLC machines come with a diode array detector (DAD).

This will let you look at the chromatogram in wavelengths from 190nm to 900nm. If you know what compounds were flushed out, you can single out 1 or a few wavelengths. For example, cocaine shows up at 254nm.

6. HPLC Can Show You What Components Make Up a Compound

Compounds within a sample will stick to the nonpolar stationary phase. Or they’ll come out during the mobile phase when their polarity matches. The detector measures each compound which means you can use the chromatogram to identify each compound.

7. What Are the Applications of HPLC?

You can use HPLC in medicine and biology to analyze biological and environmental samples. It’s used to look for the presence or absence of known compounds such as:

  • toxins
  • metabolites
  • pesticides
  • drugs

It can also help identify any unknown compounds found within samples.
In chemistry, it’s often used to track chemical reactions and check the purity of various compounds. You can also use it as preparative HPLC which will purify specific compounds so you can use them further.

As it can analyze drugs, biological material, and food, it’s common in scientific fields. Even more so in the pharmaceutical field. There, HPLC trained employees are worth their weight in gold!

Understanding HPLC is as Easy as 1,2,3…

So there you have it, HPLC isn’t as scary as it sounds. It’s actually a fascinating technique used in a wide variety of scientific fields. From the food industry to big pharma, it’s ability to analyze drugs, biological material, and food means technicians are in high demand.

If you found this article interesting, check out our other blog posts. At we have tips and information for all your industry needs.

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