What is High-Performance Liquid Chromatography?

Analytical chemistry is full of complex techniques. One such technique is high-performance liquid chromatography (HPLC), previously known as high-pressure liquid chromatography. It’s used across numerous fields, including for manufacturing, legal, research, and medical purposes.

Below, we’ll explain everything you need to know about high-performance liquid chromatography.

What is high-performance liquid chromatography?

HPLC is an analytical technique used to separate chemical compounds in a mixture, identifying and quantifying each solute. The technique uses a pump to force a pressurised liquid solvent – containing the dissolved solute – through a column of solid adsorbent material.

(Adsorption is the adhesion of molecules to a surface.)

Because each compound reacts differently to the adsorbent materials, the flow rates are different. This makes it possible to separate the components as they leave the column.

The process was first used to separate coloured compounds in the early twentieth century. Mikhail S. Tswett, a Russian botanist, used high-performance liquid chromatography to separate plant pigments using their interaction with a stationary phase—critical to chromatographic separation.

The stationary phase was often powdered chalk and alumina. Tswett would then pack the mixture into a glass tube and run the plant pigment/solvent through the column. The plant pigments separated into bands of pure components as they passed through the stationary phase.

Today, the process has been significantly refined. Still, modern HPLC owes its origins to this first crude technique. The solid particles are now often made of silica or other polymers, measuring 2–50 m in size.

It’s also differentiated from traditional ("low pressure") liquid pressure by the immense pressures used — 50-350 bar. Liquid chromatography, like Tswett’s technique, relies only on the force of gravity. The additional pressure improves the resolving power of HPLC—making it a popular chromatographic method.

Why is it called high-performance liquid chromatography?

Let’s break the name down.

High performance was formerly high pressure. Though the technique hasn’t changed—it still involves increasing the pressure—the name was altered to better describe the outcome. That’s especially true considering the gradual increase in pressure over time.

Indeed, since the initial breakthroughs in the 1960s, the particle size of the stationary phase has continually declined. However, higher pressures are needed to force the mobile solvent through the column with a smaller particle size.

That means every time the particle size drops, new instruments must be created to handle the higher pressures.

The second part of the name is less self-explanatory. Liquid refers to the mobile phase: the solvent forced through the column. whereas chromatography was derived from the first use of separating coloured pigments (chroma meaning colour and graphy meaning writing).The name just stuck.

What are the advantages of high-performance liquid chromatography?

After over half a century of advancements, HPLC is now a quick and highly accurate technique. But it can be costly, and it does not work for every compound. High-performance liquid chromatography has so many uses that it has many benefits. Here are a few:

1. quickness and precision No other chromatographic technique—such as TLC—is as efficient and rapid as HPLC. Using a pump rather than gravity speeds up the process and ensures a higher resolution. In as little as 10 to 30 minutes, you’re analysing the results of a sample. And, these days, it’s largely automated — so little training is needed to get started.

2. Versatile HPLC is used across a variety of fields. Here are some examples:

-Manufacturing, e.g., production of pharmaceuticals

-Legal, e.g., detecting drugs in a urine sample

-Research, e.g., separating biological compounds from a sample

-Medical, e.g., measuring blood vitamin D levels

3. resolution and sensitivity using the latest innovations, HPLC is extremely accurate at identifying and quantifying chemical compounds. That’s primarily due to a large number of steps, making the technique highly reproducible. Nevertheless, HPLC is better at detecting some compounds than others. Volatile substances, for instance, are better separated via gas chromatography.

What are the disadvantages of high-performance liquid chromatography?

Despite its speed, accuracy, and versatility, HPLC isn’t a perfect technique. It’s critical to know what the downsides are when using the technique, as it can lead to erroneous or inaccurate results if used incorrectly.

Here are the few negatives of HPLC:

1. COELUTIVE The speed of HPLC relies on the different polarities of compounds. Therefore, compounds with similar polarities and structures will exit the column at the same time. This is called coelution, and it makes it difficult to determine which portion of the mixture is eluted and when.

2. Cost. HPLC doesn’t come cheap. Although advancements have speeded up and automated the process, it’s still more complex than more affordable techniques like solid-phase extraction and capillary electrophoresis. That’s because HPLC requires large quantities of costly organics.

3. Adsorbed substancesThe technique works because of the relative binding strength of solutes to the stationary phase. Some compounds barely bind to the beads at all. But if the reverse happens and the compound binds strongly to the beads, it may never be released. If it’s not released, it’s not measured. That can underestimate or ignore certain compounds from a solution.

4. Difficulty. At its most complex, HPLC can even isolate specific proteins from a cellular matrix. To do so, beads are coated with particular antibodies, which require a highly skilled technician to monitor. Otherwise, you can waste expensive equipment on a failed column—it must run exactly as planned.

Types of high-performance liquid chromatography

There are broadly eight different types of HPLC:

1. Chromatography by partition

2. Chromatography in normal phaseSeparates compounds using their affinity for a polar stationary surface.

3. Chromatography of displacementBased on the idea that molecules with a strong attraction to the "chromatography matrix" compete with molecules with a weaker attraction better and push them out of the way.

4. Reverse-phase chromatography. It uses a non-polar stationary phase and an aqueous, moderately polar mobile phase to separate out compounds.

5. Size-exclusion chromatography (SEC). You can use a gel to separate particles based on the size of their molecules. This is called gel permeation or filtration chromatography.

chromatography based on ion exchangeBinding happens when compound ions and charged sites in the stationary phase are attracted to each other.

chromatography based on bioaffinityuses biologically active substances to create stable, specific, and reversible complexes with compounds.

Normal-phase aqueous chromatographyuseful for achieving high selectivity for hydrophilic compounds.

used stationary phase types

Different organisations will often use different equipment for the same technique. That’s also true for HPLC. The stationary phase can change depending on the compound or just based on preference. These are the most common stationary phases used:

1. Sodium silicate gel.Silica, or silicon dioxide, is often used as a dehydrator in food packages. You’ll see it in a small paper bag. However, it also works well in HPLC.silica gel’s pores are on the surface, increasing the surface area available for compounds to bind. The beads used today are mostly spherical and are around 5 m in size.

2. gel polymerIncreasingly, newer gels are becoming popular, including polymer gels. The most common polymers include polyethylene and polypropylene. There are numerous types available:

-Polystyrene

-Polymethacrylate

-Polyhydroxy Methacrylate

-Polyvinyl alcohol

3. Additional gelsAside from the two broad categories, other gels are used on occasion. These include natural substances like cellulose, agarose, chitosan, and dextrin; or ceramics, such as hydroxyapatite and zirconia.

How is high-performance liquid chromatography used in practice?

As mentioned, many industries rely on HPLC for a reliable way to measure compounds in solution. Here are a few of the most common examples of HPLC in practice:

Manufacturing

When producing pharmaceuticals, a reliable technique is needed to obtain the pure compound and ensure no impurities. That’s where HPLC comes in.

Indeed, the technique is so popular that over 15% of all syntheses rely on HPLC in Europe and 44% in the US.

Legal

HPLC is the go-to technique when testing for illicit compounds. It could be athletes being checked for performance-enhancing drugs or a defendant having their urine checked for a recreational substance.

HPLC can detect various illicit compounds, including doping agents, LSD, cannabis, pesticides, benzodiazepines, ketamine, cocaine, opioids, amphetamines, and more.

Research

The sheer number of ways HPLC is used in research is staggering. HPLC is a commonly used technique, from simple tests detecting antifungal drugs to distinguishing species through different compounds.

Medical

Like in the legal field, HPLC can also test for drugs in a person’s system, both illegal and legal. But it can also detect levels of important proteins and biological compounds like vitamin D concentrations. Indeed, HPLC is unparalleled in its accuracy—despite the price tag.

Conclusion

High-performance liquid chromatography, or HPLC, is an expensive but commonly used technique across multiple high-profile fields. It uses a stationary phase to help bind to the compounds in a solution and separate and measure them.