Chromatography is an experimental operation for the separation of substances. One of the most common experimental operations in chromatography is HPLC (high performance liquid chromatography).

High performance liquid chromatography is used in almost every laboratory, including physical chemistry, organic chemistry, and biochemistry. Although the experiments performed in different laboratories vary, HPLC is the analytical technique most likely to be used in any experiment.

So what is the measurement principle of HPLC? High performance liquid chromatography requires an understanding of stationary and mobile phases and detectors. You can then separate the compounds and measure the concentration of the compounds they contain.

HPLC is the most used method of chromatography, and we will explain the principles and concepts of how HPLC is used to measure compounds.

Chromatography Is a Method of Separating Substances

Often multiple substances are mixed together in a solution. It would be nice if only a single compound was present, but often this is not the case.

In a situation where several compounds are present, scientific experiments will not provide correct data. So we have to separate the substances. Chromatography is a technique for separating multiple substances in a scientific experiment.

HPLC is called liquid chromatography. There are two main types of chromatography: gas chromatography and liquid chromatography. HPLC uses a liquid as a solvent to separate substances.

There are several types of liquid chromatography and the simplest operating technique is thin-layer chromatography (TLC), a technique in which a compound is spotted on a thin plate called TLC and the compound is separated as the solvent rises.

Similarly, HPLC separates substances by the movement of liquids.

A Pressure Pump Moves the Material at High Speed to Save Time

Among these liquid chromatography methods, HPLC uses a pressure pump. You can wait patiently for the liquid to move by natural forces, but that would take a lot of time. A pressure pump is used to move the liquid quickly and save time.

The use of a pressure pump to save time is called high performance liquid chromatography. The following are HPLC instruments.

High performance liquid chromatography not only separates substances by applying a certain pressure, but also detects substances.

Reversed-Phase Chromatography Is Commonly Used for Stationary Phase Columns

So what are the principles of HPLC? Only by understanding the principles will we be able to understand how to efficiently separate materials.

In HPLC, the stationary phase must be prepared. The stationary phase is the column, a small cylinder. As the solution passes through the column, it allows the materials to be separated. This is because different compounds move through the column at different speeds.

Why does the stationary phase (column) move at different speeds depending on the compound? This is because the stationary phase has a unique polarity.

There are two types of stationary phase: normal-phase chromatography and reversed-phase chromatography. They are as follows.

  • Normal-phase chromatography: uses substances with high polarity in the stationary phase.
  • Reversed-phase chromatography: uses highly hydrophobic materials in the stationary phase

Depending on which column is used for HPLC, the substances that can be separated are different. In general, however, high-performance liquid chromatography uses reversed-phase chromatography.

Reversed-Phase Chromatography with ODS Columns

In normal-phase chromatography, silica gel is used as the stationary phase. Silica gel has the following structure and is very polar.

The hydroxy group (-OH) of silica gel is combined with an octadecylsilyl group (ODS: C18H37Si), which is called an ODS column because of the binding of the ODS (octadecylsilyl group).

As shown in the structural formula, octadecylsilyl groups are highly hydrophobic. Therefore, ODS columns have a high affinity for highly fat-soluble compounds. Including these ODS columns, reversed-phase chromatography is a type of chromatography that uses a highly hydrophobic stationary phase.

-Highly Polar Compounds Flow Quickly

Water and oil do not mix. This is because they have very different properties. This difference in properties is used in chromatography.

When using an ODS column, the more hydrophobic a compound is, the more it will interact with the stationary phase. Highly hydrophobic materials attract each other, so the more hydrophobic (less polar) a material is, the more it will be adsorbed onto the ODS column.

On the other hand, highly polar and water-soluble compounds are less likely to be absorbed by the stationary phase. They do not interact with the stationary phase and pass quickly through the column. Flushing the solvent will allow the compounds to emerge from the end of the column at an early stage.

Depending on how fat-soluble the compound is, the speed at which it moves through the ODS column will vary. Reversed-phase chromatography is the way to focus on those high polarities and high lipid solubility.

-It Is Difficult to Separate Substances with Large Molecular Weight Such As Proteins

For reference, chromatography is used to separate compounds with relatively small molecular weights. It is difficult to separate substances with large molecular weights, such as proteins.

If you run a protein through a typical ODS column, it will only clog up. The protein will never be detected. Therefore, if you want to measure proteins by HPLC, you must use a special column.

Vary the Retention Time with the Mobile Phase Solvents of Acetonitrile, Methanol and Water

The stationary phase of reversed phase chromatography is largely determined by the ODS column. On the other hand, you are free to decide on the solvent for the mobile phase. Acetonitrile and methanol are most frequently used in the mobile phase of reversed-phase chromatography.

Water is added to these solvents to create the mobile phase.

  • Acetonitrile / Water
  • Methanol / Water

Mix acetonitrile or methanol with water. The ratio of water to organic solvent depends on the nature of the experiment.

The higher the ratio of water, the less soluble the organic material is in the mobile phase. As a result, the speed at which they move through the column will be slower. On the other hand, if the concentration of acetonitrile or methanol is high, the compounds adsorbed on the ODS column will be more soluble in the solvent and will move forward more easily.

The time it takes for a substance to be detected is called the retention time. It is important to understand that depending on what kind of mobile phase you are creating, the retention time for the target compound to be observed by HPLC will vary greatly.

-The Longer the Retention Time, the Higher the Separation Capacity

The retention time can only be determined by making the mobile phase and performing HPLC. The faster the rate of movement and the shorter the retention time, the lower the separation ability. When multiple compounds are in solution, the peaks of the compounds being detected overlap.

In this case, lower the percentage of organic solvent (acetonitrile or methanol) and increase the retention time. Many experiments will take longer, but the compounds will be easier to separate.

For example, if there are no obstacles in a 100m run, the time difference is small. On the other hand, if there are a pond and a maze and a number of obstacles during the 100m, the time difference is large. In the same way, the more obstacles (longer retention time), the greater the separation ability.

Alternatively, you can change the organic solvent you use. In some cases, changing from acetonitrile to methanol will result in a higher separation capacity.

Measuring Compounds with a Detector: Detection with an Absorbance Detector

Another feature of HPLC is the use of a detector. We mix water and organic solvent to make a mobile phase, and the solvent flows through it, and the detector is located at the end where the solvent comes out.

The detector to be used depends on the type of compound you want to measure. In general, however, an absorbance detector is used. Absorption detectors detect compounds that absorb ultraviolet and visible light, and can detect compounds that absorb light in the range of 190 to 900 nm.

When the structural formula has a conjugated structure, it absorbs UV light. The most famous conjugated structure is the benzene ring. Since many organic compounds have a benzene ring, when these substances pass through the HPLC detector, UV light is absorbed and spectral peaks are observed.

For example, the following peaks appear.

Source: Partial modification of the TOSOH database

Although the retention time varies depending on the pump pressure, temperature, and solvent of the mobile phase, the peak is observed as a result of the absorption of UV light.

Looking at the structural formula of the compounds shown in the above figure, we can see that the retention time is shorter for more polar compounds and the peaks are observed more quickly. On the other hand, a compound with higher fat-solubility has a longer retention time and a slower speed of advancing through the ODS column.

When the peak of a compound is observed, only the target compound can be separated by collecting the solvent.

-The Stationary Phase Is Not Disposable and Needs to Be Cleaned

Note that the stationary phase is not disposable, and the column must be cleaned after the experiment is complete. Stopping the HPLC immediately after the target compound peak is detected will result in other impurities remaining in the column.

To prevent contamination, empty the column, for example, by running a 50:50 methanol:water mobile phase for 20 minutes. In this way, organic matter and acids are removed and the column is cleaned.

If the column is clogged by proteins or otherwise contaminated, a new column is better.

After Separation by HPLC, the Amount Contained Can Be Seen from the Calibration Curve

What we have described so far is the principle of HPLC. Because of these properties, the substances contained in it can be separated.

If the conditions of the mobile phase, such as solvent and temperature, are the same, the retention time is the same. Therefore, the retention time is checked in advance by running an HPLC of the reference substance. After that, run a solution containing multiple substances. If a compound is found to have a peak at the same retention time as the reference substance, you know that it is the same substance.

In some cases, unknown compounds may be measured instead of known compounds. In such cases, even if peaks are detected, it is impossible to know which peaks are the target compounds. Therefore, it is necessary to collect compounds for each peak and analyze them in more detail.

However, when examining known compounds, we can predict and separate the peaks of the target compounds simply by comparing retention times.

Also, the higher the amount of compound in the solution, the larger the peaks will be. To be more precise, the area value (integral value) becomes larger.

A calibration curve is drawn to see how many integral values will be detected by running a solution of different concentrations using reference material. By using this calibration curve, the amount of a compound contained can be measured from the observed peak area values of the target compound.

In other words, HPLC allows you to

  • Qualitative: check for the presence of the target compound by comparing retention times
  • Quantification: Measure the amount of a compound from its area value (integral value).

Because of its ability to do these things, and because of its superior ability to separate compounds, HPLC is the most frequently used method of chromatography in scientific experiments.

There Are Many Situations in Which It Is Used to Measure the Purity and Isolate Unknown Substances

HPLC is used in all types of laboratories. This is because there are so many uses for it.

For example, when you want to determine the purity of a compound, high performance liquid chromatography is used. If you dissolve a compound and do HPLC, you will be able to measure the compounds contained in it. The higher the purity, the more only the peaks of the reference material will be observed.

On the other hand, if the purity is low, other peaks are observed in addition to the reference substance. By measuring the peak areas of the other substances, the purity of the compound can be determined.

Other uses include the isolation of unknown substances. For example, after a compound is administered to a mouse, the compound does not remain in its original state in the body, but reacts in the body to change its form, which is called metabolism. By taking a mouse blood sample and running HPLC, we can isolate the metabolized compounds.

Then, by measuring the molecular weight of the compound and analyzing the structural formula, we can investigate what kind of substance it was metabolized into and obtain research data.

These are very different studies, such as the determination of purity or the isolation of an unknown substance. However, the chromatographic instrument used is HPLC, which has become the most frequently used chromatography because of its wide range of applications.

Understand the Benefits of High Performance Liquid Chromatography (HPLC)

HPLC has many advantages among chromatography and is used in many applications. As a result, high-performance liquid chromatography is used in all fields of medical, food and environmental applications.

HPLC can not only qualify a target compound, but also quantify it; HPLC can measure the concentration of a substance and even separate it from the compound.

However, to use HPLC, it is necessary to understand the principles of HPLC before using it. The understanding of the nature of HPLC allows us to determine the solvent for the mobile phase. By drawing a calibration curve, the concentration of a compound can be calculated from the area value. In order to use an instrument, you must learn its features as well as its advantages and disadvantages.

It is important to learn the concepts of HPLC because it is used in a wide range of laboratories. Here, we have described the principles of HPLC in as clear a manner as possible. Once you understand these features, you can then use high-performance liquid chromatography.