It is important to investigate what compounds are present in the solution by performing a structural analysis of chemicals. Structural analysis of these compounds allows us to investigate the molecules and proteins that are present.

HPLC (high performance liquid chromatography) and MS (mass spectrometry) are frequently used to determine what substances are present in these solutions. A combination of these methods is widely used.

LC/MS, MS/MS, and LC/MS/MS are examples of these methods. To understand the difference between these analytical methods, let’s understand that they are a combination of HPLC and MS.

So, what is the principle behind the use of these methods to examine substances in solution? We will discuss the differences and principles of each method of analysis in this article.

HPLC and MS (Mass Spectrometry) Frequently Used in Analytical Instruments

HPLC and MS are two instruments that are used in a large number of laboratories. Including organic chemistry and biochemistry, it is difficult to find a laboratory that does not use these instruments. HPLC and MS are used in almost every field, including medicine, food, and the environment.

HPLC is a type of chromatography. When multiple substances are mixed together, it is difficult to analyze them in their original state. If a compound is not a single compound, the data will be affected by other compounds and will contain a number of noises, making it impossible to analyze the data correctly.

Chromatography is the technique used to separate multiple compounds from a mixture of substances into a single compound. HPLC is the most widely used method of chromatography.

After separating a substance by chromatography, it is necessary to determine the nature of the compound. MS is used to determine the molecular weight, which allows us to investigate the weight of the target compound. The combination of HPLC and MS is called LC/MS.

High Performance Liquid Chromatography (HPLC) Is the Most Common Type of Chromatography

Why do HPLC separate substances? The reason for this is that it utilizes a compound-adsorbing substance as a stationary phase.

High performance liquid chromatography is also known as reversed-phase chromatography. Reversed-phase chromatography utilizes a highly hydrophobic material as the stationary phase. This is called an ODS column. Think of an ODS column as being packed with highly hydrophobic materials.

Water and oil do not mix. However, substances with the same properties will strongly attract each other. For example, a compound with high fat-solubility will strongly adsorb to the ODS column, which is also highly hydrophobic. On the other hand, highly polar compounds are less likely to be adsorbed on an ODS column and will pass quickly with the liquid (mobile phase).

The more hydrophobic a compound is, the more it will move forward gradually as it is adsorbed by the ODS column. In other words, understand that different compounds move through the ODS column (stationary phase) at different speeds.

In HPLC, the organic solvent (mobile phase) will continue to flow. As a result of the compounds moving at different speeds, we can separate the materials as follows

Source: Partial modification of the TOSOH database

High performance liquid chromatography has these characteristics.

Determine the Molecular Weight of the Compound by Mass Spectrometry (MS)

An entirely different analytical method than HPLC is mass spectrometry; think of MS as measuring the molecular weight of a target compound. MS is a device that analyzes the mass of a molecule after it has been ionized.

In mass spectrometry, positive ions are created. When a magnetic field is applied to the ionized material, it curves. Different molecular weights have different degrees of curves.

So we change the strength of the magnetic field a little at a time. The magnetic field is changed one by one, and when the detector is able to measure the molecule, a peak will appear. This is called a mass spectrum.

For example, when acetic acid is measured by the mass spectrometry, the following mass spectrum is obtained.

In the mass spectrum, the molecular weight peak of acetic acid can be observed. However, because MS bombards the molecule with high-energy ions, it produces radical cations.

Radical cations are highly reactive and can cause the bonds in the molecule to cleave. As a result, not only the molecular weight of acetic acid, but also the molecules after the cleavage of the acetate structure can be observed in the mass spectrum. These cleaved structures are called fragment ions.

LC/MS Combined with HPLC and MS

HPLC and MS are completely different in nature, but LC/MS (liquid chromatography mass spectrometry) is a combination of both. Let’s understand that LC/MS is an instrument that can perform both HPLC and MS at the same time.

When MS analysis is performed, substances must be separated by HPLC or other chromatography prior to the analysis. Even if the molecular weight is measured when multiple substances are mixed together, it is not clear whether the peaks that appear are the target compounds or not.

However, after separating the substances by HPLC, it is time-consuming to analyze all substances by mass. In order to save time, the molecular weight is measured by MS immediately after the separation of the compounds by HPLC. As a researcher, it is very convenient to perform both separations by HPLC and molecular weight measurement at the same time.

-Single Molecule Peaks Are Observed in LC/MS Mass Spectrometry

Earlier, we discussed mass spectra using common mass spectrometry methods. Electron ionization (EI) is frequently used in mass spectrometry. By bombarding a compound with high-energy electrons, it causes it to be ionized. This causes multiple molecules, including fragment ions, to be measured.

However, there are a number of ways to use MS. For mass spectrometers used in LC/MS, think of it as observing only the molecular weight of the target compound, without producing fragment ions.

For example, acetic acid, with a molecular weight of 60, will only show a peak at 60m/z (molecular weight 60) when measured by LC/MS. LC/MS does not break molecular bonds by creating radical cations. This means that multiple fragment ions cannot be produced.

While there are various methods of MS, the mass spectrometry used in LC/MS measure molecular weight in a way that does not produce fragment ions.

MS/MS Measures the Molecular Weight and Fragment Ions of a Compound

However, LC/MS/MS is also frequently used in analytical instruments; LC/MS is a combination of HPLC and MS, as I explained earlier. In contrast, what is LC/MS/MS?

In order to understand the concepts and characteristics of LC/MS/MS, you must learn about MS/MS first.

In the most common mass spectrometry, multiple peaks are observed in the mass spectrum. This is because the creation of radical cations causes bonds in the molecule to cleave and produce multiple fragment ions. However, we don’t know which peak is the molecular weight of the original compound.

In the mass spectra I mentioned earlier, I gave the example of acetic acid. Because we measure a known compound called acetic acid, we already know its molecular weight.

However, what happens when we perform MS without knowing that the substance we are measuring is acetic acid? In this case, it is not possible to distinguish which of the peaks in the mass spectrum is the molecular weight of the original compound and which is a fragment ion peak.

To solve this problem, a technique called MS/MS is used. In MS/MS, think of it as literally two mass spectrometers attached together.

In the first MS, only the molecular weight of the target compound is measured. When measuring a single compound, only one peak is observed.

However, knowing the molecular weight alone is not enough to determine the structure of an unknown compound. Therefore, fragment ions are created by applying high energy to the molecule. By cleaving within a molecule, multiple molecules are generated.

In a collision cell, the compound is bombarded by an inert gas. The resulting collision energy causes the molecules to dissociate into ions. In the second MS, we observe the fragment ions that are produced.

In materials with unpaired electrons, such as oxygen and nitrogen, the collisional energy tends to break the bonds and produce fragment ions. Once the molecular weight of the fragment ion is known, it is possible to estimate how the bonds within the molecule are formed.

MS/MS is a technique that measures the molecular weight of the original compound and then observes what kind of fragment ions are produced by the second MS.

Perform All Analyses Simultaneously with LC/MS/MS

Once you understand what we’ve discussed, it’s easy to understand what an LC/MS/MS instrument is: LC/MS/MS is a combination of HPLC and MS/MS.

Analytical instruments are useless when multiple compounds are mixed together. The compounds are then separated by HPLC.

The compounds are then measured with the first MS at the HPLC exit. Mass spectrometry is used to determine the molecular weight of the compound. We then cleave the bonds between the molecules in the collision cell and look at the fragment ions with a second MS.

LC/MS/MS can do all of these things at the same time. LC/MS/MS is capable of separating a compound, measuring its molecular weight, and observing its fragment ions at the same time.

Proteins Can Be Identified After Isolating to Peptides

When doing LC/MC/MS, the principles and concepts we have discussed so far are the principles and concepts. For small organic compounds, including the analysis of food and environmental substances, the constituents they contain can be examined.

On the other hand, there are further things to consider, especially in the study of biochemistry. This is because these scientific experiments frequently involve the use of proteins.

If you run a protein through HPLC without treating it in any way, you won’t be able to measure it. Proteins are macromolecules that can clog the column.

In LC/MC/MS, proteins are broken down into small pieces. Proteases are known to be enzymes that break down proteins. Proteases usually utilize trypsin. By adding trypsin, it cleaves the proteins of the macromolecules.

When the protein is cleaved, it becomes a peptide. Peptides are made up of several amino acids that are linked together. Peptides can be passed through an HPLC column. The resulting peptides are then analyzed by LC/MC/MS.

By analyzing the peptides cleaved by the protease (trypsin) by LC/MC/MS, we can obtain a number of MS/MS data. We can then analyze the MS/MS data to see what peptides were detected.

Different proteins have different locations where they are cleaved by proteases. In other words, the peptides produced are different. So by comparing the observed data with the known data (protein database), we can identify which proteins are present.

It doesn’t matter how many proteins are in the mix; HPLC will separate each peptide from the other, and then MS/MS will identify the proteins that are present.

However, it is not possible to identify all the proteins in the solution. It is also not possible to study minor components that are not present in the database. While the advantages of LC/MC/MS are great, it is important to understand that LC/MC/MS has these disadvantages

The study of proteins for structural analysis is called proteomics (proteomic analysis). In proteomics (proteomic analysis), the use of LC/MC/MS is indispensable.

Important LC/MS/MS Techniques for Chemical Analysis

In scientific experiments, we may do chromatography or mass spectrometry. However, it takes a lot of time to perform each method one by one. It would be more efficient if we could obtain multiple data from a single experiment.

LC/MS is a combination of HPLC and MS. Not only that, but LC/MS/MS is also being used to observe fragment ions. LC/MS/MS is much more sophisticated than LC/MS.

In many cases, multiple substances are mixed together and cannot be chemically analyzed as-is. HPLC is used to separate compounds, and MS/MS is used to confirm the details of the compounds.

These methods enable us to detect unknown compounds. As proteomics, we can also be used to identify proteins. LC/MS and LC/MS/MS are frequently used in any field.