Chromatographic Column Selection in HPLC Method Development (Part 1)

In the development of reversed-phase liquid chromatography (RPLC) methods, the selection of chromatographic columns is generally approached through two methods: one based on practical experience and the other rooted in theoretical knowledge, encompassing both the stationary phase and the chemical properties of the molecules. Today, let’s delve into the process of choosing an appropriate chromatographic column based on theoretical knowledge. First and foremost, it is essential to comprehend the functional groups present in the target molecules and understand how they interact with the stationary phase.

1.The force of common groups in compounds

• Methylene Group: In cases where the methylene chain is CH2 and coexists with other functional groups, the polarity of the additional functional group is generally low or nearly nonpolar. Nonpolar molecules can interact with the stationary phase based on London dispersion forces, and the retention mechanism is primarily determined by London dispersion forces.

• Phenyl Group: The phenyl group exhibits London dispersion forces and, additionally, a minor amount of π-π interactions, contributing to the retention of compounds.

• Halogens: F, Cl, Br, I: Fluorine (F), chlorine (Cl), bromine (Br), and iodine (I) all possess strong electronegativity, rendering these compounds polar in nature and capable of inducing dipole-dipole interactions. If a molecule contains halogen atoms, it can interact with the stationary phase through dipole-dipole interactions. Moreover, when combined with methylene or carbonyl chains, additional London dispersion forces are introduced.

• Ether Group: Ether, with oxygen being a highly electronegative atom, introduces dipole-dipole interactions, along with London dispersion forces due to alkyl groups on both sides of oxygen. Additionally, if the other compound contains hydrogen, the oxygen in the ether group can form hydrogen bonds. Therefore, ethers can be retained on the stationary phase through London dispersion forces, dipole-dipole interactions, and even hydrogen bonding.
• Nitro Group: The nitro group is also a polar functional group; nitrogen is a highly electronegative element, leading to dipole-dipole interactions and hydrogen bonding.
• Ester Group: Ester groups exhibit London dispersion forces, dipole-dipole interactions, and hydrogen bonding.
• Aldehyde Group: Aldehydes possess London dispersion forces, dipole-dipole interactions, and hydrogen bonding.
• Ketone Group: Ketones, due to alkyl chains, involve London dispersion forces, and the high electronegativity of the oxygen atom introduces dipole interactions. Oxygen in the ketone functional group can also form hydrogen bonds.
• Amino Acid: Due to the highly nonpolar nature of the alkyl chain, amino acids can be retained through the mechanism of London dispersion forces at the stationary interface. Additionally, the presence of nitrogen in the amino group introduces dipole-dipole interactions, and this nitrogen can form hydrogen bonds with compounds containing hydrogen atoms.
• Acid-Base Functional Groups: If the stationary phase contains acid-base functional groups, such as hydroxyl groups (R-OH), where R contributes to London dispersion forces, and oxygen is a highly electronegative atom capable of inducing dipole-dipole interactions, it can also form hydrogen bonds with another hydrogen-containing compound. The London dispersion forces in carboxylic acids result from the dipole moments of the alkyl chain, and the dipole moments are influenced by the presence of oxygen atoms within the carboxylic acid functional group, facilitating hydrogen bonding.

2. Case Analysis – 1

Now, let’s delve into a practical example to better understand the separation of prednisone and prednisolone. First, let’s examine the chemical structures of these two target molecules, highlighting distinctive structural features. It is crucial to identify and understand these structural features or different functional groups within the two molecules to discern the differences.

Identifying variances in their chemical makeup will guide us in choosing an appropriate chromatographic column that can effectively separate these compounds. Let’s proceed with a detailed analysis to determine the optimal conditions for successful separation. Stay tuned for further insights into the chromatographic strategies employed in this case!

Upon closer examination, we notice that Prednisolone contains a hydroxyl group, while in the structural formula of Prednisone, a carbonyl group is present at the same position. In the context of the most widely used stationary phases, the first type is alkyl-bonded phases such as C8 and C18, the second type is amino, the third is phenyl pentafluorophenyl (PFP), followed by cyano, and finally, non-bonded pure silica.

Therefore, we will proceed with an analysis of the retention behavior of prednisolone and prednisone using these common stationary phases. This approach will help us determine the optimal chromatographic conditions for effectively separating these two compounds. Stay tuned for the results of our retention analysis!

We are aware that C18 stationary phase is inherently highly nonpolar and hydrophobic, selectively retaining highly nonpolar or nonpolar molecules. Considering that Prednisolone and Prednisone contain numerous π bonds, double bonds, and oxygen – highly electronegative atoms, these compounds are highly polar. Therefore, C8 or C18 would not be the preferred choice.

On both C8 and C18 stationary phases, the retention times for Prednisolone and Prednisone are nearly identical, and retention is weak. This is expected as the likelihood of interaction between these compounds and alkyl-bonded stationary phases is minimal due to their highly polar nature. As a result, C8 or C18 may not be the optimal choice for the separation of Prednisolone and Prednisone. Let’s explore alternative stationary phases to enhance our chromatographic separation. Stay tuned for further analysis!

The phenyl stationary phase has a benzene ring acting as a Lewis base, allowing interaction through π-π interactions or hydrogen bonding. Compared to Prednisolone, Prednisone lacks hydrogen suitable for forming hydrogen bonds. As a result, the phenyl stationary phase is likely to interact more favorably with Prednisolone. This interaction facilitates the separation of the two compounds. However, the phenyl phase is not strongly polar in nature, potentially leading to shorter retention times. Consequently, the phenyl phase may not be the preferred stationary phase.

Now, let’s examine the pentafluorophenyl (PFP) phase. The PFP phase is highly polar due to the presence of five fluorine atoms, making it a strong polar stationary phase. Fluorine, being a highly electronegative atom, exhibits strong dipole-dipole interactions, attracting polar compounds intensely.

Therefore, on the PFP phase, both Prednisolone and Prednisone are likely to achieve good retention. The hydrogen on the hydroxyl group can form hydrogen bonds with the fluorine atoms, facilitating the separation of these two compounds. PFP proves to be a promising choice for this separation due to its strong polar nature and potential for effective hydrogen bonding interactions.
The cyanopropyl (CN) column is a highly polar chromatographic column, and cyanopropyl groups can also form hydrogen bonds. Therefore, based on the different hydrogen-bonding capacities of the two compounds, separation is possible, making it a viable choice.

Finally, silica gel, a highly polar stationary phase, can also be considered. Both compounds are polar, so silica gel could be another option, although a normal-phase mode might be required for separation.

3. Case Analysis – 2

Let’s consider another example. In this case, we have two different compounds, toluene and ethylbenzene, that need to be separated. What distinguishes toluene from ethylbenzene is the alkyl chain. Ethylbenzene has an additional CH2 unit in its alkyl chain, imparting hydrophobic characteristics.

In this scenario, let’s explore which stationary phase is preferable. The primary difference between toluene and ethylbenzene lies in their hydrophobicity, and alkyl-bonded stationary phases are based on hydrophobic retention. Therefore, C8 and C18 can be optimal choices for this separation. The alkyl chain in the amide phase also introduces some hydrophobicity, but it is not the best option compared to C8 and C18. Benzene is nonpolar in nature and may not be suitable, and PFP, being highly nonpolar, is undoubtedly not a suitable choice. The cyano phase is highly polar and may not contribute significantly to the separation of these two compounds.

Now, it is evident that C8 or C18 would be most beneficial for the separation of toluene and ethylbenzene, as they provide the crucial hydrophobic interaction needed for the separation of these two compounds.

4. Case Analysis-3

Now, let’s consider a slightly more complex scenario. This involves a mixture containing six different compounds that need to be separated. We have learned that compounds with different functional groups are easily separable, but when dealing with compounds that have similar functional groups and structures, separation becomes challenging.

Firstly, let’s identify two structurally very similar compounds and determine the differences in their functional groups. Understanding the different interactions of these distinct functional groups can aid in their separation. Now, considering the stationary phases, alkyl-bonded phases like C8 or C18 control the hydrophobicity of molecules, but both compounds in this case are highly hydrophilic and polar in nature, making C8 or C18 less preferable.

The amide stationary phase assists in retaining polar functional groups, with the NH2 in Compound 1 forming hydrogen bonds with the amide phase, resulting in longer retention compared to Compound 2. Therefore, the amide phase is a preferred stationary phase. Benzene also plays a role since Compound 1 contains a benzene ring, while Compound 2 lacks one. Compound 1 can engage in π-π interactions with the phenyl stationary phase, leading to better retention. Additionally, due to its polarity, the benzene ring introduces dipole-dipole interactions, providing some retention for Compound 2 as well. Pentafluorophenyl (PFP) is an interesting stationary phase; the two benzene rings in Compound 1 can form π-π interactions with PFP, and it can also form hydrogen bonds with NH2. Consequently, Compound 1 has a higher affinity for the PFP stationary phase, allowing for excellent separation of both compounds.

Another option is the cyano phase, which is intriguing as it is a strongly polar stationary phase, aligning with the high polarity of our target compounds. The cyano phase induces dipole-dipole interactions and its C-N triple bond can form hydrogen bonds with NH2 in Compound 1. Silica gel is not the preferred choice here as normal-phase chromatography might be necessary.

5. Conclusion

In the development of reversed-phase liquid chromatography methods, a theoretical analysis of how the functional groups of compounds interact with the functional groups on the stationary phase can effectively narrow down the selection of chromatographic columns. By understanding the nature of these functional group interactions, the choice of an ideal chromatographic column can be optimized through experimental exploration. This systematic approach ensures a more targeted and efficient development of chromatographic methods.