What is the Difference Between C8 and C18 Column in HPLC

The main difference between C8 and C18 column in HPLC is that the C8 column contains octyl carbon chain (C8)-bonded silica as the stationary phase, but the C18 column contains octadecyl carbon chain (C18)-bonded silica. Furthermore, the C8 column is less hydrophobic, and it has less density, but the C18 column is more hydrophobic and has a high density. Therefore, the C8 column has a low retention time, while the C18 column has a high retention time. And thus, the separation of the C8 column is low, but the C18 column gives a higher separation. 

In brief, C8 and C18 are two types of straight-chain alkyl groups used for the surface modification of silica in the reverse-phase chromatography of HPLC. The reverse-phase chromatography uses an aqueous, moderately polar mobile phase. 

Key Areas Covered 

1. What is C8 Column in HPLC
     – Definition, Characteristics, Importance
2. What is C18 Column in HPLC
     – Definition, Characteristics, Importance
3. What are the Similarities Between C8 and C18 Column in HPLC
     – Outline of Common Features
4. What is the Difference Between C8 and C18 Column in HPLC
     – Comparison of Key Differences  

Key Terms 

C8 Column, C18 Column, HPLC, Hydrophobic Stationary Phase, Reverse-phase Chromatography

What is C8 Column in HPLC 

C8 column is a type of column used in the reverse-phase chromatography. Generally, the reverse-phase chromatography uses a hydrophobic stationary phase. However, the solid support stationary phase or the column contains modified silica. Here, silica is a hydrophilic stationary phase. Besides, it contains alkyl groups, covalently bonded to silica, creating a hydrophobic silica stationary phase. At the same time, the mobile phase of the reverse-phase chromatography is aqueous. Therefore, this stationary phase has a high affinity to nonpolar compounds in the sample.

Figure 1: Retention Time is Dependent on the Relative Strength of the Analyte Interactions with Stationary and Mobile Phase

Especially, C8 columns contain octyl carbon chains with eight carbons as the alkyl groups for the modification of the silica column. And, these carbon chains are comparatively short. Therefore, this shortness reduces the hydrophobicity and the density of the stationary phase. Hence, the retention time of the nonpolar compounds in the C8 column is low, giving a lesser degree of separation. 

What is C18 Column in HPLC 

C18 column is the cheapest and the commonest forms of columns in the reverse-phase chromatography. Besides, the type of alkyl groups used in the modification is octadecyl carbon chains in the C18 column. In comparison, its carbon chain is longer. Also, the density of the column is higher in the C18 column due to the higher number of carbons in the alkyl group. Accordingly, the hydrophobicity of the C18 column is also high. Thus, it makes the retention time for the nonpolar compounds longer, giving a better separation. Also, it reduces the tailing of the separation curve.

Figure 2: HPLC Column

Moreover, C18 columns are better for the separation of more hydrophobic compounds such as long-chain fatty acids. In contrast, C8 columns are better for the separation of small organic compounds. 

Similarities Between C8 and C18 Column in HPLC  

• C8 and C18 column in HPLC are two types of straight-chain alkyl groups used for the surface modification of silica in the reverse-phase chromatography of HPLC.  
• Generally, silica is the most common type of stationary phase in the normal phase chromatography.  However, it is a polar stationary phase. Still, the above surface modifications make the stationary phase nonpolar to be used in the reverse-phase chromatography.  
• Additionally, the mobile phase of the reverse-phase chromatography is aqueous and moderately polar.  
• The pore size of both columns is similar. Also, both columns are end-capped.
• Besides, the nonpolar stationary phase increases the retention time of nonpolar compounds. 

Difference Between C8 and C18 Column in HPLC 

Definition 

C8 column refers to a type of column used in the reverse-phase chromatography, containing octylsilane as its stationary phase, while the C18 column refers to another type of column used in the reverse-phase chromatography, containing octadecylsilance as the stationary phase. 

Type of Alkyl Groups 

C8 column contains an octyl chain, but the C18 column contains an octadecyl chain. 

Structure 

The structure of the C8 column is OH-Si-C8, while the structure of the C18 column is OH-Si-C18. 

Carbon Length 

C8 column has a short carbon chain, but the C18 column has a long carbon chain. 

Hydrophobicity 

C8 column is less hydrophobic, but in contrast, the C18 column is more hydrophobic. 

Density 

C8 column is less dense, but the C18 column is denser. 

Retention Time 

C8 column has a low retention time, but the C18 column has a high retention time. 

Elusion 

Sample elutes quickly in the C8 column, but the sample elutes slowly in a C18 column. 

Degree of Separation 

C8 column gives a relatively low separation, but the C18 column gives a greater separation. 

Types of Samples 

C8 column separates small organic compounds better, while the C18 column separates long-chain fatty acids well. 

Conclusion 

C8 column is one of the columns used in HPLC in the reverse-phase. Generally, it contains octyl carbon chains, which are shorter. Therefore, its hydrophobicity is comparatively low. Also, this makes low retention time of nonpolar molecules in the column. Hence, C8 columns are better for the separation of small organic compounds. In contrast, the C18 column is the commonest form of columns in reverse-phase HPLC. However, it contains octadecyl carbon chains, which are longer. Hence, the hydrophobicity, as well as the density of the stationary phase, are high. Thereby, the retention time and the degree of separation are high in the C18 column. So, the main difference between the C8 column and C18 column in HPLC is the type of alkyl groups present and the retention time. 

References:

1. “High-Performance Liquid Chromatography.” Wikipedia, Wikimedia Foundation, 1 Feb. 2020, Available Here.

Image Courtesy:

1. “Reverse Phase Gradient Elution Schematic” By Nategm – Own work (CC BY-SA 4.0) via Commons Wikimedia  
2. “Hplc kolom jsw” By Imgupl – Own work (Public Domain) via Commons Wikimedia