C18 vs. C8 HPLC columns: What’s the difference?

February 18, 2025
Reviewed by Our Phenomenex Team

High Performance Liquid Chromatography (HPLC) is a routinely used analytical technique for the identification, separation, detection, and quantification of analytes from samples. HPLC systems commonly use octadecylsilane (C18) and octylsilane (C8) columns. C8 and C18 columns primarily differ in the stationary phase chemistry rather than specific column hardware characteristics.

Both phases consist of alkyl chains chemically bonded to silica particles packed inside the HPLC column. The key difference between C8 vs. C18 HPLC columns is the alkyl chain length. The C8 phase columns contain 8-carbon chains. In contrast, the C18 phase columns contain longer, 18-carbon chains. This structural variation vastly influences stationary phase hydrophobicity, analyte retention, and separation selectivity. Hence, the proper understanding of C8 and C18 column differences is crucial for optimizing analyte retention time, sample resolution, and overall chromatographic performance.

What is a C18 column?

A C18 column is the most widely used stationary phase in reversed-phase HPLC. It creates a hydrophobic surface due to its 18-carbon hydrocarbon chain bonded to silica particles. Analytes interact with the C18 surface mainly through hydrophobic interactions. Non-polar and moderately polar compounds show stronger retention.

Many reversed-phase HPLC separation methods rely on C18 chemistry, including robust options such as Phenomenex’s widely adopted Luna C18(2) column. It has become an industry standard for pharmaceutical, environmental, and food analyses. When comparing a C8 vs. C18 column, the C18 stationary phase offers longer retention times, higher resolving power for complex samples, and highly reproducible analysis across a broad range of pH and solvents.

What is a C8 column?

A C8 column uses an 8-carbon-containing alkyl chain bonded to a silica particle. The C8 stationary phase is less hydrophobic compared to the C18 phases. Its lower hydrophobicity offers fewer potential carbon and hydrogen interactions on the stationary phase, resulting in much shorter retention times than a C18 column.

In a C8 vs. C18 column comparison, C8 stationary phases, such as Phenomenex’s reliable Luna C8(2) column and core-shell Kinetex C8 column, are generally preferred for faster separations and to resolve analytes that bind too strongly to C18 columns. The C8 columns are particularly effective for large biomolecules, peptides, and moderately hydrophobic compounds. For these samples, excessive retention on the C18 column can result in broad peaks or long run times. Overall, the C8 stationary phase columns offer a balance of speed and resolution without sacrificing efficiency of chromatographic separation.

Key differences between C18 and C8 columns

The key difference between C8 and C18 HPLC columns lies in the extent of the hydrophobicity of the stationary phases. C18-containing columns are more nonpolar due to 18-carbon alkyl chains, leading to stronger hydrophobic interactions with analytes. This results in longer retention times for hydrophobic compounds on C18 columns. On the other hand, C8 phase columns provide milder hydrophobic interactions, offering faster elution and shorter overall run times.

Retention time is one of the most noticeable practical differences for C8 vs. C18 HPLC columns. When switching from a C18 to a C8 column, it can significantly reduce retention times for nonpolar analytes under identical mobile phase conditions.

Selectivity and peak behavior also differ between C18 and C8 column phases. C18 columns are often preferred for resolving complex mixtures containing closely related hydrophobic compounds due to their stronger retention and higher resolving power. However, C8 columns frequently provide improved peak shapes for larger molecules such as peptides, proteins, and certain biomolecules. The reduced surface interaction in C8 phases minimizes secondary interactions with residual silanol groups, thereby decreasing peak tailing and improving reproducibility.

Solvent consumption and run time further highlight the C8 C18 column difference. Shorter retention on C8 columns provides the opportunity to work with steeper gradients. This reduces the need for high concentrations of organic solvents. Additional benefits include shortened analysis time and lower solvent costs.

Analyte compatibility plays a major role in column selection. Highly hydrophobic compounds may be strongly and excessively retained on C18 columns, leading to long run times, poor peak shapes, and possible carryover. Whereas, polar or moderately hydrophobic analytes may elute faster on C8 columns, compromising resolution. Understanding these trade-offs enables analysts to select the most suitable stationary phase for their specific analytical objectives.

When to use a C18 column?

A C18 stationary phase is preferred when strong analyte retention and higher resolution are desired. It is highly effective for small to medium-sized unknown hydrophobic compounds such as pharmaceuticals, pesticides, steroids, and lipophilic metabolites. The longer 18-carbon alkyl chains in C18 columns provide a highly nonpolar environment, allowing sufficient interaction time to achieve efficient separation of closely related analytes.

C18 columns are especially suited for analyses requiring method robustness, consistency, and reproducibility. Due to the widespread use of C18 columns across analytical laboratories, related standardized protocols are also readily available. This simplifies method development and method transfer, which is important in regulated environments such as pharmaceutical quality control and regulated testing laboratories.

When comparing C8 and C18 column performances, it is clear that C18 columns offer advantages for gradient elution methods requiring gradual and controlled separation. The increased retention on C18 phases enhances peak capacity and improves resolution in complex sample matrices. This longer retention also supports more accurate quantitation by reducing co-elution and improving signal clarity, making C18 columns a reliable choice for demanding analytical applications.

When to use a C8 column?

C8 columns are advantageous for analyzing large or strongly hydrophobic molecules that may interact too strongly with C18 phases. Excessive retention on C18 columns can lead to broad peaks, long run times, or poor peak symmetry. In such cases, C8 chemistry offers more controlled retention and improved peak shapes. Analysts working with peptides, proteins, macromolecules, or polymeric compounds often observe better performance when comparing the two phases, with C8 providing more consistent and reproducible results.

Overall, C8 columns are a practical choice when speed, peak quality, and method efficiency are more important than maximum retention strength.

FAQs

Can I substitute a C18 column with a C8 column in my method?

No, a C8 column is not a direct replacement for a C18 one. Method development will be required if you would like to go from one phase to another. Since C8 phases are less hydrophobic, analytes typically elute faster, which can lead to reduced retention and altered selectivity. To compensate, adjustments to gradient slope, mobile phase composition, flow rate, or column temperature are usually required. In regulated environments, method revalidation or at least partial validation is strongly recommended before adopting the change for routine analysis.

Which column gives better separation, C18 or C8?

Neither C18 nor C8 columns are universally superior in terms of separation. C18 columns generally provide higher resolution for complex mixtures containing structurally similar and highly hydrophobic compounds due to their stronger retention characteristics. In contrast, C8 columns often deliver faster separations with improved peak symmetry, particularly for moderately hydrophobic or larger molecules. The optimal choice depends on analyte properties, separation goals, and acceptable analysis time.

Are C18 columns always better for complex mixtures?

Not necessarily. Although C18 phases are commonly selected for complex samples, excessive retention can sometimes reduce separation efficiency. Over-retained analytes may exhibit broader peaks, longer run times, or co-elution under gradient conditions. In such cases, the reduced hydrophobicity of C8 columns can improve peak capacity and enhance overall chromatographic performance, especially when strong interactions are undesirable.

What solvents work best with C18 and C8 columns?

Both column types typically use water combined with organic modifiers such as acetonitrile or methanol.

Do I need to adjust my mobile phase when switching columns?

Yes, mobile phase optimization is almost always necessary when switching between C8 and C18 columns in HPLC. Differences in hydrophobicity and selectivity mean that solvent strength, gradient profile, pH, flow rate, or temperature may need fine-tuning. These adjustments help achieve comparable retention, resolution, and reproducibility, ensuring reliable performance after the column change.