Silica is a versatile material with many scientific applications, and serves as the basis for most columns used for reverse phase and normal phase high performance liquid chromatography (HPLC). However, not all silica columns are identical – far from it – and there are many different terms and varieties you will come across during method development and shopping for HPLC stationary phases. This article breaks down some of the main difference between common silica packing types to help lead you toward the right choice for your chromatography experiments.
Reverse Phase vs Normal Phase Columns
Pure silica is naturally very polar and for many years was the main material used for liquid chromatography applications. This is the most common stationary phase used in normal phase HPLC (NP-HPLC), in which polar analytes are retained longer in the column through the use of a polar stationary phase and nonpolar mobile phase. The mobile phase solvents used for normal phase chromatography are often 100% organic solvents like hexane, heptane or chloroform. Today, NP-HPLC is mainly used for the separation of isomers and other analytes that cannot be efficiently separated by reverse phase chromatography.
Reverse phase HPLC (RP-HPLC) uses a nonpolar stationary phase and a polar mobile phase such as water, acetonitrile or methanol. The stationary phase in this case is modified silica – because silica is naturally hydrophilic, alkyl chains are bonded to the silica particles in order to create a hydrophobic stationary phase. RP-HPLC is much more popular than NP-HPLC today for several reasons, one of which being increased reproducibility due to less sensitivity to water. In NP-HPLC, traces of water from the environment can be absorbed onto the silica phase, potentially skewing the results and leading to poor reproducibility. This is not a problem in RP-HPLC due to a hydrophobic stationary phase – the ability to use water or aqueous substances as solvents also accommodates a wider range of compounds than the polar solvents used in NP-HPLC.1
When to Choose Reverse Phase vs. Normal Phase Silica Columns
For most routine analysis purposes, reverse phase HPLC is suitable due to its high reproducibility and compatibility with most common analytes. However, you may run into problems with some analytes such as very hydrophobic compounds that fail to elute. In these cases, normal phase HPLC can be used to resolve problems with analytes that are not soluble or stable in an aqueous mobile phase.
Another situation where normal phase is often preferred is in the separation of isomers. This is because the position and polarity of functional groups in a molecule can make a difference in its retention to the polar silica surface through adsorptive interactions. So while isomers may have a similar polarity and elute at the same time in RP-HPLC, their retention in a normal phase column can differ based on shape.2,3
C8 vs C18 Silica
The alkyl chains of reverse phase HPLC columns can come in different lengths, meaning a different number of carbon atoms can be present in different types of modified silica. In short, C8 silica contains eight carbon atoms while C18 silica contains 18 carbon atoms, hence the naming scheme, but what does this difference mean for your analysis? Firstly, C18 columns are more hydrophobic and will more strongly retain hydrophobic compounds than C8 columns. Longer alkyl chains also provide a longer interaction time between analytes and the stationary phase, which translates to a longer runtime and greater separation between peaks.4
When to Choose C8 vs C18 Silica Columns
C8 and C18 silica are both widely applicable to common analytes and have similar selectivity, so the choice will come down to your specific needs. C18 is the most popular style of reverse phase silica column as it provides adequate retention and separation for a vast range of compounds and may provide better separation of some analytes than C8. When a more rapid analysis time is preferred and stronger retention is not needed, C8 can be suitable; shorter alkyl chains can also be used for very hydrophobic compounds that bind too strongly to the C18 stationary phase.
3 µm vs 5 µm Particle Size
Particle size refers to the diameter of the spherical silica particles that make up the HPLC column packing, with 3 µm and 5 µm diameters being among the most common sizes. Particle size affects separation efficiency and resolution due to the surface area available within a given column. Smaller particle sizes will increase the available surface area, increase the number of theoretical plates and improve separation efficiency and resolution; therefore, 3 µm particle size columns provide higher resolution than 5 µm columns. To be precise, a 3 µm column will typically increase efficiency by about 20-25% and increase resolution by about 9-12% compared to a 5 µm column with all other factors kept the same.5 However, decreasing particle size also increases back pressure , and smaller particle size columns can be prone to clogging.6 Specifically, pressure is inversely proportional to particle diameter (dp) to the power of 2 (dp2). Thus, with all other parameters kept the same, a 3 µm column will result in more than twice as much pressure as a 5 µm column.7
When to Choose 3 µm vs. 5 µm Particle Size Silica Columns
Typically, 5 µm particles are suitable for routine analyses where samples are not particularly complex. Though they provide lower resolution than 3 µm columns, 5 µm also require much lower pressures and can require less maintenance than smaller particle size columns, making them a good choice when superior resolution is not necessary. A smaller particle size column can be chosen either to increase overall resolution for more complex samples or to increase analysis speed by shortening the column length while maintaining the same resolution. Because the number of theoretical plates (N) is roughly equal to L/2dp, where L is the column length, a longer column with larger particles can have the same number of plates as a shorter column with smaller particles. When column length is kept the same, smaller particles increase the number of theoretical plates and provide better resolution than larger particles in the same runtime.
References
- “What are the Main Benefits of Reversed Phase HPLC?,” Article, 2014, Chromatography Today. https://www.chromatographytoday.com/news/hplc-uhplc/31/breaking-news/what-are-the-main-benefits-of-reversed-phase-hplc/30467
- Naushad, M.; Khan, M. R. In Ultra Performance Liquid Chromatography Mass Spectrometry: Evaluation and Applications in food analysis; CRC Press: Boca Raton, 2014; pp 6.
- “Peak Purity,” Blog, SeparationScience. https://blog.sepscience.com/liquidchromatography/hplc-solutions-84-peak-purity
- “What Is C18? A Guide on the Basics of HPLC Columns,” Develosil. https://develosil.us/what-is-c18-a-guide-on-the-basics-of-hplc-columns/
- “Things You Should Know About Your HPLC Column, Part I - Pore Sizes and Particle Diameters,” The LGCC Blog, 2013, LCGC. https://www.chromatographyonline.com/view/lcgc-blog-things-you-should-know-about-your-hplc-column-part-i-pore-sizes-and-particle-diameters
- “Picking the Perfect HPLC Column,” Article by Caitlin Smith, 2014, Biocompare. https://www.biocompare.com/Editorial-Articles/168795-Picking-the-Perfect-HPLC-Column/
- Majors, R. E. Column Pressure Considerations in Analytical HPLC. LCGC North America 2007, 25 (11), 1074–1092. https://www.chromatographyonline.com/view/column-pressure-considerations-analytical-hplc-0