The different phases joined for reverse phase separations have different combinations of possible solute-stationary phase interactions. For the bound phase, the variety of separation mechanisms will affect the overall selectivity. This predominance depends on the properties of the analyte and the chromatographic conditions used. The main interactions between the solution and the stationary phases that contribute to the selectivity of the column are listed below.
- hydrophobic interactionit is a dominant retention mechanism for all reversed phase columns and the most significant interaction of the alkyl phases. For a given phase, the retention time is proportional to the hydrophobicity of the molecule.
- hydrogen bonding capacityA phase usually involves the interaction of a basic solute group with an acidic group within the stationary phase, possibly unbound silanol groups.
- p–p interactionsbetween an aromatic or unsaturated solute and an aromatic stationary phase.
- steric selectivityIt is a measure of the accessibility of solutes to the stationary phase. Larger solute molecules can be excluded from the stationary phase.
- Dipole-dipole interactionsbetween a dipolar solute group and a dipolar group in the stationary phase are most important in the case of cyano- and PFP-bonded columns.
- Cation Exchange Interactionscan occur between a cationic solute and an ionized silanol within the stationary phase.
A general summary of these interactions for typical reverse-phase connected phases is given below. Different interactions may be dominant for different analytes, and the strength of the interaction varies between bonded phases from different manufacturers. However, this table provides a useful indication of the possible forces of interaction.
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Hydrophobicity is the main mechanism of interaction of the analyte with C18 and other alkyl-linked stationary phases. In addition, the phase polarity also contributes to the observed overall selectivity.
The strength of the hydrophobic interaction can be measured by the retention of neutral (nonpolar) molecules. The k values (retention factors) for a neutral species for a given C18 phase provide an indication of the surface area and surface coverage (binder density) of the silica.
The percentage of carbon in the material is a simplified but useful indication of the hydrophobic retention properties of a column. In Figure 1, this weak correlation is demonstrated by the observed increase in retention as alkyl chain length (ie, carbon charge) increases. This increase results from an increase in the hydrophobicity of the stationary phase. Likewise, an increase in retention would be expected when going from a low carbon to a high carbon C18 phase.
The hydrophobic selectivity can be determined from the ratio of the retention factor between two neutral species. This is a better measure of surface coverage than carbon content, since surface area and porosity can vary from silica to silica.
The second key property of C18 materials is their silanol activity, which is often discussed in the context of polarity. This can be determined by measuring the retention factor ratio between a basic and an acidic compound. At pH > 7, the total ion exchange capacity corresponds to a measure of the total activity of the silanol. At acidic pH (eg pH 2.7) an indication of the acidic activity of the silanol groups can be obtained. The presence of metal ions in the silica base increases the activity level of the silanol. Earlier generation silicas have higher and less controlled levels of metal ions and are therefore higher
Silanol activity compared to new generation alkyl bond phases. For this
and for other reasons, it is highly recommended to develop new methods
it must be treated with newer generation, higher purity silica.
Deactivated phases with high purity base
Modern alkyl-linked phases have very low cumulative concentrations of metal ions within the base silica (<10 ppm), which means that the number of isolated silanol groups and therefore the polarity of the silica surface is also reduced. the silica. Combined with more efficient and reproducible binding processes, these next-generation reversed-phase materials result in significantly improved chromatography for the most basic polar solutes. The use of pendant alkyl groups containing hydrophilic (ie, incorporated polar) substituents may enhance this effect and/or provide alternative selectivity.
Figure 2 illustrates the relationship between polarity change and hydrophobicity for typical C18, C8 and C4 materials, showing a decrease in hydrophobicity with decreasing alkyl chain length. Higher density of binders and therefore
lower polarity is also observed when the alkyl chain length is reduced. However, changing the alkyl chain length can shorten the analysis time but does not have a significant impact on selectivity. Changing the chemistry to an alternative bonded phase is a more powerful tool to achieve this.
"Traditional" stages of the previous generation
The older "traditional" C18 phases are hydrophobic and have high polarity due to the lower purity silica containing a higher proportion of the acidic silanol groups on which they are based. The use of state-of-the-art high purity silicas reduces the silanol activity of the resulting phases and improves reproducibility. The use of polar embedded functionality can also result in a material with reduced polarity. For basic solutes that strongly interact with surface silanols, phases of lower polarity are generally recommended. However, for certain analyses, the additional interactions provided by the surface silanols of a "traditional" C18 material may be beneficial for the overall separation.
Phenyl-linked silica phases offer alternative reverse phase selectivity to alkyl-linked phases. They show less hydrophobic retention than their C18 counterparts, with retention properties similar to those of C8 bonded phases. Phenyl stationary phases interact with compounds containing aromatic groups or unsaturated bonds through the participation of π-π interactions. For aromatic solutes that contain an electronegative atom or group (eg, F, NO2), the degree of π-π interactions with the phenyl phase increases.
Due to the rigid nature of the phenyl ring, the shape of the solute can also affect selectivity.
Conventional phenyl phases tend to be less stable than the corresponding C8 or C18 reverse phases. In addition, the greater steric volume of the phenyl group reduces the surface coverage, leaving a greater number of silanol sites exposed. The recently introduced phenyl phases show higher stability. The use of a purer silica base, more efficient and reproducible coupling methods, and the availability of a sterically protected phenylsilane all contribute to higher phase solidity and reduced column bleed.
Conventional phenyl phases are attached to the silica through a propyl spacer. Incorporation of the longer chain hexyl spacer results in greater hydrophobic retention and aromatic selectivity.
Polar bonded silica phases offer alternative selectivity for alkyl bonded materials. In general, they have lower hydrophobicity but higher polarity. The bonded cyano, amino, and diol phases can be used in either normal or reverse phase mode. In the normal phase, they equilibrate with the eluent faster than the silica itself and are not deactivated by traces of water.
Cyano-bound phases exhibit unique selectivity for polar compounds and are more suitable than neat silica for normal phase gradient separations. The cyan functional group is a strong dipole that can interact with other dipoles or induce dipoles in solutes. These phases also show moderate hydrophobicity due to the alkyl linker.
Amino acid-bound phases show alternative normal phase selectivity for unbound silica, especially for aromatics. Amino acid columns are also used in HILIC mode for the analysis of carbohydrates and other polar compounds. Its weak anion exchange properties can be used in the analysis of organic acids and anions.
Diol-bonded phases are a versatile alternative to unbonded silica for normal phase separations. The hydroxyl groups provide good selectivity without excessive retention, since the H bonds with the diol layer are weaker than with the silanols. Some diol-linked phases have been specially developed for HILIC applications. Size exclusion separations use materials with different pore sizes.
What C4 C8 and C18 columns are meant for? ›
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.What are the differences between C4 C8 and C18 columns in HPLC? ›
C18 has 18 carbon atoms while C8 has only 8 carbon atoms. C18 has a longer carbon chain, but C8 has a shorter one. C18 has higher retention while C8 has shorter retention. C18 has higher hydrophobicity, but C8 has a lower hydrophobicity.How do I choose the right column for HPLC? ›
The key to good column selection is knowing the dominant characteristics required from the chosen column set which drive the separation of your analyte types, or covering the widest selectivity range possible if screening unknown or unfamiliar analytes.What is the difference between C4 and C8 column? ›
YMC-Pack C4 (Butyl) columns are less hydrophobic than C8 or C18 packings, and generally utilize more aqueous eluents than either of these reversed phase column types. When compared to C8 and C18 columns using the same eluent, C4 columns show significantly shorter retention for non-polar compounds.Is C4 more polar than C18? ›
C4 is slightly more polar than C18 and is typically recommended when purifying either large (>40 amino acids) or very hydrophobic peptides.Which is more polar C18 or C8? ›
C8 is a silica-bonded octadecyl group, and C18 is a silica-bonded octadecyl group. In comparison, the polarity of C18 is smaller than that of C8.Can you run 100% water through C18 column? ›
It is definitely not recommended to run a common C18 column with pure aqueous mobile phase. Because of their length the linked chains have hydrophobic properties and might collapse if used with 100 % water.What is C4 column in HPLC? ›
Reversed phase C4 HPLC column is a liquid chromatography separation mode with the surface nonpolar carrier as a stationary phase and solvent with stronger polarity than the stationary phase as the mobile phase.How do I choose a column size in HPLC? ›
The choice of the length of the column depends on the resolution needed, therefore, on the efficiency: shorter 50, 75 or 100 mm columns ensure fast analyses with a low solvent consumption. Longer columns (150 or 250 mm) are used when higher resolution is needed or when 3 and 5 µm particles are used.Why is C18 column mostly used in HPLC? ›
Because of the extra carbons, C18 has a larger surface area that the mobile phase has to travel across. This offers more interaction time between the bonded phase and the elutes. Thus the sample elutes more slowly and has more separation.
Which column is used in reverse phase HPLC? ›
Reversed-phase HPLC (RP-HPLC) on C18-bonded columns eluted with methanol–water mixtures provides the most widely used system.How do you decide which column position to use? ›
Maintain equal distance between the centres of two columns. If using the minimum size of column 8” x 8” (200mm x 200mm) distance should not more than 3.5 m centre to centre of column distance. If the distance between the two columns is more than 3.5m than you should going for large column.What is the difference between C4 and C18 column? ›
C4 is generally used for proteins and C18 is generally used to capture peptides or small molecules. The idea here is that the larger protein molecule will likely have more hydrophobic moieties to interact with the column and thus a shorter chain length is more appropriate.What is C4 column for protein? ›
The BIOshell™ A400 Protein C4 column is a high-speed, high-performance liquid chromatography column based on a wide-pore (400 Å) Fused-Core® particle design.What is C18 column used for? ›
C18 columns are HPLC (high performance liquid chromatography) columns that use a C18 substance as the stationary phase. C18 HPLC columns are used in environmental sciences and chemical analysis, as well as industries such as pharmaceutical and environmental sciences, to analyze individual parts of chemical mixtures.Which HPLC columns are best for polar compounds? ›
Hypercarb columns can be used successfully for the retention and separation of extremely polar compounds, which are problematic to separate in reversed-phase conditions. This application has demonstrated that: Hypercarb columns offer good retention of polar compounds. Methods developed on Hypercarb columns are robust.Is c4 polar or nonpolar? ›
All the outer atoms are the same - the same dipoles, and that the dipole moments are in the same direction - towards the carbon atom, the overall molecule becomes non-polar. Therefore, methane has non-polar bonds, and is non-polar overall.What type of column would you prefer in HPLC analysis? ›
The reversed-phase HPLC column is the most versatile and commonly used column type and can be used for a wide range of different types of analytes.Is C18 hydrophobic or hydrophilic? ›
The beauty and simplicity of a C18 stationary phase is that it offers a very simple hydrophobic interaction.How do you determine which isomer is more polar? ›
The bulky groups for cis isomers are on the same side of the alkene. This makes the cis isomer less symmetrical and the electron cloud distribution is more unequal. Therefore the cis isomer is more polar than the trans isomer and has a slight permanent dipole - permanent dipole attraction between molecules.
How do you tell which is most polar? ›
The larger the difference in electronegativity between the two atoms, the more polar the bond. To be considered a polar bond, the difference in electronegativity must >0.4 on the Pauling scale.Is a C18 column polar or nonpolar? ›
A C18 column is an example of a "reverse phase" column. Reverse phase columns are often used with more polar solvents such as water, methanol or acetonitrile. The stationary phase is a nonpolar hydrocarbon, whereas the mobile phase is a polar liquid.What happens if you let the column run dry? ›
If you let the column run dry the silica will start to crack and you will get poor separation of your compounds. As you run the column, never let the level of solvent go below the level of the silica gel or you will get poor results.What are the different reverse phase columns? ›
Our reversed phase column portfolio includes options for C18, C8, C4, C30, polar-embedded phases, and aromatic group phases like phenyl, biphenyl, phenyl-hexyl, and PFP. These stationary phases also come in different sorbents such as porous silica, solid core silica, and polymeric resin.Are all C18 columns the same? ›
Although the USP column code (L1) is the same for all C18 phases, there are a lot of differences which must be considered when choosing the right column. C18 always is a good choice for an initial try but one must bear in mind that not all C18 phases have the same separation characteristics.What is Ace C18 column? ›
Chromatography Columns. Avantor® ACE® C18 columns provide great reproducibility and column lifetime. These columns are available in a wide range of particle sizes and dimensions from capillary to preparative. Ultra high purity, base deactivated silica with exceptional reproducibility.How do I select a column size? ›
- Select the column or columns that you want to change.
- On the Home tab, in the Cells group, click Format.
- Under Cell Size, click Column Width.
- In the Column width box, type the value that you want.
- Click OK.
- You need to find the minimum clear cover of the column to find the size of the column.
- If the size of the column is less than 200mm, then the clear cover should be 25mm.
- You will need to add the diameter of the column with the dia of Stirrup.
Size doesn't always matter in science, but it certainly does for chromatography columns. That's because the size of your column will affect how particles move through it, the volume of those particles required, and how reactions occur within the column.What is C8 column in HPLC? ›
C8 column is a form of column present in some HPLC apparatuses, and it has Octylsilane as its stationary phase. And, this compound in stationary phase has 8 carbon atoms in its alkyl chain. Further, it tends to retain components of the analyte less than that of the C18 column.
Are all C18 columns reverse phase? ›
A C18 column is an example of a "reverse phase" column. Reverse phase columns are often used with more polar solvents such as water, methanol or acetonitrile. The stationary phase is a nonpolar hydrocarbon, whereas the mobile phase is a polar liquid.What solvent is compatible with C18 column? ›
mRP-C18 material is compatible with water and all common organic solvents, including N, N-dimethyl-formamide (DMF), and dimethylsulfoxide (DMSO). Maximum protein loading (4.6 × 50 mm mRP-C18 column) is 380 µg. Avoid use of this column below pH 0.9 or above pH 8.0.Why is column choice important in HPLC? ›
“Good sample prep is a key factor in obtaining consistent and reliable results.” Ultimately, matching your best-prepped sample with the most appropriate HPLC column will yield the highest quality results.What is C3 column? ›
C3 Guard columns are expendable small columns designed to remove anything that may interfere with the separation or shorten the lifetime of the primary column.What does conditioning A column do? ›
Conditioning involves establishing a flow of carrier gas through a column and then heating the column to drive off contaminants. This makes the column fit for analytical use. This step is crucial to the optimal performance of any analytical method.What does a protein A column do? ›
Protein A columns are used for the purification of antibodies from complex mixtures such as serum, ascites, and hybridoma culture media. Several protein A column and cartridge formats are available.