Reversed-phase columns usually take silica gel, organic/ inorganic hybrid silica gel or polymer as the matrix, while C18 as the bonded phase. Based on the hydrophobic interaction between analytes and alkane chains, typical reversed-phased column is able to separate the components of samples according to the different polarity of the components. This separation mode depends on the free-stretching conformation of the C18 bonded phase in the porous matrix of the column.
For organic acids, alkaline compounds, polyhydroxy compounds, azo compounds and other polar compounds which contain carboxyl groups, amino groups, hydroxyl groups and other dissociable parts, their polarity is too large to be separated by the classical reversed-phase C18 bonded phase. This often manifests as too small capacity factor K, which leads to the rentention time of the compounds is near the dead time.
For these polar compounds, we can reduce the proportion of the initial organic phase or even use the pure aqueous phase as the initial mobile phase to increase the capacity factor when we are developing a gradient analysis method. However, broadening peak or Dewetting in the stationary phase of C18 column may occur. When the pump is stopped and the flow rate is resumed, method reproducibility problems may emerge such as decreased retention time of the compounds or abnormal peak shape (peak bifurcation, peak tailing), etc.
So how can these polar compounds be better retained?
In reversed-phase HPLC, water is generally used as weak elution phase while methanol, acetonitrile and other organic reagents as strong elution phase. The proportion of organic phase can vary in the range of 5%-100% in order to meet the separation requirements of different samples. For polar compounds, even 5% organic phase on a typical C18 column fails to effectively retain them. But further reducing the organic phase proportion (down to zero) will cause Dewetting of the porous inner surface of the column and the stationary bonded phase.
As shown in Fig. 1 A, when containing 5% or more organic phase, the mobile phase can be easily immersed into the porous matrix of the stationary phase. Then wet the inner surface and the C18 alkane chain, in this time, C18 chain is in a free-stretching conformation and appropriate retention can be achieved by well interaction with the hydrophobic part of the sample component. When using 100% aqueous phase, as shown in Fig. 1 B, we can choose the way to increase the front pressure of column, making pure aqueous phase immerse into the porous matrix, but the effective immersion of internal surface and C18 chain can’t be realized. Moreover, due to the phenomenon of pressure drop (Fig. 2), wet state of the whole column varies greatly. When front column pressure is reduced (such as stopping the pump), the pure aqueous phase is “discharged” from the pores due to the strong hydrophobicity inside the porous matrix, thus Dewetting occurs, as shown in Fig. 1 C.
Before and after Dewetting, the stretching conformation of C18 chain in the porous matrix is shown in Fig. 3. Under typical reversed-phase HPLC mobile phase (aqueous phase ≤95%), the C18 chain manifests as the free-stretching conformation in the porous matrix. In the 100% aqueous phase, the C18 chains are “cross-linked” with each other and close to the inner surface to the maximum extent, forming a hydrophobic shielding layer and losing the ability to interact with the hydrophobic part of the component to be separated. Thus, retention time is shorter.
2. The Challenge of Separating Polar Compounds
When the polar compounds are separated by a typical reversed C18 column, the retention time is often too short and the tailing phenomenon occurs. The reasons for too short retention time are as follows. ①Compound itself has excessive polarity ②Compound has excessive polarity in the system after dissociation ③The oil-water distribution coefficient is too small for compounds to interact with the hydrophobic selective groups sufficiently, so compounds flow out of the column directly with the initial mobile phase.
For example, when the pH of the mobile phase is greater than organic acid compounds’ pKa, organic acid compounds dissociates and are negatively charged, having electrostatic interaction with metal ions remaining on the silica matrix. When the pH of the mobile phase is less than organic base compounds’ pKa, the organic alkaline compounds dissociate and are positively charged, having interaction with the weakly acidic silanol group. Hydrogen bond interactions between polyhydroxyl, hydroxy-amine organic compounds, and undissociated organic acids and bases with silanol group. The above three “secondary retention” interactions all result in the tailing of organic polar compounds. In addition, if choose an unsuitable solvent for dissolution of polar compounds, it can also lead to the phenomenon of tailing. Typical polar compounds are shown in Fig. 4.
Therefore, the development of analytical methods for polar compounds on reversed-phase columns is mainly confronted with the following problems:
(1) How to ensure the appropriate retention time of polar compounds?
(2) How to avoid the problem of Dewetting (phase collapse) when using high water proportion or pure aqueous phase?
(3) How to minimize the trailing factor to obtain excellent peak shape?
3. Separating Strategy of Polar Compounds
Using pure aqueous phase elution method to separate of polar compounds may cause Dewetting (phase collapse), but modifying classic reversed-phase C18 bonded phase, using other bonded phase or using Hilic separation mode can reduce or avoid the occurrence of the phenomenon as much as possible. At the same time, polar compounds are effectively retained.
The occurrence and degree of Dewetting in the column are related to the pore size of the packing materials, the bonding density of the bonded phase surface in porous matrix, the length of the alkane chain used, the number of silanol group exposed on the surface of the substrate and the type of end-capping.
3.1 Not-endcapped short alkanes chain bonded stationary phase
There are two main characteristics of this type of reversed-phase column: the silanol group which exposed on the surface of the silica matrix is not end-capped and the length of the stationary phase chain is less than C8. Since the length of the stationary phase alkane chain is much smaller than that of C18, the hydrophobicity in the porous phase becomes smaller. Moreover, no end-capping of the silanol group reduces the contact angle between the pure aqueous phase and the porous matrix. Therefore, compared with typical C18, the possibility of Dewetting is greatly reduced. At the same time, due to the small length of the bonded alkane chain, adsorption and hydrogen bond interaction of silanol group play a dominant role in the separation of sample components for this type of column. For example, Welch Ultisil® XB-C1.
3.2 Polarity end-capping and enhanced polarity type of stationary phase
This type of column adopts polarity or hydrophilic end-capping reagent to end-cap silanol groups which are exposed on the surface. C18 has low bonding density, this modified method increases the wettability and low bonding density also makes the hydrophobicity inside the pore be relatively reduced, thus 100% aqueous phase can be used. For example, Welch Ultisil® ALK-C18.
3.3 Polarity embedded alkyl stationary phase
Polar modified functional groups such as formic acid ester group, urea or amide group and thiamine group are embedded into the long chain alkyl end where nears the inner surface of silica gel. Due to the polarity embedding, the hydrophilicity of the whole bonded phase is enhanced and the hydrophobicity in the porous matrix is reduced. Under the condition of 100% aqueous phase, the alkane chain with the polarity embedded in it still maintains the free-stretching conformation. In addition, the embedded polar groups also have a certain shielding effect on the exposed silanol group, which reduces the trailing factor of polar compounds, especially alkaline compounds, making the peaks more symmetrical. For example, Welch Ultisil® Polar-RP.
4. 100% Water Compatible Column
Welch provides 100% water compatible columns of the reversed-phase including:
When a typical reversed-phase HPLC column uses 100% aqueous phase for separation and analysis of relative polar compounds, the phenomenon of Dewetting (phase collapse) inside porous silica gel is prone to occur, resulting in the decrease of retention time of compounds and problem of method reproducibility. By modifying the surface of silica gel or alkane chain, for instance, increasing the polarity of inner surface, embedding polar functional groups in alkane chain, the ability of porous inner surface and the stationary phase to be infiltrated by water can be increased, thus, the phenomenon of Dewetting can be reduced or avoided. In addition, the retention factors and peak shape of polar compounds can also be adjusted by changing the operating way of HPLC and the conditions.
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