Preventing Hydrophobic Collapse: Demystifying AQ Columns

Chromatography enthusiasts are well aware that reverse phase chromatography (RP-HPLC) is one of the most widely used separation modes. It can analyze a majority of compounds ranging from low to medium-high polarity. Commonly used bonded phases include C18, C8, and phenyl. However, reverse phase chromatography has a limitation: highly polar compounds tend to have weak retention in conventional reverse phase settings, making subsequent qualitative and quantitative analysis difficult.

What measures should we take in response to this?

First and foremost, the easiest solution is to increase the proportion of the aqueous phase in the mobile phase to reduce elution strength, thereby increasing the retention of polar compounds. Generally, the higher the polarity of the analyte, the higher the proportion of water required in the mobile phase. However, this leads to a problem: conventional C18 columns do not tolerate high proportions of water or pure aqueous phases well, often resulting in pore drying, commonly known as "hydrophobic collapse." Once this occurs, issues such as reduced retention on the chromatographic column are likely to arise.

Why does hydrophobic collapse occur in pure aqueous phases?

The most widely accepted explanation is the "stationary phase dewetting" theory:

In simple terms, 100% aqueous phase cannot penetrate the hydrophobic pores of the packing material. Silica gel is a fully porous spherical packing material with various tiny pores inside, where hydrophobic C18 long chains are bonded. If the mobile phase consists of less polar solvents like methanol or acetonitrile, it can easily penetrate these pores and wet the packing material (as illustrated below).

Wet pores

But if the mobile phase is pure water, once the flow stops after the experiment, the pressure driving the aqueous mobile phase into the silica pores decreases. Due to the repellent action of the hydrophobic stationary phase surface on water, the water inside the pores is "squeezed" out, causing the pores to dry. During the next experiment, the high surface tension between water and the hydrophobic stationary phase prevents water from entering the pores (as illustrated below). Since almost all the surface area of the silica gel spheres is inside the pores, if the silica surface is not wet, this "hydrophobic collapse" occurs. This means the sample cannot come into contact with the bonded phase, leading to reduced retention performance of the chromatographic column.

Generally, C18 columns with smaller pore sizes (pore size <160Å) are more prone to "hydrophobic collapse" because the C18 long chains spread out within the pores, leaving very little space for pure aqueous phases to pass through, effectively "blocking" the small pores with the C18 long chains. On the other hand, C18 columns with larger pore sizes (pore size ≥160Å) are not susceptible to hydrophobic collapse.

To address the issue of small pore size C18 columns not tolerating pure water, Welch Materials developed the classic Ultisil® AQ-C18 chromatography column. This column uses polar functional groups as end-capping reagents, making it resistant to 100% aqueous phases without experiencing "hydrophobic collapse." It maintains a longer lifespan in high-water-content mobile phases.

Product Features

• Made from >99.999% pure B-type ultra-high-purity fully porous spherical silica gel.
• Moderate surface coverage and complete end-capping provide excellent compatibility with high-water-content mobile phases.
• Carbon load of 12%, pore size of 120Å, and particle sizes of 3μm, 5μm, and 10μm.
• Excellent peak shape, higher plate count, and appropriate column pressure.

Application Case 1

Determination of Four Organic Acids

Chromatographic Conditions

• Column: Ultisil® AQ-C18 (4.6×250mm, 5μm)
• Mobile Phase:
  • A: Phosphate buffer
  • B: Acetonitrile
• Column Temperature: 30°C
• Detection Wavelength: 210 nm
• Flow Rate: 1.0 mL/min
• Injection Volume: 20 μL
• Gradient Elution:

Granules derived from the aqueous extracts of traditional Chinese medicinal herbs contain highly polar components such as nucleosides, organic acids, water-soluble vitamins, and alkaloids. In this case, using an AQ-C18 column can also be considered, starting with a high proportion or even 100% aqueous phase for gradient elution.

Application Case 2

Determination of Banlangen Granules Content

Reference Standard: Chinese Pharmacopoeia 2020 Edition, Part I

Chromatographic Conditions

• Column: Ultisil® AQ-C18 (4.6×250mm, 5μm)
• Mobile Phase:
  • A: Water
  • B: Methanol
• Column Temperature: 30°C
• Detection Wavelength: 254 nm
• Flow Rate: 0.8 mL/min
• Injection Volume: 5 μL
• Gradient Elution:

Friendly Reminder: If you occasionally use a high aqueous phase method or accidentally flush your chromatographic column with pure water, causing "hydrophobic collapse" on a regular C18 column, don't worry too much. This does not mean the column is permanently damaged. You can usually restore the column to normal by flushing it with a high organic phase for a while. For example, flushing it overnight with pure methanol or pure acetonitrile at a flow rate of 0.1 mL/min can restore the column's performance.