Preventing Hydrophobic Collapse: Demystifying AQ Columns

Table of contents

Introduction A Common Dilemma: Weak Retention of Polar Compounds Understanding Hydrophobic Collapse in 100% Aqueous Mobile Phases A Reliable Solution: Ultisil® AQ-C18 Application Spotlights: Analysis of High Polarity Compounds Application 1: Determination of Organic Acids Application 2: Determination of Boric Acid in Eye Drops Formulation Final Tip: Recovering from Hydrophobic Collapse

Introduction

Reversed-phase high performance liquid chromatography (RP-HPLC) is one of the most widely used separation techniques in analytical laboratories. With its versatility in handling a broad range of analytes from low to moderately high polarity, RP-HPLC serves as the backbone of routine analysis. Common bonded phases include C18, C8, and phenyl groups.

However, despite its widespread application, reversed-phase chromatography does have limitations—particularly when it comes to highly polar compounds, which often exhibit insufficient retention on standard reversed-phase columns. This poses a challenge for both qualitative and quantitative analyses.

A Common Dilemma: Weak Retention of Polar Compounds

A typical solution to enhance the retention of polar analytes is to increase the aqueous content of the mobile phase. In general, the higher the polarity of the analyte, the greater the proportion of water required in the mobile phase to achieve sufficient retention.

However, this presents a practical issue: traditional C18 columns are often not stable under high aqueous or 100% aqueous conditions. Extended use under such conditions can lead to a phenomenon known as hydrophobic collapse—a condition that compromises column performance by reducing retention.

Understanding Hydrophobic Collapse in 100% Aqueous Mobile Phases

The most widely accepted explanation for hydrophobic collapse is the "stationary phase dewetting" theory.

C18 columns are based on fully porous spherical silica particles. These particles contain internal pores, where hydrophobic C18 alkyl chains are bonded. Under standard reversed-phase conditions using organic solvents like methanol or acetonitrile, the mobile phase readily penetrates these pores, keeping the stationary phase wetted and functional.

Wetted pore

However, when 100% aqueous mobile phases are used, particularly when flow is stopped after a run, the driving pressure that keeps water within the hydrophobic pores diminishes. The hydrophobic nature of the C18 chains repels water, effectively pushing it out of the pores. As a result, the pores dry out.

Once dry, reintroducing water is problematic due to the high surface tension between the hydrophobic surface and water. Since the majority of the silica’s surface area lies within its pores, a dewetting event results in loss of contact between the analytes and the bonded phase—causing decreased retention and poor chromatographic performance.

Dried pore

Hydrophobic collapse is particularly prevalent in C18 columns with small pore sizes (<160 Å), where internal spacing between C18 chains is minimal, leaving little room for water molecules to pass through. In contrast, C18 columns with larger pore sizes (≥160 Å) are generally more tolerant of 100% aqueous conditions.

Small pores vs. large pores

A Reliable Solution: Ultisil® AQ-C18

To address the challenge of pure aqueous compatibility in small-pore C18 columns, Welch Materials developed the Ultisil® AQ-C18—a robust column specifically engineered to withstand 100% aqueous mobile phases without experiencing hydrophobic collapse.

This AQ-type column incorporates polar end-capping groups that improve wettability, enabling sustained retention and extended column life under high aqueous conditions.

Key Features of Ultisil® AQ-C18:

Manufactured using ultra-high purity B-type fully porous spherical silica (purity >99.999%)
Moderate surface coverage with complete end-capping for excellent aqueous compatibility
Carbon load: 12%; pore size: 120 Å; particle sizes: 3 μm, 5 μm, 10 μm
Excellent peak shape, high theoretical plate number, and appropriate column back pressure

Application Spotlights: Analysis of High Polarity Compounds

Application 1: Determination of Organic Acids

Organic acids are widely present in medicinal plants (herbs) and fruits—especially in the leaves, roots, and fruiting bodies of botanicals like Fructus Mume and Schisandra chinensis. They are also commonly used as preservatives and stabilizers in food and beverages. HPLC is the most often used determination and quantification method for organic acids.

Due to their high polarity, organic acids typically require mobile phases with high water content—often up to 100%—for adequate retention when ion-pairing reagents are not used. In such scenarios, the Ultisil® AQ-C18 column delivers optimal performance, ensuring stable retention and reproducible results.

Conditions:

Column: Ultisil ® AQ-C18 (4.6×250 mm, 5 μm)
Mobile Phase: A: Phosphate Buffer, B: Acetonitrile
Column Temperature: 30 ℃
Wavelength: 210 nm
Flow Rate: 1.0 mL/min
Sample Load: 20 µL
Gradient:

Time /min	Phase A /%	Phase B /%
0	100	0
20	90	10
21	100	0
30	100	0

Chromatogram and Data

Application 2: Determination of Boric Acid in Eye Drops Formulation