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Sugar Analysis

Superior Separation with SeQuant® ZIC®-HILIC HPLC Columns

Analysis with SeQuant®

SeQuant® ZIC®-HILIC HPLC columns are suitable for the analysis of sugars, sugar alcohols and other carbohydrates in many different type of samples. By using higher pH and higher temperatures the speed of mutarotation can be increased to avoid peak-splitting for reducing sugars.

Silica and Polymer Core Base Particles

When using a bonded zwitterionic HILIC stationary phase, the elution of polar compounds under high percentage organic mobile phase conditions results in maximum analyte sensitivity and superior ionization. SeQuant® ZIC®-HILIC stationary phases are available with either a silica or a polymer core base particle. The latter offers higher pH stability, thus it is an advantage for carbohydrate analysis, as many analytes of interest are pH dependent. Using a silica core particle, the working pH range is typically 3-8, while polymeric material tolerates both more acidic and basic conditions.

Isocratic Elution at Neutral pH

The use of a basic buffer component, like ammonium hydroxide, in the mobile phase aids separation and enables controlled elution of anomers of reducing sugars. This is illustrated with simple carbohydrates separated under both neutral conditions and at an elevated pH using electrospray ionization mass spectrometry (ESI-MS) detection. The figure below demonstrates how two of the model compounds, fructose and sucrose, are easily separated at any pH using a SeQuant® ZIC®-HILIC column.

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Isocratic Elution at High pH

When separating reducing sugars, such as glucose and lactose, mutarotation at neutral pH occurs slowly, resulting in peak splitting and potential elution as isomeric pairs. In contrast, a polymer based SeQuant® ZIC®-pHILIC column with higher pH tolerance, combined with a basic buffer component, ammonium hydroxide, in the mobile phase results in the collapse of glucose and lactose anomers. This aids both separation and detection. As seen in the figure, high pH conditions allow fast mutarotation between the isomeric forms of glucose and lactose. This results in a single, although broadened, peak width. All four sugars are easily baseline separated in less than 4 minutes.

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Gradient Elution at High pH

Merck:/Freestyle/LE-Lab-Essentials/Chromatography/Diagrams/LE-Gradient elution at high pH -200x159-11252013.jpg

To sharpen the peak width for all model compounds, gradient elution was explored. However, an obstacle with gradient elution is that column pressure changes with time as the mobile phase composition alters. To overcome this, column temperature should be increased, and the viscosity contribution of the changing mobile phase composition should be reduced. At 55°C, the speed of mutarotation was increased, thereby collapsing peak width during separation. As seen in the figure, high temperature combined with high pH conditions during gradient elution using a SeQuant® ZIC®-pHILIC column resulted in optimum baseline separation of all four analytes within 3 minutes.

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Identification of Carbohydrates in Difficult Matrices

The chemical stability of SeQuant® ZIC®-pHILIC columns allows for direct ESI-MS quantitation of simple and complex carbohydrates at high pH and elevated temperatures. Using high pH mobile phases, it is possible to collapse anomers of glucose and lactose. This facilitates identification of carbohydrates in various types of samples, even those with difficult matrices. Combined with simple sample preparation procedures, such as protein precipitation or liquid-liquid extraction, efficient analytical work schemes can be developed to monitor sugars, sugar alcohols and other carbohydrates in different formulations and matrices.

SeQuant® ZIC®-HILIC Columns for Sugar Analysis

SeQuant® ZIC®-HILIC columns
SeQuant® ZIC®-HILIC (3,5µm, 100 Å) 20×2,1 mm
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SeQuant® ZIC®-HILIC (3,5µm, 200 Å) 100×2,1 mm
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SeQuant® ZIC®-HILIC (5µm, 200 Å) 150×4,6 mm
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SeQuant® ZIC®-pHILIC (5µm) 100×2,4 mm
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HPLC Application Collections

HPLC Application Collections

Application Notes and Methods

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HPLC Workflow Solutions - Stationary Phase

HPLC Workflow Solutions

Stationary Phase

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