Sugar Analysis using ZIC®-HILIC
EMD Millipore’s SeQuant® ZIC®-HILIC HPLC columns
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.
Click here to find some example applications and links to scientific references within this area of analysis.
Suitable SeQuant® HPLC columns for sugar analysis:
|
1.50439 SeQuant® ZIC®-HILIC 20 x 2.1 mm, 3.5 µm, 100 Å |
|
Analysis of sugars, sugar alcohols and other carbohydrates has been accomplished using several methods such as ion chromatography, reversed-phase chromatography or gas chromatography. However, Hydrophilic interaction liquid chromatography (HILIC) is an attractive alternative because it offers superior separation of polar, hydrophilic compounds like carbohydrates. HILIC is easy to use and works well where traditional reverse phase methodology fails and offers greater flexibility than ion chromatography. The elution order is typically the opposite with the most polar compounds eluting after the non-polar compounds, resulting in an alternative selectivity.
The use of a bonded zwitterionic stationary phase under HILIC conditions enables chromatographers to maximize analyte sensitivity by eluting polar compounds under high percentage organic mobile phase conditions, which is ideally suited for analyte ionization and increased sensitivity. SeQuant® ZIC®-HILIC stationary phases are available with either a silica or a polymer core base particle, 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 a 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 is a key to 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. In the figure you see how easy two of the model compounds, fructose ans sucrose, are separated at any pH using a ZIC®-HILIC column (1.50439). |
|
|
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 potentially elution as isomeric pairs. Changing to a polymer based ZIC®-pHILIC column (1.50462) that has a wider pH tolerance, and using a basic buffer component, ammonium hydroxide, in the mobile phase results in the collapse of the anomers of glucose and lactose, to aid both separation and detection. As seen in the figure , under high pH conditions mutarotation between the isomeric forms of glucose and lactose is fast, resulting in a single, although broadened peak width. All four sugars are easily baseline separated in less than 4 minutes.
|
|
|
Gradient Elution at High pH To sharpen the peak width for all model compounds, gradient elution was explored, but an obstacle with gradient elution is that the column pressure changes with time, as the mobile phase composition alters. To alleviate this, one may increase column temperature and reduce the viscosity contribution of the changing mobile phase composition. At 55 degrees Celsius, 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 ZIC®-pHILIC column (1.50462) were found to optimize baseline separation of all four analytes within 3 minutes.
|
The chemical stability of the 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, simplifying identification of carbohydrates in different types of samples even with difficult matrices. Combined with simple sample preparation procedures like protein precipitation or liquid-liquid extraction, efficient analytical work-schemes can be developed and used for monitoring of sugar, sugar alcohols and other carbohydrates in different types of formulation and matrices.
Click here to find further suggested reading, including application notes, technical application notes, and scientific references.
Related information
| SeQuant® Applications | ||
| Applications by Topic | Glycomics and Glycoproteomics | |
| Ion Analysis | ||
| Melamine | ||
| Peptide Mapping | ||
| Sugar Analysis | ||
| Application Notes | ||
| Scientific Papers | ||
| Technical Notes |
