Quantifying glucose using HPLC with a C18 column and PDA detector is ineffective due to poor retention and lack of chromophores for UV detection. Instead, ion-exchange or HILIC columns are recommended for better separation, often requiring derivatization for detection.
The C18 column is typically used for non-polar compounds, and glucose, being a polar molecule, might not be ideally separated on a C18 column.
Chromatographic Methods for Glucose Determination
1. High-Performance Anion-Exchange Chromatography (HPAEC): Effective for separating and quantifying sugars, often coupled with Pulsed Amperometric Detection (PAD).
2. Reversed-Phase HPLC: Less common for glucose but can be used with appropriate derivatization.
3. Gas Chromatography (GC):Used for volatile derivatives of glucose, requiring derivatization to make glucose volatile.
4. Liquid Chromatography-Mass Spectrometry (LC-MS):Provides high sensitivity and specificity, useful for detailed analysis and quantification of glucose.
5. Hydrophilic Interaction Liquid Chromatography (HILIC):Suitable for polar compounds like glucose, offering high sensitivity and compatibility with MS.
Detectors used for Glucose
1. Refractive Index (RI) Detection:Commonly used for sugar analysis due to its simplicity and effectiveness in detecting non-volatile compounds like glucose.
2. Pulsed Amperometric Detection (PAD):Highly sensitive and selective for carbohydrates, often used with HPAEC.
3. Evaporative Light Scattering Detection (ELSD):Suitable for detecting non-volatile compounds and less sensitive to changes in mobile phase composition compared to RI detectors.
4. Mass Spectrometry (MS):Provides detailed analysis and high sensitivity, especially when coupled with HPLC or HILIC.
5. UV Detection:Possible if glucose is derivatized to a UV-active compound, though less common.
Each method has its advantages and is chosen based on the specific requirements of the analysis, such as sensitivity, specificity, and the nature of the sample matrix.
Derivatization of glucose to introduce chromophores could improve PDA detection. 1-Phenyl-3-Methyl-5-Pyrazolone (PMP) is a common reagent for derivatizing glucose. It reacts with the reducing ends of glucose, forming stable products that absorb strongly in the UV range (typically around 245-250 nm).
The glucose is reacted with PMP in alkaline conditions (e.g., with sodium hydroxide), and the resulting derivatives are stable for HPLC analysis.
You can use a C18 reverse-phase column after derivatization. Since PMP makes the glucose derivatives more hydrophobic, a C18 column would provide good retention and separation.
A typical mobile phase might be acetonitrile with water or a buffer system (e.g., phosphate buffer) for gradient or isocratic elution.
I hope you are doing well. I have ordered 1-Phenyl-3-Methyl-5-Pyrazolone (PMP) for HPLC analysis, but it may take some time to arrive. You mentioned that GC-MS could be used for sugar detection, and I would appreciate your recommendation on a suitable method.
Thank you for reaching out. I’m glad to hear that you’ve been able to order PMP for HPLC analysis, and I understand that shipping may take some time. In the meantime, using GC-MS is a good alternative, and the columns you have could work effectively for glucose analysis.
Article Measurement of Glucose and Fructose in Clinical Samples Usin...
This research article describes a GC-MS method for the determination of glucose using a Phenomenex Zebron-5® capillary column. This column is quite similar to your HP–5MS UI, as both are 5% phenyl, 95% dimethylpolysiloxane non-polar columns, making them well-suited for general-purpose applications in GC-MS, including sugar analysis.
Please review the method outlined in the article to see if it aligns with your setup. I believe it should provide a solid starting point for your work. Let me know if you have any questions or need further assistance with method optimization.
Hello Mahmoud Elshahawy Sir, I have completed the PMP derivatization of the glucose standard, but I am not getting a linear range in the peak area. Could you please provide the correct protocol for PMP derivatization of glucose? I have tried 3–4 times.
Our detector is a PDA, and the column is C18. The mobile phase I used is phosphate buffer (pH 6.9) and acetonitrile in a 70:30 (v/v) ratio.
Please let me know if this is okay or if I need to use a different method.