Which column do I need and the temperature,flow rate ,which detector to be used for good results .
Hello vishal,
As per my knowledge, to detect different molecular weight PEGs in a binary mixture using HPLC Agilent 1260 infinity, you will need to choose a suitable column, optimize the temperature and flow rate, and use an appropriate detector. Here are some guidelines to help you get started:
Column selection: The choice of column will depend on the characteristics of the PEGs you are trying to separate. You may want to consider using a size-exclusion chromatography (SEC) column, which separates molecules based on their size, or a reversed-phase column, which separates molecules based on their hydrophobicity. Some popular columns for PEG separation include Agilent Bio SEC-3 (for size exclusion), Zorbax Eclipse Plus (for reversed-phase), and Agilent Bio HILIC (for hydrophilic interaction chromatography).
Temperature and flow rate: The temperature and flow rate will also depend on the column you choose. For example, with a size-exclusion column, you may want to use a higher flow rate and lower temperature, whereas with a reversed-phase column, you may want to use a lower flow rate and higher temperature. You may need to experiment with different conditions to optimize the separation of the PEGs.
Detector selection: To detect PEGs, you may want to use a refractive index detector (RID) or a UV detector. A RID can detect all PEG species, regardless of their UV absorbance, while a UV detector can only detect PEGs that absorb at the specific wavelength used for detection. For example, if you use a UV detector at 214 nm, you will only be able to detect PEGs with a certain number of repeating units. You may also want to use a multi-angle light scattering detector (MALS) in combination with a RID or UV detector to obtain more accurate molecular weight information.
Method development: Once you have chosen your column, temperature, flow rate, and detector, you will need to develop a suitable method for your separation. This will involve optimizing the mobile phase composition, column equilibration time, and injection volume. You may also want to use standards with known molecular weights to calibrate your method and confirm the accuracy of your results.
Overall, the choice of column, temperature, flow rate, and detector will depend on the specific PEGs you are trying to separate and detect. It is recommended to consult with experts in the field and consult the user manual for the HPLC system and column used to ensure proper operation and accurate results.
I hope this clear up your doubts,
Good luck with your research,
Best wishes & regards,
Vishwajit
Hello Vishal,
I agree with Vishwajit.
I believe the simplest method would be size exclusion chromatography with RI detection.
The chromatography conditions here are limited to flow rate and temperature. The separation is purely isocratic and therefore needs little optimization.
The RI detector is also not critical when working isocratically. It detects the PEGs in all cases. A UV detector, on the other hand, needs chromophoric molecule components such as double bonds, which are unfortunately not present in common PEGs. Since I do not know in which concentration range they have to work, the UV detector can only be a useful tool in very high concentrations.
The choice of column depends mainly on the molecular weight range of the PEGs. Standard size exclusion columns range from ~ 2000u up to 100000u, but these columns are quite expensive.
On RP-phases you can easily separate PEGs by EO chain length by chromatography with high water fractions, e.g. water/ ACN 90:10, but in my experience quantifications are very difficult under these conditions, because PEGs can form clusters and adducts with water. This experience refers to the quantification of emulsifiers in vaccines with HPLC-ITMS.
Therefore, for a simple molar mass determination, I prefer size exclusion chromatography.
Good luck with your work and best regards
Joachim Horst
Dear sir
Vishwajit Chavda & Joachim Horst.
I just wanted to reach out and thank you for taking the time to answer my question. Your response was incredibly helpful and provided me with the information I needed to move forward with my research.
Thank you again for sharing your knowledge and expertise with me. I appreciate your time and willingness to help.
Best regards,
Vishal
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