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Sample load and concentration limit for a Preparative LCMS Column (ZORBAX Eclipse XDB 80Å C18, 9.4 x 250 mm, 5 µm HPLC column)

Yuhang

I am currently using Preparative LCMS to purify my sample (around 150 mg). The column type is Part Number:990967-202 (ZORBAX Eclipse XDB 80Å C18, 9.4 x 250 mm, 5 µm HPLC column)

I have tried a 0.1mL injection of 30mg/mL sample (3mg sample load) and it worked fine. 

However, I am not willing to separate my samples dozens of times whilst I am a bit concerned about blocking the column if I inject more than it can accommodate (i.e. 30mg in 1mL). 

So, I would really appreciate it if someone could help me with this obtaining the information on the maximum sample load and concentration for the following column, please?

 

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  • 0

    Hi  

    Your question is difficult to answer!  The challenge here is all about the solubility of your sample in the mobile phase and how well the target compound is retained.  Normally, with that amount of material, I would be looking at using a larger diameter column.

    Just a few points to help you with what you have:

    You will not block a column by overloading it UNLESS your sample precipitates on injection.  Overloading the column normally changes the peak shape.

    If 0.1mL works fine, how about trying 0.2mL.  Watch the pressure trace with each injection.  A significant rise in pressure, above the normal starting pressure, after injection indicates a solubility issue.  With the larger injection volume, is your separation acceptable?  As each increased injection volume works successfully, go larger!!

    Hope that helps.

    /Andy

  • 0 in reply to Andy Wright

    Dear Andy,

    Thank you so much for the suggestion! My separation is acceptable. But, my sample did precipitate upon contact with 95% water 5% MeOH (at the starting gradient), thus I suppose a low concentration and amount might be suitable? Like 25 mg/mL concentration and inject between 100uL-1mL.But Yes, your suggestion is totally helpful! Thank you so much!!!

    Best,

    Yuhang

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  • 0 in reply to Andy Wright

    Dear Andy,

    Thank you so much for the suggestion! My separation is acceptable. But, my sample did precipitate upon contact with 95% water 5% MeOH (at the starting gradient), thus I suppose a low concentration and amount might be suitable? Like 25 mg/mL concentration and inject between 100uL-1mL.But Yes, your suggestion is totally helpful! Thank you so much!!!

    Best,

    Yuhang

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