A strange gradient elution

[zlb215]

Dear all;
Recently,a strange gradient elution method has been transfered to me. It starts in the proportion of ACN-Buffer(60:40) ,then changes to ACN-Buffer(45:55),and gets to ACN-Buffer(75:25) at last. As a result of this gradient, I get an acceptable resolution and a "rainbow" baseline(the mid baseline is higher than the begining and end) .
I never met a gradient like this, in the past, I always increase the proportion of organic modifier by the time.
So,my questions: Is here someone whom have experience about this kind of gradient? Why a similarly gradient can be used?What is its advantages and disadvantages?
Terry Z

[mohan_2008]

This is not a strange gradient as it may seem.

The initial 60:40 ACN/Buffer is to equilibrate your column with a higher % organic. The 45:55 ACN/Buffer is the actual gradient run - leading to the elution of your peak/ and other relatively less hydrophobic components as in your method. The stronger 75:25 ACN/Buffer is done to elute the more hydrophobic peaks, and also as a part in rinsing your column.

You can also equilibrate your column with the 45:55 ACN/Buffer, however a more stronger organic gets a faster equilibration.

I find this gradient perfectly alright.

[zlb215]

mohan_2008;
Maybe, I did not express my situation clearly yet.My gradient is:0-10min, 60%ACN-45%ACN;10-25min,45%ACN;25-27min,45%ACN-75%ACN;27-37min,75%ACN;37-40min,75%ACN-60%ACN;40-45min,60%ACN.
So, I am not agree with your opinion about equilibrium.Thank you for your attention!
Best regards!
Terry Z

[danko]

Hi zlb215,

Are you running HILIC? In other words; what is the stationary phase?

Best Regards

[reyoungs]

This gradient may be designed to maximize resolution for peaks in the middle of the chromaogram while avoid excessive dead time in the beginning and end that a "conventional" chromatogram might provide.

You do not discuss peak elution, but I would guess that some of the peaks of interest elute in the first 60% section, the two minute 45% section is strange, but effectively shuts down elution with that much of a change of organic, allowing remaining (close) peaks to move apart, whether they elute in that window or not, and then the 75% stage restarts the elution, but reataining the increased resolution from the 45% for a period. The last peaks elute in the 75% stage and the column is cleaned for the next injection.

Your gradient reequilibration time is not defined relative to flow rate, but is probably just enough enough to reequilibrate the column ot the starting conditions. If you are running on an Acquuity or similar low dead volume system, this makes good sense for tweaking resolution in the middle of a chromatogram rather than working to find what other parameters would create the same seapration for the difficult or critical pair.

Gradient elution is powerful enough that it allows separations without understanding the "design space" rigorously. The 15% absolute drop in ACN is a relative change of 25%, or enough to change k' by a factor of about 10 in an isocratic separation. This would allow you to see very small differeneces in lipophilicity, at the expense of very long run times.

We just ran a separtion in which we separated "overlapping" peaks by cutting solvent strength about that much and then kicked the gradient up just after the "unknown" peak eluted so we didn't have to live with the three hour injection that the original profile would have provided. OUr seapartoin was a bit different, in that we were pulling apart coeluting peaks at the beginning of the separation, but the principle is the same, especially if you are on a low dead volume system.

Let me know if the logic fits your peak elution.

[zlb215]

Hi zlb215,

Are you running HILIC? In other words; what is the stationary phase?

Best Regards

danko;
The stantionary phase is PFP.
Best regards!

[danko]

Hi again,

danko;
The stantionary phase is PFP.

I see. It’s a kind of RP column, but hydrophilic enough to retain polar compounds. So, you may have some hydrophilic interactions that are stronger than the hydrophobic ones and then the gradient is sort of logical. It should be noted though, that I’m only speculating – not knowing the nature of the analyte.

Best Regards

[zlb215]

Dear reyoungs;
I agree with your logics after inspection the peak elution again. Thanks a lot.
However,I still have some questions about your words:
1."Your gradient reequilibration time is not defined relative to flow rate, but is probably just enough enough to reequilibrate the column ot the starting conditions." Do you indicate that I could change flow rate to get a faster reequilibration?
2."If you are running on an Acquuity or similar low dead volume system, this makes good sense for tweaking resolution in the middle of a chromatogram rather than working to find what other parameters would create the same seapration for the difficult or critical pair. " I know that dead volume will affect the reequilibration time, but how does it "make good sense for tweaking resolution in the middle of a chromatogram"?Or did you mean flow rate make sense?
3."Gradient elution is powerful enough that it allows separations without understanding the "design space" rigorously". I think it describe that operator can develop a good gradient method and get a perfect resolution without understanding the "design space" rigorously.Did i get the point?

[reyoungs]

1."Your gradient reequilibration time is not defined relative to flow rate, but is probably just enough enough to reequilibrate the column ot the starting conditions." Do you indicate that I could change flow rate to get a faster reequilibration?
You generally need to run at least 5-10 column volumes (CV) through the column to achieve reequilibration. You simply inidcated the times for the profile without the flow rates. If you calculate the volume of the column, then multiply the flow rate by the time and divide by the CV, you can determine if you have passed enough solvent through the column to achieve equilibirium. A stable baseline is a good indicator many times, but we should go back to basics and know WHY we are doing what we are doing in the gradient. You want to run just enough volume through the column to achieve equilibraium, but not waste time with extra volume. Some people do run a higher flow rate to get faster reequilibration, but you need to return to the original flow rate condiitons a couple of minutes before the start of the next injection to allow the pressure to adjust within the column as well. We are dealing with complex equibrium here and flow rate and CV are only part of the issue.

2."If you are running on an Acquuity or similar low dead volume system, this makes good sense for tweaking resolution in the middle of a chromatogram rather than working to find what other parameters would create the same separation for the difficult or critical pair. " I know that dead volume will affect the reequilibration time, but how does it "make good sense for tweaking resolution in the middle of a chromatogram"?Or did you mean flow rate make sense?
I meant that the gradient profile made sense if you are getting the resolution of a difficult pair without wasting extra time throughout the entire run. If your two minute 25% organic is enough to pull apart the difficult peaks, then stay with it. I don't think increasing the flow rate would make a positive difference.

3."Gradient elution is powerful enough that it allows separations without understanding the "design space" rigorously". I think it describe that operator can develop a good gradient method and get a perfect resolution without understanding the "design space" rigorously.Did i get the point?
Yes, you did.

Whoever designed your separation should have explained more about why they did what they did so that you had a better understanding of what you could change and what was important to the separation. It sounds as if you have a fairly efficient separation, but just weren't told why it was developed the way it was.

I also mean that there are many ways to solve a separation, and some are more robust than others. Gradients are powerful, but much more tricky to transfer between users and equipment. I believe we need to know as much as possible about our molecules to design the most productive and robust separations. Many people do not wish (or have) the time.

I am glad I may have been of assistance.