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By Alex on Friday, August 20, 2004 - 01:15 am:
Hi Everybody,
I recently developed a method for separation of leucine oligomers that where synthesized through solid phase peptide synthesis.
My aim is to extract the Hexaleucine, which elutes in 25 Min. using following procedure:
Gradient
MeCN/H2O (2:8) + 0,1 %AcOH --> MeCN/H2O (1:1) + 0,1 % AcOH in 30 Min. (Flow: 0,5 ml/Min.)
Now I want change to a preparative HPLC and I would like to know how I could estimate the new retention time.
Analytical Column: RP-18 EC 100/5 µm (Dimensions: 250x4mm)
Preparative Column: RP-18 EC 100/16 µm (Dimensions: 230x50mm)
Another problem is that the UV-Signal is very weak (best at 220 nm) and I am afraid not seeing it later. Because of the gradient, using the refraction index won't make sense.
I greatly appreciate every help or hint solving this problem.
Thanx in advance
Alex
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By tom jupille on Friday, August 20, 2004 - 10:08 am:
Matching retention times is straightforward:
1. Since the gradient delay times (from pump to column) will undoubtedly be different on the prep vs the analytical systems, you'll need to measure the delay volumes on the two systems. If you download and install an evaluation copy of the "DryLab" program from Rheodyne (the link is near the upper left corner of the Forum) and look in the help file, there is a fairly detailed procedure.
2. Instead of retention time, think in terms of a "corrected" retention time (retention time minus the gradient delay time); it will make the rest of the job a lot easier. At the end, you can add in your gradient delay time to get the actual tR.
3. Keep your gradient time constant, and scale your flow rate to the column volume (L x dc^2)
- L = length
- dc = column diameter
For the column dimensions you gave, the factor is 143. The flow rate on the prep column should be 143 times the flow rate on the analytical column (i.e, 72 mL/min).
Under those conditions, you should get the same "corrected" retention time on both columns.
4. Add back the gradient delay time to get your actual retention time.
By keeping gradient time constant and scaling the flow to the column volume, you're running under "equivalent" conditions.
Predicting what will happen as you change flow and gradient time (i.e., under non-equivalent conditions) can be done using the DryLab software I mentioned earlier with data from two experimental runs on your analytical column. The math involved gets complicated (hence the reliance on the software), but the predictions have been shown to be quite accurate (for peptides, typically better than 1%).
By way of "truth in advertising": I was involved in developing and supporting the DryLab software up until the beginning of this year.
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By Uwe Neue on Friday, August 20, 2004 - 03:17 pm:
One thing remains simple though:
Assume that the flow rate is too high for your preparative conditions, or you want to run at a slower flow rate: the gradient will remain the same, if the gradient volume (= gradient run time * flow rate) remains the same. If you want to run at 36 mL/min, you should run the gradient for 60 minutes, and your hexaleucine peak will elute at 50 minutes. Everything else will be the same, unless your prevolumn volume (= gradient delay time) has a giant influence.
Hi Everybody,
I recently developed a method for separation of leucine oligomers that where synthesized through solid phase peptide synthesis.
My aim is to extract the Hexaleucine, which elutes in 25 Min. using following procedure:
Gradient
MeCN/H2O (2:8) + 0,1 %AcOH --> MeCN/H2O (1:1) + 0,1 % AcOH in 30 Min. (Flow: 0,5 ml/Min.)
Now I want change to a preparative HPLC and I would like to know how I could estimate the new retention time.
Analytical Column: RP-18 EC 100/5 µm (Dimensions: 250x4mm)
Preparative Column: RP-18 EC 100/16 µm (Dimensions: 230x50mm)
Another problem is that the UV-Signal is very weak (best at 220 nm) and I am afraid not seeing it later. Because of the gradient, using the refraction index won't make sense.
I greatly appreciate every help or hint solving this problem.
Thanx in advance
Alex
-------------------------------------------------------------------------------------------------------
By tom jupille on Friday, August 20, 2004 - 10:08 am:
Matching retention times is straightforward:
1. Since the gradient delay times (from pump to column) will undoubtedly be different on the prep vs the analytical systems, you'll need to measure the delay volumes on the two systems. If you download and install an evaluation copy of the "DryLab" program from Rheodyne (the link is near the upper left corner of the Forum) and look in the help file, there is a fairly detailed procedure.
2. Instead of retention time, think in terms of a "corrected" retention time (retention time minus the gradient delay time); it will make the rest of the job a lot easier. At the end, you can add in your gradient delay time to get the actual tR.
3. Keep your gradient time constant, and scale your flow rate to the column volume (L x dc^2)
- L = length
- dc = column diameter
For the column dimensions you gave, the factor is 143. The flow rate on the prep column should be 143 times the flow rate on the analytical column (i.e, 72 mL/min).
Under those conditions, you should get the same "corrected" retention time on both columns.
4. Add back the gradient delay time to get your actual retention time.
By keeping gradient time constant and scaling the flow to the column volume, you're running under "equivalent" conditions.
Predicting what will happen as you change flow and gradient time (i.e., under non-equivalent conditions) can be done using the DryLab software I mentioned earlier with data from two experimental runs on your analytical column. The math involved gets complicated (hence the reliance on the software), but the predictions have been shown to be quite accurate (for peptides, typically better than 1%).
By way of "truth in advertising": I was involved in developing and supporting the DryLab software up until the beginning of this year.
-------------------------------------------------------------------------------------------------------
By Uwe Neue on Friday, August 20, 2004 - 03:17 pm:
One thing remains simple though:
Assume that the flow rate is too high for your preparative conditions, or you want to run at a slower flow rate: the gradient will remain the same, if the gradient volume (= gradient run time * flow rate) remains the same. If you want to run at 36 mL/min, you should run the gradient for 60 minutes, and your hexaleucine peak will elute at 50 minutes. Everything else will be the same, unless your prevolumn volume (= gradient delay time) has a giant influence.
