Q1: one method I developed has very good repeatability on my system, but it is different on other systems. What’s the matter?

Answer: Sometimes there are some problems with the gradient method when changing the chromatographic system. Unless the system is exactly the same, the retention time always changes. Usually, the change of this time is within the range of resolution. Ignoring it, the method can still be used. In addition, if the potential causes are properly understood, the gradient can be adjusted to obtain the same effect in different chromatographic systems.

Another problem related to this problem is the gradient lag volume. It is the volume from the gradient mixture to the stigma. When you start analysis after injection, the gradient does not reach the column head, and it will not arrive until the lag volume is emptied. This means that the sample migrates equivalently at the beginning of a period of time. Because the lag volume of different systems is different, the retention time is different, and sometimes it still affects the degree of separation.

Another reason is the gradient itself, which is caused by the component difference between systems. For most chromatographic manufacturers, this problem is not very serious. However, in general, the accuracy is the highest when transporting the mobile phase with equal proportion (50% a and 50% B), and the accuracy will be impaired when the proportion difference is large (5% a or 95% a).

Q2: How can I know what kind of situation I am?

Answer: The simplest way is to compare the gradients of the two systems. Remove the column and connect the double-pass. (connect UV detector after gradient solution b) if reversed-phase chromatography is used, 10mg / L propyl p-hydroxybenzoate can be added to solution B. Both systems started running gradients and recorded baselines. Then compare the two maps. To find where the gradient begins, measure the gradient map. If the gradient is linear, just measure the slope of the gradient.

It is likely that you will find that the gradient start time of the two instruments is different, the map is very similar, and the gradient end time has changed to a certain extent. In this case, the gradient lag volume of the two instruments is different.

Q3: If this is the case, is there a simple way to offset the difference in lag volume?

Answer: There is one method that can be adopted most of the time. If the gradient lag volume of the new system is less than that of the system before transfer, an equal degree can be added at the beginning of the gradient. If the lag volume of the new system is large, this situation is more difficult. In principle, you can open the gradient first and delay the injection for a period of time, which should be consistent with the lag time difference between the two instruments. However, this cannot be done in the automatic system. In the automatic system, the injection triggers the gradient. At this time, it may be necessary to manually zero the baseline or redevelop the method.

Q4: I’m not happy to encounter this situation after I spend time developing methods. What can I do to avoid it in the future?

Answer: You can develop methods on the final instrument. This may require some foresight and planning, but it is usually feasible. What you need to do is to understand the performance of the new system. You need to know two basic conditions of the system: gradient lag volume and proportional accuracy. You can get these two data in the same experiment: as mentioned earlier, add an ultraviolet detector after solution B and set a multi-level gradient. Solution B changes from 0% to 100% in the range of 5%. The flow rate is the same as usual. It should be 1ml / min. Each gradient is maintained for about 5min. Then connect the two-way operation gradient and record the map. The difference between the setting time of each gradient and the actual gradient time is the gradient delay time. The height of the stratum can be used to measure the proportion of mobile phase. These layers may be a little fuzzy, which is caused by the mixing volume in the system.

After testing the system, we can design the method according to its performance. As I mentioned earlier, the biggest problem is the gradient lag volume. If you know that the lag volume of the new instrument is larger than that of the instrument developing the method, you should consciously add a section of equivalence before the method to offset this difference. If the lag volume of the target system is small, you should add a gradient delay time at the beginning of the method when transferring the method.

If the proportion difference appears in the middle of gradient operation, I believe you can also offset this difference by adjusting the gradient map.

This discussion assumes that the column has been fully balanced with the initial mobile phase. Sometimes I encounter situations where the gradient changes rapidly in daily analysis, and the column is not well balanced with the initial mobile phase. In this way, the first stitch is always different from the later one. This may be a disadvantage of the acceleration method, but some problems may arise when moving to a system with different lag volume.

If you have any problem or require further information, please contact info@welchmat.com

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