John W. Dolan
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As I write this, I have just finished a week of teaching a liquid chromatography (LC) training class in Chongqing, China.
On the last day, we spent several hours looking at problems that the students brought to class, and it reminded me how chromatographers
worldwide encounter the same type of problems. One problem that came up was related to the transfer of a method from one instrument
to another. This kind of problem is one that all of us encounter at one time or another, so I would like to dedicate this
month's "LC Troubleshooting" installment to method transfer problems.
The Rule of One
It is strange how we automatically apply the scientific method to most of our work in the laboratory, but somehow we discard
it when it comes time to troubleshoot an LC problem. For troubleshooting purposes, I summarize the recommended technique as
the of One." This reminds us to change just one variable at a time when investigating a problem.
Often a problem goes something like this: We notice that something is wrong with the chromatogram, so we replace the column.
There is a guard column in use, so we might as well change it, too. Then we observe that the mobile phase reservoir is almost
empty, so we make up a new batch of mobile phase. Then we look at the clock and see that it is almost quitting time, so there's
no way we will get a run started before going home. We use the extra time to change the pump seals, because it is about time
for the periodic service of the pump. The next morning we come to work and start up the system. It works great! But the same
problem happens again the next week — are we going to repeat all the same changes? By changing one thing at a time, it often
takes longer initially to identify the problem source, but with additional knowledge about the system, we will be able to
solve the problem much more quickly the next time. Restrict the Differences
In one method, I observed that the separation changed noticeably when the method was moved from one instrument to another
in the same laboratory. It is easy to assign the difference to the instrument, but is this the only variable that has changed?
Often it is not. For example, in the present case, the original method was developed on a two-pump high-pressure mixing LC
system in which the mobile phase was prepared by on-line mixing. In the new instrument, a single-pump system was used with
manual mixing. The same brand and model of column was used and the same temperature was set on the column oven, but, although
we assume these conditions are identical to the original conditions, they might not be.
In a case such as this, I like to further restrict the variables, if possible. First, make up a batch of hand-mixed mobile
phase and run it on the original system. Then take the same bottle of mobile phase and the same column and move it to the
second system. Now are the results the same? Look at the retention times. If they differ between the two systems, one possible
cause is that the temperature of the column is different. For reversed-phase methods, retention changes by approximately 2%
per each degree Celsius. If the ovens are calibrated differently, this can account for the difference. Often a change in temperature
will change the relative retention between two peaks. Estimate the amount of change in temperature necessary to result in
the retention difference. Now adjust the oven setting to see if this will correct the problem.
On the other hand, in isocratic runs, a difference in flow rate will affect the retention time of all peaks by the same proportion,
so relative retention will stay the same. The pump flow rate is easy to check. Just use a 10-mL volumetric flask and measure
how long it takes to fill the flask for each system. You can determine quickly if there is a difference in flow rate.
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