The gradient linearity and step tests are two of the most useful performance tests that can be made for a liquid chromatography
(LC) system. These check the linearity of gradient generation and the accuracy of mobile phase proportioning. These tests,
and examples of problems detected as a result of these tests, have been the subject of at least seven "LC Troubleshooting"
columns (1–7) over the last 18 years. We strongly recommend that every LC system undergo these tests at least on an annual
basis, and preferably semiannually. When a new and different example of a problem detected by these tests is discovered, it
is hard to bypass the opportunity to share it with our readers. So this month, you get yet another example of how an LC system
can fail.
The Test
Just as a reminder, the test is quite simple. Replace the column with a piece of capillary tubing (for example, 1 m of 0.005-in.
i.d. PEEK). Place water in the A-reservoir and water plus 0.1% acetone in the B-reservoir. Set the UV detector to 265 nm.
Adjust the flow rate so that there is sufficient backpressure (≈35 bar) for good check-valve operation, such as 1–2 mL/min.
The first test is a gradient linearity test — just run a blank gradient and record the baseline. This will give you information
about gradient linearity, and you can use the results to calculate the system dwell-volume (also called the gradient holdup
volume). The second test is a step test, in which isocratic steps are programmed between 0 and 100% B, generally at 5% or
10% increments. More details about these tests can be found in references 6 and 7, which you can access on-line (8).
Initial Data
Figure 1: Results of gradient linearity tests obtained (a) for the initial test and (b) after sonicating one inlet check valve.
Dashed line between the two plots is for reference. Plot of 0–25 min. See text for details.
We run the two gradient tests as a part of a semiannual instrument performance test suite. These are programmed to run unattended,
and data are analyzed after the tests are complete. First, we ran the gradient linearity test as a linear gradient of 0–100%
B over 15 min at 1 mL/min. The results for the initial test are shown in Figure 1a. The second test is the step test. We run
this with steps at 0, 10, 20, 30, 40, 45, 50, 55, 60, 70, 80, 90, and 100% B. We add the 5% steps near 50% B, because, in
our experience, if problems are going to occur, they occur near 50% B more often than not, especially with low-pressure-mixing
systems. (A good example of low-pressure-proportioning-valve failure was presented in reference 6.) The program is set up
to generate each step for 4 min and is run at 2 mL/min. The results of the initial step test are shown in Figure 2a.
A well-behaved linearity test should appear similar to that shown in Figure 1b. Clearly there is something wrong with the
run of Figure 1a (dashed line added for reference). The initial and final isocratic sections appear to be OK, but the center
portion is quite nonlinear. Remember that the apparent isocratic hold at the beginning of the gradient linearity test is due
to the dwell volume of the system, approximately 2.3 mL for this system (see reference 6 for determining dwell volume from
such plots).
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