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Agilent recently published information on decreasing the delay volume on the 1100. I like to save time whenever possible so I decided to give this a try. The method in question is the same one that I described here. The method was modified with an injector program to switch the valve to bypass at one minute into the run. This is before the gradient starts so switching back to main-pass before during the next injection will not effect the retention times or the second sample. As you can see the elution time of the last peak decreased significantly.

This decrease and setting the autosampler to pre-fetch the net vial saves about 50 seconds per sample. This can add up fast when you have 20 or more samples to run.

A reference is great for correcting for baseline changes but is not always needed, and when set incorrectly have detrimental effects. Here is a comparison of the same sample analysis with different collection parameters. Two sets of collection parameters were collected simultaneously: The first being a sample wavelength of 210nm with the reference turned off. The second used the same sample wavelength parameters but with the reference set to a typical 360nm with a band width of 100nm. We can see the reference-off chromatogram looks normal, while the chromatogram with a reference of 360nm shows many negative peaks.


The sample in question contains multiple compounds that absorb through the entire UV and into the visible. Here is the spectrum of one of the peaks with the reference highlighted.


When compounds elute that absorb in the reference range (310nm to 410nm) Chemstation offsets the absorbance at 210nm accordingly. This is obviously an extreme example, but demonstrates the need of the analyst to be on guard for cases where the impact of incorrect reference is less apparent.

Here is an example of pressure ripple in a binary pump. Purging the pump did not reduce the pressure ripple as it did in "Troubleshooting High Pressure Ripple Part 1". A little more work is required this time. As with most flow processes, we can segment the system step wise to find the source of the problem. Isolating the origin of the ripple starts with:

1. Installing a restriction capillary
   In order to effectively troubleshoot we need to keep the pressure above 50bar, but also want to eliminate having to equilibrate the column after each step. A restriction capillary is ideal for the purpose.
2. Run 100% A:
   
   
   
   After changing to 100% A the pressure ripple is gone. This eliminates the A channel from the list of possible sources. This also eliminates all the flow path components that the A and B channels share.
3. Run 100% B:
   
   
   
   We can see that the pressure ripple is isolated to the B channel.
4. Run 100% B2:
   
   
   This eliminates the pistons and seals as the cause of the ripple. (Note that the initial pressure drop is air being purged from the B2 channel.) We now know that the cause of the pressure ripple is on the B side of the pump and specific to the B1 channel. There are three remaining suspects: the solvent selection valve, the inlet filter or the B1 mobile phase. Changing the inlet filter is the easiest so that's a good place to start.
5. New Inlet Filter:
   
   From ripple to stable in only four steps. This final graphic was squished to show all the steps in one and the ripple looks a little higher than it is. A closer view shows no more than pressure noise:
   

Many times high pressure ripple is caused by air. Air can be introduced via so called micro-leaks that allow solvent to evaporate and air to enter the system without visible signs of a leak. This typically happens as the instrument sits idle, over the weekend for example. Air can also be introduced form mobile phase that has not been properly degassed. In the first case, simply purging the system is enough to eliminate the air. Here is an example where air was introduced through the solvent selection valve. The instrument showed high ripple. The purge valve was opened and the system purged at a high flow rate.
After the air has been purged, the ripple disappears and pressure stabilizes shortly after that as seen here.