HTEC Drop in sensitivity
If you are experiencing a drop in sensitivity and are using an autosampler, please rule out issues with the autosampler by performing a manual injection and compare peak height. If you are unfamiliar with the manual injector, please watch the following video for assistance. Please ensure the syringe is cleaned prior to use.
If there are no peaks at all (including the solvent front peak), please check the following:
1. Ensure the pump pressure is within a normal range. Zero pump pressure means there is no flow and a possible leak..
2. Confirm there is a signal by observing the real-time window in the Clarity software. Pressing the ‘zero’ button typically generates an observable response. If there is no signal, check wire connections for loose wires, check USB connections, and confirm the USB drivers are up to date.
3. Please ensure the ECD is turned on by checking the display on the HTEC.
*ECD* means on
ECD means off
4. Check that the red, white, and black wires connecting the electrodes to the potentiostat are in place.
If all peaks are present, but all peak signals are lower than usual (including the solvent front peak), please check the following:
Confirm the applied voltage is correct on the HTEC display.
Confirm the correct gasket and working electrode are being used for the application. Inspect the gasket for wrinkles or signs of wear and replace as needed. Inspect the working electrode. Typically, a residue can build up over time which inhibits a good signal. Please see the following article on proper care and cleaning of the working electrodes. If cleaning the working electrode does not solve the problem, it might be time to replace it.
Check the reference electrode for signs of damage or drying out. Please see this article for proper storage. We recommend replacing about once a year regardless of use. Check the serial number on the reference electrode. The first 2 digits indicate the year it was made.
Please check for contamination in the instrument/tubing.
**Do not recirculate mobile phase**
Always prepare fresh mobile phase and run directly to waste. Recycling the mobile phase will cause contamination and bacterial buildup over time. Quality of the mobile phase can be checked by inspecting the precolumn packing material. Unscrew the inlet side of the precolumn with 2 crescent wrenches to expose the packing material. Packing material in the precolumn should be bright white as shown below.
Any off-white or tan color indicates it is time to repack the precolumn. Also, premature discoloration indicates poor mobile phase quality. A pinkish color could indicate bacteria growth. For more information on the precolumn including when and how to repack, please see this article.
5. Check that the correct volume is being injected by the autosampler. This can be done by using a pipettor to measure how much sample is left in the well/vial after an injection is made.
If all other peaks are the same height as usual (including the solvent front peak), except your analyte of interest, please check:
1 The standard/sample itself (ex. degradation/contamination). Please always prepare fresh standards the day of analysis following our protocol outlined in the application manual. Please double check concentration levels of your standards. Samples need to be prepared using our preparation protocol.
2 Runtime may be too short. Please extend the runtime longer than anticipated and check if the analyte is eluding at a later retention time.
3 Double check the set flow rate and pump pressure. Confirm the flow rate is correct by measuring the waste for 1 minute using an Eppendorf tube.
5 For amino acid analysis: Make a fresh batch of derivatization reagents. The thiols/reduction agents used for derivatization oxidize quickly.