The negative peak is caused by the change in matrix from the carrier to the sample. There is almost always a negative peak and this is normal. However, an abnormally large negative peak can cause problems. Below are possible causes for a large negative peak:
1. The column, precolumn, or both are old. Repacking the precolumn, washing the separation column, or replacing with a new one.
To wash the separation column, remove a precolumn from the line and disconnect the tubing from the outlet of the column. Deliver water for 30 min after priming the pump and replacing the contents of the pump to water from the carrier. Then deliver methanol for 1 hour. Switch back to water for 30 min, and then deliver carrier for at least 8 hours. The separation column, NO-PAK, requires 8 hours to reach equilibrium with the carrier. Pump speed of the carrier is 0.33 ml/min for all steps. Check the column condition with a standard sample diluted with carrier. Then replace the re-packed precolumn to the line between the injector and the NO-PAK. Before connecting the line to the NO-PAK from the precolumn, deliver the carrier for at least 3 min. Then try a sample injection.
2. Sample overload. If the sample is loaded to more than 20 µl, the negative peak can be larger. As specifications state, the ENO-20 accepts at most a 50 µl sample volume, but Eicom recommends injecting 10µl. This volume capacity can vary depending on the types of samples.
3. Autosampler settings are not optimized. If the autosampler switches the valve position to the load position after injections, a negative peak can occur. When the valve is set to the load position, the loop can be emptied and at the next injection, a large volume of air or water can be injected. Maintain the loop contents only up to 20 µl of sample. A 2-4 µl air segment can be used to separate sample and carrier. The loop size should be 100 µl or smaller. To identify if the negative peak is from the autosampler, inject the sample from the manual injector. When you inject the sample from the manual injector, keep the autosampler valve at load position.
4. If the peak shape is asymmetrical, this is an indication of poor separation of the column. To confirm the quality of the NO-PAK separation column, remove the precolumn from the line and inject a standard sample. From this process you can see the quality of the separation column.