4.5. HPLC separation of standards

Once the ECD cells equilibrate, standard samples can be run on the HPLC system.

1. Prior to injecting any samples, prime the HPLC by injecting 10 µl of 0.2 M perchloric acid (PCA).

2. After this and every injection, be sure to flush the syringe with polished water several times.

Any residual PCA would crystallize, causing the plunger to become frozen in the barrel. As an added precaution, store the plunger out of the syringe when not in use.

3. If the PCA run looks clear of peaks, sample analysis can begin.

Standards and samples should be processed in this order: Standards G to B (lower to higher concentration), Standard H, 5-7 samples, Standards G/D/H, 5-7 samples, Standards G/D/H, PCA Blank.  Standard H and the PCA clear the HPLC of residual amine peaks. Standards should always be run at the start, middle and end of any series to ensure that the sensitivity of the system has not changed. The cycle can be repeated as necessary. Duration of the run will depend on the flow rate, which in turn is determined by the pressure registered within the HPLC. Adjustments to flow rate and run length may need to occur over the course of the day as contaminates injected with the sample cause a gradual pressure increase.

Because of the sensitive nature of the amines, an autoloader cannot be used to inject the samples, as they would degrade while waiting. Instead, use a microsyringe to directly deliver the sample into the injector port.

While standards are being run on the HPLC, tissue samples can be prepared for analysis.