By Bob Forbes on Thursday, April 1, 2004 - 12:40 pm:

I'm developing a gradient method using pH 6.5 ammonium phosphate/acetonitrile. My HPLC systems are typically used with lower pH mobile phases. When switching over to ph 6.5, I see multiple peaks in blank chromatograms (with and/or without injecting anything)that appear to be due to the HPLC. I've worked to rule out mobile phase and/or column contamination, and the peaks did not diminish when the injector was bypassed. I suspect something is leaching off the stainless surfaces of the tubing and pulse-flow dampeners. What cleaning procedures can anyone recommend to reduce the peaks in my blank?

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By DR on Friday, April 2, 2004 - 07:43 am:

Search the boards for a link to or mention of such a procedure from Waters, or check their www site.

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By lilcloud21 on Monday, April 5, 2004 - 04:29 pm:

This is a procedure recommended by my Waters rep: flush lines with 6N Nitric acid for about an hour. Wash lines overnight with water. Because you're using salts on your HPLC the lines may be corroded. According to my rep, the Nitric helps to clean the corrosion. Not sure whether this is true, but the cleaning seems to work.

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By Durk on Monday, April 5, 2004 - 11:52 pm:

Cleaning with 6N Nitric acid is to me the last procedure to clean a HPLC system. It is a very rigorous method and you must be carefull using this method. We clean our systems with hot water for several hours (the flask with water is thermostatted during cleaning at boiling temperature (use carborundum!!!)). After this procedure we rinse the HPLC system with 2-propanol at low flow.

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By Anonymous on Tuesday, April 6, 2004 - 05:18 am:

This is my list, how far you proced through it depends on how clean you need the instrument.
It is important to remember to exercise all valves. Also, if there are parts that can contaminate and easily be changed (frits, filters), do that too.


1: Hot water
2: propanol
3: 30% H3PO4
4: 6N Nitric

Of course, with the acids, it takes a lot of flusing with water to remove the last traces of acid, but the system will be clean when you are done. As well, the nitric will passivate the stainless steel surfaces, important for very high sensitivty work with some detectors (ECD in particular).

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By Chemist on Tuesday, June 1, 2004 - 05:33 am:

I agree with lilcloud; flushing a system with 6N Nitric/ Isopropanol is recommended procedure from Waters, however they also suggest to run water for a long time after that. They also suggest when you running water to increase the oven temperature to 40-50C. You don't need to use boiling water.

P.S. Never mix 6N Nitric and Isopropanol in a single bottle. Pressure can be quite significant.
That's from experience and damaged ceiling above the instrument is the remainder.

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By xiao2 on Wednesday, June 2, 2004 - 04:00 am:

You also can try the following procedure:
1. water
2. ethanol
3. water
4. HNO3 (1N)
5. water
6. EDTA.2Na (10mM)
7. water
Run each solution 10-20min at the flow rate of 1ml/min.