Stainless Steel Tubing

[TLK]

If I wanted to use stainless steel tubing for an LC, how often would the lines need to be replaced?

Also, I've heard mention of treating the lines before they're used in an analysis--what is the purpose of this, and how is it done?

Is there any difference in how I would treat/maintain the lines if they're being used on an LC with a UV-vis detector or an MS detector?

Thank you!

[shaun78]

stainless steel should very seldomly need to be replaced. The most likely cause of replacing the line would be because it broke because someone tried to bend it too much/too many times.

Some analytes are sensitive to metals. If you work with analytes that will degrade in the presence of metal cations then you need to go through a passivation process for the system. The passivation is the same no matter if you are using a MS or UV detector because you always disconnect the tubing that runs into the detector. The passivation process will make using your detector a nightmare if you leave it inline.

Anyway passivation:

1. Wash all lines with water.

2. Wash all lines with 0.1M citric acid.

3. Wash all lines with water.

4. Wash all lines with 0.1M EDTA.

5. Wash all lines with water.

6. Wash all lines with nitric acid (concentration is really up to you, I have seen people use anything from 1% to 50%. I would recomend staying closer to 1% then 50% [I generally use about 5%]).

7. Wash all lines with water until the pH of the eluent is the same as the source water.

I hope this helps...

[HW Mueller]

shaun78,
where does your passivation recipe come from? Is it something you tried and it worked for you, or is it based on unequivocal study results?

I am asking, because I can see that you may remove some metal ions with citric acid and/or EDTA, but passivate? Usually one has EDTA permanntly in the mobile phase to control metal ions?
Then there was an article in LC.GC, June 2000 (in Dolan´s Troubleshooting) which presents evidence that nitric acid in lower concentrations can dissolve stainless (some stainless?). Anyway, at least a 50% HNO3 is recommended there.
This has been discussed several times here, maybe someone has something like a summary?

[TLK]

We're not analyzing proteins using this LC/MS, but we are analyzing biological samples (plasma, tissue extractions, etc.) that contain proteins. Would the treatment be necessary (i.e., would there be a concern of proteins actually sticking onto the lines, or would they just pick up metals and move along "quietly")?

Would something other than stainless be better? We do not want to use PEEK because phthalates leech off into the mobile phase (we often quantitate phthalates). Titanium or teflon, maybe?

What problems would titanium or teflon present?

[Mark Tracy]

The treatments with EDTA/citrate are to remove loose corrosion products and iron and nickel at the metal surface. New SST components should not need this step. The treatment with nitric acid oxidizes the chromium and molybdenum in the 316 SST to form a protective oxide layer. It takes at least 2M HNO3 to efficiently form the oxide. This oxide layer is vulnerable to halide ions, especially at low pH. When passivating, treat the entire system, excluding the column and MS, and be sure to flush it until all the nitrate ions are gone.

Any exposed iron in the system will corrode and this can bind a variety of complexing agents including phosphorylated molecules of biological interest. Also the iron can migrate onto your column and cause trouble there.

I routinely passivate my UV detectors since the flow cells are made of SST, quartz, teflon and PEEK. I find that the HNO3 cleans the windows. Some of my colleagues however find that freshly cleaned silica surfaces also bind proteins.

Titanium is also suitable for work with proteins or protein-containing samples, but there is one thing to watch for. Titanium forms a protective oxide layer on exposure to air, but this oxide can bind phosphorylated molecules. (Ferric complexes and TiO2 are the basis of class separations for phosphopeptides by IMAC.)

PEEK does not contain phthalates or any other plasticizers, so if you are seeing them, it is probably coming from somewhere else. See www.victrex.com for the facts. (Where did this bit of urban legend come from?) It has low protein binding, and does not shed objectionable metal ions. Tubing made of Teflon or other fluoropolymers like Tefzel or Kynar have lower pressure ratings than PEEK or metal tubing; also it is easy to accidentally crimp the tubing.

[TLK]

Thank you.

I'm not sure about the phthalates--I've heard from others who use LC/MS that they get in the way because of PEEK tubing. It's pretty common in plastics, but if it isn't coming from PEEK, then it must be coming from elsewhere. We frequently see it when we're not supposed to.

So if we don't analyze proteins (but our samples contain them) and the phthalates aren't coming from PEEK tubing, is there any reason to switch to stainless steel? Is there any advantage of stainless steel over PEEK for biological samples?

[Mark Tracy]

If you are seeing phthalates when they are not expected, you have to hunt down the contamination. As you know, phthalates are omnipresent in the modern environment; worse yet in the biomedical environment. Since you are worried about wetted materials, systematically replace them and analyze reagent blanks. Capillary tubing is cheap, but it might not solve the problem. (I had to do do this for a project to analyze perfluorinated acids. Teflon = bad.)

[rhaefe]

shaun78,
where does your passivation recipe come from? Is it something you tried and it worked for you, or is it based on unequivocal study results?

I am asking, because I can see that you may remove some metal ions with citric acid and/or EDTA, but passivate? Usually one has EDTA permanntly in the mobile phase to control metal ions?
Then there was an article in LC.GC, June 2000 (in Dolan´s Troubleshooting) which presents evidence that nitric acid in lower concentrations can dissolve stainless (some stainless?). Anyway, at least a 50% HNO3 is recommended there.
This has been discussed several times here, maybe someone has something like a summary?

Hans,

here are some literature references you might find useful:
(1)K.E. Collins, C.H. Collins and C.A. Bertran: "Stainless Steel Surfaces in LC Systems, Part I-Corrossion and Erosion"; LCGC Magazine 18, 600, 2000
"Stainless Steel Surfaces in LC Systems, Part II, Passivation and Practical Recommendations, LCGC Magazine, 18, 688, 2000

(2) R. Shoup and M. Bogdan: "Passivation of Liquid Chromatography Components", LCGC Magazine, 7, 742, 1989

(3) US Patents: 5,997,742 (1999);
5,772,889 (1998); 6,017,457 (2000); 6,030,527 (2000)

There was an old application note for the HP 1090 dealing with stainless steel and LC separation of proteins. I do have a copy somewhere.

I always use 50 to 70% nitric acid for passivation and usually leave the UV detector in line. The nitric washes out quite nicely.

reversed phase ion-pairing chromatography of ds DNA (with triethylammonium acetate) is VERY sensitive in regard to transition metal contaminations. We found that titanium frits and passivated ss frits are best. Some PEEK frits were completely useless for ds DNA separations.

If you are interested in more info, contact me directly at
rhaefele@hamiltoncompany.com

[Bruce Hamilton]

The first treatment of any stainless steel tubing, unless it's been specifically identified as being chemically cleaned, is to thoroughly clean the internal surfaces, removing all oils and greases using solvents, preferably warm.

I prefer a good mix of oxygenated and halogenated solvent, but environmental regulations may constrain your choices. You can chemically descale the tubing if you wish, but the stronger nitric acid treatments will also remove such smut.

Needless to say, using high concentrations of nitric acid to passivate should only be performed after you have ensuring there is no residual organic solvent present. Otherwise, bad things might happen...

If you increase the tubing and nitric acid temperature to 50 - 60C, you obtain a much more robust protective film with about 30 minutes of contact time.

Ferritic, and most free machining, grades of SS also require the addition of sodium dichromate to the nitric acid passivation treatment, which can create wastes that are difficult to dispose of.

If there are restrictions on the use of nitric acid passivation, a similar coating can be obtained on most stainless using 10% mass citric acid at about 65C for 30 minutes. In general, the citric acid passivation is slightly less robust than nitric, but should be OK for most purposes.

Note that if there is any chance of nitric acid being retained in crevices, after water rinsing, treat with a dilute NaOH solution, as nitric acid in crevices will rapidly produce active surfaces. Some passivation processes have a post-treatment alkaline rinse to ensure no acid remains.

There is an ASTM specification for Stainless Steel Passivation, ASTM A967.

Bruce Hamilton

[HW Mueller]

Bruce, sorry, but now I have this question again: Do you have unequivocal evidence for something like citric passivating? Or high temp. nitric? Problems with a bit of organic in a capillary tube? Na dichromate? NAOH (how do you get rid of NaOH in the crevices if water couldn´t remove HNO3? Flushing with HNO3?)
Is that from ASTM?

[shaun78]

HW Mueller:

Sorry for the delay in responce. I do have what you might call unequivocal evidence for doing something such as I described.

If you have access, lookup the current USP method for Hydralazine (analytical assay, not impurities assay).

If not, here are the basic parameters, at least as far as this discussion is concerned. Mobile phase is water/organic with SDS and tertbutylammonium bromide as ion exchange reagents (sorry I can not be more exact as I am going from memory). Hydralazine is exceptionally sensitive to light and metal ions.

If anyone has ever treid to run this assay, I am sure that they have gotten exceptionally fustrated due what appears to be random degradation of test sample.

I looked at everything from vials used to ordering low residual metal acids used in the assay. Nothing stopped the random analyte degradation (well, vials were a major player because of residual metals [Fe if I recall correctly] in abmer vials. However, vials did not solve this alone).

Because I was unable to change the method (ie add edta to the mobile phase) I had to find some way to passivate the system. I found that the procedure I outlined above seemed to hold up for about six months before it has to be done again. The combination of using that passivation procedure and rinsing/drying all amber glassware/vials before use stopped the "random" degradation I observed in the hydralazine assay.

[HW Mueller]

shaun78, ok, by unequivocal I meant that you proved that you removed the metal ions for about 1/2 year and that the metal ions present prior to and after your "passivation" did the degradation. The reason I think it´s important to know is that we, the ones who use EDTA in the mobile phase or passivate with 50%+ HNO3, should do some sort of penitence.

[Mark Tracy]

When I passivate, I use the UV detector to determine when the nitric acid is removed. First flush with deionized water until the baseline is flat (254 nm or below), and zero the detector. Replace the water with HNO3 and passivate for 20 minutes. Rinse the solvent dip tube and filter device. Then change back to water. I change the water reservoir at least twice. Then let it flush at high flow rate until the baseline is again flat. The baseline should be zero or less, if not, change the water again and keep flushing. It also helps to exercise the injection valve while passivating and flushing.

One nice feature I like on the Dionex pumps is that the fluid inlet channel in the head is angled to flush out the piston seal. It makes solvent changes less of a nuisance.

[Bruce Hamilton]

Bruce, sorry, but now I have this question again: Do you have unequivocal evidence for something like citric passivating? Or high temp. nitric? Problems with a bit of organic in a capillary tube? Na dichromate? NAOH (how do you get rid of NaOH in the crevices if water couldn´t remove HNO3? Flushing with HNO3?)
Is that from ASTM?

Sorry, it's Saturday morning and I can't access the library till Monday sometime to confirm my recollections of ASTM or NACE protocols. I'll put it on my " to do " list, but may take a couple of days. There were two ASTM documents, ASTM A967 and A380 ( Standard Practice for Cleaning, Descaling, and Passivation of Stainless Steel Parts, Equipment, and Systems ).

An abstract of ASTM A380 that I have notes that passivation is “the removal of exogenous iron or iron compounds from the surface of a stainless steel by means of a chemical dissolution, most typically by a treatment with an acid solution that will remove the surface contamination but will not significantly affect the stainless steel itself.â€

[HW Mueller]

Bruce, I skimmed over the link you gave, except for the dichromate (which they obviate themselves by suggesting 50% HNO3) it seems to contain about the same info as the other refs. above. It is interesting that right in the beginning they make a clear distinction between cleaning and passivation (I think it was Mark who gave a good explanation of passivation, no need to repeat it).