When using buffers you have to be careful about the amount of organic in the mobile phase. For this reason you have to be careful running gradients since there is a point where the buffer may start to precipitate. Phosphate buffers can be particularly problematic. Be sure to flush the system with a high aqueous (95%H20:5% ACN or MeOH) mobile phase (without buffer) after use. I would not recommed 100% water as this can have potentially damaging effects on RP columns. If needing the column for continued use (within a few days), it is a good idea to program in a flush after the sample sequence is complete with a high aqueous mobile phase as mentioned above without buffer at a very low flow and to prevent any type of growth in the system. For longer term storage, once the buffer has been removed, flush with 100% ACN or MeOH.

Although it depends on what pH range you need for your separation it might be possible to use a different type of buffer such as ammonium acetate, adjusted with acetic acid and ammonium hydroxide, which I know can be used to hold a pH at 4.5 (or in that range) and is less likely to form problematic precipitates.

SEC columns can be stored in aqueous (at least the ones I have used) but you want to be sure to include some type of bacterial growth inhibitor such as sodium azide at a conc of about 0.05%. Be sure to check though with the column manufacturer.

Best option is to use plenty of warm water and flush the system without the column. Ideally best to carry this out.

If using salts always make sure you are continuously flowing (even overnight) and this will limit that amount of salt precipitation.

Please consult the manual supplied with your column (or available online at the website of its manufacturer). Generally, if your sattionary phase permits, you could wash the chromatograph by pumping an aqueous eluent compatible (miscible well) with the eluent you using for your routine separation (but do not leave the water-containing eluent in the HPLC for long--finally wash the system with isopropanol or methanol). Or, if your HPLC pump is capable of gradient formation, wash the system starting from highly aqueous eluent and finishing by pure methanol or isopropanol.

Hello Zheng

Simply rinse your system with an eluent containing a high degree of water (buffer-free, of course) on a regular base, say, after every sequence. You might automate it using a method doing so for a while and eventually re-equilibrating your system afterwards. In heavy cases you could apply warm water to get use of precipitates, but at best you shouldn't let it come to that.

Concerning your pump, use back piston flushing; in case of high buffer content on a permanent base. If your pump doesn't provide this option already, think about an upgrade. On the long run it's cheaper than permanently replacing pistons and seals.

Try with warm water at low flow rate 0.2-0.5 ml/min, but first remove the column.

I can suggest you 2 options:

1\ Continue to fed mobile phase for quite long

2\ invert the column for some hours with mobile phase supply

Hope this helps

Thank you all for sharing the answers. Is there any auxiliary devices that can be coupled with Pump system to help the removal process. I would think decreasing the flow rate is another good approach to refresh the HPLC system since the normal flow rate (1 mL/min) caused the damage of the flow cell in FLD with the limit pressure of 300 bars.

After every sample run, make 1-2 100% Acetonitrile injections. Create a method in which you have continuous gradient change every 4-5 minutes. Purging the whole system (without column) with Iso-propanol will help too.

Flush the system with water at 40-45C at a flow rate of 0.5ml/min for 5 hours at least and you can do this with the column on and.or without the column attached.

Use warm water or remove connections and use ultrasonic bath.

Hi Zheng!

Unfortunately, the most common problem I´ve seeing during my time on HPLC maintenance is due to buffer salt precipitation and the only certain way to avoid that problem is time consuming!.

A sudden salt precipitation into the tubes or column is quite rare but anyway you should always check before (in a test tube) how much organic solvent can be added to your buffer before precipitation (in general you just do it once when you set the protocol). Never flush a buffer line with solvent or vice versa, always intercalate water.

On the other hand, to avoid “chronic” salt accumulation in the lines, filters and fits you should spend some time on properly filtering your mobile phase (even if you use HPLC solvents and on line degasser) and after working, purge the buffer line with filtered water and wash your system with 20 times its volume (as an empty cylinder) with water:solvent 95:5 (don´t use 100% because you damage your column). Then, purge all the lines with solvent.

Never let the buffer flux to stop (even if it is diluted) and never let buffer lines unused overnight (it is better, if you finished late, letting everything at very low flow and then “first thing in the morning!”)

Spend some time taking care of you HPLC and you will see better results and less troubleshooting!!

Well, I hope it works!

Cheers

Luciano

After HPLC runs (with Size exclusion superdex column) in a Tris-HCl buffer, I pump water like hell through my system for a few hours, do the same with 100% methanol for about 3-4 hourse to be safe (after cleaning up my column appropriately, obviously) and then simply leave the system in methanol. This leaves a very minor chance of any salt in the system!

Run a high water content flow thru the system, if need be remove the column and flush with 100% water

Hey Luciano,

Thanks for your nice response. That seems to be a good way for removing salts out of HPLC system. But I do have a concern about the mobile phase. As you may know, the use of buffer as the mobile phase is to keep the condition in a constant pH condition. Normally, after filtering the buffer, a unexpected increase on pH would be definitely observed. This may affect the experiment, especially the buffer related study.

If the column is C18 column; Wash the column with 100% methanol with low flow rate (about 1 ml/min) for long time (3-7 hours) until you have a clear baseline on the system. If this did not work try flushing warm water (40 ˚C) for long time until you notice a clear baseline on the system, this would dissolve the salt crystals in the system.

Also, it is important to wash the column with a solution of 80% methanol and 20% water for about 30 min after finishing the runs for the day, this would maintain the column and the HPLC system for long time.

NOTIC: methanol is an extremely dangerous chemical, it is important to read about how to deal with it.

I hope this would help

Only for the tubes and pump, but without the column.

If you use in your buffers Na, K or ammonium salts, you can use aqueous acetic acid (1 % v:v) and you need to pass at least 500 mL. You decide at very low flow rate (0.2 mL/min) and more time, or higher flow rates and shortener times.

The suggestion of Dr. Angelov of warm water is good alternative.

When using buffers you have to be careful about the amount of organic in the mobile phase. For this reason you have to be careful running gradients since there is a point where the buffer may start to precipitate. Phosphate buffers can be particularly problematic. Be sure to flush the system with a high aqueous (95%H20:5% ACN or MeOH) mobile phase (without buffer) after use. I would not recommed 100% water as this can have potentially damaging effects on RP columns. If needing the column for continued use (within a few days), it is a good idea to program in a flush after the sample sequence is complete with a high aqueous mobile phase as mentioned above without buffer at a very low flow and to prevent any type of growth in the system. For longer term storage, once the buffer has been removed, flush with 100% ACN or MeOH.

Although it depends on what pH range you need for your separation it might be possible to use a different type of buffer such as ammonium acetate, adjusted with acetic acid and ammonium hydroxide, which I know can be used to hold a pH at 4.5 (or in that range) and is less likely to form problematic precipitates.

SEC columns can be stored in aqueous (at least the ones I have used) but you want to be sure to include some type of bacterial growth inhibitor such as sodium azide at a conc of about 0.05%. Be sure to check though with the column manufacturer.

Flush the HPLC system with hot water(80-100°c) through all channels using a union in place of column with 5.0 ml/min flow for more than 2.0 hours and also use 50% Methanol after water flushing which also helps to remove unnecessary organic

content. carrying this exercise immediately after completing analysis would give a trouble free operation also there are chances for eliminating carryover problems.

If the buffer crystallizes inside the column you can try dissolving it and flushing it out but there is a chance that column is "toast."

With regards to your comment above: "Normally, after filtering the buffer, a unexpected increase on pH would be definitely observed." I am surprised that filtering would change the pH unless some components of the buffer were not completely dissolved.

Wash your system with a mixture of solvents similar to that employed for running your simple. Do not leave salts within your column.

To remove salts from HPLC simple and effective solution is wash HPLC with hot water (80-90C) followed by MeOH etc.

Zheng, What buffer are you using and what pH do you want your mobile phase to be? Different buffers have different pH ranges where they are most effective. Another consideration is to make up a stock solution of your aqueous buffer at for instance 10 times the concentration. When needed dilute appropriately to the working concentration with DI water and filter. Double check the pH if necessary but it should not change by any significant amount. If a slight shift in pH effects your chromatography then you may be to close to the pKa or isoelectric point.

I'd suggest using triethyammonium bicarbonate (TEEB) buffer, especially if your compounds are acid sensitive (like DNA, RNA oligos and NTPs and dNTPs). This buffer would never clog anything and is very easy to remove on speedvac or lyophylizer afterwards.

Flush the HPLC system with hot water(80-100°c) through all channels using a union in place of column with 5.0 ml/min flow for than 30 min and also use 50% Methanol after water flushing which also helps to remove unnecessary organic

content. carrying this exercise immediately after completing analysis would give a trouble free operation also there are chances for eliminating carryover problems.

Generally many organisation follow this system and also make a SOP for this thing and every weekend they make this schedule and my personal experience if we follow this then never any problems comes with salt.

An extra pump can be be applied to suuply the mobile phase to the detector while the column elution swithed to waste. This is specially helpful when high salt content samples such as plasma or blood samples being analysed.

Thank all of you for the comments I would ask my students tomorrow to learn by hard before they will touch again my beloved FPLC machine;) The one I'm using in my Institute is general use, so people did already all the crimes there. Since I'm not afraid to repair it again, colleagues ask me as a first what to do (or rather could you do it). I never know what is in the system, since logbook is not popular there. First I'm doing is the temperature increase by switching of the cooling system. When my FPLC starts working in the refrigerated cabinet it is warming all the system by them own heat. The most cases of damage were the sucrose gradients made in FPLC instead the proper equipment. The unknown idiot is doing it usually by manual runs until the pressure increases over 3 MPa is a default alarm limit (fortunately didn't found yet, it is possible to change the limit). Crystallization of the sugar occurs due to the temperature decrease from RT to 4 deg. is there. I had the same situation with the phosphates ago. Sugar left in the system is a perfect medium for bacteria. To wash everything off my beloved is 8M urea (Attention! is precipitating below 20 deg.) gives nice UV peaks (rather Himalayas) at first few ml of the wash. Also nice is 1M HCl or 1M NaOH done occasionally, since I'm running milligrams of crude mammalian extracts, there is always some contaminants left in the system.

Buffers are in almost all mobile phases, usually there is no problem to use till finish the samples, then wash column, unless you switch from almost 100% aquous phase to almost 100% organic phase. In this situation, salt crystalisation forms. Avoid sudden phase change.

Prime pump with water. Flush water through column and entire system for a couple hours. Also have water wash kit to flush back of pump seals to eliminate any salt residue. Wash exterior of fittings with water as wel.

I had used sodium sulfate buffer for the HPLC. After completing my work I tried to wash the system with hot water (80 C), even after that I observed some precipitate in the pipes.

How can I remove the precipitate from the pipes?

How can wash the column after using with sodium sulfate buffer?

Hey Vinyas Mayasa,

I would suggest using a large amount of water to rinse out the tubing of the whole system with a low flow rate. That would help to remove the salt from the system. Try not to connect your FLD or DAD when washing out the tubing since extremely high back pressure would damage your detector.

If you need to run your sample with buffer as the mobile phase, probably you may need to get an accessory device for your pump system.

Hope this would help you solve your problem

One approach is to remove the salts before contaminating your system. Thermo has a lines of IC columns that can be used inline-it is actually an ic package . We also have used BIORAD IC columns as a precolumn to prevent ion issues. Running HOT water can dissolve many of these in column, but it is a slow process!