This is common for chromatographic columns due to contamination / precipitation from sample and/or buffer. As stated in previous answer using in-line filters is a good strategy to increase your column life and avoid back pressure problems. I am sure you must be filtering your sample and buffers before loading on column. Please refer to the column manual, you can find the cleaning procedures when columns is giving high back pressure.

you can connect the column as reverse position for cleaning purpose for ten min using 100% metoh or ACN. Then reconnect it as ussual. If you have guard column take it out and put it in sonicator for 10 min. If not these and above ones are enough, you may find another similar column to check as to whether system upto columns is problematic or not. I have a pdf copy of solving HPLC problems, if you want i can send you ([email protected])

If you have run a buffer based mobile phase, wash with 100% MeOH for 10 minutes followed by 100% water for another 10 minutes. This will remove all the polar debris on the column which accumulate with time. Alternatively you can flush with 50/50 Water/MeOH followed by a flush with ACN to get a clean column.

Take care that after used of buffers you don't flush your column with a less polar solvent like ACN as this precipitates the salt in the column raining the back-pressure.

Also check for blocking of transfer capillaries as that can also increase the back-pressure.

Yes, I know that loading samples in general leads to increase of back-pressure, but I meant, whether is it more serious if I run many and many samples in a row without giving pause to the machine and column, because it seems so to me.

Depending on what type of material is building up at the column inlet a number of things can happen. The obvious effect is that the back pressure increases - by its self this is not going to affect your assay provided the pressure does not exceed the set upper pressure limit. Though generally the higher the back pressure - the more stress on the mechanics of the pump etc - so it is generally best to remove particulates before injection. I use a 0.2 µm PTFE syringe filter - whatever type of filter matrial you use, you will need to check it's efficiency i.e does it retain any of your analytes?

The second effect is peak shape. The material that accumulates at the column inlet may interact with the analytes, usually causing peak tailing and in exteme cases causing peaks to tail into each other.

So in a nut shell - best filter the samples unless you are prepared to accept short column life.

Cheers

Neville

I'm actually filtering samples through 0.22 um Costar filters (however, recycled). The same applies for mobile phases (buffer/MeOH)

That's a good start.

What is the mobile phase buffer?

Many reasons. Mainly:

1) high pressure causing inhomogeneous packing density, hence breaks in particles (especially those above 3µm) and lower column permeability;

2) unfiltered samples: suspended solid can accumulate at the inlet filter or, if it is smaller, inside packed bed;

3) unfiltered mobile phases (same concept as above)

Increase in the back pressure is due to contamination in the column or guard column. Regarding your query that you run many samples in a row without any stop. The only solution is to put a blank run after every three runs using the same gradient conditions. Hopefully this will solve your problem.

There is a normal accumulation of microscopical particles present inside any mobile phase slowly blocking the inlet frit...hence the observed pressure increase. Proposed solution are OK. I usually disconnect the column return it and reconnect it only to the high pressure side (not to the detector side) and flush it with methanol for five-ten column volumes. Flushing backward has a chance to push some particle stuck on the frit liberating it. Next you put your column back in the indicated normal way and check for back-to-normal pressure drop.

First change your frit and if it persist change the column. I did that and it worked for me. The back presure changed from 8000 to 4500.

In my case, after about 200 runs or more, the pump will clog.....but it´s all related to maintenance and cleaning and contamination all related to this pressure.....just one idea in your mind is that you have to FILTER with syringe tip filter 0,45 or 0,22 the stuff you are injecting in HPLC....bye....

While setting the program include column washing in between each sample load.

Could be a lot of things. Most modern LC's have a filter to purge which is in line with the column. Frequently this plugs up. Then there is the precolumn (I assume you use one) . This also plugs up and needs to be changed. Finally there is the column (hope this isn't it). The best way to diagnose each of these is to remove or replace these one at a time and monitor the pressure.

Also columns CAN be regenerated per manufacturer's guidelines.

To my experience, especially with continuous HPLC separation during several days, you can "blocked" your column, ie if mobile phase and sample when mixed can precipitate.

Also if you use a guard column, it can be blocked more rapidly than a higher size column.

In general, after long term use, it's better to wash the column before continue (ie pure acetonitril if C18).

Increase in colum pressure can be solely physic: blockage on the flow.

I hope it's usefull for you.

Best regards

Didier J

One general reason for increase of back-pressure might be clogging of column, but I am not sure about that without much information on your case...

My column is Zorbax Eclipse Plus C18 (1.8 micron; Agilent). Buffer is 15 mM ammonium formate. In each gradient I go to 100% of Methanol and then re-equilibrate to 20%, which are my starting conditions. It's not recommended to turn the column.

Agree with all comments especially, Alan Bethod's. If the pressure is not coming from the column (if the pressure is still high even after disconnecting the column), the pump check valve could be dirty and may need to replace it. You need to systematically troubleshoot to see where the pressure bulidup is by disconnecting column, guard column or tubing. Do not change the frit, unless you know how to do it. Otherwise, you can loose the packing material.

If it's just an acute problem you can try "unclogging" your colum and LC-pathways as already suggested by the others. However, if you are concerned about the general lifetime of your colum with this particular method (e.g. colum keeps clogging after 200 samples instead of 2000 as with other methods) you might want to consider changing your method. that includes sample preparation as well as LC conditions like switching from protein precipitation to solid phase extraction, changing your LC buffer/pH/organic portion etc. I once had a similar issue with a method for colchicine/demecolcine, where my colum kept clogging after like 200 samples. It took me several months to finally resolve this issue when I switched pH from slightly basic to acidic, when I realized by chance, that my old clogged colums still worked well for my colleague, who was using acidic conditions in his methods and backpressure even decreased. That way I was even able to rescue my old colums.

Good Luck!

First of all, work systematically through your system to see where abnormal pressure might come from.

Generally, when you use buffers, especially when the gradient goes to 100% organic solvent, it might be a case of salt precipitation. Flush with water, low flow, for about 30min. Nothing bad will happen, even if the column is C18. Also, if the issue is clogging due to sample precipitation at the head of the column, turn the column around - again, nothing will happen. mind you, use it in reverse just for flushing, not for running samples. as other people have mentioned, use a guard column to save your analytical column. if you already use one, just change it or clean it. In the latter case, ultrasonication in isopropanol works very well.

Okay, its obvious that it's most likely due to some blockage somewhere as most others have alluded to...my question/suggestion starts from the the very basic (which you probably do already). Do you filter your sample and eluents before running? If not then you should.

Also, go through your whole system to see if there are any areas that could cause backpressure...e.g a line that has a smaller diameter than it should and things like that.

How is your sample preparation? Do you use SPE for your extracts ? Depending on the complexity of your sample matrix, good sample prep is mandatory. Generally biological samples are more "cleaner" than food samples.

You are using 1.8 micron particle size ? What is your flow rate like? And how old is your column already ? Have you tried to flush your column with MeOH overnight at low flow rate?

I would like to add another posibility. Maybe it's because some of the particles from the column just move a little cloging the column, it's a standart method to prepare the columns with pressure so that this won't be a mayor problem, but it' may still happen given enough time.

Cleaning the column each n samples will help with the contamination and if your system it's not isocratic you should also check for precipitations, but sometimes just letting it rest so the particles can move a little to unclog the column may be a solution. And if that doesn't solve the problem maybe it's time to change the column.

I have not had that problem in HPLC yet but in MPLC it can be a problem if you don't pack well enough your column.

1. If the baseline falls under the selected threshold, uncontaminated solvent is directed back to the solvent reservoir.

2. Shift in the retention times that are caused by increase of backpressure in the column.

3. Increased backpressure may be due to contamination in column.

4. Viscosity of mobile phase also the reason.

Below link may provides the information abot troubleshooting

http://www.waters.com/webassets/cms/library/docs/wa20769.pdf

Are you using a guard column? and if yes, after how many injections you normally replace it? The high back pressure might be caused by contamination of the guard or even the analytical column.

Do you also filter your liquids for separation? small particles especially when you use an buffer in the gradient with high salt content can cause clogging and increase back pressure.

Since the hplc system works for several days non stop as you mentioned. You need to check for any clogging in the system and check the lines, the pump,contamination or parital clogging of the column. You need to read on reasons causing increase of back pressure in hplc trouble shooting toutorials.

all the comments are corrects, I want to add some based on my experience, one of my method require me to equilibrate my buffer for 1 day and if I did not do that i'll experience back pressure during running. another is you cannot simply change directly from buffer system to 100% organic solvent when flushing or gradient because it can cause precipitation on your system. and I also agree with pH of the buffer, you have to check your column pH stability, there is limitation on the pH where your column can easily broken if you use beyond the limit.

This is an acute problem, the reason could be e.g. a bubble in the system, or an overload of the column. try to rinse it in opposite direction or with clear isopropanol

One of the major reason could be precipitation of Buffers .I do not agree to put the column in reverse direction to clean. If a column is used for a fairly long time a void at the end is generated. This will get disturbed on reverse ruining and splitting of peak head can occur.

Bottom line is :

1 Clean sample preparation and injection of clean sample

2 Check for the precipitation on Plunger heads when using a gradient with buffers

3 use guard columns

I agree with Mr.Ajmal Nasir,

and Mr.Tomáš Hluska one more thing is also left in all above discussion that are you using Mobile Phase Sparger? If sparger is there then do you know the porosity of your spargers? If you have filter the mobile phase with 0.45 µ and spargers you are using less than 0.45 µ it may also get choked. And just washing of these spargers can resolve this issue.

I agree with Markus most of the time if problem persists up-side down can be the best option

Dear Saima Memon,

Please read the comments of Markus twice and then agree with him. He is not talking about to reverse the column for analysis. He emphasizes to wash the column after detaching it from detector and pass IPA from reverse side in it.

Regards

AMIR HAIDER

Remove your guard column and sonnicate in MeOH.

or wash your your column with hot (70-80C) distilled water. then wash with MeOH. your problem will be solved of back pressure.

Try it

gudluck

:)

Are you getting back pressure that stops your system, and then upon restart, your column runs smoothly again, or, does your column cause an increase in back pressure and continue to increase after a few days whlie using the same column. If the first, then that is normal, get used to it, its most likely precipitation of the buffer or protein in the mobile phase. If the second, then your column is getting clogged up. You either need to get a suitable washing method that is suitable towards the re-usability of your column, or use a buffer in the purification that is suitable to the re-usability of your column.

I think the reason is the composition of gradient and the 1,8micron of particles of stationary phase. You can use a bigger dimensions of particles (3,5; 5microns..) or to use 2 kinds of mobile phase in gradient (e.g. phase A with 15mM ammonium formate - MetOH(90:10) and a phase B with 15mM ammonium formate - MetOH(10:90) )

Column back pressure can increase if the stainless steel frits in the column becoming clogged or if there is deposition of material on the stationary phase. The former will occur if you do not filter your mobile phase or if sparging gas is introducing particulate. The latter will occur if your mobile phase, or samples, contain substances that are capable of precipitating or aggregating on the stationary phase, as is common in separating protein digests or polymer-containing solutions.

In addition to back pressure increase, I would like to share one of my experince. I was using HPLC system Agilent 1100 with manual injection. There was a PTFE frit within the purge knob. In this system, screw off the purge bulb and take out PTFE frit in it, white in color 1 cm in length, then replace it with new one if you have. This frit is very important for high pressure and avoids the mobile phase leakage through the purge bulb in manuel systems (probably others sholud have similar way too). Best way you can, the lesser evil, follow instruction manuel of your HPLC system...

You are running samples non-stop. How clean are your samples and how often do you change/clean your filters?

Sometimes the back pressure could also be due to block in the connecting tubings, which may be due to dirty samples or solvents containing particles. This may also happen before the column. Sometimes, if we are using buffer, and after running, the lines are not washed properly, or if the buffer is left overnight in the system, precipitation may take place, especially in cold weather or temperature.

If you have run a buffer based mobile phase, wash with 100% MeOH for 10 minutes followed by 100% water for another 10 minutes. This will remove all the polar debris on the column which accumulate with time. Alternatively you can flush with 50/50 Water/MeOH followed by a flush with ACN to get a clean column.

Take care that after used of buffers you don't flush your column with a less polar solvent like ACN as this precipitates the salt in the column raining the back-pressure.

Also check for transfer tubings/capillaries as any blockage over time can also increase the back-pressure.

Are you sure that is the column that gives the back pressure? Did you test the system without column? Because some time it is the online filter in the injector that clog. What kind of HPLC system are you using?

Second point, i use the same column and it is recommended to filtered buffer with 0.2µm filter specially when you have salts in you buffer !!!

That true is not recommended to reverse flow in the column but only the running flow rate, if you using low flow rate with low pressure, it is not a big deal.

So just for next time verify the pressure without the column and keep this data, not only column gives back pressure.

How long have you used the column for? you need to wash it out after every run and ensure that the pressure prior to injection is stable. If the mobile phase show a stable pressure and your test sample increases the pressure to an extent that the system shuts down, you probably not filtered the test solution properly. Columns become unstable for a variety of reasons, the number of runs, the washing of columns , the mobile phase, the test solution and only you will be able to pin point the actual cause.

This thing happens while using the buffer in the mobile phase

Increase of back pressure is mainly due to dirt in your final samples-fisrt thew dirt will be collected in your filters, pre-column and finally on your column; if you change the filters and/or the precolumn you will see the difference; anyway a samll increase in pressure is normal depending on your samples and doesn't mattre as long as your peaks are fine...

The likely cause of your issue would be an accumulation of particulates in your sample. This is particularly a problem in biological samples that would have initially contained a significant amount of protein (if the precipitation step is not efficient/complete). Referring to one of the suggestions above that advises you to run 100 % water through your column to remove polar molecules, this is a bad idea if you are using a C18 (ODS) column as it could result in phase collapse (which will ruin the column). I would suggest a minimum of 5-10 % organic content on a C18 column.

Your column head may be clogging

If running a non-stop sampling program, you should build in an automatic hydrophilic /hydrophobic rinse or flush (5min could be enough) after a number of samples. Most chromatographic software allow this

The best practice is to use a guard colum, and properly filter the sample through 0.45 micro meter pore size filter, and also recondition the column for every run. If your are using buffer in the mobile phase then perform the run in isocratic as well in the gradient mode and you will feel the difference

when the column will block it will so back pressure, Do one thing, change the side of your column i.e. opposite direction at same flow rate

There are many reasons due to which column back pressure increases.

1. prolong use without washing.

2.Buffers

3. Debris in the mobile phase

4. particulate matter from wear and tear of pump seals.

and many more.

So, if you want to prolong the life of the column then

1. you have to use guard column to protect your main column.

2. Wash the column in regular interval say like after every 50 injections

3. Use filtered mobile phase always.

4. Use simple mobile phase as possible like water:methanol. if not possible then use buffers prepare in mili-molar concentration.

5. Always monitor rhe pressure of your system and keep record of the same.

if you do this you can save the your column and get rid of column back pressure as well.

There are many reasons which cause the clgging of the column and thus an increase in the back pressue and all these issues were discussed in the replies of other scientists above.

Always use a guard column for the long life of the column and filter the sample through 0.45 micro meter pore size filter paper; Additionally, if you are using a solvent system containing buffer then carefully wash the column. Besides, there are so many issues which are mention above.

My experience despite filtering our samples is the the blockage of the frite can cause increased back pressures. Also, accumulation of particles in the column or exposure of the internal surface of the silicon in the column results to increased back pressure. In any case we had to change the column most of the cases or change the frite.

Probably column head is clogging

Dear all, I've really appreciate the previous comments, they are very interesting.

I've to add a method for recovering a column having high back pressure. Someone wiil change the direction of the flow mounting the column back to front, but I prefer another method. As Theodor Kaufmann wrote, the accumulation of particles will occurr in the head of the column, then we have to unscrew the cap, remove a layer of material for about 1 or 2 mm and substitute it with a paste made using the same compound plus a bit of CH3CN. After that we only must screw again the cap and start for cleaning the "new" column.

That's possible only if the column has two unscrewable caps at its extremities; I worked on HPLC from 1988 till to 1998 and then I'm not aware regarding todays' items.

Regarding the frites, I remember they were washed using a solution of HNO3. The life of the column was prolonged two or three times.

Good Work and a Happy New Year!

It may happen due to following reason

* Poor sample quality ( may contain suspended material or some fiber particle)

* all component of your compound may not fully soluble in your desire mobile phase [ for that some method required some specific solvent column washing to clear unwanted, probable less soluble and high retained compound to maintain column pressure and its efficiency]

* Buffer solution create same problem [ it may be due to minor leakage or improper solvent mixing at low flow rate (due to pump inefficiency) ]

* if you use are using buffer or mobile phase for long time from the same reservoir, mobile phase have some algae growth and it create problem in the term of back pressure.

i experinece this problem always after hundreds of injection; but today for my new coloumn the backpressure increases in  a very abrupt way, i thought the problem was in the coloumn, but once i change the port filter, which i used to insert it in my mobile phase the pressure goes back to normal, it should be sonicated with 6 molar nitic acid then methanol then distelled water, try it i wish it will help.

best regard