Hi, I agree with Hediye, What was the last mobile phase that you used? If non polar (CH2CI2), I think try first wash with CH2Cl2, than CH2Cl2: MeOH (50:50), than, MeOH, MeOH: Water (50:50), Water; but if you used (semi )polar solvent than I did first using water, water:MeOH (50:50), MeOH, MeOH: CH2Cl2 (50:50), CH2Cl2; CH2Cl2: MeOH (5O;50), MeOH, MeOH:Water 50:50 than Water, I hope it can work. If it is still does not work than I will reverse the direction of the flow with those above solvents

Hi Sukanta,

Are you sure that it is the column and not one of the fittings?

First, try and trace where the back pressure point is by systematically removing the components, one at a time.  Is this a reverse-phase HPLC column?

If you confirm that it is the column that is causing high back pressure, try to clean it by running high organic followed by 50:50 organic and water and finally water.  During thr organic washes, your back pressure should be lowest, compared to aqueous washe back pressure.  Since most columns are flow direction dependent, I would first  try and clean it in the direction that it was packed. 

Best,

Hediye

Hi, I agree with Hediye, What was the last mobile phase that you used? If non polar (CH2CI2), I think try first wash with CH2Cl2, than CH2Cl2: MeOH (50:50), than, MeOH, MeOH: Water (50:50), Water; but if you used (semi )polar solvent than I did first using water, water:MeOH (50:50), MeOH, MeOH: CH2Cl2 (50:50), CH2Cl2; CH2Cl2: MeOH (5O;50), MeOH, MeOH:Water 50:50 than Water, I hope it can work. If it is still does not work than I will reverse the direction of the flow with those above solvents

Often the high back pressure comes from parts in front of the column. As example, if you run mixtures of aquous and organic eluents, micro crystals can form on along term in the system and at some point block it and cause pressure increase , e.g. the needle seat of the autosampler or in connecting fittings. Also, if you do not properly filter the eluent, small particles might enter the system and cause pressure increase as well. To check for this and clean your HPLC, remove the column and open the system at these sensitive points one after the other and check the back pressure. If it still high, increase flow rate. Eventually you are lucky and can flush out the blocking agent. Otherwise replace tubings and fittings by new ones. sometimes you can clean them by microwaves.

If it is really the column, which is contaminated, I have to say that we never managed to rescue the full separation efficiency and decrease backpressure to normal by column reversal - despite we tried all kind of washing steps with column in both directions.

Generally, we run our ystems with pre-columns and we routinely include maintenance intervals, where columns are equilibrated and cleaned by stepwise pumping of different organic and aquous mixtures. Before you try this, check the range of eluents your column tolerates as not all columns stand such a treatment.

I agree with the previous responses. First ensure it's your column then if it is reversing it sometimes works. Also remember that MeCl2 is not miscible with water so you need to flush with IPA then MeCl2 and then to return back to aqueous flush the MeCl2 out with IPA. IPA also produces a high back pressure so a slow flow rate is to be expected. I have had some success reversing a column and flushing with DCM, ethyl acetetate and cyclohexanes. 

Thank you all! Certainly your inputs helped! Column reversal is the last thing that I would consider doing. I would try it out only after exhausting all the other options but was curious to learn from others experience.

@Hediye and Christoph: I did remove the components individually and have checked their effect on the backpressure and am sure that it is the guard column and the column (back pressure is zero if I remove these two components). The column that I am using is a C18 column. Thanks Christoph for sharing your experience with the decreased separation efficiency on column reversing.

@Hediye, Gunawan and David: Thanks for the inputs on the mobile phase composition and compatibility. It helped. It was good to know that David got some success by reversing the column and flushing it with DCM. I shall try it if every other thing fails. 

Once again, thank you for the inputs! Will keep you posted on how it went.

Best,

Sukanta

Sometimes the only way to clean a C18 column is opening it. It´s easy, just open the inlet side, remove the initial dirty phase and refill it with a clean c18 phase from an old column. You must clean the inlet metallic filter whit HNO3 50% in a ultrasonic bath.

All suggestions are good but after you systematically ascertain the cause of the back pressure: reversing the back washing works for me with C18 using 50:50 Aq + Org

Most importantly, obtain the cleaning instructions from the column manufacturer. As intimated above, some columns are no longer built to run either direction. The front frit may be different from the rear. Also, columns are packed at such high pressures now that if you open the column, the bed will never be the same. Also, John Dolan and other experts will remind us that columns are consumables and may not be expected to last more than several hundred injections in some cases. It depends on your needs and your budget. If at all possible use a precolumn next time. Inexpensive cartridges are available that match your stationary phase. Catch the gunk on the precolumn, which can then be replaced for as little as 20-30 bucks.

I agree with many of the suggestions above. Try cleaning with a range of solvents of varying polarities first. If cleaning with solvents doesn't really work. Like Sidney Pacheco suggested, I would suggest carefully opening the inlet by unscrewing the nut and ultrasonicate the inlet screen or frit in methanol to remove fine particles which may be causing the blockage. However, before you do it, make sure the column is flushed with an appropriate storage solvent first. if it is C18, then flush it with HPLC Methanol prior to opening the inlet. Refill any void in the inlet with similar packing material, making sure to wet each addition of packing material with droplets of Methanol. Dry material expand with addition of methanol. Once the void is filled, you can put on the clean frit/screen and screw tight the nut again. Hope this helps. Sometimes, it is better to use a new inlet screen/frit than reusing the old ultrasonicated one because some of the very minute particles doesn't really come off even with ultrasonication..  

Yes, you can backflush the C18 column to try and see if that corrects the issue. It should be washed in either direction with a solvent mixture that you know will fully dissolve all of the compounds you have been injecting into it.

Once the problem has been fixed, or the column replaced, please add an appropriate wash step to your HPLC methods. If you are running in an isocratic mode, you need to follow each method with a gradient which utilizes a solvent which is much stronger than the one you use in the method to flush all late eluters and bound material off the column. If you are using a gradient method for analysis, then here too you need to either end the method with a stronger solvent than used to elute your compounds or better yet, add a second separate method (preferred) after the analysis method which flushes and washes the column down after each use. Failure to wash and flush the column each time often results in the column being fouled over time. This is considered basic maintenance in chromatography.

Other tips would include:  always filter your samples before you inject them. Use fresh, HPLC grade solvents, high resistance filtered water, and replace any system filters or frits (bottle pickup or internal frits) on your HPLC on a regular basis.

Here are two articles on the topic which may be useful to you.

• "Two Common HPLC Problems and their Causes (Sudden changes to either the HPLC Backpressure or Peak Shape) " ; http://hplctips.blogspot.com/2013/12/two-common-hplc-problems-and-their.html
• "HPLC Mobile Phase Filtering & Solvent Inlet Filters" : http://hplctips.blogspot.com/2012/09/hplc-mobile-phase-filtering-solvent.html