I agree with All previous answers

You have large molecules stick to the column inlet

I recommend to invert column elute with methanol for 1 hr inject 50ul dmso then 50 ul chcl3 x 5 times elute with acetonitrile then gadient to 100% water for 1 hr then 50:50 methanol acetonitril

All elutions at 50c

Bri g your column to correct direction wash and test the pressure using 100% acetonitrile

Yes I did .

I agree with Angel, prevention is the best medicine. I would recomend a combination of different sample prep approaches, such as protein precipitation + SPE. It can be sometimes imposible to clean a column after multiple plasma injections. Oh, and the use of a guard column is strongly recommended.

You must precipate the plasma proteins before injection of the sample to hplc. Acetonitrle is known to cause protein precepitation. So do not inject the plasma sample directly into the column. There are some coulmns that you can directly inject plasma samples it is known as pinkerton columncheck it out.

I use anion exchange columns and in such a case I clean them with 1% HNO3 in methanol (10-20 ml at most). And I never use them without precolumn. Be also careful about losses on the column after plasma injection - I found out that all arsenite is ireversibly bound to the column after a single plasma injection.

The problem is not with the sample preparation but with the plasma and most of the time your mobile phase may have buffers. So, I think if you wash your column after complete analysis with 100% water for at lest 45 min. and then 15-30 min with methanol. It will be fine. Additionally you have to filter the samples before each injection.

Are you using a guard column? If you are, you should change it. If you are not or you but the problem is in the column you can try flushing the it with several volumes of the strong solvent of the mobile phase (usually B). If the problem remains you can try connecting the column in the opposite directions (backwards) and flushing it. If you try this, you need to disconenct the column from the detector to avoid any damage caused by the debris.

since you mention using phosphate buffer, note that above ~~80% acetonitrile and greater than 10 mM phosphate buffer, the phosphate salts starts to precipitate.

I advise you to take purification techniques as spe, zip tip, precipitation.

You need to filter the sample prior to HPLC analysis with 0.45 micron filters and then with 0.2 filters

Read this pdf for the best cleaning in your column.

Personnaly, I will suggest to use online filter 0.2 µm that are easily changed after several injections. Precipitate the proteins with methanol or acetonitrile. Be carefull to not use too much acetonitrile with phosphate buffer as Syed pointed out. I am not keen on guard column usually this is a waste of money.

Use 2-3 harsh MeOH: H2O gradients; 80-20 to 20-80 in a span of 15-18 minutes. For me the best mobile phase (least carryover and precipitation) was MeOH-H2O 80-20, with wash in between as stated above. ACN h2o with buffers, did create problems for me.

In my opinion, sample preparation must be performed before injecting to your HPLC system. It a good idea to prepare sample in the mobile phase or a weaker solvent than the mobile phase and must be sure sample, solvent and mobile phase are miscible to avoid sample or buffer precipitation. Finally, filter sample with at least 0.45um membrance before injecting.

Sample preparation is vital here. You need to precipitate the proteins and clean up the sample as much as possible. If you have precipitated the proteins and removed a lot of the matrix you will still get deposits at the top of the column. If you can remove the frit then that could be sonicated and cleaned. If not then i would suggest that you consider rinsing the column with water initially. You can then increase the organic modifier to the recommended storage conditions for the column and evaluate the back pressure. remember too that buffer salts may also collect at the top of the column if there are changes to the mobile phase during long runs.

Thanks for all of you, I figured out the problem was more simple than I expect. Since I am using stainless slip free connector, the blockade was in that part. I sonicated the slipfree connector and every thing returned to normal.

Plasma sample or debris contains proteins. They are only soluble in water. Organic solvents mostly ineffective to clean plasma material.

When you performed blank, check out the intensity of plasma peaks. It will confirm the effectiveness of cleaning method used.

If blank sample contains lot of plasma peaks, there is need to improve cleaning method, use guard column and take other measures to clean the sample.

Now, as blank contains less plasma peak. Proceed for analysis. After analysis you have to wash the column. Replace the portion of buffer with equal portion of water. Keep the organic content same.

Then wash column with high proportion water, use high temp if HPLC system contains column thermostat at 0.2 to 0.4 ml/min flow rate.

Still the plasma peak remains, use the combination solvent as 2-propanolol:water:methanol:ACN in 1:1:1:1 proportion, reverse the column and wash with 0.2 to 0.4 ml/min flow rate. Mostly it will work.

You can try reading the following articles

http://www.nestgrp.com/pdf/colcare.pdf

and

http://www.lifescience.ca/DATA/DOCUMENT/75~v~DocNom.pdf

and

http://www.chem.agilent.com/cag/cabu/ccleaning.htm

Hi Ayman

Pls read the information in attachment. Not even for plasma samples, but it will be useful for column restoration for many kinds of samples frequently used in HPLC analysis.

Gud Luck

Regards

I am suggesting you to use the solvent first in which your sample is dissolved. and monitor the base line if anything elute from column. Store your column in ACN.

Hi, try to use a filter and improve the clean-up step

Plasma proteins are rather "sticky" and tend to plate out on the interior surfaces of not only your column but all of your tubing, etc. We perform depletions on a routine basis (no organic solvents involved in those runs), and will see our system backpressure (without any columns on the HPLC) rise over time. We often clean this out by running 100% MeOH through the system followed by plenty of water. We filter our samples prior to injection, therefore even with filtration, those proteins are plating out on the surfaces of the tubing and fittings and anything that they come into contact with. Flush with Methanol and possibly store your column in MeOH (especially after flushing it out first).

Dear Ayman,

I hope your problem have been sorted out. Yet there are some precautionary measure that you should take in your account while injecting the plasma samples in the column.

The plasma samples should be properly deproteinated using proper protein precipitating agent (trichlor acetic acid, ACN, etc.) as suitable to your study. This will cause the precipitation of protein which can cause blockade of the column pores. The precipitated proteins can be easily separated by cetrifugation and filtration.

After the above protocol the plasma sample is ready to inject in the column.

Washing of the column should be done with water and then MeOH and ACN.

I hope with the precaution in the first step of preparation of sample will reduces the chances of column contamination.

If column is contaminated with the plasma sample then again go for the water washing at slower flow rate (0.2-0.4 ml/min). If this works and reduces the back pressure then good otherwise go for the column regeneration process.

Thanks Dear Awanish, I do appreciate your suggestions.

I suggest to clean up the column using a gradient of water99.9 +0.1% of formic acid to MeOH 99.9+0.1% of formic acid in order to dissolve all the precipitates and to release the compounds that have been retained into the column. After the cleaning procedure you can reequilibrate the column with MeOH-water (1:1) without formic acid.

Good luck.

There are some additonal steps you should take care. If the plasma samples you prepared are not clear, go to filter or centrifuge at higher rpm. But doing filter, the recovery may lost. So, I think centrifuge is better, if you can get clear solution, otherwise go to filtration of sample. This also contaminate the column.

Next is you are using C18 column. So, while washing C18 column for longer time with water can also deteriorate the column perfromance. So, wash with 100% water only for 20-30 minutes, followed by 50% methanol for 30 minutes and by 100% methanol for 45-60 minutes. It will also prolong the life of column.

Are you using a guard column? If not, you should use a guard column, to increase the lifespan of your column. Preventing the accumulation of salt and waste on your plasma separation column.

First was several time with HPLC grade water followed by acetonitrile.

First used good quality of Guard Column it will reduce your problem

I agree with All previous answers

You have large molecules stick to the column inlet

I recommend to invert column elute with methanol for 1 hr inject 50ul dmso then 50 ul chcl3 x 5 times elute with acetonitrile then gadient to 100% water for 1 hr then 50:50 methanol acetonitril

All elutions at 50c

Bri g your column to correct direction wash and test the pressure using 100% acetonitrile

first with your mobile phase, then ACN: Water (50:50), Then Methelene dicloride, Then again ACN: Water (50:50) and finaly with your mobile phase if it is without buffer.....

1. Always using Guard Column when every your work is related to the plasma because plasma's debris always block the column.

2. Wash the column with 50:50 ACN:Water for flow rate 0.5 ml/min for at least one hour.

2 The wash with 50:50 ACN:warm water 60 C for atlease half an hour then wash with 50:50 Methanol : warm water 50 c for at least one hour.

3. The if problem is not solve then reverse the column and wash with 70 :30 ACN: Water for at least half hour and then reverse it and wash it again.

4. Sonicate all in line filter for 2-3 min.

Filter samples through a syringe filter to start with.

To clean a fouled RP column, first check with the column manufacturer for the recommended method. For generic RP columns a good approach is to use a sequence of washing solvents. I typically wash the column down with 10 to 20 column volume equivalents in this order: DH20; MeOH; ACN; IPA; Heptane/IPA; IPA; ACN with 0.1% formic acid; MeOH and then finally with 80 % MeOH/ 20% DH20.

*To remove bound proteins from RP silica based supports you can also wash with 5M Urea solution too, then with acidified MeOH/Water and acidified ACN/Water rinses (both with 1% acetic or formic acid; optionally, 1% or less of TFA). Pure organic solvents do not work well in this application. An acid, base or ion-pairing reagent is often needed.