You have not provided enough information about the column, sample or method. As you are a student and new to liquid chromatography, please review the instruction sheet which came with the HPLC column for advice and suggestions which are specific to YOUR column. The manufacturer often provides information regarding which chemicals to avoid and which can be used to wash it. If you can not find this document or need more info, please contact the column vendor for a copy of it (or download it from their website). Always start with the information provided to you for free from the manufacturer.

One of the most common reasons a "chromatogram contained undesirable peaks" is due to it being a poor quality method (not properly retaining, holding and eluting all peaks) and/or not having an appropriate wash method developed (leaving material to build up on the column over time) which uses a stronger solvent than your mobile phase to elute off any remaining material. A properly developed HPLC method will retain ALL sample material on the column (K prime > 2), then hold and elute it ALL off over time, while baseline separating ALL peaks. This is fundamental to basic HPLC. Get some help from your teacher with your method or from someone local to you at your school.

What is the source of contaminant?

You have not provided enough information about the column, sample or method. As you are a student and new to liquid chromatography, please review the instruction sheet which came with the HPLC column for advice and suggestions which are specific to YOUR column. The manufacturer often provides information regarding which chemicals to avoid and which can be used to wash it. If you can not find this document or need more info, please contact the column vendor for a copy of it (or download it from their website). Always start with the information provided to you for free from the manufacturer.

One of the most common reasons a "chromatogram contained undesirable peaks" is due to it being a poor quality method (not properly retaining, holding and eluting all peaks) and/or not having an appropriate wash method developed (leaving material to build up on the column over time) which uses a stronger solvent than your mobile phase to elute off any remaining material. A properly developed HPLC method will retain ALL sample material on the column (K prime > 2), then hold and elute it ALL off over time, while baseline separating ALL peaks. This is fundamental to basic HPLC. Get some help from your teacher with your method or from someone local to you at your school.

There are oh-so-many ways to mess up a C18 column. Every user should keep a strict diary of samples injected and instrument conditions, precisely for the purpose of answering the question. Much more information is needed before anyone can answer the question.

Well follow the following sequence flushing

Water - methanol - IPA - methanol - water

Many many times this work for me

Please do not use a generic trio of solvents or "80-90% acetonitrile" as a generic column cleaning solution. Unscientific and Not recommended for two reasons:

(1) ACN has very poor solubility characteristics for so many compounds, so they will not be dissolved and eluted off the column (MeOH is far better, but the best solvents for your sample(s) will depend on what it is);

(2) Please do not specify a specific wash solution without first identifying which compounds you are trying to wash off the column. We should base answers and methods used on the information acquired about the specific problem we wish to solve.

The correct choice of wash solution is always based on the properties of the compound(s) you are running through the column. Applications vary, so the most appropriate wash solution will too. This information is combined with any known column support precautions and a solution which is stronger than the original mobile phase and which dissolves all of the fouling material is used (as stated before).

I want to chime in to support the answer by Bill Letter. It is very important if at all possible that you know with what the column is contaminated. Once you know that, then you can approach column cleaning scientifically using solvents that are strong enough to elute the contaminants off the column without damaging the column or the HPLC system. I also find that methanol and IPA are better than acetonitrile for cleaning up our contaminated columns, but YMMV. It depends on what has contaminated your column.

There is the perfect way and there is the practical way.

Unless you are the only one using the system or you know that you are the one who contaminated it, you will likely not be able to identify the contaminant.

First, I would not assume that the contamination is from the column, there are plenty of other sources, such as contaminated solvents, injector, tubing etc.

1. Start by making sure all the solvents are freshly made and in clean bottles. In a university environment this is not an uncommon problem.

2. There is often at least one filter in the flow path, check it for contamination, check the user manual if you cannot find it. Often there is one filter in the solvent bottle and one near the pump head. Clean them or better just change them.

3. Do you have a precolumn? Change it. If you don't have one, you should for your next column.

4. If you can you test with another column, do that, but blank run only. This helps you isolate the source.

5. Are you running a gradient elution? Where do the undesirable peaks appear, this might give you a clue as to what to use for cleaning. If not, run a gradient elution, typically with the solvents/buffer system you already use and see what happens to the undesirable peaks.

6. If you still have no idea, try water - methanol - IPA and back again as Tabrez suggests above. You will probably have to reduce the flow and let it run longer.

7. Stop wasting time and buy a new column (and precolumn).

8. Clean your samples so it doesn't happen again.

Dear Chingyeh Ong,

I have obtains for good results washing the volume with ACN and T2O mixture,

Start at 5% HCN and increased to 95% within 1 hour and keep it about 30 minutes for another 1 h.

Piyal

Higher proportion of water or ACN may collapse stationary phase

Dear Mr. Tabrez Shaikh,

I think that you have to learn more.

ACN and H2O ratio up to 95:5 (V/V) is recommended for C-18 column washing and cleaning for HPLC systems.

Please refer the reference attached.

Thanks

Amended on 12 May 2019

Surely, column cleaning is essential for repeatable and reliable analysis, and the gradient elution method becomes to be prerequisite.

Our gradient elution method contains the important column-washing step for usually 5 min, and returning to the initial condition has occurred within 5 min with 5-cm-length short-type column (please see file; Lysozyme by RP-HPLC). The gradient elution method washes the column for one time in one analysitical run. The original gradient elution method has been developed using long 30-cm column (please see file; Monch Protein separation).

High concentration of AN is very dangerous to injure the plunger seal of HPLC pump, and I have chosen the alcoholic three-phase eluent system; i.e., acidic sodium-phosphate buffer (0.1M, pH 2.0; solvent A)-2-propanol-ethylene glycol (1 : 3 : 1 (v/v/v)) as solvent B. Utilizing of TFA is also very dangerous to ODS column (please see file; PTH Roma RP-HPLC).

Therefore, I am now considering that relative Mr. smaller than 100,000 should be collected by HPLC-SEC method (please see file; SEC column 300A silica).

Further, cysteine residue should be pretected by the pyrydylethylation.

Hello,

Washing with 80:20 (water:ACN) for 3 hours at 1ml/min and 80:20 for other 3 hours(ACN:water) works fine.

Otherwise there are more precise procedure (source: chromacademy)

Reversed phase columns include C30, C18, C8, C4, C1, CN and phenyl stationary phases. To clean and regenerate a reversed phase column flush it with the following volumes of solvent.

20 column volumes of water/acetonitrile (95:5 v/v)

20 column volumes of acetonitrile

5 column volumes isopropanol

20 column volumes hexane

5 column volumes isopropanol

20 column volumes acetonitrile

20 column volumes of water/acetonitrile (95:5 v/v)

Re-equilibrate with the required mobile phase

Be aware that in the case you are using phosphate buffer an overnight washing of the system (removing the column) with 100%water is required. This is done in order to prevent precipitation of salts

Regards

Davide, a vendor biased website such as 'chromacademy' may not be the best resource to use. For better suggestions in column washing (and for what questions to ask first) please refer to the previous answers provided above, many months ago (and review them before posting). Please skip suggesting the use of ACN or ACN/Water as a column wash solution (or the other less than ideal long list of solvents which are likely to make the problem worse). Generic answers like those often have little practical use.

As a student first starting to use HPLC, one of the fundamentals that will help you early on is that we choose or select the column wash solution based on the solubility of the compounds used. This is critical if the goal is to remove material which may foul the column over time. Please do not be fooled by some of those websites with generic suggestions or answers which do not utilize solvents and mixtures which are better at dissolving material (such as MeOH, not ACN) and which are safer for the column.