Not knowing what your normal operating pressure is, two thoughts come to mind. The most obvious reason is that your pump(s) may be failing - they could just be having a hard time sustaining your initial pressure. The second is that, assuming you use a gradient elution system, your analyte may be fairly insoluble in the first solvent (contributing to high backpressure on the column), while it is much more soluble in the second (thus reducing backpressure as the gradient increases).

Not knowing what your normal operating pressure is, two thoughts come to mind. The most obvious reason is that your pump(s) may be failing - they could just be having a hard time sustaining your initial pressure. The second is that, assuming you use a gradient elution system, your analyte may be fairly insoluble in the first solvent (contributing to high backpressure on the column), while it is much more soluble in the second (thus reducing backpressure as the gradient increases).

Without any experimental details, nobody can suggest an answer. Please provide all details of mobile phase composition and other conditions.

What is your Flow rate? are you running gradient or isocratic? what solvents? what are the column dimentions column dimensions? what type of sample do you have?

You said the initially pressure was normal.....what do u mean by normal? Try to be specific. How about u say the pressure was at......MPa but gradually dropped to .....within so so minutes.

If your analysis pressure dropped to 0.1 MPa during running time with reasonable flow rate, it means there could be a hardware problem such as leakages or ur column was not properly installed.

Check your system carefully. The system is likely to leak from somewhere and subsequently, the pressure has fallen over time.

Although as Prof. Katt mentioned, using gradient elution may also results in changes of backpressure

Could be either a failing pump seal, a defective oven, a leaky fitting (this would be obvious) or void development in the analytical column - common in hand packed columns but rare in commercial ones.

Given that the column is in good working condition, as suggested by everyone, check for any leakage from the column or inside the HPLC for pump seal. I once faced this problem because our pump seal got bad and we had to replace it. Also check your inlet filters for mobile phases. We recently faced very similar column pressure fluctuation problem and we figured out it was one of the inlet filters. You can clean these filters with water nitric acid mix using sonicator. Also look for contamination in the mobile phase. Whats the pH of your mobile phase? If it’s neutral then check for microbial growth and try to prepare fresh everytime before running the batch.

Kindly clarify the detailed conditions of your analysis and also the brand of inustrument you used (Waters, Agilent, Shimadzu,......). The problem may be related to your chromatographic conditions or related to the inustrument it self. May be pump seals or active inlet valve to be checked

The most likely cause, without more details, may be buildup of excipients from your formulation, if you are testing a pharmaceutical product. These may be clogging your column. An example would be Noveon Carbopol polymer, which precipitates when exposed to high concentrations of acetonitrile during gradient runs. We all need more details, to be helpful.

Carbopol can seriously ruin your day, and causes huge pressure increase problems. Please provide more details, so we can help. :)

Increase or decrease of pressure means the column is having some matter from sample ijnected which either get accumulated in the column or getting released from column based on the mobile phases properties.

Oh, decrease of pressure. If you are running a gradient run, pressure does generally decrease as the organic fraction is added, in reverse phase HPLC using for example high concentrations of acetonitrile in mobile phase B.

Pressure will also generally decrease slightly as the column equilibrates, even in isocratic HPLC.

I must sadly and repeatedly say that RP-HPLC should be performed without using acetonitrile (AN), which is dangerous to pump-seal ring or plunger seal.

Gradient elution with 2-propanol by RP-HPLC is surely stable and reliable (please see file; Lysozyme by RP-HPLC).

Dr. Hayakawa,

What about using methanol instead of acetonitrile? I know methanol gives higher back pressure than acetonitrile as former is more viscous. So for higher flow rate methanol might be a problem. Is 2-propanol is a better alternative to both acetonitrile and methanol?

Interesting discussion.

RP-HPLC should be performed by using the gradient-elution method as shown in the above mentioned paper.

Gradient elution, which washes the ODS column everytime, should be performed by using a short column (such as 50 x 4.6 mm I.D.) in order to get the quick analysis time.

Therefore,any alcohols are surely successful when used in the RP-HPLC photometric-gradient analysis.

By the way, it is noteworthy that HPLC-MS is not a quantitative method, since MS is not a quantitative detector at all (personal information kindly communicated by an engineer employed by the Shimadzu Co. to me via the telephone).