Might be due to air bubbles within the system causing not continuous flow and pressure!? Try to purge the pump(s).

You should first check for air bubbles and secondly clean your column by running 50% ACN or MeOH to get straight base line. Sometimes preliminary cleaning of column plays very important role,specially in case of peptides.

I tried purging the pump. I even tried washing of column first with methanol: water (50:50) then with acetonitrile: water (50:50) but it doesnot work. 

Which detector are you using? MS? UV-Vis? Did you try introducing eluent without the HPLC (by syringe... or only without column)... just in case it is the detector? Try to localize the problem to a component of your setup (pumps, column, some connection (leaks)...).

Large wavy baseline generally occurs at low wavelength.Which wavelength you are using on UV visible detector? Acetonitrile sources vary in their UV cutoff, and supplier literature is not always reliable.

I am using PDA (UV-Vis) detector at 220nm and 254 nm

Which column you are using? I suggest to prefer C8 Symmetry 3.9x150mm and then try for baseline . You may also try by making gradient changes. Also use your detector at a single wavelength 254nm and then observe.

I am using C18 4.6x250mm column,

Check the pump status, may be some choking, keep on washing by changing the gradient upto 6ml/min(if Shimazu) by rising and lowering the same then caliberate the auto zero by running standard sample. Check column is stabilished or not properly in solvent u r using, otherwise keep the system on washing overnight for column stablishation. Thank u

Check air bubbles in the pipe line, if some presents then degassed ur filter (in acetone) and solvent in ultra sonication bath for half and hour. Thank u

Thanks

May be ur solvent not upto the HPLC grade, may be chocking in pump and column, detector cell contamination, air bubble trap inside the system, so u have to go for degassing ur solvent, and continous washing ur system by increasing and decreasing the gradient of solvent upto 6-8ml/min gradually., then run a blank and std. solvent.

Proper column wash is required with water and ACN (50:50v/v). keep the concentration of buffer below 50mM. often it happens with acidic or basic sample. so prepare solution in mobile phase.

If you run an gradient you should try to use an online cleaning column like the Ghost-Guard-LC. This absorber removes traces of the impurities from the mobile phase and you get an good baselien!

Two of the most common causes of an unstable baseline are: (1) Poor (or no) degassing of the mobile phase. When the liquid is compressed, the dissolved gas inside comes out causing gas bubbles to form which results in poor pump performance. Use proper in-line degassing. Example: An inline electronic vacuum degasser or low pressure continuous sparging of the mobile phase using Helium gas. (2) Sticking Check Valve(s) in pump head. Valves pick up particulate debris and/or buffer salts, causing them to cavitate, "float" and/or backflow liquid. This results in unstable flow. Valves can be flushed with fresh solvent, removed and sonicated / cleaned or replaced to restore function.

For other common causes of an unstable HPLC baseline, please refer to these two free articles:

"Diagnosing & Troubleshooting HPLC Pressure Fluctuation Problems (Unstable Baseline) " http://hplctips.blogspot.com/2014/01/diagnosing-troubleshooting-hplc.html

"Common Causes of Baseline Noise in HPLC, UHPLC"  http://hplctips.blogspot.com/2014/09/common-causes-of-baseline-noise.html

http://hplctips.blogspot.com/2014/01/diagnosing-troubleshooting-hplc.html

The probable causes are

1. Quality of the buffer and solvents you are using.

2. Insufficient degassing of mobile phase or system built-in degasser problem.

3. Compared to 254 nm the noise and baseline drift will be more at 220 nm. For Low wavelength operations the absorbance due to gradient change will be more. This can be controlled by adjusting the gradient method.

4. Buffer concentration, keep the buffer concentration low and check peak shape and baseline noise or drift. High concentrated buffers not only cause baseline drift but also cause shorter column life and frequent pumping problems.

5. Buffers are solvents used are of high UV cut off value.

6. UV lamp hours might be more.

Thanks you all for the response.

wavy baseline conveys that pressure of the HPLC pump is not stable. this may be due to multiple reason like:

1. The solvents/ buffers are not of HPLC grade.

2. In-Line Degaser is not in proper working condition.

3. the column has been choked due to higher concentration of the analytes.

you can purge the whole system with ACN:Water (70:30) to remove this problem. you may also try pure water to remove any contamination. also try to filter (using 0.45 micron filter) and sonicate (2-3 min) the solvents prior to use.

1. wash your system with hot water at least 2hr

2. wash with ACN: H2O (70:30) for 1-2 hr

3. Check solvent quality or filter it properly

4. use HPLC grade water or milli Q water

5. Proper degassing of solvent or buffer system required

6. Dilute the sample upto the limit of column detection

7. After complete run of sample wash the system as mentioned above.

1. The most common causes of a rhythmic or wavy baseline are related to the pumping system. Typically, your pump will have two pistons and seals. If one is more worn than the other, this can cause flow and pressure variations in a very rhythmic pattern, which will also be seen by the detector. You can confirm a pump seal problem by varying the flow rate; the frequency (in time) of the baseline fluctuations should increase/decrease proportionally to an increase/decrease in the flow rate if the pump seals are worn. The solution is to replace the pump seals.

 2. The problem may also be a function of insufficient mixing or of a malfunctioning proportioning valve.

3. Finally, another possibility may be the detector itself, though fluctuations would not tend to be rhythmic. Again, varying the flow-rate as mentioned above can confirm pumps are the issue, but if adjustment of flow does not have an effect on the baseline fluctuation, the detector itself (e.g. aging lamp if UV) could be the cause.

If you have a multiple pump system (binary, ternary, or quaternary), the problem may also be a function of insufficient mixing or of a malfunctioning proportioning valve.

Finally, another possibility may be the detector itself, though fluctuations would not tend to be rhythmic. Again, varying the flow-rate as mentioned above can confirm pumps are the issue, but if adjustment of flow does not have an effect on the baseline fluctuation, the detector itself (e.g. aging lamp if UV) could be the cause.

bubbles in the mobile phase may cause wavy or noisy chromatograph , next time whenever you do hplc first properly degas the mobile phase and then use

The most common causes of a rhythmic or wavy baseline are related to the pumping system. If you have a multiple pump system (binary, ternary, or quaternary), the problem may also be a function of insufficient mixing or of a malfunctioning proportioning valve. Finally, another possibility may be the detector itself, though fluctuations would not tend to be rhythmic. Again, varying the flow-rate as mentioned above can confirm pumps are the issue, but if adjustment of flow does not have an effect on the baseline fluctuation, the detector itself (e.g. aging lamp if UV) could be the cause.

Thanks