Hi, do you use identical columns in your 2 HPLCs? Age of column could affect the results. You wrote the peak was appeared in one HPLC, Have you checked the identity and purity of that peak using DAD? Have you checked the stability of your compound in the solvent? Did you inject into 2 HPLC the same solution in the same time? Best regards

Hello Dr. Indrayanto, Yes same column in the two HPLC systems. but not same solution in the same time.

Dear Mohamed,

This is happend to me when inject diffrent dilutions of malonaldehyde with an old HPLC.  You may also check your mobile phase concentration/ try to increase the concentration of the one that does not appear. Another idea, make one dilution for a standard material such as vanillic acid and inject in both systems and see how is going. Also try to use several  wavelngths

Good luck

Aly R Abdel-Moemin

Helwan University

Cairo Egypt

The two instruments are probably not set up the same. There is no reason to ever assume that two HPLC systems would provide the same result, unless they were specifically configured with the same exact modules and setup in an identical manner.

First step: Start by taking the same injection vial (with fresh sample) and using it and the same column to compare BOTH HPLC systems. You should also test a known standard (not your sample) on BOTH HPLC systems too for a more reliable comparison.

Too many possible variables to list here, but some obvious areas which can cause different results would include:

• Different plumbing (the ID and length of all interconnecting tubing used is very important);
• Flow cell choice. The manufacturers offer many different flow cells for these instruments. They vary in flow cell volume and flow cell path length. Path length changes can have a dramatic effect on sensitivity! For more info: http://hplctips.blogspot.com/2011/03/flow-cell-volume-path-length.html
• Detector Settings: Failure to standardize on the same detection wavelength and detection bandwidth may also have a dramatic effect on sensitivity! For more info: http://hplctips.blogspot.com/2011/09/uv-vis-hplc-detector-signal-bandwidth.html
• Detector lamp output: A weak lamp can introduce noise to the system. This reduces the overall dynamic range of the detection system (sensitivity is poor). Always monitor and evaluate both lamp intensity output (using proper S/N ratio tests of a std) and wavelength calibration. Differences in lamp energy can directly effect sensitivity.
• Use of Reference Wavelength on a DAD/PDA: Reference wavelengths should always be turned OFF. If the Ref Wavelength feature was turned ON on one system and not the other (or was ON and has different settings), then your raw data has been modified and your results may not be vaild. For more info: http://hplctips.blogspot.com/2011/03/reference-wavelengths-as-used-in-hplc.html
• Injection Technique(s): Samples must be dissolved in a solution that is the same as the mobile phase or weaker than the mobile phase. Injection volumes should be within the 20-80% range of the injection system.
• Poor quality integration settings and/or high baseline noise levels which make proper integration of peak areas unreliable. Good quality methods have clean baselines with peak integration start and stop points at appropriate points.

These are just a few things worth checking. I assume from your statement that the exact same HPLC column was used in all cases. If not, then the column is another variable. Mobile phase preparation (the person who prepared it too!) AND the actual condition of the HPLC system are also huge variables which must be evaluated too. Never assume something is operating properly until after it is fully evaluated using standardized tests and methods. As we sometimes say, compare "apples to apples"

Hi Fuad, in addition of all comments above, you wrote, you have injected in different time. Did you use same solution for these 2 HPLC? if Yes, How long is the difference? Have you checked the stability of your compound? Best regards

It could be PDA problems (lamp lifetime). Try the same analyte solution but highter concentration and check detector documents, may be it is time to change a lamp on second HPLC. 

Hi Fuad,

additionally to the former contributors with extremely valuable answers, you may prepare fresh solvents for both instruments (use from the same perepared mixture to be sure you do not have any differences in solvent composition). Using the same column for both HPLCs is a must in this case. Do not use the same column type from the same supplier - even columns from the same batch number do have different properties since the particle packing is slightly different (buth within a certain range which fits the respective company standards). With that, you can not run the samples in parallel but you are very sure you do not have any column effect.

All of the above is excellent advice.  Another factor to keep in mind (probably not a problem here) is "carryover" from sample to sample.  If you injected a very high concentration of a standard of the impurity before injecting the actual sample, it can be an artifact to see a trace amount in a subsequent sample.

Hi Fuad,

Peter is right - to avoid that you can set up an injector program (for Agilent I know it is possible, I'm sure it is possible for all companies), where the needle is washed in 1 or 2 vials with pure solvent after injection - the washing solution is then being discarded and not injected to the column.

Alternatively, we sometimes do that just by injecting 1 µl of e.g. 50% acetonitrile/ 50 % water mixture between the runs, where we put 100-200 µl in the microvolume insert of the vial to ensure the needle is clean for the next sample.

I agree with Bill Letter. He has clearly set out all points which can cause different results. As you see there are too many variables that can affect your results.

From my experience, I would say that the detector lamp is definitely important to control.

Differences in lamp energy, which depends on the lamp running time, can directly affect sensitivity (S/N ratio).

Moreover, the monochromator slit width of the diode array instrument can also affect the resolution of the istrument. You must also check this parameter. 

I see just one thing that no one has mentioned yet.  You say the two HPLC systems are from the same company ...... but are the two systems actually the same model ?  If not you can certainly expect that they will have different performance  (and as Bill has already well documented - even if they are the same model there are plenty of other variables.....)