Chromatography Forum

I am getting a peak plit on Waters HPLC but not on Agilents with same chromatographic conditions, (column, mobile phase etc.)& same standard preparation. What could be the reason?

can you give more details of the instruments type
the mobile phase
the sample solution used
the injection volume
the flow rate

it will help us a lot in order to get what is going on

mobile phase is water:acn:opa 14004
sample solution is in 0.01N HCl (dissolution sample)
injection volume is 500mcL (0.5mL)
flow rate is 1.5ml/min

But i think it is irrespective of these things,sum hardware diffference or sumthing because everythi ng is same except HPLC system. What could be the reason?

actually there is the possibility of 2 things that could cause the peak shape difference

you have a sample solution that is significantly different than the mobile phase.
100% buffer solution and a mix of about 60:40 for the mobile phase
and the injection volume is very high, 500ul

it is possible that in the water system for this application, there is not enough tubing length, or maybe the tubing is made of a smaller capillary ID.
this maybe does not allow for a good mixing of the sample in the mobile phase prior to getting to the column.

if you compare, in which of those 2 systems, is the peak having a later retention time?

Thanx for immediate help unmgvar. The second reason could be right because pH is almost same because of OPA in mobile phase.
Plus, as my senior suggested. the tubing in Waters is having more internal diameter than agilent, which May cause bandbroadening & split. Both can contribute. What say?

if waters has more tubing and more volume then the retention in the waters will be later then with the agilent,
still this should actually help for a better mixing of your sample in the mobile phase and decrease peak splitting
is the bad shaped peak also creating a bad tail in the back? this what you should be seeing because the sample is made of 100% water/buffer

have you tried running something else on those systems?
it still looks like an application related issue of the high injection volume

is your application a gradient or an isocratic one?
and what are the retentions on both systems?

now if you have another peak in the chromatogram
even the small peak at the beginning of the chromatogram, the t0 peak.
see if it is also split.
if yes then the problem might be of a void volume somewhere in the system

What are you using in the rinse bottle on the Waters system - same thing as on Agilent? If the rinse solution is more polar than your mobile phase, it can cause peak shape problems.

even mobilephase polarity also not working ? is there any more idea for avoiding peak splitting?Also any suggestion for 100 microlitre volume of injection?
Agilent HPLC- Peak sharp,symmetry in nature,but water's HPLC- Peak broadening & peak splitting at peak top.
Note: Method setup is same for both HPLC
Any suggestion ?
1.for instrument compatability
2 If any

Thank you

As was discussed above, if the diluent is stronger than the mobile phase, there is always the potential for peak shape problems. Solutions:
- use the (initial) mobile phase as the sample diluent
- limit the injection volume
- add a coil of tubing between the injector and the column to allow the injected solvent to mix with the mobile phase before it gets to the column.

Peak shape problems can also be caused by extra-column volume (a poorly assembled tube end, for example), in which case narrow early peaks (isocratic) will show the problem more severely than wider late peaks, or by flow-profile anomalies at the column inlet (e.g., a trapped air bubble, a partially plugged frit, or a head space), in which case all the peaks will show the same problem.

Chances are that the column was not washed properly after analysis on one system which reflected in the form of split peak in the second run on another system. If you are observing peak splitting in all the peaks, it must be the column issue.

What model of Waters HPLC do you use?

The later models inject some needle wash with the samples.

Thank you. Splitting in main peak only.
Model of HPLC : water's alliance e2695
THANK YOU

One more thing to check... is the column being properly installed on the Waters system each time? Is it possible that someone has a stainless ferrule & nut on there (or a PEEK ferrule that has been overtightened on a PEEK tube int he wrong place) and that the tubing on the inlet end of the column is not seated properly in the column's inlet? If there is a gap there, splitting due to the void can occur.

Have you measured the void volumes on both systems? Differences there could explain things. Also, I might consider reducing injection volume on the Waters unit and see if the chromatography improves. 100-uL is a pretty good sized slug and a system with a larger void volume would be more tolerant of sample solvent variations than one with a smaller void volume at that level. If 100ul is crucial, make sure that the level of solvent in your sample is no higher than in your starting mobile phase.

Good luck!