Bad tailing

nenaine

<p> I am using a column SunFire C8 4.6X150 mm 3.5µm with a gradient (see below).  Specification for the tailing is between 0.8-2.5 but even after many injections (on many days equilibration), the better I've got was 3.2.  I've tried to increas the flow rate to 1.5 mL/min but still too high.  It is a tech transfer and the other lab had tailing around 2.</p><p></p><p></p><p>Gradient A=0.5 mL TFA in 2000 mL Water   B= 0.5 mL TFA in 2000mL ACN</p><p>0     82 A     18B</p><p>15     82A     18B</p><p>22     33A     67 B</p><p>26     33A    67 B</p><p>28     82 A   18 B</p><p>33     82A    18B</p><p></p><p>Flow rate at 1.25 mL/min</p><p>Column at 40°C</p><p>20µL injection</p><p></p><p>Standard prepared in a mix of: 200mL  ACN: 0.5mL H3PO4:800 mL water</p><p></p><p>Retention time between 11-14 min.  Modification of 1% gradient shift 1.5 min</p><p></p><p>Any suggestions</p><p></p><p>Thanks!</p><p>Elaine</p>

dougm

Hi,

I have a few questions.

Are you running this HPLC column on a UPLC system?  What was the original LC system? Are the retention times similar? Do all the peaks tail, or just one?

How many SunFire columns have you tried?  How many SunFire columns were run in the other laboratory as part of method validation?

Thanks,

--Doug

AnalMD

How old is your column compared to the column at the other lab?  It could be that your column has reached the end of it's life for that test and you need a new one.

nenaine

Hi Doug;

It is a brand new column and the only one we have.  The column was equilibrated for many hours. For the moment, I've no idea about the other site.  My retention time is the same compared to the othe site.  I've tried on Waters H-Class and Agilent 1100 with the same results.

Thanks!

Elaine

dougm

Hi,

Have you contacted any local Waters person regarding this issue? The reason I ask is that you may want to look into getting a replacement column and they can help facilitate that. A new column will determine if the problem is related to the particular column you are using (if a new column DOES NOT tail) or if the problem is the system or method (if the new column DOES tail).

Once a replacement is obtained, would it be possible to send YOUR tailing column to the lab that developed the method so that THEY can test it?

Does that make sense?

Thanks,

--Doug

nenaine

The person I've contact at Waters is on vacation until next monday.  So, to be continue next week!

Thanks for your input.

Elaine