Chromatography Forum

Dear Members,
We want to determine active ingredient dissolved in dissolution medium (HCl 0.1N). The system as follows:
Column: 150 x 4.6 mm, contains C18 column packing.
Mobile phase: Phosphate buffer, pH 8.0 – acetonitrile (73:27)
Flow rate: 1.5 mL/minute
Column temperature: 55 C
Detection: UV 210 nm
Injection volume: 50 uL (1 – 15 ug/mL)

After several hours, the retention time still unstable (tend to decrease). Here is the retention of last 25 injections:
6.959
6.955
6.918
6.886
8.899
6.877
6.844
6.857
6.837
6.834
6.833
6.800
6.769
6.755
6.758
6.758
6.725
6.753
6.715
6.688
6.673
6.660
6.658
6.618
6.575

We use the same system in assay for tablets, but in lower injection volume (10 uL) and higher working concentration (100 ug/mL). Solvent in assay is mobile phase. The problem is not occurred in assay.
Any explanation please why the retention could not be stable…

Best regards,
Siswanto Tanuatmojo

Is this understood correctly: It occurs only with standard? Did you run your samples after this standard? Did you run standard some hours later again (with constant flow on the column all the time?)? Did the peak area change? What is the matrix of the injected material?

Dear Mr. Muller,
All of them are standard solution in 0.1N HCl (dissolution medium). The peak area are relative constant.

Have you tried neutralizing your samples before injecting them? You're injecting 5x your regular assay volume of pH 1 media into a pH 8 stream...

Dear Siswanto,

I had similar problems with retention times variability and I thought it could be the mobile phase multichanel because I have a quaternary pump bot I think your problem could be the strength of your phosphate buffer (concentration up to 50 mM is OK) or other possibility is the reduction of the quantity injected. Maybe the area is small but you can choose other wavelength. I hope you could solve your analitical problem.
Best regards,

Diego

I go with DR on this one, phosphate buffer isn´t so hot at pH = 8, your standard is probably "overtaxing" it. The equilibrium of SiO- + M+ is touchy and could influence the r.t. if disturbed.

Sorry, I have made a mistake. The pH of mobile phase is not 8.0 but 2.0... I was confused by another method.
But I will try to use higher concentration of the buffer up to 50 mM.

The concentration of the buffer had been increased to 0.05M and the salt was changed to ammonium dihydrogen phosphate. The movement of the retention time is still occurred but slight reduced.

If this really only occurs with standard (higher volume of HCl) then you just proved that the mentioned pH incompatibility is the cause.

It occurs on standard and sample solution. The RSD of retention time from 30 injections is 1.2% and 0.8% using 2 different columns.

Confused?

Is it ok? The retention is shorter and shorter though it have been run for hours. I never see like this before.
But I think the result is better now than the earlier results, in this threat (RSD=6.35%).