Rentention time shifting towards right

[mskishore13]

Hello,
I have been working on method development for the Active drug from dissolution samples of Extendend release suspension. The Rentention time keep on shifting to right day by day ( I usally run 110 samples/dissolution plus working standard). But the peak shape and the area are very good and linear. In between run I make to wash the column by establshing a wash method. And the drug is in resin and coated. The dissolution filter probes are 10um.

I am assuming that the resin/ bigger parciles are contaminating the column. Do you have any ideas? Please let me know.

Thanks,
Kish

[zokitano]

Kish,

tell us something about your HPLC method, instrumentation, etc. Do you use freshly prepared mobile phase every day. Slight change in buffer preparation/mobile phase can shift the retention times. What about your column? Did you do all of your analyses on one single column? Is it new or being recently opened? Maybe it is close to its lifetime end. You said that you run 110 samples + standards per day. So degradation of the stationary phase can provoke change in retention times.

Do you eventually using an ion-pair chromatography (IPC) method? I have seen rapid change of the retention times toward right (bigger values) when running numerous analyses in IPC. The problem is I assume, faster degradation of the column efficiency, which also affects the chromatography. The columns used in IPC require appropriate cleaning and regeneration after use.

Best regards

[tom jupille]

As zokitano indicated, it's difficult to make specific comments without knowing more about the details of the separation. That said, HPLC columns have a finite lifetime. Particulate contamination per your suggestion is one possibility.

If you can find the "dead time" ("t0", "solvent front") peak or baseline deflection on your chromatograms, see if that is shifting by the same percentage as your peaks. If so, that suggests a flow rate problem; that might come from a partially plugged column, or from a leaky seal or malfunctioning check valve on your pump.

If the dead time is staying constant, but the retention times of your analytes are increasing, then you have a problem with system chemisty. As zokitano suggested, it may be a mobile phase problem. It may also be the result of strongly bound material from your sample sticking to the column, or to a change in column chemistry caused by the mobile phase (extremes of pH, long-chain ion-pair reagent building up, etc.).

If you go back and look at other threads on the LC board, you'll find that column lifetime is a fairly frequent issue. In general, a couple of thousand injections is a reasonable expectation. There are things that can be done to increase the column lifetime, but they aren't free; in many cases, it's more cost-effective to just replace the column when the chromatography no longer meets your specification ("your mileage may vary" )

[mskishore13]

Hello zokitano and tom,
Thanks for your reply. I am using Waters Spherisorb CN (Reverse Phase) 4.6X150 mm. The flow rate is 2 mL/min and column kept at 40C, the mobile phase contains mixture of 15 % of 5mM Dibasic Phosphate (pH Adjusted to 5.3), 10 % Methanol and 75% ACN. I use 2 mL/min. With in the day, The RSD of Rt is with in the limits but day to day is increasing i.e 5.5 to 6.4 (right now). The samples I am dealing is Drug treated with Ion exchange resin and then coated with polymer , suspended in aqueos base.

The only reason I can think is the dissolution probe filters are 10 um and usually for the LC we filter 1um or less filter. I can not think of any chemistry in this. Please pour your thoughts.

Thanks,
Kish

[zokitano]

Dear Kish,

when I'll take into consideration all the information you've already posted, I'd say that this looks like stationary phase problem. I mean, let's take a look on your method parameters: temperature (40C), pH (5.3), phosphate buffer...

All parameters have led me to think that your stationary bonded phase hydrolyses with time. Increasing the column operating temperature, increasing the pH of the mobile phase, using phosphate buffers (which can induce hydrolysis of the bonded phase more than other types of buffers) shortens you column lifetime. It wouldn't be unexpected if your column looses the stationary phase from day to day. Not to mention the high number of column applications (that means also that the column is set under these "unfriendly" conditions for a longer time).
As you mention, the purity of your samples can additionaly contribute for the column failure.

If you have another cyano-column, try to run one of your analyses and see the difference between the new one and your "problematic" column.

Regards

[Dan]

Kish,

You may have already identified the source of the problem: those 10 um filters!

There is a style of 10 um dissolution filters that fit onto the end of the dissolution sample probe/cannula. These filters should not be used!

1) The filter pore size is too large for use with HPLC. You will get a large amount of particulate matter from the dissolution sample that can accumulate on the head of your HPLC column. Have you noticed an increase in pressure on the column after some usage? You can back flush the column to try to clean off the particulates.
2) If the filter is left attached to the cannula and you leave the cannula in the dissolution vessel continuosly, then you add unwanted turbulence to the dissolution vessel and, thus, you do not get the appropriate results.

For #1 above, you can have problems with the HPLC run.
For #2 above, you just have problems in your dissolution technique.

I recommend using a 0.45 um syringe filter, either in-line (outside of the dissolution vessel) or just manually filter the dissolution samples after collection.

OK, that's my 2 cents looking at things from the dissolution/sampling end of things. There may be other issues to look at for just the HPLC side.

I have an additional request for info. Can you tell us the dissolution medium and the injection volume? That info may help to further troubleshoot.

Regards,
Dan

[Peter Apps]

If you are using pre-mixed mobile phase, and degassing with helium the organic fraction of the mobile phase will decrease with time, which will increase retention. Turn off the helium when the instrument is not running.

Peter

[mskishore13]

Thanks for your responses. I was tryin at room temperatue but the peak tailing is too bad. Thats why I come up with 40C and the 20ul injection. I will send the update.
Thanks for your help and this forum is reallly helpful.
Kish

[mskishore13]

Hello All,
As I asked about the retention time shifting using the waters spherisorb CN (RP) 5 u , 4.6X150mm column. Here are more details and what I found. I still need your valuable information to trouble shoot this phenomena.

As per many suggested I installed the guard colum 4.6X10mm with holder and new column as well and kept the column at room tem. All other parameter are same. Once again, I put the details here
Conditions
Acetonitrile 75%
MeoH 10%
DBPB 15% (pH~5.3)+59mM TEA

Flow rate 2 mL/Min
Column Temp RT

And the Retention time are

Date Day 1 Day 2
Samples Rt(min) Rt(min)
Start 4.812 5.104
End 4.989 5.194
Mean 4.915 5.151
STDDev 0.05 0.02
RSD 1.02% 0.40%
Sutability 4.736
Sample Inj 12X2 6X2
Ws Inj 6X2 2X2

I observed the same phenomenon like at the begining aroung 5.1 min and when I changed the column it was~6.7 min.Most of the injections it stays at 5.9 min . The difference was the colum tem (40C) and guard column.I am worried even at room temperature this column behaves same.

Please have a look and let me know

thanks,
Kish