I used a 150X4.6mmX5um C8 column to test a single sample for 5 continuous injections. Flow rate was 1.8 ml/min, and a UV detector was used. Strangely, the retention time decreased continuously, i.e., each was shifted to the front of the previous one while the void peak remained unchanged.

I wish someone can give me ideas about the case. Thanks a lot!

Equilibration? Temperature? What's the mobile phase? pH?

Mobile phase is 0.03M KH2PO4:ACN:MeOH=53:37:10, PH around 5. Equilibration lasted more than 2 hours at 1.5 ml/min, then to 1.8 ml/min for about 1 hour. It happened at 25C for injection while column was exposed to room temperature of 23.4C.

Phosphate is not a buffer "around pH 5". Why do you keep the injector warmer than the column?

The final PH of the mobile phase is 5.57. The temperature for the injector might be close to the room temperature. Any suggestions?

Thanks for the timely response!

We can exclude a flow rate change, since the void peak remained at the same retention. I would turn off the heating of the injector. Also, I would look into equilibrating the column at the same flow rate as the final analytical run. Both of these things should exclude unusual temperature effects. I am not yet convinced that the problem is temperature, but I have seen on occasion extraordinarily large temperature effects.
The pH is a little on the hairy side, but not as bad as I first thought. This could be OK. If you have flexibility with the method, I would adjust it closer to pH 7 or increase the concentration of the buffer.

At pH 5.6, silanol is partially ionized. If the analyte is a cation at such pH, it will absorb somehow on the column, causing peaking. As more injections are made, the silanol tend to be more and more saturated, and the RT will be shorter and shorter.

If this was true, you would see that the peak areas were increasing with later injection.

What kind of sample? How much is injected?

It's drug containing N and S in five-membered ring. Injection is 50ul.

Thanks for all valuable ideas!

Sorry! I forgot to mention that the peak ares didnot show significant chnages.

Use internal standar for retention time.

Check piston seal and pressure.

Did you premix the individual mobile phases manually or thru a multi-channel gradient valve (MCGV)?
The retention time will be all over the place if you have a faulty MCGV.

It sounds as if the column phase is changing. Is there anything in the sample matrix,used in sample preparation or the eluents that could be stripping or attaching to stationary phase. I have seen this before with both ion and RP columns. In the IC case, sulfonated hydrocarbons were attaching to sites on the column and reducing capacity. With one RP column case the C-18 was being stripped off by injection of the sample prepared in a high pH solution. In the other case the column had previously been used with an ion pairing agent. The ion pairing agent slowed the analytes progress through the column. As remanants of the ion pairing agent were being removed from the column the retention time of the analyte was reduced. After several weeks it became stable.

Is the column new? If not what has been used previously for elution? It could be that the sample injection itself or the eluent is cleaning the column, permitting faster elution of the analyte.

Prepare a solution containing both a late and an early eluting compound with retention times before and after your peak of interest. Make duplicate or triplicate injections of that solution between a couple of your regular injections. If the retention times for these compounds is constant between sample injections but decreases after sample injections, then the sample itself or the solution in which it is prepared is causing the problem. If all retention times decrease for for all peaks after each injection then either your column or mobile phase is the culprit. The column stationary phase may be unstable and decomposing or the eluent may be destroying it.

Regarding the final pH of the mobile phase. I was taught that salt should be added to water and pH adjusted before addition of organic solvents when preparing mobile phase for isocratic elution. The apparent pH of a solution containing organics was not considered to be a valid pH. I agree with the individual who suggested trying to work at pH 7 if possible. Are your chromatographic conditions, eluent, column, flow rate, etc. from an existing separation similar to your own or did you decide on them yourself after trial and error?

I've seen this happen as well with samples that had significant quantities of hydrophobic material that remained on the column between injections. What worked in the short-term was careful rinsing of the column with a more hydrophobic solvent after sample injections. Later on, I developed a proper SPE sample clean-up procedure and the problem disappeared.

If you wish to remain at the pH you're currently using, is there any good reason not to use an acetate buffer?

Good Luck!

Chris

Could anyone tell me what are the basic considerations to be taken while selecting buffers and their concentration

Am also facing problem of decreasing retention time with a phosphate buffer - methanol gradient of 60 minutes. Buffer range is 100 - 60% (1hr step wise). The RT comes down from 35 minutes to 18 minutes while the last eluting peak RT does not change. Same phenomena observed with different columns and different HPLC systems. Same column sometimes gives the problem, sometimes not. Any clue?

I assume that you are using a reversed-phase column, maybe a C18. If it is a good column with good endcapping, the problem could be related to the time that the column is exposed to 100% water, including reequilibration time. If the separation allows you to do this, you could start the gradient with 95% water instead of 100%. This would solve the problem, if indeed this is the problem. Alternatively, you could use a column that is not sensitive to this, either a modern one with incorporated polar group or an ancient one with plenty of silanols (non-endcapped).

I just saw your previous question. The key concern is to stay away from concetrations where the buffer precipitates in the presence of the organic solvent. You are safe at 50mM, and this is also a sufficiently strong buffer.

Thanks for the info Uwe Neue. Though starting with 95% affected separation, when tried with 98%, reasonable separation was achieved and Retention times were reproducible over 12 hours of injections.

Try a polar embedded reverse phase column such as Discover Amide C16 column. You can use 100% aqueous on it.

Decreasing retention times could also be caused by a clogged frit on your aqueous solvent inlet line, effectively increasing the organic content of the mobile phase. Remove the inlet filter on the aqueous line and see if the retention times are restored.

I want to know why some type of analysis are practical in GC and other in HPLC. Or what is the advantages and disadvantages of the 2.

GC (gas chromatography) requires that you be able to vaporize your analyte(s). Many (if not most) organic compounds will decompose before they will vaporize. LC (liquid chromatography), which only requires that analytes dissolve, is recommended for those analytes.