Problema with new column Hamilton prp-1

[ChromaQuestion]

Hi everyone! I am new to the field and I dont know many things yet so I would be very grateful if you could help. I am currently running a related substances analysis on a new column. It s a Hamilton prp-1, 5 um, 150x4,1 mm. My mobile phase is a phospate buffer pH 6.8 one with 95:5 v/vphosphate buffer:acetonitrile and the other one with 34:66 v/v buffer:acn. Gradient elution. Sample preparation with phospate buffer pH 2.5: acetonitrile 95:5 v/v. The problem is that first the pressure is very high, the specificity was tried for 4 days because the pressure kept on rising past 5000 psi. We washer the column with HCl 0.1 M and the pressure was better and could carry out the analysis after all. The problem now is that we need to carry on precision on an impurity containing sample and the impurity doesnt eluate properly and becomes somehow integrated in a peak thats present in the sample preparation mixture. How can i separate this peak properly? Lower flow? Is it ok to wash this column with HCl? It doesnt work otherwise, we tried different washes. Thank you very much!

[Multidimensional]

We need to know what the flow rate is. We need to know the exact sample type, concentration, injection volume, detection method and settings.

You asked, "How can i separate this peak properly?" This requires the process of HPLC method development, which is best solved if you can get some local help from your school. No one can learn all of the needed skills in a week, month or even year and if you had someone knowledgeable nearby, they could make sure your proposed method and column type are suitable for your sample application.

Other comments: You stated that you were concerned about the pressure rising. From what to what during which changes in mobile phase? During a gradient analysis, pressure does changes when the mobile phase composition changes (that is normal). It should be repeatable for every run. If you are observing a trend of increasing pressure after each run, then there is a method or sample preparation problem. You must solve this problem and prove that the method is both reproducible AND is selective for the sample(s) being analyzed. Column fouling is a common problem for novices and it often results in loss of resolution and poor reproducibility. Be sure to create a method with acceptable peak K primes, be sure to wash the column after each analysis (and 0.1M HCl is pH 1.0, which is safe with your column, but may not be a very good wash solution to use at all). Any impurity peaks must be clearly baseline resolved, repeatedly. Follow good chromatography fundamentals (review the classic texts from Snyder & Kirkland). If the method or peak retention times vary during the run, then no scientific data can be obtained and the method fails. You should not use it until the problems have been solved. This process takes time and experience. Please do not proceed with testing until you understand how to develop a proper method and also have shown it to be selective and reliable.

[ChromaQuestion]

We are developing a method based on the European Pharmacopoeia for Enalapril maleate. Flow rate is 0.8 mL/min, decreased from 1mL/min because of the column pressure too high. When i said the pressure is too high I meant that it increases to the max, 5000 psi and it stops. This happens in the first injection. We use Waters Alliance separation module 2695, PDA detector. Injection vol is 50 microL, gradient run time is 30 min. Sample preparation is the exact method in the pharmacopoeia using a phosphate buffer pH 2.5 :acn 95:5 v/v. You are right, I know next to nothing about developing a method, I am just an analist, trying to understand and learn by asking questions. I found this forum and i asked here cause most people are well trained and experienced. I only have basic knowledge from college about cromatography and dont have any other materials to study. Thank you very much for your reply.

[Multidimensional]

I think it is admirable that you are trying to learn chromatography, but unrealistic that you will be able to do so following this or any other forum so early in your pursuit. Without an understanding of the basics, you will find yourself being led in all directions. Many will try to help, but will be unable to since they have no way of really knowing what the condition of your system is in or what you are actually doing. You need to be there in person to really help. It takes many years of practical, hands-on training just to learn the basics of chromatography. When you are first starting out and do not know any of the basics or terminology, you are in for a steep learning curve. If you want to be successful in the shortest amount of time, find an experienced local chromatographer to help you (or get a job at a lab that has experienced chromatographers that might train you).

Regarding the high pressure problem you have observed. Begin basic troubleshooting to find the cause. Pressure that rises until the max pressure is reached indicates a plug or that the operator has selected settings which are not within the range of the system. Test one-thing-at-a-time. Use logic. Learn the entire flow path of the HPLC, in DETAIL. You need to understand how everything works to use it. Start by removing the HPLC column and replacing it with a quality zero dead-volume union or capillary restrictor line (which you can purchase). With the column safely stored away, begin flowing mobile phase without any buffers or salts in it through the Alliance system and monitor the backpressure. Select a flow rate that provides > 60 bars of pressure. Monitor the pressure. Does it stabilize, rise, drop ??? Monitor it for 10 or 20 minutes if needed. If it continues to rise, you have a plug and now need to troubleshoot the source of the obstruction. If liquid flows through the system and stabilizes fine, then repeat the test with your column in-line and run using an acceptable mobile phase and acceptable initial flow rate (maybe 0.5 mL/min) and observe what happens. If the pressure is stable w/o the column, but rises until it it reaches the system max with the column, then the column may be plugged. Put the column aside, try and get another column to practice with and find someone local to help you.

[HPLCaddict]

EP doesn't include reequillibration in the gradient table - did you include a respective reequillibration step in your gradient? If no, the method could yield all sorts of weird stuff. If yes, is it maybe too harsh? - rushing back from high% ACN to low% in an instant could possibly crash out your buffer, resulting in pressure buildup...
Back pressure should be absolutely no problem with the method running at 70°C, as in the EP. You should solve the problem concerning pressure buildup first before thinking of tweaking the method to get better resolution.