Problem with Impurity.Please help me!

[ultima_86]

I have analyzed Omeprazol 20mg for 4 days. My sovent is prepared by adding buffer (0.1M NaHCO3 in 1000ml water then adjust pH 7.0 with HCl 1M): Acetonitrile = 70:30.

Everything went smoothly from the Blank to Reference and the first injection of my sample. But with the second injection on that sample, I recieved a result with the same numbers of peak but the area of peaks originated the second injection always higher than the first one. So:

+ Why do the Unknown Peaks' area always increase after 40 min ( my run time). Can I have some ideas to stop or reduce this problem.

+ Another question, I put 2 subtances in my reference. In the previous injestions, the separation was very good, one peak with RT is 4 and another one is 10. But today, I did the same things, but I recieved only 1 peak with RT about 6,6. My seniors said because the clogged pump affect to Mobile phase. Do you have any idea else?

+ I read a book that said: A solution of KH2PO4at pH 4,5 is not a buffer but a salt solution because pKa of a phosphate is about 2 to 7,5. And my desired pH is in the middle of phosphate's pKa. But I still don't understand, can anyone give me a clear explanation?

[Mattias]

A couple of possible causes:

- The unknowns increase beacuse the are coming from the mobile phase and not the sample. If you run a gradient these mobile phase peaks typically elute late in the gradient.

- Omeprazole is unstable in acidic environment. Are you sure that your samples keep their neutral pH during storage? Is baking soda OK to use as a buffer salt?

[nschwartz]

Peaks increasing in height or intensity with each injection might indicate that the injected sample is not being fully rinsed from the autosampler or injector and accumulating in your system. To check this, run a solvent blank after a couple of these and see if your analytes show up there also. If they do, you might be injecting levels that are too high and need to decrease the concentration or you may just need to adjust your rinse solvent, making your analytes more likely to be dissolved by it for better cleaning. The worst case scenario would be long term/permanent contamination that might require replacing some autosampler parts and/or tubing.

Regarding your questions about buffer solutions, here is a pretty good explanation:

http://download.5117.com/download-action-30.htm

[ultima_86]

Thanks so much for your answers, especially the ones from nschwartz, they helps me a lot.
By the way, I wanna ask one more question, some of my samples will have more impurities if I keep it in a long time ( a few days) or when they contact directly the light (It can be from sun or lights) and under a long sonication. I wonder if those above factors work as a catalyst and they motivate reactions in my sample. As a result, more impurities appear???

[nschwartz]

Yes, it is possible in many cases for UV light to trigger reactions and degradation. I have seen this happen with some things. It is also possible to happen with sonication for an extended period of time.

[ultima_86]

Thanks so much for your answer! I aslo have got another query, here is its detail :
"When an acid or a base is ionized it becomes much less hydrophobic and will elute much earlier. Acids lose a proton and become ionized (negative charge) as pH increases. Bases on the other hand, gain a proton and acquire a positive charge as pH decreases."
1. This rule is easy to learn, but I still don't understand. When is pH called increased or decreased, what is pH compared with to know that it is increased or decreased ?

2. How does buffer concentration affect to the analysis? If I use a high buffer concentration and a very low one. What will happen?

[gabriel85]

You are using salt solution but not buffer solution. You can refer back to the definition of the buffer. However, i think the molarity of your salt solution is quite high in your mobile phase. Have you seen any cloudiness when you mix salt solution and ACN? If your want to try using phosphate buffer, you must set the molarity of your salt solution is below 0.02 M because phosphate salt has low water solubility compared to others. Besides, increasing buffer strength will reduce the lifespan of your column. Try to optimize the buffer strength for new method development.
When a compound become polar, it will elute faster in non-polar column (phenyl, C18, C8...) because it less react with the non-polar stationary phase. pH plays important role in method development stage. It will affect your result whether you want to retain your compound longer or shorter in your column. (mainly based on ionization concept).
Buffer concentration will affect selectivity and symmetry of your peak because buffers tend to even out the selectivity between stationary phases.

I have two troubleshooting guidelines for you and absolutely for free: HPLC troubleshooting guide from Waters and LC handbook from Agilent.

[ultima_86]

Thanks so much for your answers.
1. I have fixed those problems, but other problems' occur. I injected six times on 1 vial of my Standard Reference. The first injection was okay with right Retention time ( 7 minutes) but from the second injection on, RT reduced gradually and at the sixth injection, RT was only 5 min. Surprised and I injected again but I got the same result. RT changed gradually with the first RT was 7 and the last RT is 5. Can you tell me some reasons and preventions.

2. I analyze many Impurities in my teo samples, but some initial peaks' retention time of two sample is not correct ( at 10 initial mintute) and after that time, RT came back normal. The next time, I incresed my analyzing method 10 mintutes more than usual. Now the RT become the same.

3. I tested my Standard Reference, I thought the the column was ok and I started my batch. But my blank vial was infected by the Standard Refernce. Then I injected the blank sample again and I saw the infected peak in my blank is gone. Can you help me to find solution? Thanks in advanced!

[gabriel85]

Have you verified your instrument recently? Flow rate consistency is one of the tested parameter. Did you monitor the pressure of the system because pressure might able to tell you the problem? If the retention times change randomly from one run to next run, pump(s) and the solvent mixing devices might be possible cause. If both of these parts are working well, check the prime seal of your instrument because there might be leakage in this part. Are you using oven to control the temperature of your column?
There are so many reasons causing the drift of the retention time. Wish you can figure out your problem soon.

[tom jupille]

"When an acid or a base is ionized it becomes much less hydrophobic and will elute much earlier. Acids lose a proton and become ionized (negative charge) as pH increases. Bases on the other hand, gain a proton and acquire a positive charge as pH decreases."

I've pulled a "chalk talk" recorded during one of our web courses and posted it here:
viewtopic.php?f=1&t=17917

1. This rule is easy to learn, but I still don't understand. When is pH called increased or decreased, what is pH compared with to know that it is increased or decreased ?

Increased or decreased from whatever you started with! It's all relative!

2. How does buffer concentration affect to the analysis? If I use a high buffer concentration and a very low one. What will happen?

As long as you have enough buffer to keep all the ionizations under control, any further increase usually has little effect (I'm assuming that there is no ion-pair chromatography going on!). If you have too little buffer, then you will likely see peak shape problems and, often, retention time variability. Most reversed-phase work is done with buffer concentrations in the 10 - 100 mM range; 25 mM is probably the most common.

[HW Mueller]

gabriel85, you have some claims in one of your posts (Oct. 6, 9:01) which I have not seen before:
-phosphate salt has low water solubility compared to others"
-"Besides, increasing buffer strength will reduce the lifespan of your column."
-"Buffer concentration will affect selectivity and symmetry of your peak because buffers tend to even out the selectivity between stationary phases."
I would surely like to see explanations of these.

[ultima_86]

Hi everyone!Here is my another query and I need explanation.
I want to control pH of my phosphate buffer at pH 6.5. I intend to use Phosphate 2 because it has pKa near my desired pH so that my buffer will have a high buffer capacity. But should I use NaH2PO4 or Na2HPO4, some of my friends said that NaH2PO4 is Photsphate 1 and Na2HPO4 is Photsphate 2. Which means I will use Na2HPO4 ????

And if the above things are correct, if I use Photsphate 1 and adjust to pH 6.5. Are there many differences between those methods??

[tom jupille]

If you simply prepare a solution of a salt, the pH will be approximately halfway between the pKa of the weak acid and that of its conjugate base (but will be poorly controlled). Monobasic sodium phosphate (NaH2PO4) by itself will give a pH around 4-5. Dibasic sodium phosphate (Na2HPO4) will give a pH around 8-9.

What you want to do is to blend equimolar solutions of those two as necessary to get the pH you want.

In practice, you could also start with the monobasic and use concentrated NaOH to raise the pH (you will get to approximately the same place, but the phosphate concentration will be a tad lower than what you started with).

In principle, you could start with the dibasic and add a concentrated strong acid to drop the pH. If you use phosphoric acid for that purpose, you will end up with considerably higher phosphate concentration than you started with. If you use something else (e.g., nitric or hydrochloric), then you will have a considerable amount of the other anion.

Of course, you could always go to one of the many "buffer calculator" web sites (do a Google search). Here's an example:
http://www.liv.ac.uk/buffers/buffercalc.html

[ultima_86]

I had a query just in the morning. I injected a sample and I got 2 peaks. One is my principal peak and one comes from the sovent, I think! My principal one is alright after several injections ( about 6 times) but the one coming from sovent is very strange. It is large in the first time and get smaller and smaller from the next five injection. From the sixth injection on, the peak's area appears to be stable and do not change. My partner tells me that he got the similar problem in the previous operations with the same machine and column. Could you please explain it for me??? Thank you so much!!!