I am trying to analyse a certain material in a certain food product via HPLC. However, I crossed problems and tried to correct them but problem goes on.
After i see baseline in HPLC, i put standards and then my sample vial.
My sample showed a peak more area than standard peaks area. As first correction, i sent more concentration standard to HPLC column, Then i recognised that my sample peak got increase. As i increase standard concentration, peak of sample getting increase
As second application, i tried diluting sample concentration. Firstly i sent standards then i applied samples which are different concentration. But it didnt work well. For each diluted sample i read two times but machine didnt read peak of sample accurately. While first parallel showed a peak, second parallel didnt show any peak.
I cleaned HPLC column with mobile phase but it didnt change results. After cleaning of column i get a baseline but while reading samples, it doesnt show peak each time.
What is your recommendation for this problem?
Dear Elif Öztürk,
As expertise in HPLC I would like to go with you step by step. You said you gonna to analyse certain material in a food product? are you sure a material or a compound?. you can not inject a material onto HPLC. The sample must be in a purified liquid state. Also which compound you interested in to analyse in HPLC? and which food material you work on? if your sample increase according to increasing the concentration of standards. That means either you spike your sample with the standards or your sample is contaminated with the standard!!. As a matter of fact there is no correlation between sample and standards if they injected separately. See Elif you need to develop your HPLC method and to have a satisfied results? So ask yourself how can I develop my HPLC method? the answer is reading enough literature. I have several clues you can work on to develop your method: check the following: your mobile phase isocratic or gradient elution?, your wavelength ultraviolet or visible region, your detector DAD or what, the concentration of your standards. The food material that I work on do I followed the right steps until becomes purified and ready to inject onto HPLC? you need some time usually PhD students take 3 months to develop a methodology. Be patient follow my steps and insist to do it successfully. I have attached something that my students do
Best regards
Aly R Abdel-Moemin
Thesis Chapter 2-TO INVESTIGATE THE ANTIOXIDANT PROPERTIES OF DIETA...
Thesis Chapter 3: TO INVESTIGATE THE ANTIOXIDANT PROPERTIES OF DIET...
Hi Elif,
From the little information you have posted, I would think you may have some carryover in your system. You can quickly check that by injecting a blank (the diluent you use to dissolve your standard) followed by the standard, followed by another blank. If the first blank did not show a peak but you see a peak in the second blank you know that you have carryover.
Carryover occurs when you work with "sticky" compounds. Make sure you have a decent needle wash that can wash your compounds of the needle. And if necessary run a blank between standards and samples to ensure that all of your compound is gone from the system.
Good luck!
Dear Sabine,
Exactly, ı have this problem. I am agree with you. I will try it. I suspected that needle doesnt work well during reading sequence. Thank you for your attention.
Yours Sincerely,
Elif ÖZTÜRK
Dear Aly R Abdel-Moemin,
Thank you for your attention. Your references are useful for me.
Yours Sincerely,
Elif ÖZTÜRK
I am using C18 column. And my mobile phase includes acetonitrile and water. I diluted extracts of my samples and standard solutions with water. İs it better or not to add mobile phase when i prepare dilution of samples and standards.
While HPLC results are evaluating there is used two way.One of them is determined by standard solutions curve. y=ax+b. y shows area of standards and x shows concentrations of standards.
,and the other is determined by formula
caffeine,ppm=(A2/A1)*(C1/C2)*dilution factor
A2 is area of sample
A1 is area of standard
C1 is conncentration of standard, ppm
C2 is sample amount, g
but i cant decide which way is better. I would like to show my results as mg /g sample. I am applying for y=ax+b method to be able to determine results.
(Note; mobile phase is isocratic elution. I prepared 100 ppm standard stock solution and obtained standard curve from 5ul to 40 ul volume.
Standard peak area was not high volume and then i recognised there was recommendation on prospectus of standard. It is said that 20 mg caffeine standard should be used while i am preparing stock solution. If i use 10 mg standard i have to use U=k.Uc as mass fraction (g/g) and k is 2. U is expanded uncertainty. I still dont comprehend how can i apply this formulation to my results. Or i will prepare new stock solution with 20 mg standard, 200 ppm solution.
What should i do ?
Because the sample is choosing the micro-environment (concentration of target in mobile phase versus stationary phase) elution conditions. You can redevelop the method (stronger conditions) OR just dilute the sample in mobile just prior to injection (1/10).
The problem as I see it might be because of the carry over contamination at the auto injector or needle
Area of sample is not between standard areas. It is lower than standard areas. It has to be between standard areas, hasnt to it? However, i prepared 100 ppm stock standard and i didnt obtained peak when i injected 5 ul stock standard. I started to observe peak after 10 ul volume of standard.
Multiple issues going on here.
1. Initially it sounds like carry-over. Can be diagnosed by running a blank between injections but that is not the solution. The cause could be a number of things: Standards and/or samples are too concentrated, or your chromatographic conditions aren't optimized, or you need to run at a higher temp to fully elute the compounds, or need a gradient.. BUT, it could also be that your sample diluent is creating a condition upon injection where the compound is getting stuck because the solubility in your diluent and mobile phase is different. And the viscosity of the two solutions also is different. Try diluting in your mobile phase and see what happens. Depending on your compound, an excess of ACN in your mobile phase could cause the compound to crash out up on injection and only slowly re-dissolve throughout the run.
Based on what you say, I believe your standards are too concentrated/or your compound is crashing out. In either situation, your standard is being retained on the column and is only eluted when the next injection comes through and pulls it out. This means that the sample you analyze AFTER the standard injection will have a HIGHER peak height/area than it should. However if you inject the sample or another similar sample immediately after this it will have a MUCH LOWER peak because there isn't the retained material to add to it.
"As second application, i tried diluting sample concentration. Firstly i sent standards then i applied samples which are different concentration. But it didnt work well. For each diluted sample i read two times but machine didnt read peak of sample accurately. While first parallel showed a peak, second parallel didnt show any peak"
Yes, exactly! Because the retained material had been flushed out by the first sample injection.
If you get the carry-over under control, either by reducing concentration or modifying your diluent or modifying your mobile phase, you should hopefully see more consistent results on your peaks. Once you are seeing consistent peak areas for repeat injections, then you can proceed with the next problem...
2. Samples being outside your curve. I don't believe your samples are too concentrated, I think your curve is the issue. First, if material is being retained on column, then your curve is garbage because you're not "seeing" all the analyte for each injection. That said, are you preparing standard samples are different concentrations or are you using a stock solution and injecting that on column at different injection volumes to achieve your curve? I strongly suggest making standard solutions and running everything at the same injection volume.
If the problem is that your samples are too LOW in concentration, you will either need to dilute them less or inject a higher volume on column and adjust the calculation/dilution factor accordingly.
3. What chromatography software are you using here? It should be able to do the math for you provided that you have set the processing method up correctly.
I hope this helps! Good Luck!
Dear Sara Kuhl,
I used 100 ppm standard stock solution and injected different volumes to achieve concentration curve. I obtained R2 of curve approximately 0,99. I also used HPLC-DAD. I recognised that as i washed needle some intervals, i obtained more stationary results. Your statements are so useful for me. Thank you for your attention and explanations.
Yours Sincerely,
Elif ÖZTÜRK
If your samples aren't very high, then it wouldn't take a great deal of carry-over from your standards to see it there. It's entirely possible that you are seeing most of the analyte, enough to get a "good enough" curve, and still have carry-over. Most systems perform some sort of needle wash each time anyhow. But it's never a bad idea to include it in the process.
Other things to consider are your instrument method itself. You may need to include additional time in the run to flush the column, or slow down the flow rate to allow more residence time for the mobile phase.
Keep at it.. You'll get there.
I think Sara kuhl is right. Apart from instrumental part there is one more problem with food material. Bse it contains lots of components. It would not be specific. If u increase the concentration then peak area increase but if u dilute then peak area is not properly decrease so there may be the concentration below loq of component. There may be washing related issues and pressure related issues.
Right.
And I guess we are making an assumption that because you're calibrating with caffeine that you are analyzing caffeine in your samples? There hasn't been any discussion about your sample prep, but I certainly HOPE you aren't injecting.. uh... a beverage? There's a relatively easy SPE procedure for extracting caffeine.
The primary problem here seems to be carry-over coming from the standards, either from solubility problems or concentration problems. Get that resolved, THEN look at your samples. If you still can't see the peak in your samples, alter their dilution.
when i applied hot water treatment to my samples instead of room temperature water treatment, then signals could be observed better. And i applied my sample from 5 ul to 20 ul and then also i observed signals more clear. I obtained standard signals respectively according to its concentrations. I suppose that i solved my problem.
What on earth are you analyzing? I'm not sure why hot water would make a difference, unless you're analyzing a dry powder and using hot water is getting it to dissolve better. If you're referring to the mobile phase, it's better to apply heat via the column oven then to heat the mobile phase.
At any rate, assuming that carry-over is no longer an issue, and that your peak areas are consistent between duplicate injections, and your samples are now falling within your calibration range, then I would say yes. Is this what you are saying?
I extracted coffee powder (turkish coffee) with tridistilled water. I read an article which was written hot water makes caffeine more soluble in coffee powder during extraction process. So i tried to extract it via hot distilled water. And i started to observe signals otherwise i couldnt observe any signal. Duplicate injections is shown consistent. But there is a problem. sample area is not between standard areas. Sample area is less than standard areas. I prepared my sample more concentrated. But its area is still less than standard calibration range.
Yes, you need to use hot water to extract the caffeine. You will either need to inject a higher sample volume, or make them less dilute, or reduce your calibration curve. What is the lowest concentration of standard you can inject and still get a good peak. Start from there.
Did you use the same solvent as in standard solutions? You could try evaporate water after extraction and dissolve solids in the same solvent as the standard.
I think this problem related to the cleaning of your column and decrease your flow rate to allow more residence time , and try again.
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