Hello everybody!
I am initiating my research on HPLC. I have used a C18 Accucore column, A gradient program of 40 min (60min in total) using as eluents MeOH (A) and Water(B), both acidified with formic acid 0,1%.
My samples have 0,5mg/ml of concentration. My standards(11) concentration are 1mg/ml...of course the peaks i see in my samples are quite smaller. I injected a sample with the mixture of all standards in order to identify them in the run.
Ladies and gentlements...what is now the best procedure to analyse and identify (if possible) some of the peaks?!
All were injected in 3x, but it seems to me that are some deviations of peaks in some situations....not very happy with this?
Can anybody suggest me a way of solving my problem? Also, is there a free software to treat chromatograms (actual software is chromeQuest) and possible do statistical treatment?
Thank you very much
Hi Joana,
que tipo de detector estás a usar? PDA ou MS? Se injectaste os padrões à partida só vais conseguir identificar pelo PDA os compostos através do tempo de retenção e que tenham o mesmo espectro UV. Se tiveres um detector MS disponível a identificação é muito mais fácil.
Em relação ao software não conheço nenhum que seja gratuito mas se pretendes uma análise estatística depois da identificação dos picos podes proceder à quantificação (com as respectivas curvas de calibração) e depois usas os dados quantitativos num programa estatístico para obter algumas conclusões.
Se tiveres mais dúvidas podes enviar mensagem.
Cumprimentos.
Hello Joana,
Find a proper LC specialist at your university or start to read literature.
Hello Joana,
You first need to run your standards to figure out number of components and retention time including relative retention time of each peak.Once you have run your standard you can run your sample to match RTs and RRts. Once this matching is done you can spike your sample with standards to check the overlapping and recovery.
If you are only using HPLC you sholuld be able to compare retention time of standards with your sample if they are same the compounds or else you have to use LC-MS to identify the components of your interest.
Hello!
Abdur and Vítor, I am using a UV-DAD analyser, so i will just have the option of comparing the RT and UV profile!
I thought there would be available a software for analysis of chromatograms FREE...anyone knows one?
Hi Joana
What software did you used to run your sample and standards on HPLC ?If you have used some software to run your standards and sample, the same software should be using for processing chromatograms.
.
1.You can check & compare the UV spectra from DAD.
2.For identification go for RT & RRT.
3. Spike the sample with individual standard & confirm the peak which shows increasing area.
4.You have standard so after identification you can perform assay too.
5. If RT change in every run then change the method like mobile phase, additives etc.
You have a spectrum of standards, save them in a library spectra and compare your sample with the spectra patterns and retention times. If the time is shifted, then release the sample from one pattern (internal) and you have your answer what is the impact of your plant matrices for the duration of the pattern. The rest is all. You write that you have just DAD, well, that not only UV or UV-WIS, then it is only because you are working on retention times without spectra of the substance. And besides poświącic you take the time to read the literature, it is inevitable and highly recommended, the rest is practice and hundreds of hours at chapciu to understand where it hurts. I'm sorry, but that is the reality. And what pozinnaś tell us the kind of substance that ocznaczasz and raw material, because the same phase and gradient, it's a little bit to get more information. Best regards and wish you a pleasant read and spent time on the equipment.
Hi Joana,
normally, the software controlling your instruments and aquiring the DAD Spectra offers also data evaluation with all feature you need. Which system do you use? Nevertheless, there is a free chromatographic software "OpenChrom" (Open Source) which is primarely ment for GC- or LC-MS data. But as far as I know, they will offer soon also DAD support (maybe with the next release in July 2015)
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