Science topics: ChemistryBiochemistryBiochemical MethodsAnalytical ChemistryAnalytical Chemistry TechniquesChromatographyScience methodChromatography - Science methodTechniques used to separate mixtures of substances based on differences in the relative affinities of the substances for mobile and stationary phases. A mobile phase (fluid or gas) passes through a column containing a stationary phase of porous solid or liquid coated on a solid support. Usage is both analytical for small amounts and preparative for bulk amounts.Questions (1,072)Publications (622,241)Questions related to Chromatography12345 Vikram Kumarasked a question related to ChromatographyProtein Folding during Elution step of Affinity Chromatography ?Question3 answersJul 15, 2021During Elution of protein (say an Antibody) from Protein A column using Elution buffer which is at low pH (3 to 4) and immediately adding Neutralization buffer to the collected fractions to prevent the negative effects due to this extreme low pH. I ld like to know during this process will the protein remains in native form only (or) It will first unfold during low pH elution fallowed by re folding during neutralization ?Relevant answerDominique LigerJul 16, 2021AnswerHi there,Neutralizing the pH as quick as possible limits the denaturating effect of low pH on proteins. It s not about denaturation/renaturation processes, it s rather about preventing denaturation.View13 Recommendations Rawaah Y. Al-sharrabasked a question related to ChromatographyWhy this peak in the picture appear in every run although the wavelength is fixed? Question15 answersJul 11, 2021As seen in the picture, the peak appeared in several retention times which is very weird (it has no fixed retention time) !!Also, it appearedis later at 7 min and disappeard from 5 min !! IMG_20210711_141646.jpg8.78 MBRelevant answerWilliam LetterJul 13, 2021AnswerRawaah Y. Al-sharrab wrote: I just put an empty vial in a certain position and name it as blank and run it between sample runs. An empty VIAL is NOT a proper HPLC BLANK ! Please obtain some training and assistance in using the HPLC system. Incorrect conclusions will be reached without years of formal training and experience. Data gathered will be invalid. Time, materials and money wasted.Please inject a real blank (inject the same volume of mobile phase as you normally inject for the sample, but without the sample inside, just 100% mobile phase). At identical wavelengths, compare the blank run to a real sample analysis chromatogram (overlay the two chromatograms). If you still see the peak , then you have a contaminated flow path (column fouling or carryover, as noted earlier).View7 Recommendations Aleksandra Bjelosevicasked a question related to ChromatographyDoes anyone have method suggestions to run hydrophobic interaction chromatography (HIC) on antibodies?Question5 answersJun 30, 2021HIC chromatography of antibodiesRelevant answerJeff CallJul 1, 2021Answerbut why HIC? lots of easier/better ways for Ab purification. ( https://cdn.cytivalifesciences.com/dmm3bwsv3/AssetStream.aspx?mediaformatid=10061 destinationid=10016 assetid=11660 ) covers lot of options. View4 Recommendations Simon Menanteau-Ledoubleasked a question related to ChromatographyWhat is the best way to purify compounds from the supernatant?Question6 answersJun 28, 2021I am studying the effect of algal supernatant, and, ideally, I would like to purify/separate individual molecules to test molecule/compound s separately.What methods would you folks recommend? Chromatography? Any idea welcome.Relevant answerWilliam LetterJun 29, 2021AnswerFirst, you must identify you goals and purpose. Algal supernatants often include a mixture of trace metals, vitamins and other bio-molecules which were present in the original growth media. You can expect many of those to be present. Techniques used to analyze the mixture should be based on the source. Liquid chromatography coupled to appropriate detection modes would provide a good starting point. *As a consultant, most of the microalgae production projects that I have worked on started with having the client identify the ingredients used in the growth media. You can try a simple key-word search on the web to find useful resources (i.e. Google).View2 Recommendations Priyanka Satiasked a question related to ChromatographyI would like to know which mobile phase and spraying reagents are best for analyzing terpenoids?Question7 answersApr 25, 2014I want to analysis terpenoid by thin layer chromatography?Relevant answerHamid Reza FarhangJun 29, 2021AnswerI think that if you could have done your test by LCMS, you would get better results for identifying the terpenoids. Meanwhile, it is better that to use n-haxane for the first time. View4 Recommendations Ali Alahmadasked a question related to ChromatographyHow to do chromatography for dispersion waterborne adhesive ?Question4 answersJun 21, 2021How to do chromatography for dispersion waterborne adhesive ?Which kind of method used to get rid of the remaining adhesive in the tube?Relevant answerWilli GlettigJun 22, 2021AnswerYou have to separate polymers (PVA, EVA etc) from the Additives (Sufactants, admixtures, colourants, fragrance, maybe external plasticisers etc). 1. Dilute the mixture and centrifuge or precipitate polymers. 2. Run LC-MS on dilluted additive mixture. Do you know what kind of polymer you are looking for? You may be able to measure MW by Size exclusion. Do you have IR for polymer chemistry?View0 Recommendations Morgan Currierasked a question related to ChromatographyCan I use expired Chelex-100 (100-200 mesh)?Question1 answerJun 16, 2021Hi all,I have been using a bottle of Chelex-100 in a chromatography column to separate dicationic Cu and Zn. However, when I am trying to set up my columns, the Chelex keeps pulling away from the side of the glass, sometimes all the way down my column. Would this be a result of it having expired? The date on the bottle was from 2013 and it was also stored with a Scoopula inside the bottle. Thank you!Morgan CurrierRelevant answerPierre Valery Kemdoum SindaJun 18, 2021AnswerI don t think it would work. Since Chelex 100 is a chelating material from Bio-Rad used to purify compounds by ion exchange. With its good ability to bind to transition metal ions. Therefore if it is expired it risks not having chelation with different constituents of the mixture since its chelating properties would be modified. But without chelation there will be no separation. So you have to look for a good new Chelex to work.View0 Recommendations Iona Shearerasked a question related to ChromatographyHow should I make acidified methanol for separating ommochrome pigments out of crab eyes using TLC plate methods?Question3 answersJun 16, 2021I have to use a variety of chemicals to identify and separate out the different red pigments within certain species of crabs eyes, one of which it is suggested to used acidified methanol for ommochrome pigments. However we do not have any premade in the labs and they have told me to make some myself and i have no clue what the best method (or acid) to use for this is. There are some papers that say to use hydrochloric acid and some that say to use sulfuric acid.Relevant answerSteven Collins Njonté WouambaJun 17, 2021AnswerHello Iona. I can t figure out your question. what do you want exactly? -Do you want the protocol for making a TLC plate? or -Do you want the protocol to separate your pigments on TLC?View0 Recommendations Fatima Marinasked a question related to ChromatographyWhat statistical methods can I use to test the reproducibility of my data?Question6 answersJun 11, 2021I am analyzing data obtained from chromatography analysis done on samples collected by two different users. The first user repeated the experiments 3 times and the second user 2 times. Each analysis consists of 10 components. The attached picture illustrates the number of data points. i need to test the reproducibility between both users. Capture.PNG27.53 KBRelevant answerDavid Eugene BoothJun 14, 2021AnswerI believe I understand now and in the morning I will try to message you. Thanks for the explanation. Best wishes, David BoothView3 Recommendations Adeseye Adeyigaasked a question related to ChromatographyI need a recommendation/suggestions for HPLC machine for my research Lab? What is the cost for this?Question9 answersMay 10, 2021Here are my specifications:Quarternary (4-ch) HPLC pump (0.000-10.000ml/min), Vacuum Degasser, Diode Array detector (UV/Vis), Autosampler / Autoinjector and a high end chromatography, Data System for data anaylysis. Relevant answerEdward MunteanJun 9, 2021AnswerShimadzu - very good price/ quality balance .Be careful with issues regarding training, technical assistance, maintenance, especially if you have no experience in the field.View3 Recommendations Rawaah Y. Al-sharrabasked a question related to ChromatographyHow to solve an internal standard problem?Question19 answersMay 26, 2021I analyzed the internal standard 2-methyl-L-cysteine hydrochloride using HPLC and the chromatogram shows three peaks instead of one which is weird!! I repeated the preparation process two times to check if there is a contamination problem but the chromatogram still showing three peaks. So what do you suggest?! Is there a possibility that the internal standard is converting to other compounds?! Relevant answerM. D.H. ProdhanJun 8, 2021AnswerDear Rawaah Y. Al-sharrab,Thank you so much for your interest. Actually, it is very difficult to solve the problem of internal standard. That is why, most of the researcher are now developing their method using external standard and to use matrix matched standard for avoiding the use of internal standard. Anyway, if you interested to work with internal standard, try to use the solvent as same as mobile phase, it may helps to minimize the problem. I read the answer made by William Letter, he described many things, you may follow his advice. Thank you.View45 Recommendations Ram Gopalasked a question related to ChromatographyWhich chromatography is preferred for purification of hydrophobic peptide?Question5 answersMay 30, 2021my fermentation broth has 30ms/cm conductivity,need to purify small peptide,I tried with phenyl sepharose.suggest any another way to isolate or purify the small hydrophobic peptide.Relevant answerFrank T. EdelmannMay 30, 2021AnswerDear Ram Gopal many thanks for your interesting technical question. For a useful guide to reverse-phase HPLC please go through the following link:A Guide to the Analysis and Purification of Proteins and Peptides by Reversed-Phase HPLChttps://www.hplc.eu/Downloads/ACE_Guide_Peptides.pdfThe article can be freely downloaded as pdf file.You might also want to have a look at the answers given to the following closely related RG thread:Does anybody have an idea of how to purify hydrophobic peptides by HPLC which are hard to solublize in initial HPLC conditions?https://www.researchgate.net/post/Does-anybody-have-an-idea-of-how-to-purify-hydrophobic-peptides-by-HPLC-which-are-hard-to-solublize-in-initial-HPLC-conditions(22 answers)Good luck with your work!