Chromatography - Science method
Techniques 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 related to Chromatography
I have a data set of peak areas from gas chromatography I would like to run on a PLSR model. Generally, for PLSR I would center and scale the data, is that appropriate here?
As the peaks differ in scale between compounds on a magnitude of 100, running the model on unscaled data is unfeasible.
Is it standard to scale these peak areas? Is there a scaling method that will reduce overfitting the model and avoid introducing extra noise?
I am trying to purify a chimeric protein (his-tagged) via affinity chromatography for which I am using 30mM binding buffer (couldn't purify using 40mM)..but each time I am getting a contamination of many other bacterial proteins...i also tried using a 50kda cut off filter as concentrator, but didn't work...what else can i do?
I purify my recombinant his-tagged protein (40 kDa) using Akta Pure 25.
However, I have been through a couple problems.
I use Lb medium, but when expressing using TB medium (only 250mL pellet resuspended in 20mL lysis buffer), my 1mL HP column (Ni+) seems to get clogged and the back pressure increases by the end of sample application, specially after only a couple uses.
I am aware of the need to use DNAse as it helps with viscosity, but I have never used it to my cultures. I usually prefer LB, but I am trying out TB medium for higher cell concentration.
1. Bypassing flow restrictor helps decreasing the pressure, but I have bypassed it once and the column seemed to crash even under pressure limit. The flow restrictor module seems to be increasing a lot of the pressure, cleaning it up would be enough?
2. Has anyone experienced any limit for protein concentration of the lysate (not his tagged protein) or maximum volume of culture pelleted for 1mL HP columns?
3. Besides filtering lysate and buffers, any recommendations to avoid high pressure? Such as avoid using TB medium, high volume cultures…
4. I’ve been able to reuse 1mL HP columns for 5x using LB medium before it crashes (the beads seem to collapse and a “void” is visible in the column). Any recommendations to increase column lifetime?
Can anyone help in the field of packing steel chromatography columns with ion exchange resins?
High-Performance Liquid Chromatography (HPLC) is a technique used for the separation, identification, and quantification of components in a mixture. It provides rapid separation and high-accuracy results. Despite the advantages, interferences might occur and can affect the samples.
High Performance Liquid Chromatography. (2020, August 16.). Retrieved from https://chem.libretexts.org/Bookshelves/Analytical_Chemistry/Supplemental_Modules_(Analytical_Chemistry)/Instrumentation_and_Analysis/Chromatography/High_Performance_Liquid_Chromatography
What is the reason for peak time shift for a single sample in digital chromatography? (for Example: one day in 3 minute , one day in 2 minute , another day in 6 minute and...
I need the help of a chemistry researcher who will be able to identify chemical compounds through HPLC-UV-MS/MS mass spectra in the context of a collaboration (he will be mentioned as one of the authors of the work).
Thank you very much in advance
I've been routinely using Dialysis for removing salts after we do protein purification using Ni-NTA or any other chromatography. But I'm curious to know how economically and operationally feasible this process is when we move to Industrial set ups?
Since we have lower volumes (15-20mL) of protein in R and D lab it's easier to perform in beakers, how would it affect the process when we move to 15-20L of the same protein?
I'll be happy to read if anyone has any literature in this area.
I need to separate 25kda scfv from 50kda scfv. Which sephadex should I use?
After removing His-tags with enterokinase from mEGFP in pet32b(+) without TEV. I'm measuring protein concentrations with BCA assay and it seems I lost 2/3 of my proteins. I did his-tag removing with enterokinase. After that did affinity chromatography purification with His-trap column. After chromatography concentrated and desalted the protein(mEGFP) with vivaspin 3000 MWCO 20 ml. The changed buffer was phosphate buffer without salts. When doing BCA assays proteins seems to disappear. What could be wrong?
What are the Secondary Metabolites that present? Is it necessary to do the tests to prove them in the lab by color changing chemicals?
The cost is about $ 4,500.00 USD and I would like your feedback and know if it is worth the expense. Thank you!
I want to use size exclusion chromatography for drug-encapsulated PLGA nanoparticles. I am going to use Sephadex. but I couldn't find any suitable procedure for nanoparticle separation using SEC. did anyone use SEC for nanoparticles?
My protein was found to have a high nucleic acid peak when it was further purified by exclusion chromatography. How can I remove the nucleic acids when purifying my protein?
i have two samples of my protein: (1) sample after dialysis (2) sample after dialysis and ion-exchange chromatography. I ran a SDS-PAGE followed by western blotting. Sample (1), after dialysis, is at the correct size (expected MW), but my samples (2), after chromatography seems to be at the wrong size, my protein appears in a higher MW. Why is that?
I am working on a structural protein. It is a membrane protein. I have purified the protein by affinity chromatography (my protein has histidine tag). But the yield is very low. To perform size exclusion chromatography and other experiments I need more protein. I'm able to purify only with 1ml Ni-NTA matrix. If I go for 2ml and more I see more non-specificity in elution. I first denature the protein using 0.5M urea and allow it to bind to the Ni-NTA beads. Then I collect Flow- through and wash and after that I wash out the urea from the column with 20CV of chilled buffer. Then I collect elution fractions with 0.3M Imidazole. I'm stuck at this point and don't know how to increase its yield. Please help me. Does increase in IPTG help me to solve this problem?
What are the various factors that can impact the binding of antigen to immuno affinity chromatography
I am working with a Lem12 Legionella pneumophila protein (14 kDa). The protein was expressed in E. coli BL-21, filtered through a polyethersulfone (PES) membrane pore size of 0.22 µm then performed Affinity Chromatography using 5 ml HisTrap column charged with Ni2+ this is the first time for me to use this technique.
The Ni2+ column was recharged by stripping the nickel using EDTA, washed with distilled water, recharged with nickel and went for a final wash using distilled water.
When running SDS-PAGE gel electrophoresis (4-12% Bis-Tris) I observed impurities along the lanes. I would like to perform different purification methods, like size exclusion chromatography and Ion exchange chromatography.
I looked into size exclusion chromatography and I am wondering if the following method would be applicable to my protein. Superdex 75 increase 10/300 GL column with eluent PBS; 10 mM phosphate buffer, 140 mM NaCl, and 3 mM KCl with pH 7.4. Flow rate 0.8 ml/min.
And for Ion exchange chromatography my protein has a theoretical pI of 6.33 with a molecular weight of 14408.44. I could use Mini S 4.6/50 PE with a starter buffer of pH 6.0 and an elution buffer 1 mM NaCl pH6.0 with a flow rate of 0.80 ml/min.
This is my first time using these purification methods and I would really appreciate it if someone could recommend me something better or if I’m at least on the right track?
Any comment will be helpful to me, thank you in advance.
I have prepared carbon dots now i want to use chromatography to purify instead of dialysis
Hey everyone. I am trying to find a cheap mix of lipids to use as a system suitability standard to make sure the instrument and chromatography is working as expected. What do people use? And most importantly, how much (volume/concentration/amount) do people load for different sized columns and flow rates?
mAbs are purified using Pro A chromatography, eluted with acetate buffer pH 3.0 and pH was adjusted to 6.0, stored in fridge.
During thawing, entire protein gets precipitated.
Please share thoughts.
Does someone twice use columns for mini-SEC (Econo-Pac Chromatography columns from BioRad)? Can you recommend me something similar? Unfortunately, the waiting time for BioRad columns is more than three months ((.
If yes, I have some additional questions:
1. How to clean it?
-Do you use the same column (packed with sepharose), just clean it 3-5 times with PBS or something else? Or do you discard the old sepharose and filled with a new one?
2. Do you have some side effects?
3. Do you check the target fraction after the second use, is it have the same properties?
Thank you so much in advance for your appropriate answers.
I have been working with overexpression of a protein of 29KDa and purified it through affinity chromatography. I used 20mM imidazole concentration in the lysis buffer and the protein got eluted at 100mM imidazole concentration. However, I got a single band and the size decreased to 25KDa. Further, after dialysis of the sample, I ran the SDS page where I got multiple bands for the same protein. I don't understand where I am wrong.
I am curious if column bleeding is still "a thing" on modern UHPLC columns (reversed phase and hydrophilic interaction columns), such as the HSS T3 C18 column from waters or the iHILIC column from hilicon (or any other UHPLC colum). Did anyone observe such bleeding? If yes, which masses did you observe for which columns (if you used MS)?
I'm looking for the best/most suitable code of column /resin to use in chromatography steps for separation of freeze-dried peptides in the ÄKTA pure. The manufacturers brochure has about 5 to 6 options, but I could not find what is the best for may case. Someone has experience int this case and this device? The fractions were ultafiltraded in MWCO raging from 10 to 3 kDa. and kept freeze dried in -80 °C
I can express it by IPTG inducer but when I purify with affinity chromatography, it doesn`t have any activity. I have to add this point that my protein express in plete not sup.
I perfomed a column performance test, which showed an abnormal peak shape. when I was opening up my column packed with daiso cilica gel, I noticed That the column bed was extemely jelly-like. I have never seen this before. what could be the reason? What have I done wrong?
In recent years, the progress of chromatography has been dizzying.
I am confident that the use of sustainable quantitative chromatography, which uses less solvent than is currently used, will become more popular in the future.
One of the candidates is UHPLC or SFC with supercritical CO2, but is there any well organized information on the advantages and disadvantages of them?
We would like to develop analytical methods to meet future global current trends.
Everyone, if you could point us to any literature that better summarizes the situation, we would appreciate it.
In response to a reviewers comments, I want to measure the following:
- The presence/ratio of different nucleotide di-/tri-phosphates produced by an enzymatic reaction (E.g ATP vs ADP and dATP vs dADP)
I thought this reaction could be measured via some sort of tandem mass spec, where a chromatography step is used to seperate small molecules from large proteins, and then native mass spec can indentify the different nucleotide species.
This experiment is somewhat outside my wheelhouse, so I am looking for suggestions as to how to measure such a reaction.
Hi, I'd like to ask whether it is necessary to precipitate MMLV Reverse transcriptase before affinity chromatography purification (Äkta)? My colleague must do this step with his Taq DNA polymerase. He use (NH4)2SO4 or Na2SO4 + PEG. Without this precipitation is polymerase inactive.
Thank you for your responses.
we want to determine effect of different compounds in cell culture harvest on protein A chromatography.
We intend to separate the mixture of three glycosilated flavonoids having two sugars units, each, according to their LCMS profiles.
I wonder what is the principle behind the usage of core-shell technology (SPP) columns (2.7, 2.6 um particle sizes with various lengths at micro, capillary, or analytical flows (IDs)) at LC separation prior to Orbitrap HRAM spectrometer analysis at shotgun metabolomic or proteomic approaches? I read lots of papers that preferred to use SPP with >2um size even the UHPLC system configuration is available. Since we were acknowledged the fully porous or SPP version of the sub 2um particles can supply sharper and more efficient peaks, why SPP version of the >2um particles are being chosen for omic-based (I meant discovery mode/non-targeted modes which the higher resolution is more amenable (AIF, DIA, DDA)) investigations recently?
Is that about the trapping principle of the Orbitrap?
Accumulation of the ions in ion routing multipole along with C-trap and therefore low data cycle times can cause the sensitivity loss when combined with fast chromatography application such as UHPLC? Not enough data points can be recorded with UHPLC pressure and flow at the Orbitrap system when an increased resolution was selected for accurate mass detection at discovery modes?
I'm looking forward to purify my compound from the impurities.But, I couldn't find an appropratie solvent to dissolve it!
I tried, MeOH, Acetonitrile, Water and mixture between those solvents.
How can we handle with this kind of samples?
I used reverse-phase for the first time, and as you could probably notice the amount of water is already very high! Although, most of the compounds come earlier during the first 5 min!
To estimate the concentration of PFOS, we use mass-labelled PFOS (MPFOS). We generally spike the standards and samples with unknown concentrations with MPFOS. To estimate PFOS, we first make a standard curve by plotting peak height or peak area ratio vs concentration ratio of PFOS and MPFOS. Now, my question is, how do we differentiate the peaks of the mass fragments with the same m/z (say 99) generated from PFOS and MPFOS from the LC-MS/MS MRM chromatogram? Do they have the same retention time?
Thank you in advance for your praiseworthy answers.
I am using zeba desalting column to purify my protein + plasma sample in the beginning step of protein enrichment procedure to get rid of all unwanted stuff. After performing desalting process, I am going through the enrichment and digestion procedure but on LC-MS my peptide peak shape getting poor as the more number of injections injected. Initially peak shape is perfect but after injecting about 30 to 40 samples, chromatography getting poor. Earlier, samples prepared without zeba desalting have not shown any poor chromatography. Anyone have any idea about troubleshooting?
I am developing a gradient method for the estimation of 3 drugs, two are water-soluble and one is hydrophobic. I am using Dionex HPLC system equipped with P680 HPLC pump, ASI-100 automated sample injector, UVD340U detector. The mobile phase is composed of 10mM KH2PO4 buffer pH 6.8 and ACN. A gradient is applied from 97:3 (or 90:10, depending upon the column used) Buffer: ACN up to 5 mins, followed by 50:50 buffer: ACN up to 16 mins, followed by 97:3 Buffer: ACN up to 20 mins. Columns tried are Inertsil ODS 3, Inertsil ODS 3V, Eclipse XDB c18, Phenomenex Gemini C18. Samples prepared in 50:50 Water: Methanol.
After 30-50 injections, the pressure rises to 170-200 bars (initial pressure 96-106 bars). After back flushing or washing at 50 deg celsius using 60:40 ACN water, the pressure reduces. However, again with increasing the aqueous content beyond 60%, the pressure starts rising and the peak shape is also not good. What could be the reason?
We are experiencing some corrosion/peeling of the white paint inside the Akta F9-C fraction collector. We suspect that this is due to overflow while washing the fraction arm under high flow rate after runs with high salt (6M urea). We have since fixed the overflow issue, but are curious if anyone else has experienced this paint corrosion/peeling before and if so, did it cause any further problems? For example, did the metal under the paint begin to corrode? Was there anything you did to re-seal the corroded area?
Thank you in advance for any comments/advice!
Chromatografy is recommended for epigenetic studies with organisms lacking a reference genome
To have the chance to find new structures from plants, we need to collect all the fractions and isolate even the most minor compounds.
But, the problem is that we recapitulate at the end only a very small mg!
Is this kind of work still significant? or not worth!
Could we publish in high journal with only elucidation of new structures from plants?
I ran my methanolic extracts (flavonoids compounds) over silica gel, then no separation took place due to the complexity of the mixture. I collected all the test tubes fraction together and I removed the solvent. Consequently, the first color was white turned yellow! While, I didn't separate any fraction and all the fractions were concentrated together!
How can we explain the change of colors, whereas we didn't make any isolation?
As we know Like dissolves Like; polar analytes are dissolved in polar solvents.
I noticed in an article that they use the soxhlet apparatus with a temperature in the range of 70-80° to recover methoxyflavoinds (polar class of compounds)!
I'm just questioning if the temperature might plays the role to extract polar compounds even with apolar solvent?
Hello! I am trying to extract chlorophylls from microalgae, and I would love to ask how can I separate chlorophyll a from chlorophyll b? I want to get each of them separately. Thank you in advance.
We are using a C18 reverse-phase column in liquid chromatography for purification and separation of oligonucleotides. However, it seems that they got crystalized inside and the column is stuck now. We have tried elution with acetonitrile, methanol and water but it doesn't work. Is there anything else that could save my column? Thx.
Generally HPLC, we can use it for qualitative and quantitative analysis.
What is the main difference while using it with PDA or with MS detector?
What are the advantages of MS to PDA and vis-versa?
I isolated this protein from priplasmic area by TES & TES/4 protocol. Our protein has a 10 kDa taq containing two His tag in N-terminal and a His taq in C-terminal. Approximately, 80% of protein in priplasm has no 10 kDa tag because of autocleavage. But 20% of protein contain a 10 kDa tag in N-terminal. When I perform affinity chromatography purification, protein with 10 kDa tag was purified but protein without N-terminal tag wasn't purified and whole protein got out of ni-nta column and enter in unboud vial. I must mention that we do purification of protein in danaturing form but hadn't any purified bond on SDS-PAGE.
Our purification condition:
1. Equilibration with Tris 50 mM pH 8.5
2. Sample loading and incubation in 4C for 3-4h
3. Washing with Tris 50 mM pH 8.5; Tris 50 mM pH 8.5 + imidazole 10 mM
4. Elution with Tris 50 mM pH 8.5 + imidazole 150 mM
I have a problem with my internal standard, it seems that there is a peak has the same retention time of my internal standard, this peak appeared in human plasma chromatogram although I made a derivatization step and SPE and my internal standard has no appearance in human body as the reference article saying ...
There are two photos ... one illustrates how the peak of my internal overlapped with the other peak which I don't know ... and the other photo shows the derivative of my internal and it's a cyclic derivative ...
I tired playing with mobile phase ratios but no change not even a small change ... they move with each other and they are totally attached to each other ... I'm thinking of using external standard method to solve the problem but is that logical while working with biological samples ....
My internal standard is 2-methyl cysteine and I'm using % acetonitrile in % acetic acid as mobile phase
My current work is producing a reasonable amount of dibenzyl ether contaminated with a small amount of dichloromethane, approximately 10% vol. I would rather reuse the reagent than throw it away, so can anyone recommend a simple method for extracting the dichloromethane.
I have a solution containing both 1-naphthol and 2-naphthol and I want to separate them. Is there any way other than chromatography to do that?
Thanks in advance for your answer
I'm trying to purify a protein with a StrepII tag using Strep-Tactin XT. I discovered that 2/3 of my protein was eluting in the flow-through. I think its an issue of kinetics. That my protein doesn't have enough time to bind to the resin. Does anyone know of a way to reduce the flow of my protein extract through a column? I'm using a Econo-Pac® Chromatography Column and a three-way stopcock.
I have recently started working with Alzheimer's disease. So, I have been expressing the amyloid precursor protein (APP) in a mammalian system. APP is a transmembrane protein that can be processed by a protease to release the cleaved peptide (that is ab-42) into the conditioning media. Most papers are expressing the peptide itself in bacteria and directly purify it. However, I used HEK293T cells to express the full-length protein (APP) alongside the protease that processes it. I have also used ELISA to confirm the presence of the peptide in conditioning media. So, is it possible to purify the peptide directly from the conditioning media? The size of the peptide is around 4 kDa, so would size exclusion chromatography be a good approach to purify the peptide from conditioning media? any other suggestions?
My mobile phase is 40 mL water ( with 0,112 g sls and 0,64 microliter of orthophosphoric acid), 56 mL acetonitrile and 4,8 mL methanole. A C18 column is used for the analysis. Detection is at 233 nm with UV. HPLC system is thermo finnigan surveyor. The standard solutions are prepared in mobile phase without sls. After analysing 3 repetitions of same standards with good similar results, peaks were smaller or disappeared in fourth repetition. What could be the possible reason for these results?
P.S: I prepared new standards and mobile phase but the problem persists?
I see some researchers doing some isolation of already known and available compounds!
Does this isolation still meaningful?
I have been asked to estimate the maintenance cost of a chromatography lab. The lab has a GC/MS and a UPLC. The cost includes service contracts and calls.
How will I be able to estimate that? What are your annual chromatography maintenance costs?
I need to do UPLC-MS for an structure unknown compound. Is there any service in india to do so?
How will you approach to find the quantitation limit in case of size exclusion chromatography depending on whether the testing method is purity or impurity method. Will your approach and focus will be same Irrespective of testing method? Please share your thoughts and why do you think so?
I would like to get my results in Word files in order to use it in my article.
And thank you for your cooperation.
Has anyone had experience working with Exo-spin columns? They propose to use a combination of precipitation and size exclusion chromatography resulting in pure exosomes. Can someone tell me what the performance of this kit looks like? Has anyone been successful in obtaining high enrichment of exosomes using this kit? Any suggestions or replies would be highly appreciated!
Hello dear researchers,
I would like to know if there is a possibility to purify antibodies using ionique exchange chromatography or gel filtration with basic equipment.
If yes, i would like to have some tips to make it possible.
Thank you in advance.
I am new to a lab that has AKTA pure 25 chromatography machine, and I am trying to figure out how to use it. I have tried to follow the user manual religiously, but I have not had much luck.
I was wondering if any here is familiar with the machine, and would be willing to guide me through a run.
Thanks in advance
Irrespective of other noble gas elements such as Ne , Argon or the easily available gas in atmosphere i.e. N2 , why Helium is used largely ?
Hi all! In my experiment, I am trying to study the effects of treatment with EVs isolated from different mice groups on inflammation and thus need to also demonstrate that EV-depleted plasma from the same groups does not have the same effect. I isolate EVs using size-exclusion chromatography. Does anyone have any experience with EV-depleted/poor plasma and how to isolate it? Many thanks!
I 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.
Can anyone explain how to use a glass hygrometer to find the % water remaining in solvents distilled from the rotovap so that I can reuse them for chromatography? Where can I get the chart that will relate the numbers on the hygrometer to % water for common solvents such as methanol, hexane, ethyl acetate....
During 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 ?
As 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 !!
HIC chromatography of antibodies
I 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.
I want to analysis terpenoid by thin layer chromatography?
How to do chromatography for dispersion "waterborne adhesive"?
Which kind of method used to get rid of the remaining adhesive in the tube?
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.
I 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.
Here 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.
I 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?!
my 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.