Science method
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 am dealing with unspecified impurities in a pharmaceutical finished product. It contains 4 active ingredients to be analyze with 4 different HPLC-UV methods. I have EuPh impurities standard, so I'm quite sure about the peaks in the sample with similar retention time. But what about unspecified impurities? How can I state if one unspecified peak is related to one substance or to another? Chromatogram of the finished product is plenty of peaks and I'm not sure from where they are coming from. Thank you all
We utilize Shimadzu GC-2010 GC-FIDs and using a supelco SP-2560 column. We notice that we experience a significant retention time shift with injection 1 and gradually the retention time will shift back to normal in about 10-20 injections. We mainly see this after the GC sits idle for extended period of time (>1 day). Basically every Monday at start of new work week we experience this on all of our GCs.
We utilize Gas Generators for all our gases but also have gas cylinders as back up and we experience this using both type of gas supplies.
We integrate batches using Methods so this will pick the 27 peaks for us. However, with the shift the methods will not work as the parameters we set for the integration do not line up. Also, within our methods we set the retention times for the 27 peaks of interests and this shift creates issues as it will identify the wrong peaks or no peaks at all. This will create issues as we use that information to report results to clients. For the retention time shift here is the reason why it is significant. 1 picture is a sample at the start of the batch and 1 picture is the same sample at the end of a batch. The same integration method is ran through each sample. As you can see the sample at the start of the batch has no peaks integrated correctly and the identification of the peaks would be incorrect if the sample was integrated.
Example of Retention time Shift for first 15 injections of our ISTD peak.
Ret. Time (min.)
Injection 1 9.951
Injection 2 9.990
Injection 3 10.003
Injection 4 10.010
Injection 5 10.016
Injection 6 10.020
Injection 7 10.022
Injection 8 10.024
Injection 9 10.026
Injection 10 10.026
Injection 11 10.028
Injection 12 10.028
Injection 13 10.029
Injection 14 10.029
Injection 15 10.029
What might be causing this to occur? How could we correct this issue?
I try to purify a glycosylated protein that is secreted and glycosylated (expressed without histag) using Pichia pastoris. I used different ion exchange chromatography while 90% of glycosylated protein does not bind to the resins. Any suggestions on how the binding could be improved, and is there any specific protocol for purification of glycosylated proteins?
Hi everyone, i would ask if someone have experience in performing the linked methods, in particular i have some doubt about the column steps:
-"Rinse the round-bottomed flask with water R and
dilute, with the washings, to 20.0 mL. Transfer the solution
to a chromatography column about 0.15 m long and about
30 mm in internal diameter containing 15 g of kieselguhr for
chromatography R. Allow to stand for 20 min. Elute with
200 mL of a mixture of equal volumes of ethyl acetate R and
methylene chloride R. Evaporate the eluate to dryness in a
250 mL round-bottomed flask."
I'm not sure I understand the protocol correctly. My first doubt is that there are no instructions for embedding, so I'm not sure how to do this or if I should avoid it.
I've tried following the instructions but the results are quite unsatisfactory as when pouring the mobile phase the column is badly broken and the solution is eluted by preferential channels.
I am pretty sure I am missing some steps, I would be grateful if someone could help me.
Also, doesn't dissolving the sample (water solution) in diatomaceous earth conflict with a highly hydrophobic mobile phase?
Thanks in advance
I'm wondering anyone of you here have been knowing the different between sepharose high performance and sepharose Fast flow? What is the different between this two? Are they different in term of the binding efficiency, flow rate, appication or resin type? and what is the difference of these two with capto?
What role do detectors play in chromatography, and what types are commonly used?
I'm working on selecting antibodies against a recombinant protein that has a His-tag. My idea is to first bind the recombinant protein to a HisTRAP column and then use this column for an affinity chromatography to select the antibodies.
However, I haven't found any articles or protocols that describe using this column in this specific context.
Has anyone used the HisTRAP column for this particular purpose? If so, which buffer did you use to elute only the antibodies without eluting the bound protein?
Why is TLC not a form of partition chromatography and paper chromatography not a form of absorption chromatography?
Hello everyone,
I am currently working on a protein that is 10x his-tagged and positively charged (predicted pI=10.16). But when I tried to use Ni-NTA column to purify the protein, it's not binding to the resin at all. Then I switched to cation-exchange chromatography, but this protein won't bind SP-sepharose resin either. Does any one have any suggestion?
Many thanks!
I forgot to autozero during the run (Size exclusion chromatography.) and later i realised i forgot to do that and the baseline was not zero but below zero (and in some cases it above zero). I would like to present the data in a report, however, was wondering if it possible to correct or refine this absorbance values by any means.
what I have tried so far is try to subtract the absorbance value obtained at 0 ml. Any help would be appreciable!
Hi,
Just curious to know that in chromatography, a chromatogram should be zoomed to what scale? 10 times of output , 50X or ..? Is there any regulation or general chapter which describes this? Does it varies from lab to lab & test to test?
Thank you for your guidance.
Hello,
I am trying to find a protocol that looks at the extraction and purification of ACE2 inside the cell. We have the protocol for the affinity chromatography, but we are having trouble finding the protocol at what percentage of ammonium sulfate to use for a protein precipitation. We don't want to just load crude homogenate into a column so we were hoping the precipitate the proteins out, and then load them into a column.
Thanks!
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...
currently conducting a study trying to figure out the secondary metabolites from bacteria.
Question #1: does it make sense if our flow goes from: extraction > TLC > SEC > TLC > antibiotic susceptibility test > MS to identify the specific metabolite.
we lack time and would like to seek an alternative for SEC (it takes 7-14 days here in our partner lab).
Question #2: is Gravity Column Chromatography a decent alternative for SEC?
thank you for all your help 🙏
Hi there,
adding a paper or towel to the tlc chamber is told to fasten the chamber saturation in TLC.
But i could not find any information what paper can best be used for that - which works/which doesnt.
What do you use? Can you recommend a paper/towel for the chamber saturation?
I can't find a regeneration protocol for this type of resin specifically, only how to do a cleaning for size exclusion chromatography resins, in a general way.
For those working in the field of Mass Spectrometry, Chromatography and allied topics, and based in NY state, we are launching a new local discussion group !
Feel free to sign up as member to be part of it.
We will organize in-person events (main area: Buffalo, Syracuse, Ithaca, Corning, Rochester) and virtual meetings - which anybody can attend !
Soon to be listed officially among other local discussion groups on the American Society for Mass Spectrometry (ASMS) website.
Thierry
I did a synthesis of a carbonyl ruthenium complex coordinated with the 1H-imidazole-1-ylacetic acid, but I'm having problems in the purification process. All the mixture is only slightly soluble in methanol, so purification for classic chromatography is unreliable. Acid or basic addition to a water solution of this mixture does not produce any change in solubility or precipitation. Any suggestions are welcome.
HI!
Has anyone ever done protein refolding using affinity chromatography? My recombinant protein has a His Tag and usually to purify it we use IMAC on the ÄKTA machine. However the protein is mostly produced in inclusion bodies of E. coli. This means I have to do refolding, and want to try using affinity chromatography to utilize the His Tag (as I read in some journals that this can be done). But since I have no experience in that, I would like some guidance if anyone has ever done so.
There are some things that I am still confused with:
1. After the last centrifugattion (following pellet resolubilization with urea buffer) do I directly load the supernatant on the machine?
2. Should all the buffers (binding and washing buffers) contain urea as well?
3. Are there any additional steps that I need to do besides load - wash - elution?
Here is where I need guidance, as I am not really sure how the refolding will take place and what I can do to initiate this??
Thank you so much for your attention.
I will be very grateful for hints and also some discussion.
Kind regards,
Lieke
Hello,
I am analyzing purified antibodies using a superdex 200 increase 10/300 GL column. Occasionally, I observe tailing at the peak, particularly with antibodies that have high surface concentrations of Lysine and Arginine.
The figure below shows data from size exclusion chromatography analysis conducted under the same conditions, where tailing was observed in antibodies with high positive charge.
I'm wondering if increased positive charge on the surface could enhance interactions with the column.
Are there any references demonstrating this phenomenon?
Thank you!
Hello! Is it possible to identify pigments using HPLC-MS but without the use of any standards? Despite not having any pigments standards, I have performed the separation of my extracted pigments sample in HPLC-MS. I tried comparing my results with those of other studies who have used standards but I still couldn't identify which pigments I have.
Thank you in advance.
Recently I've done the synthesis of a water-soluble ruthenium complex with 1H-imidazole. I could separate the desired complex from other complexes formed during the synthesis by chromatography(silica gel), but I haven't been successful in the separation of the complex and the 1H-imidazole ligand. I already tried chromatography and washing with some organic solvents (the problem is that all that imidazole is soluble my complex is soluble as well). Does someone have any suggestions for this problem?
Since insect pheromones exhibit highly variable forms, as well as compositions, and can change based on various conditions, especially releaser pheromones, which are usually highly volatile, could someone provide information on how to prepare alive or preserved insect samples for specific pheromone analyses using mass spectrophotometry and chromatographic methods? Is there anyone who can offer insights on this topic?
Hello,
We are trying to purify proteins using a secretion system but do not have TFF cartridges for our device. Does anyone know where we can purchase these? Or is there a better replacement? We want to downscale the volume from 3 L to 100-200 mL.
I've attached a picture for reference.
Thanks,
Thomas Newton
In my schiff base there is a impurities, which is n,n-dimethylaminobenzaldeyde. I want to seperate the compounds by column chromatography. Now which solvents, and what ratio I should use? The other precursor is p-anisidine. The schiff base is soluble in water, ethanol, methanol but insoluble is n-hexane, diethyl ether.
I have liters of cell lysate and I need to separate the recombinant protein from the rest. The volume is very large for the affinity chromatography column. Any other technique? I don't need high purity, I just need a more clear sample containing just protein. I thought about TCA separation but I have concerns about protein functionality afterwords. Does anyone have experience with that?
I have been purifying proteins which have an N-terminal His-tag using Ni-NTA affinity chromatography. I carried out size exclusion chromatography using Superdex HiLoad, Superdex, and Superose columns. I also carried out dialysis of the Ni-NTA purified proteins.
Both the above experiments were performed for buffer exchange and to remove non-specific proteins.
When I ran SDS-PAGE gels for the Ni-NTA purified and dialysed proteins, I could see a single band corresponding to the protein of my interest. Multiple bands beneath the protein of interest could be seen on SDS-PAGE gel for the SEC fractions.
I used appropriate controls to rule out the possibility of degradation due to prolonged exposure at room temperature and the effect of varying salt concentrations.
Please help... to all the protein purification experts out there!!
I purified my protein of interest using Ni-NTA chromatography. The protein was eluted but it eluted with a yellowish brown color which I have experienced before. I believe it was due to excess DTT (buffer mismatch) which led to leaching of Ni with my eluted protein.
Any ideas as to how to remedy this and remove the color (and Ni) without compromising the integrity of the protein.
Thank you.
Hello,
There are several studies regarding the quantification of chemicals by chromatography and/or mass spectrometry in urine samples.
Would the same techniques work in stools (faeces)? Or one needs special purification features?
Thank you
I have tried simultaneously analyzing three chemicals, i.e. benzyl alcohol (BA), benzaldehyde(BAD), and benzoic acid using GC-FID while using TR-1, TR-5, or TR-wax columns. With all three columns, I have been able identify both BA and BAD but benzoic acid peak is not emerging in the chromatogram. I have done all possible variations with GC-FID but still no sucess. Sample is prepared in acetonitrile with concentration of ~50 ppm. Does anyone else also has experienced the same problem? And any suggestions to overcome this issue?
I performed DLS on purified exosomes with an EXO SPIN kit (precipitation and size exclusion chromatography) the Z-average was too big and the result wasn’t good I should mention that to break up aggregations I do sonication before analysis. Does anyone have any idea? Could filtering through 0.2 µm be a solver?
I use AKTA Pure for chromatography. I packed CM Sepharose resin in XK50/20 column. my loading flow rate is 28 ml/min which pressure is blow 0.07 MPa. I want to know that the maximum pressure that I can use that doesn't damage the packing, when I load 2CVs of NaOh 1M and 7 CVs of regeneration buffer (0.5 M Sodium Acetate). The CV is 200ml.
Thanks
I installed a superdex 75 pg myself, and the conductance baseline was flat at the beginning of the balance, and then the baseline was flat when the sample was eluted to 50%, and the last 50% jittered up and down, which was unstable.
I’m purifying a recombinant protein with a Hi tag via nickel affinity chromatography. If my sample has MgSO4 in it as part of the purification process, will that interfere with nickel affinity chromatography? Should I perform dialysis first to remove the MgSO4? Thanks!
Does someone have any practical experience with those columns?
The producer suggest ODS-B are more suitable for hydrophilic analytes, but without any concrete information.
Can they be used interchangeably with ODS-A columns? Are there expected changes in retention times of non-polar analytes? Are there differences in their pH tolerance?
Hi everybody. I am a student working on a research project. I would like your help to gain a deep understanding of my work. I am using a mammalian expression system to synthesise and then purify (via affinity chromatography using different resins and gel filtration) a protein with an isoelectric point of 5,3. I am using all buffers with a higher pH (7.4 or 8) for all the purification steps.
1) Could you please tell me in which way buffers could influence my protein?
2) What is the link between pI and buffer pH)? Because my proteins should be negatively charged at pH of 7.4 and 8, my question is if this negatively charged is something that I need for the better functioning of affinity chromatography and gel filtration.
3) Could I have used a buffer with pH at 5.3 like the pI, or would I have risked an aggregation?
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?
Enzyme purification The first step after dialysis is to purify the enzyme by ion exchange technique, where it appeared for me three large peaks representing three isoenzymes and some small peaks. Samples representing the isoenzymes of the enzyme that were purified by gel chromatography showed three distinct peaks representing the isoenzymes with the disappearance of the peaks The other small ... Question: - Is this result considered reasonable and can it show three peaks for isoenzymes in the gel chromatography technique?
I am looking for a method to completely remove proteins from plasma without using heat or adding salt ions. I have considered using activated carbon, but I am unsure if this is feasible. Are there any other effective methods for achieving this goal?
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 would like to get my results in Word files in order to use it in my article.
And thank you for your cooperation.
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?
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.
References:
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
Hello,
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
Hi,
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.
Thanks
I need to separate 25kda scfv from 50kda scfv. Which sephadex should I use?
Sephadex G-25
Sephadex G-50
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?
Hi there.
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 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?
Hello
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.
What are the various factors that can impact the binding of antigen to immuno affinity chromatography
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?
Thanks.
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 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.
Questions are:
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?
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?
Everyone.
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.
Bohuš
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.
Hi,
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?
Emir
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?