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.
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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
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To identify unknown materials as well as those input components in a pharmaceutical products or intermediates, one can apply Raman micro analysis. It can qualitatively and quantitatly characterise the solid/semi-solid samples.
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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?
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Thanks for the responses and sorry for the late replies, things have been little busy in the the lab.
To answer Jaemin Lee question about the method and sample properties. The samples are mainly DBS or RBC analyzed for FAMEs using BF3-MeOH for the methylation of the fatty acids. We then extract these into hexane and inject onto our GC. The GC inlet is at 230C, FID is at 290C and column starts out at 180C with hydrogen as carrier gas at constant velocity of 55cm/sec. There is an oven temperature program.
To answer Raymond Loyer post: We have a moisture/oxygen trap on our Hydrogen line (carrier gas) which we only change if we see a change in the indicators within them. We have not changed these in over a year. If we did get a leak in the line downstream from this trap and moisture/oxygen got introduced into the carrier gas we would see a much quicker deterioration of the column phase, which we have not seen. Not to say that the leak is minute enough that this deterioration is not happening but is causing this shift though, we would just have no way to identify a leak like this. Our laboratory is not pressurized and we will see this shift in retention times anytime the GC sit idle for >1-2 day, it does not have to be a Monday or be in the morning. There is a way to adjust the retention times based upon our ISTD peak however, this doe not adjust any of the integration parameters within our integration method (where we tell the software to start looking to integrate a peak at this time and stop at this time). This is the spot the software we currently have breaks down as with the shift that happens at the start none of the peaks get integrated.
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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?
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Try concanavaline A resin which is highly succesfull for capturing glycoproreins...Jacalin may also works well..Lentil lectin is the most predominantly used..you should packed your own column or must use them as gravity column or as batch mode
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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
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Abdelhak Maghchiche, thank you for your reply. Can I ask what you mean by proper packing? Do you mean packing the column with some solvent and then letting the water/methanol sample adsorb into the diatomaceous earth or do you mean just packing the column with the sample and then pouring the mobile phase. I assume that in the latter case the sample volume should be sufficient to wet all the kieselguhr, that's correct?
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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?
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For Laboratory and E.coli system you can use FF. You have a better velocity for HP when you load big volume what yeast system (liters of supernatant). For capto, the groups are linked to a high flow agarose base matrix modified with a dextran surface extender which further increases capacities and mass transfer properties. (you use less bed height). The resin choice, it's a process development for scaling up.
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What role do detectors play in chromatography, and what types are commonly used?
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In general, in chromatography, detectors are essential for identifying and quantifying the compounds that come out of the chromatography column. They convert the physical or chemical properties of these compounds into a measurable signal, which is then analyzed to determine the presence and concentration of each substance. The following are some of the types of detectors commonly used in chromatography: 1. Ultraviolet and visible detectors: These detectors measure the absorption of ultraviolet or visible light by compounds as they pass through the detector. They are widely used because many organic compounds absorb ultraviolet or visible light, making this method versatile and relatively straightforward. 2. Fluorescence: Detects compounds that glow when exposed to specific wavelengths of light. Common Uses: Highly sensitive and selective; used for compounds that are naturally fluorescent or can be derived to be fluorescent.
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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?
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Hi Jackelinne,
I don't think it will work well. First, antibodies themselves can bind on NiNTA (generally IMAC was developed for purification of natural non tagged proteins, even before his-tag). Secondly elution, highly acidic conditions will be required to remove antibodies from the antigen. At low pH the recombinant protein itself will elute from the column (firstly histidines are protonated and secondly nickel stripping occurs). Part of the problem with nickel stripping can be circumvented if you use Sepharose Excel. Nickel binds firmly there, but the binding of recombinant protein to the nickel histag will weaken anyway.
It is easier to immobilize the recombinant protein on Sepharose by any suitable method (cyanogen bromide activation, epoxyactivation - there are even ready-made activated resins) and purify the antibodies in this way.
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Why is TLC not a form of partition chromatography and paper chromatography not a form of  absorption chromatography?
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I respectfully disagree. Silica adsorbs solvent as a thin layer and so acts as liquid-liquid chromatography (partition chromatography) as well. A properly run TLC plate provides a retention factor that can be converted to column volumes- TLC is used to predict retention in column chromatography (1). This means the mechanism of chromatography is the same for both columns and TLC plates. Partition chromatography on silica has been known for a long time (2) and is used to explain aqueous normal phase (HILIC) on silica gel. Thin layer chromatography plates need to be saturated with the vapor in the chamber to give reproducible results (1) that match the column chromatography, so the adsorption of the solvents is much more subtle. With a few exceptions, such as hydrocarbon compounds, compounds won't elute on a silica column unless the surface is saturated with the strong solvent (which causes elution down the column). Reference 3 below shows this effect with hexane/ethyl acetate (3) where the compound only elutes after the column is saturated with hexane- like water, the ethyl acetate forms a layer on the silica. Adsorption does play a role, as the silica pH and surface structure does play a role. However, adsorbed cause partitioning!
1) Let Us Teach Proper Thin Layer Chromatography Technique! | Journal of Chemical Education (acs.org)
2) biochemj00969-0073.pdf (nih.gov)
3) Don’t start your normal phase gradients from 0% B! (teledynepharma.com)
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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!
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This problem is often the result of the protein being highly aggregated. It sometimes helps to reduce the rate of expression by lowering the culture temperature (for expression in bacteria) or co-expressing with chaperones.
You can find out if the protein is highly aggregated by running it through an appropriate size exclusion column and comparing the elution volume to the void volume.
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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!
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That is what I meant, but in my opinion you don't have to zero the axis at all. You can just show the chromatogram with no y-axis, just a vertical scale bar to show how much distance corresponds to a certain absorbance change. The absolute baseline value isn't important.
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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.
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There is no single universal rule, following regulatory guidance and best practices for chromatogram scaling can help ensure the data is clear, informative, and suitable for its intended purpose. The specific scale should be determined based on the analytical requirements and adjusted as needed to maintain data quality.
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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!
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To precipitate ACE2 from Calu-3 cells using ammonium sulfate, dissolve new ammonium sulfate to 70% saturation, add slowly to the supernatant with stirring, and allow precipitate for 30 minutes at 4°C before centrifuging to obtain the protein.
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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...
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I also have never heard of " digital chromatography" as a technique and have been teaching HPLC and LC_MS for 30-years. Rezvan Abedinloo, perhaps you are referring to using a computerized data system ("digital") with your HPLC or simulation software ???
In any case, real world changes in peak retention time indicate a problem with your HPLC method or instrument. Please contact someone experienced in HPLCc at your school for assistance and also refer to this free article on the topic of changing peak retention times in HPLC.
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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 🙏
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For SEC (size exclusion chromatography), can you use Sephadex LH-20? This can be run in an open column (gravity column).
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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?
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usually any filter paper grade can use. W-42 is faviourate
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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.
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Thank you so much for your help. Have a nice day.
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% rsd fail down 10%limit
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Rod's law - the more you don't tell me the more I can't help you. Your question tells me nothing. Revise it; tell us what you are doing, instrument and conditions, how you calculated your results and why you think exceeding 10% RSD is a failure.
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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
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ahah hello All !
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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.
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Would fractional freezing be an option?
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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
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I have participated in such a procedure. The washed inclusion bodies were dissolved in 6M guanidine-HCl-containing buffer for several hours. This buffer also contained dithiothreitol to make sure the disulfide bonds were fully reduced, so it was important to use a Ni resin that is compatible with dithiothreitol. The solution was centrifuged, then loaded onto the Ni column that was equilibrated with the starting buffer, also containing 6 M guanidine. After a washing step, the guanidine-HCl was reduced to zero in a long, linear gradient. Both the start and end buffers also contained 1 M arginine-HCl to assist with refolding, and a redox buffer (a mixture of reduced and oxidized glutathione) to help form the disulfide bonds. Finally, the refolded protein was eluted with 0.5 M imidazole in the final buffer.
Most of the recovered protein was aggregated, so the last step was to run it over a gel filtration column to separate the monomeric protein from the aggregate.
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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!
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I really appreciate your interest. I'll share the information you requested.
1) pH 7.4 PBS
2) 0.75 mL/min
3) Particle size, d50v : ~ 8.6 μm
Bed dimensions (mm) : 10 × 300-310
Approximate bed volume (mL) : 24
4) RT
5) 1mg/mL, 10μg
6) 280nm
7) The void volume was measured by a senior, not by me, but I'm unsure of the exact value.
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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.
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No. "MS" data is only as good as the overall method used to collect and evaluate it (The "method" includes the column, mobile phase, instrument settings, standards and proof that the method follows good chromatography fundamentals). With no standards for comparison and other orthogonal techniques used, no identification would be possible. Purely speculative.
MS data on its own has very little value as a MS system will always output "data". Data "from other studies" would be anecdotal at best as only data collected on the same instrument, by a trained operator (which takes many, many years) using standards with an approved method would be useful to draw conclusions from.
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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?
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Separating a ruthenium complex from its ligand, especially when they have similar solubilities, can be challenging. Here are a few strategies you can try:
Column Chromatography Optimization:
Experiment with different column chromatography conditions, such as changing the stationary phase, using a different elution solvent system, or adjusting the polarity of the solvent.
Solvent Gradient Elution:Employ a gradient elution strategy during column chromatography. This involves starting with a more polar solvent and gradually increasing the polarity, which may help in better separating the ruthenium complex from the 1H-imidazole.
Reversed-Phase Chromatography:Consider using reversed-phase chromatography, especially if your complex is hydrophilic. This technique can exploit differences in hydrophobicity for separation.Size-Exclusion Chromatography:
Size-exclusion chromatography separates molecules based on size. If your ruthenium complex and 1H-imidazole have significant differences in size, this method might be effective.
Recrystallization:Attempt recrystallization using a suitable solvent system. Sometimes, complexes can be encouraged to crystallize separately from their ligands.Selective Precipitation:
Investigate the possibility of selective precipitation by adding a non-solvent or changing the solvent conditions to encourage the separation of the ruthenium complex from 1H-imidazole.
Extraction:Explore liquid-liquid extraction using an immiscible solvent that selectively extracts one component. This method might require optimization of conditions.
pH Adjustment:Evaluate the impact of pH on the solubility of the complex and the ligand. Adjusting the pH may alter the solubility of one of the components, aiding in separation.
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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?
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Modern Gas-liquid Chromatography with electronic processing is so wonderful, compared to what we did having to build these machines ourselves 60 years ago. GC "Standards" were essential at the beginning, so you could figure out what those thousands of "peaks" were, and thus begin to understand the incredible mixture or compounds in your beeswax! I remember well seeing my first Mass Spectrometer attached to a GC at U. California- Berkeley, California in a chem lab.
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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
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PALL (now Cytiva) I think, used to sell TFF devices.
Tangential flow filtration | Cytiva (cytivalifesciences.com)
Another alternative could be Sartorius
TFF Systems For Ultrafiltration and Diafiltration | Sartorius
Hope it helps.
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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.
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You can try to focus on crystallization. Schiff base crystallization is generally achieved using acetonitrile, acetic acid, ethanol water, DMF water, and acetone
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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?
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TCA precipitation is a bad idea. The protein will be inactivated.
One way to deal with the large volume, if you don't have a pump or an FPLC, is bulk binding. Mix the affinity resin into the lysate. Let it incubate for a while with gentle mixing for a few hours. (Don't use a magnetic stir bar because it will grind up the resin.) Once the binding has happened, pour the whole thing into a column and let the resin settle while draining the column. Then perform the affinity chromatography.
You can also use ion exchange resins the same way to bind up the protein from the bulk lysate, then elute the column with a salt gradient.
Something else you can do is ammonium sulfate precipitation and fractionation. In this method, you gradually add increasing amounts of solid ammonium sulfate while stirring the lysate. After each addition and an incubation, you centrifuge the sample to pellet any protein that precipitated. This can achieve a small degree of purification and gives you a pellet that is a mixture of protein and ammonium sulfate. Then you dissolve the pellet in a buffer and dialyze it to remove the ammonium sulfate.
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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!!
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If the proteolytic cleavage did occur near the N-terminus, you could not see the fragments with anti-His antibodies. Thats also why polyclonal ab are better than monoclonal for this application.
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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.
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1 mM dtt should be enough to reduce disulfide bonds (or prevent their formation). Higher concentrations will reduce the Ni ions. It is easier to decrease dtt conc before running imac. Moreover some Zn particles will start clogging the adsorbent pores.
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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
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Not my area, but I think you need to extract the soluble components or otherwise the macroscopic, unsoluble parts will just jam your column.
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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?
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Thanks Bruce Neagle and Bruce Neagle . I would definitely consider trying the derivatization approach. While I'm preparing the derivatization agent, I'll also perform the same analysis using HPLC coupled with a UV detector. In this setup, I'll use a C18 column and a mobile phase consisting of ACN and water.
I've used HPLC for this analysis previously, but I've encountered an issue where the retention time of benzaldehyde varies significantly from one run to another, and eventually approaches to that of benzoic acid. Is it possible that this variation is due to the oxidation of benzaldehyde to benzoic acid?
It's worth noting that several studies have conducted a similar analysis using HPLC, but none have mentioned any difficulties in measuring benzaldehyde. In contrast, benzaldehyde consistently shows reproducible results when analyzed via GC.
I would greatly appreciate your feedback on this matter.
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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?
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Hi,
Exosome size measurements through DLS tend to be variable , thus should be repeated several times in single as well as multiple different preparations, in order to reach a cumulative particle size range . Please pay attention towards QC report at the end of each measurement ('Good' report means acceptable results). Further you may need to modify the default DLS protocol method in order to fit EV measurements.
Filtering through 0.2um could be helpful but sonication will break up the EV particles giving incorrect sizes.
NTA is a better method to quantify EV size and concentration.
Regards
Neha
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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
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This product information is for CM Sepharose Fast Flow from CytivaLifeSciences:
Pressure/Flow Specification: 300-600 cm/h, 100 kPa, XK 50/30 column, bed height 15 cm.
This is for CH Sepharose High Performance:
Pressure/Flow Specification Base matrix: 100-200 cm/h, 300 kPa, BioPilot 60/600 column, bed height 30 cm
This is for HiPrep CM FF 16/10 prepacked 20-mol column:
Pressure max. (over the packed bed during operation)1.5 bar [0.15 MPa] (22 psi)
Here is product information from Sigma for CM Sepharose FastFlow, which includes cleaning information, but not pressure limits.
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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.
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Could be a bubble stuck in the conductivity meter.
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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!
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Hi there,
According to the joint document magnesium choride has no effect up to 4M (!) on IMAC
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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?
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Sir,
How can we remove blockage of Waters Spherisorb ODS2 column? Any solution you have?
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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?
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For IMAC resin, a mildly basic pH (e.g. 7.5-8) is preferred. Acidic pH can cause the resin to lose the immobilized metal ions because metal chelation is accomplished by carboxylic acid groups.
For anion exchange chromatography, a pH above the pI is desired to impart a negative net charge. For cation exchange chromatography, a pH below the pI is desired to impart a positive charge. However, this is not an absolute rule. For one thing, you can use ion exchange chromatography as a subtractive step, whereby the protein of interest flows through but some of the contaminants bind. In such a method, the choice of pH may be different from when you want the protein to bind.
You also have to consider the stability of the protein, which can be affected by pH. Most proteins are most stable at near-neutral pH, unless they are normally found in an environment in which the pH is far from neutral.
Chromatofocusing is a chromatography technique that takes advantage of the different pIs of proteins by eluting with a pH gradient. pH gradient elution can also be used with ion exchange columns, although it is more common to use salt gradients.
In general, pH is not a factor for gel filtration chromatography, which separates on the basis of size and shape, unless the protein's oligomeric state or stability is affected by the pH.
It is usually considered undesirable to purify proteins at their pIs because of the risk that the protein will precipitate when its net charge is zero. However, this also is not an absolute rule. The key phenomenon associated with a protein at its pI is lack of mobility in an electric field, which is the principle of isoelectric focusing, rather than insolubility.
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N/A
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It is generally not recommended to store a protein A chromatography column in 20% ethanol without NaCl as this may damage the resin.
Protein A is commonly used for the purification of antibodies, and the Protein A resin is typically composed of cross-linked agarose beads that are coated with recombinant Protein A. The binding of the antibodies to Protein A is mediated by interactions between the Fc region of the antibody and the Protein A.
The presence of NaCl in the storage buffer is important for maintaining the stability of the Protein A resin. The salt helps to stabilize the Protein A on the resin, preventing denaturation or aggregation, which could lead to loss of binding capacity. Ethanol can also affect the stability of the Protein A by disrupting the tertiary structure of the protein, which could also lead to loss of binding capacity.
Therefore, it is generally recommended to store protein A chromatography columns in a buffer containing 20% ethanol and 0.5 M NaCl, or in a buffer containing 20% ethanol and 20 mM Tris-HCl (pH 7.5) without NaCl. This can help to maintain the stability of the Protein A resin during storage.
It is important to follow the manufacturer's recommendations for storage conditions for the specific Protein A resin that you are using to ensure optimal performance and longevity of the column.
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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?
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Adam B Shapiro I got correct size protein after Histrap purification and also after removing His tag. But when I later purified it through anion exchange Chromatography I get protein of lower size almost 16KD lower than expected size. I am using Tris HCL + 1M NaCL for elution. Do you or anyone else observed this kind of shift in protein after anion exchange chromatography. For Anion Exchange Chromatography I used Mono Q from Cytiva
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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?
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Hi there,
Generally isoenzymes are very close in size and differ in their primary sequence. Therefore IEC is the most appropriate approach to solve an isoenzyme mixture as the overall charge of a protein depends on its sequence.
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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?
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Dear Sir you may try the protein separation with foam fractionation coloumn. it would suppose to help you
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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?
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Firstly, you must perform scaling before you perform a PLSR or PLS-DA, PCA model. Some compounds are dominant in concentration in your sample so their peaks usually have higher peak intensity and variances. Therefore, if you don't scale your data, peaks with high intensity will be recognized as the most important differential metabolites and you will lose the information of others.
For scaling, you can choose:
1. Autoscaling
2. Range scaling
3. Parero scaling
These three scaling methods were often chosen. For more details about the advantages and disadvantages you can check the paper written by Robert A van den Berg at
although the paper was mentioning the peak table from GC-MS or LC-MS, it is the same issue as yours, the features in MS datable are m/z, in your case is rt.
Secondly, as for reducing overfitting, you can compare the consequences of accuracy when you choose different scaling methods, but more importantly, is usually the overfitting is related to the shape of your dataset. The best way to solve overfitting is to increase the number of samples. In PLSR, there is one way to reduce overfitting is to reduce the number of components you use to do prediction.
To avoid extra noise, once you do scaling, no doubt that all the noise will be amplified, remember all the scaling methods aim to regard all metabolites as having the same importance. But Parero scaling may work a little better than others. To reduce the noise, the best way is to remove the noise before scaling. Usually, we utilize QC samples. All the peaks in your QC samples with variances higher than 20% (or another number, please check papers recently published, 20% is usually used in MS data) will be recognized as noise and unstable metabolites and removed from the dataset.
Hope this will be useful for you.
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I would like to get my results in Word files in order to use it in my article.
And thank you for your cooperation.
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To extract the report of analysis, including chromatograms and composition tables, from a Shimadzu GCMS-QP 2010 SE, you can use the LabSolutions software that comes with the instrument. Here are the general steps:
  1. Open the LabSolutions software and navigate to the data folder where your GCMS data is stored.
  2. Select the file that contains the data you want to extract and open it.
  3. Once the file is open, you can view the chromatogram by selecting the chromatogram tab.
  4. To export the chromatogram, right-click on the chromatogram and select "Export Data." Choose the file format you want to export it to (e.g. PDF, Excel, etc.) and select a location to save the file.
  5. To view the composition table, select the "Report" tab in the LabSolutions software.
  6. In the "Report" tab, select the data file you want to extract the composition table from.
  7. Once the data file is selected, you can customize the report settings and choose the composition table option.
  8. Select the format you want to export the composition table to (e.g. Excel, CSV, etc.) and select a location to save the file.
These steps may vary slightly depending on the version of LabSolutions software you are using. If you are unsure how to use the software, you can consult the user manual or contact Shimadzu technical support for assistance.
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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?
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HI,
Some suggestions from me:
- Optimize binding& wash buffers by increasing imidazole concentration.
- Use screening kit to find the optimal metal ion(Ni2+, Co2+, Cu2+, Zn2+).
- Optimize the length of the His-tag. Longer tag will bind with higher imidazole concentration and you remove your contaminants that are not bonding.
- Optimize the elution step. Change from step to linear gradient.
- Increase the volume of the linear gradient.
- Change to a pH gradient (can be tricky).
- Use high quality imidazole (should be a white powder).
- Use NTA ligand (and not ISA ligand) for immobilization of the metal ion.
- Add a second purification step, e.g. SEC or IEX. This will help a lot for increased purity!
I have added some information of different prepacked 1 mL and 5 ml columns with different metal ions attached for easy screening for optimizing.
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Can anyone help in the field of packing steel chromatography columns with ion exchange resins?
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We can supply everything related to chromatography! We are the leading specialists and offer all types of columns and chromatography materials. I presume you have specific requirements? Please let us know. www.chromatographyshop.com
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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:
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For this method, interferences can either result from pressure, leakage, or chromatography problems.
In relation to the pressure problem, there are four common errors that could arise. First is the abnormal pressure, which is caused when there is a leak or air trapped inside the pump head resulting in no flow. Next is the high backpressure which is caused by a flow rate that is set too high. Another one would be low back pressure. With this, the flow rate is set too low, and can also be caused by having a high temperature, system leak, and controller malfunction. Lastly, the pressure cycling is caused by a faulty valve, system leak, seal failure in the pump, use of gradient elution, and insufficient degassing.
The problem with leakage can occur in different settings such as leakage in fittings, pumps, injectors, HPLC columns, and in detectors. While for chromatography problems, these could arise due to different peaks, drifts, retention time changes, and resolution loss.
Science Unfiltered, 2022. Common HPLC Problems & How to Deal With Them. Retrieved from https://phenomenex.blog/2022/01/04/hplc_problems/#:~:text=Using%20an%20improper%20HPLC%20column,the%20use%20of%20gradient%20elution.
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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
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You can contact me
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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
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Dialysis is still feasible with 15-20 Liters. However as much more rapid method if you are able to concentrate your material to as low as 1 Liter using either ultrafiltration or other concentration methods. You would be able to use Size Exclusion Chromatography(ie Sephadex G25-300) to quickly remove the salts. Of course another alternative is diafilration using an ultrafilration system.
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I need to separate 25kda scfv from 50kda scfv. Which sephadex should I use?
Sephadex G-25
Sephadex G-50
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Superdex is still used in FPLC columns.
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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?
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Run an SDS PAGE of your protein before and during cleavage. If the histidine tag isn't fully cleaved by enterokinase then you are not using enough enterokinase or for a long enough time and so your uncleaved polyhistidine tagged protein is sticking to Ni-NTA and that is how your losing it.
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What are the Secondary Metabolites that present? Is it necessary to do the tests to prove them in the lab by color changing chemicals?
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Olive bark is a term used to describe the outer layer of the bark from an olive tree. It is composed of layers of thick, dark-colored cork cells, which protect the inner bark from weather and pests. Olive bark is harvested for use in various products, including homeopathic medicines, cosmetics, and dyes.
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The cost is about $ 4,500.00 USD and I would like your feedback and know if it is worth the expense. Thank you!
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Regarding COST, no idea as each system is different (and costs will be too). Preventative Maintenance (PM) is not normally an option. It is a requirement to maintain the basic operation of the system. You will not know what the condition of the system is in unless you formally test it (i.e. Pressure testing, Leaking testing, Composition Testing, Lamp checks, Detector calibration, etc). Never assume it is functioning properly or wait until something breaks. Typical PM service includes the replacement of common seals and parts that "wear out" from normal use and are required to maintain pressure and normal operation. PM service should NOT be a replacement for any needed SERVICE or REPAIRS (e.g. broken parts, damage, abuse, extensive wear etc). PM's are designed to inspect for and identify areas of normal wear. When found, parts are cleaned and/or replaced, as needed. Failure to "maintain" an HPLC system may result in invalid data (invalid methods) being generated. PM services should be budgeted as part of the normal operating costs (just like supplies and consumables).
  • There are few tasks more frustrating that trying to use a poorly maintained HPLC system to analysze samples on!
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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?
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Yes
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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?
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Hey Tim,
You can include an anion exchange chromatography step in your purification process provided that your protein of interest remains as a cation in your purification buffer. Even if your protein act as a weak anion, it would be eluted at a lower ionic strength from a strong anion exchanger and nucleic acids would be eluted later at high ionic strength. But this entirely depends upon the isoelectric point of your protein and the pH of your buffer.
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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?
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Membrane proteins require detergent to keep them in solution when they are not in a lipid membrane. Once the urea is washed out, the protein will precipitate if there is no detergent to keep it in solution. Some commonly used detergents are CHAPS, n-octylglucoside and dodecylmaltoside, although there are many others.
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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.
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The elution buffer for the cation exchange column should be 1 molar not 1 millimolar. You might also want to lower the pH a bit more, to say 5 to 5.5.
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Conferences
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Please inform me any conference related to herbal medicine
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What are the various factors that can impact the binding of antigen to immuno affinity chromatography
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High pressure of more than 1bar can impact binding efficiency of Immunoaffinity resin?
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I have prepared carbon dots now i want to use chromatography to purify instead of dialysis
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Carbon dots in the range of 10-100nm
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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.
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by fractional inhibitory index
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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.
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Try a higher pH (e.g. 7.4, more physiological). Other options might be adding a carrier protein,e.g. BSA, detergents (Tween 20) or non reducing sugars (e.g. saccharose, sorbitol, mannitol). Glycerol in my experience is likely to inactivate your AB if you store it at 4degC, but good to keep it pipettable when storing it at -20.
Alternatively, flash freeze aliquots in liquid nitrogen and store at -80 or below or lyophilize it.
Add an antimaucrobial, e.g. sodium azide.
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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.
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You can use it twice. You can clean the resin if your sample is dirty by applying NaOH flow. It is a good way of regeneration. SEC resins are relatively robust and safe for several usages even if they are produced in gravity flow formats.
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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.
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Thankyou for your responses. I am very positive that I dialysed the correct fractions @Nick Gee . I thought the sixth and the seventh fractions are my purified protein and hence I dialysed those samples after two days. Also, I have a confusion that why being a 29Kda protein, a thick band appears at 25 KDa. I didn't concentrate the protein after dialysis @Chandru Subramani I am trying to purify the protein again but what should I be careful about, kindly suggest.
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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)?
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To answer your question regarding "column bleed". Not if the functional groups on the silica are BONDED to the support, as most are... If the column support consists of material that is not covalently bounded, then it may indeed slowly elute off the support and out of the column with the mobile phase. There are HPLC columns with coated supports (very delicate and require special treatment to use). These "coated" supports will wash off over time and as the coating is washed off, their retention characteristics will change too. However, "coated" phases are not common. Most HPLC column types you are likely to encounter today have fully bonded supports. Any observed "bleed" is likely to be the result of poor method development resulting in material from the flow path, fouled column, sample(s) and/or mobile phase additives being seen at the detector. With LC-MS, adducts are very commonly observed.
*I should also mention that one rare thing that we do sometimes detect when using columns packed with very small particles (~ 2.1 u or less) is the silica itself. The LC column-end fitting has a frit (filter) on it to prevent the silica from leaching out into the flow path, but these extra small particles are often accompanied by super small "fines" which get past the filters and into the flow path (and detector). Silica and the associated chelated metals that they can attract are sometimes observed.
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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?
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we avoid clogging is by dilution with lysis buffer upto 40-50 ml and after cell lysis centrifuge at 18000RPM ( using high pressure tubes, falcon tube doesn’t work), this way we get almost clear solution, which we passed through HisTRAP HP at 1ml/min flow and doesn’t cause much blockages
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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
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Can the AKTA Pure tolerate solvents used for reversed phase? Or is there a reason that you don't want your samples to be in Acetonitrile (or Methanol) and either TFA or Formic Acid? Reversed phase is going to give you the highest resolving power for separating peptides.
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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.
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That is correct. You should not expect the protein to be phosphorylated by E. coli, even if it is expressed in a soluble form.
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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?
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What was your mobile phase? What was its pH? Without knowing these, it is hard to imagine why you found gel formation. The column should have a pH range to work with. If you are within the range, then finding an answer will be difficult. At that point, you may wish to talk to the manufacturer of the column to learn more.
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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.
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The combination between these two tow top techniques and getting maximum benefits from both, enhance the field with lots of advantages.
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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.
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Remember, an MS detector cannot use a non-volatile phosphate pH buffer (use formic/formate instead because they are volatile) unless you really LIKE cleaning the ports.
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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
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the so called Pluthero method to purify Taq is quite old (1993) and while simple it can result in low yields and DNA contaminants (see "A simple and efficient method for extraction of Taq DNA polymerase" 2015)
I don't think the ammonium sulphate precipitation is required for activity (please correct me if I' wrong) but it may be worth a try on MMLV RT, as they are very different proteins. Yields are sometimes not very important and Am sulph precipitation can be a very useful method to isolate/purify and concentrate protein preps. I might just use a modern MMLV RT prep like this https://www.protocols.io/view/recombinant-protein-expression-of-mmlv-rt-h-yxmvmxmw9l3p/v1?version_warning=no
iff your constructs are similar, good luck!
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we want to determine effect of different compounds in cell culture harvest on protein A chromatography.
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I suggest using Cytiva Fibro PrismA (Dynamic binding capacity is ~ 30 mg IgG/mL matrix) at your experiments which DBC is relatively expanded.
Rapid cycling chromatography, thereby fast equilibration times is advantageous, and the wide pore structure of the cellulosic backbone enables it to be resistant against clogging which is typically seen at cell culture harvested samples (experimentally observed)...
emir
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We intend to separate the mixture of three glycosilated flavonoids having two sugars units, each, according to their LCMS profiles.
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Depending on the gradient you used for your scouting run, they may be well-resolved for preparative LC.
You didn't mention the solvents used for your analytical run, but, assuming acetonitrile, try methanol or tetrahydrofuran. You didn't mention solvent modifiers, but if you didn't use any, try TFA to keep the phenolic groups protonated, and less polar to allow more resolution from the glycosylated compounds.
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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
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Hi Ismail,
You correctly answered your question. Most orbitraps cannot stand the speed of elution of the peaks in UHPLC, especially if used at high-res and in polarity switching mode.
I am sure new technology will arrive soon and overcome this limitation!!!
BTW I am not aware about the last Orbitrap generation... it might be already a reality.
Greetings
Luca
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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?
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Read this paper it might be helpful
Ind.Eng. Chem. Res 2012 ,51,18, 6586-6590