Chromatography - Science method

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.

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.

Hi there :)

What you can also potentially use is larger purification beads. Normally they are either 40-100µm in diameter. But the smaller they are the denser the solid Phase is and the more the pressure builds up when you put them up to your settings.

If you use larger beads at ~400µm the flow rates are way higher and your columns shouldn't burst as the pressure cannot build up so much.

I suggest to give these a try:

Only downside is that due to the smaller total surface area of these beads (square-cube-law) the protein yield is smaller than the more commonly used smaller beads.

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

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.

Throughout the analyses, did you check the pressure? Your column's retention time will change if you don't stabilize it. For example, if you begin your analysis with 5% ACN and 95% H2O, you must stabilize your column with 5% ACN and 95% H2O for 2-3 minutes before beginning the analysis.

You can contact me

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.

Superdex is still used in FPLC columns.

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.

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.

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).

Yes

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.

Hello Bárbara Chagas

This could be due to several reasons. Make sure the targeted protein is properly charged during the IEC and eluted properly. You can check your chromatography and see whether any other peaks are present. It is necessary to confirm the correct fraction/s.

And, did sample 1 (dialyzed) have only one band?

Best wishes!

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.

Please inform me any conference related to herbal medicine

High pressure of more than 1bar can impact binding efficiency of Immunoaffinity resin?

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.

Carbon dots in the range of 10-100nm

by fractional inhibitory index

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.

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.

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.

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.

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.

That is correct. You should not expect the protein to be phosphorylated by E. coli, even if it is expressed in a soluble form.

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.

The combination between these two tow top techniques and getting maximum benefits from both, enhance the field with lots of advantages.

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!

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

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.

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

Read this paper it might be helpful

Ind.Eng. Chem. Res 2012 ,51,18, 6586-6590

Kindly check...

Hello Arpan,

the MRM allows you to distinguish between PFOS and MPFOS by the recording of different mass transitions: usually the mass transition 499 -> 80 and 499 -> 99 are used for PFOS, while (in our case) for MPFOS (13C4 labelled) it is 503 -> 80 (and 503 -> 99). Usually there should be no interference in these mass transitions, meaning that MPFOS does not produce peaks for PFOS and vice versa due to the specifity of these transitions. And of course they should have the same retention time, but please note that technical PFOS has several branched isomers that elute in front of the linear PFOS (and linear MPFOS).

Hope this helps and good luck for the analysis!

Thomas

Kindly, Check the estimated yield of sample in the eluate of desalting column.

In my opinion try to use Methanol instead of Acetonitrile.....

In my opinionYou have to clean up any spills or leaks quickly to prevent long term damage & fully dry any areas of damage to remove any remaining liquid.

Waters is one of the most popular

Check https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3746660/

Kindly see also the following useful RG link:

Also see kindly the following link: https://graduateway.com/separation-of-chlorophyll-a-chlorophyll-b-and-beta-carotene-by-paper-chromatography/

Back washing of column through Acetonitrile, 0.1% phosphoric acid in water.

A photodiode array detector, or PDA, is a type of absorbance detector (such as a UV detector) that is able to detect light-absorbing compounds at the PG level. However, PDA detects an entire spectrum simultaneously. UV and VIS detectors visualize the obtained result in two dimensions ( optical density and time), but PDA adds the third dimension (wavelength). The analyte or related impurities peak can be determined simultaneously during separation at all wavelengths, which is convenient for choosing the most suitable wavelength and determining the purity of the analyte or related impurities.

MS Mass Spectrometer

The analytes are detected based on their MW (Mass: charge). The obtained information is especially useful for compound structure and identification (based on the Mass: charge ratio (without standard injection)). By constructing a standard curve, a very low detection limit of analyte can be quantified.

You said that you did the purification in denaturing conditions, but the equilibration, washing and elution buffers do not contain any denaturant. What may have happened is when the denatured protein was exposed to conditions without denaturant, it aggregated instead of refolding. The aggregated protein was unable to bind to the column and ended up flowing through. You may get a better result if you include the denaturant (urea or guanidine) in the chromatography buffers. Once you have purified the protein, you can worry about refolding it.

If you can express this protein in soluble form and purify it without denaturant, you won't have to worry about how to refold it, which can be difficult.

Good luck.

Dear James,

thanks for posting this interesting technical question. As already mentioned by Salim, the boiling points of dichloromethane and dibenzylether are far apart so that a separation by distillation is easily possible. However, I would not recommend to use a rotary evaporator. This way you can easily remove the low-boiling dichlormethane, but high boiling impurities and decomposition products will remain in the dibenzyl ether. Thus I would recommend to use a classical distillation apparatus as shown in the attached picture which was taken from the very instructive Wikipedia entry on the term "Distillation". First you can distill off the dichloromethane under normal pressure. Then replace the receiving flask and distill the dibenzyl ether under vacuum (oil pump etc.). This will give you a pure product. I'm pretty sure that some brownish residue will stay behind... 😎

Good luck with your work and best wishes, Frank Edelmann

Neha Umesh Mishra

hello

Please, why this enantioselective synthesis is only possible with the 2-naphthol?

Thanks in advance

I forgot to add you can do the incubation in batch mode, which is a great approach if you can accommodate the entire sample within the Econo column. Seals the ends and rotate for 30-60 min or whatever time you feel is needed for full interaction. Or incubate the sample and beads in a large tube, and transfer the mixture in portions to the econo column until all the resin has been transferred (a slight disadvantage is that you always lose some resin this way). Then wash and elute the column.

I don't see why it won't be possible but if the fragment hasn't been expressed and purified like this before, you have the task of devising a purification scheme. As many proteins will be larger in size, a step that involves size separation seems logical. However, you will have many peptides, protein fragments and other small biomolecules still to remove. As you know the sequence you can calculate the charge and see which ion-exchange mode might be useful. Hydrophobic interaction is another option. As your protein is small, you do not have the easiest SDS gel option to track the protein, but at least you have an ELISA. Analytical hplc could be used too, as long as you can positively identify your peak. Finally, if there is an antibody that can be used for immunoaffinity purification, your task may be considerably simplified. i.e clarification of the crude extract and then direct application to the affinity column.

Thank you all for your answers. I washed needle and syringe about 10 times and now the problem seems solved. I think there was a problem with needle. Trapped bubble or sth ?

Dear Alexander, I would never do a mee too job. If you go commercial one has to invest substantial sums and somebody has to provide and invest the required funds. Nobody will invest if he can't get a reasonable return on money invested. On the other hand on everything we do we can add value so that all project participants can earn some money in various forms. To add value one requires intelligence and trustworthy partners. Unfortunately, this topic is never discussed in Universities. In a competitive world value creation is the most important aspect for everybody that wants to survive in research. What is important is also important is to create circular economies to create sustainability and multilateral trade to spread the wealth. This issue is particularly important in emerging economies.

In a general perspective, the average total cost of the analytical apparatus to chemical experiments (polar and nonpolar compounds) such as GC and its specific columns, HPLC along with specific detectors, NMR, Amino-acid analyzer coupled with its equipment and other analytes preprocessing equipment is varies between from 100 to 250 thousands dollars. The cost of maintaining such a laboratory is estimated between 15 to 25 percent of the total cost of its equipment, averagely. However, the mentioned costs do not include a comprehensive lab.

Size Exclusion Chromatography is a technique to find out average molecular weights and molecular weight distribution. Standard s are available to create calibration curves and one can find out the total permeation and total exclusion limits of the column. Many times we use multiple columns having various pore sizes to cover the entire range of molecular weight distribution of the sample.

I have an older instrument and all I can say is "Good luck with that".

Sneha Garudeswaran Julia Milliex Hi, may I have th référence of th exospin column you have tried please?

Thank you

Yes all you need is a glass tube or plastic syringe of appropriate length. Glass tube can be elongated with a gas flame to constrict the exit. Then pack down a blocking amount of glass wool ( I used aquarium filter plastic cotton wool). Pour the slurry of matrix into the tube and add more as the column flows and packs down under gravity until there is about 2cm above the gel matrix. You can then add sample and collect samples into tubes for protein measurement. Large molecules will flow round the beads and elute in the void volume of the column and small salts elute in the total volume of the column and between those two extremes your proteins will elute. With ion exchange it will be easier to manage as there will be fewer eluted samples to test...just the binding process and the elution but probably not the wash steps. You could try emailing colleagues to see if anyone uses disposable desalting columns because these can be washed and refilled many times

Interested

Van Deemter plot and corresponding equation in the calculation of HETP as Prof.Noel W Davies indicated is the answer. It is the observation for an ideal gas flow where the sweet spot is needed in terms of optimum linear velocity serving reasonable run time with the lowest HETP possible.

Emir

For EV-depletion, FBS can be ultracentrifuged at 118,000 × g for 18 h (Shelke et al., 2014) .

I think that there are researches in this field that you can get from searching on scientific sites interested in this field.

Hi Dr Sasha Nealand . I hope the following link could help you: https://www.google.com/amp/s/www.labmanager.com/product-focus/lab-evaporators-recycling-solvents-2312/amp

Hi there,

Neutralizing the pH as quick as possible limits the denaturating effect of low pH on proteins. It's not about denaturation/renaturation processes, it's rather about preventing denaturation.

Please obtain some training and assistance in using the HPLC system. Incorrect conclusions will be reached without years of formal training and experience. Data gathered will be invalid. Time, materials and money wasted.

Please inject a real blank (inject the same volume of mobile phase as you normally inject for the sample, but without the sample inside, just 100% mobile phase). At identical wavelengths, compare the "blank" run to a real sample analysis chromatogram (overlay the two chromatograms). If you still see the "peak", then you have a contaminated flow path (column fouling or carryover, as noted earlier).

but why HIC? lots of easier/better ways for Ab purification. ( https://cdn.cytivalifesciences.com/dmm3bwsv3/AssetStream.aspx?mediaformatid=10061&destinationid=10016&assetid=11660 ) covers lot of options.

First, you must identify you goals and purpose. Algal supernatants often include a mixture of trace metals, vitamins and other bio-molecules which were present in the original growth media. You can expect many of those to be present. Techniques used to analyze the mixture should be based on the source. Liquid chromatography coupled to appropriate detection modes would provide a good starting point.

*As a consultant, most of the microalgae production projects that I have worked on started with having the client identify the ingredients used in the growth media. You can try a simple key-word search on the web to find useful resources (i.e. Google).

I think that if you could have done your test by LCMS, you would get better results for identifying the terpenoids. Meanwhile, it is better that to use n-haxane for the first time.

You have to separate polymers (PVA, EVA etc) from the Additives (Sufactants, admixtures, colourants, fragrance, maybe external plasticisers etc). 1. Dilute the mixture and centrifuge or precipitate polymers. 2. Run LC-MS on dilluted additive mixture.

Do you know what kind of polymer you are looking for? You may be able to measure MW by Size exclusion. Do you have IR for polymer chemistry?

I don't think it would work. Since Chelex 100 is a chelating material from Bio-Rad used to purify compounds by ion exchange. With its good ability to bind to transition metal ions. Therefore if it is expired it risks not having chelation with different constituents of the mixture since its chelating properties would be modified. But without chelation there will be no separation. So you have to look for a good new Chelex to work.

I believe I understand now and in the morning I will try to message you. Thanks for the explanation. Best wishes, David Booth

Shimadzu - very good price/ quality balance .

Be careful with issues regarding training, technical assistance, maintenance, especially if you have no experience in the field.

Dear Rawaah Y. Al-sharrab,

Thank you so much for your interest. Actually, it is very difficult to solve the problem of internal standard. That is why, most of the researcher are now developing their method using external standard and to use matrix matched standard for avoiding the use of internal standard. Anyway, if you interested to work with internal standard, try to use the solvent as same as mobile phase, it may helps to minimize the problem. I read the answer made by

William Letter, he described many things, you may follow his advice. Thank you.