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Liquid Chromatography - Science method

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Questions related to Liquid Chromatography
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Hi, I am trying to separate bifenthrin from timber extract using HPLC. However, the peak I obtained was not symmetry, and had a small shoulder. The shoulder peak came out in when I ran the bifenthrin standard and also my sample.
Below were the parameter and column I was using:
Mobile phase A: Deionized H2O + 0.05% Trifluoroacetic acid
Mobile phase B: Acetonitrile + 0.05% Trifluoroacetic acid
Column: Hypersil Gold C-18, 250mm
Solvent for samples and standard: Acetonitrile:H20 (80:20 v/v)
Parameter: Isocratric (80:20 of Acetonitrile:H2O)
Flow rate: 1mL/min
Injection volume: 20uL
(No temperature control, my HPLC is not equipped with oven, ambient temperature around 23C)
May I know is this the bifenthrin peak should look like? Or is because of poor resolution due to enantiomers of bifenthrin?
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hi Dharmendra Kumar , my HPLC does not have temperature control. I have a dedicated room with air condition (door and windows are shut), so I assume fluctuation in temperature could be low
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Hi team,
Appreciate someone’s insight on my query. I am extracting vitamin B2 (riboflavin) and FMN analytes from infant milk formula and identifying/analyzing it in reverse phase liquid chromatography using linear regression. We run multiple injections of pure working standards (WS’s) before samples and quality controls (QCs) in between samples of riboflavin and FMN to check if the conditions are same and consistent throughout the run and whatever results we get are true results. I am facing a problem in which QC’s of FMN analyte peak area start getting increase as the injections reach to the end of the sequence. It is the same vial, same position in the autosampler but peak area gets increased. Does anyone faced the same type of issue?
Thanks
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yes bubbles can affect the reproducibility of the peak because when there are bubbles in the syringe, the volume of mobile phase that is aspirated from the syringe (which is used to inject and aspirate the sample) is not correct due to the bubbles of air so the volume of the sample is corrupted.
To monitor the carry over try to run a sample and then do another one by injecting only the mobile phase: you should not see peak or see a very small one
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I want to determine low molecular organic acid in Agilent Infinity II HPLC with DAD detector. Can Anyone say what is the detection limit of that instruments? Thanks
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Hi Tamjid, according to my understading, these limits of detection and quantification should be determined for every method, as every method will have different conditions (the column, the analytes to analyze, flow, mobile phase, injection volume, etc). These limits are determined through the validation of the analytical method. After method validation. you can continue and quantify your samples in a reliable way. As an example of method development and validation, you can see this publication. I hope this helps
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Dear colleagues,
I wanted to ask your inputs/ideas for a problem we are currently encountering in liquid chromatography.
We have developed a method for the analysis of a peptide at 1 mg/mL having a size of about 40 amino acids. The method is quite conventional, with the use of a Waters BEH C18 peptide column, together with a mobile phase composed of ACN and water + 0.1%TFA in both solvents. Obviously, we are working under gradient conditions.
We have performed more than 500 injections on the column without any issue. However, after this number of injections, we have begun to observe a strange peak distortion. Therefore, we believed that the column was too old, and we decided to order a new one.
With the new column, we have performed less than 20 injections of the sample without special problem, but after these few injections, the problem came back and similar peak distortion was observed. You can see the enclosed picture to have an idea of the very strange peak distortion observed.
Therefore, in order to ensure that there was no issue with the column batch, we have re-ordered a new column of the same type, but the problem came back after only 10 injections.
To be honest, the peak shape looks very special, and to the best of my knowledge, I have scarcely seen such shouldered peak shapes in liquid chromatography.
If you have already observed such a behaviour during your career or if you have an idea of what could be the reason for this peak distortion, please don’t hesitate to share your thoughts.
It is sometimes great to think outside the box, and this is why I have posted this message here.
Many thanks in advance for your help.
Davy Guillarme
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Dear Davy,
If contaminations or sample degradation are excluded, shouldered peaks are the sign of an LC issue. Your peptide is probably retained somewhere in the LC, which explained the slight RT shift inducing the second peak.
As you’ve changed the column, it’s likely not a column problem. Therefore, I suggest you to :
  1. Make fresh wash and mobile phases.
  2. Use an old vs freshly made standard sample.
  3. Check carryover and pressure
  4. Check all the tubings in contact with the sample, maybe unplug and replug them to ensure there is no dead volume.
  5. Check that the valves work properly.
  6. Check the injector/needle.
  7. You can also use a surfactant directly in your reconstitution solution to limit the sample adsorption.
Points 1-3 will help understand the problem origin.
Hope this will help! I'm looking forward to see what was the trouble!
Best,
Jonathan Maurer
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Hello,
Yesterday I measured some samples on Orbitrap IDX with LC Vanquish- measurement ended with nasty leak on top of IDX source, in the connection between PEEK and needle. After various solutions I tried to look at the capillary under magnifying glass and found completly corroded inner end of the needle (viz picture).
Mobile phases used were Water with 20 mM Ammonium formate, pH 7 (adjusted with MS grade ammonia) and acetonitrile with 20 mM ammonium formate.
Is it possible, that some of the additives is responsible for corrosion? All of them are used quite worldwide for LC/MS systems, so I'm really not sure.
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Hello researchers,
I am planning to do a SEC on a polysaccharide extract, but I dont know which column I should use. The polysaccharides should all have a weight larger than 3,5 kDa, because I dialyzed them with a MWCO 3,5 kDa tubing before.
My choice is now between
- Superdex 200 10/300 Increase (Fractionation range 1-100 kDa for dextrans)
OR
- Superose 6 10/300
So the thing is, in most publications I have seen the Superose 6 is used for polysaccharide analysis. But the superose 6 gives me only a flow rate of 0,25 ml/min at the maximum tolerated pressure of 1,5 MPa, while the superdex can go up to 0,75 ml/min and tolerates 5 MPa. Also the superose here is old, and not the "Increase" version.
So basically my question is: Is there a reason NOT to use the Superdex 200?
Thanks!
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I am detecting the polysaccharides by resorcinol-sulfuric acid-assay.
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I am developing a gradient method for the estimation of 3 drugs, two are water-soluble and one is hydrophobic. I am using Dionex HPLC system equipped with P680 HPLC pump, ASI-100 automated sample injector, UVD340U detector. The mobile phase is composed of 10mM KH2PO4 buffer pH 6.8 and ACN. A gradient is applied from 97:3 (or 90:10, depending upon the column used) Buffer: ACN up to 5 mins, followed by 50:50 buffer: ACN up to 16 mins, followed by 97:3 Buffer: ACN up to 20 mins. Columns tried are Inertsil ODS 3, Inertsil ODS 3V, Eclipse XDB c18, Phenomenex Gemini C18. Samples prepared in 50:50 Water: Methanol.
After 30-50 injections, the pressure rises to 170-200 bars (initial pressure 96-106 bars). After back flushing or washing at 50 deg celsius using 60:40 ACN water, the pressure reduces. However, again with increasing the aqueous content beyond 60%, the pressure starts rising and the peak shape is also not good. What could be the reason?
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In my opinion try to use Methanol instead of Acetonitrile.....
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I want to extract certain lipids, which are prone to (per)oxidation, from mitochondria. To prevent them from oxidizing, I want to add BHT (Butylated Hydroxytoluene) to the isolation buffer after harvesting the cells.
What is the best concentration of BHT? I have found values ranging from 50 µM to 6500 µM, and I am confused as to what is the best amount. I want to dissolve the BHT in DMSO, is that good? Or would ethanol be better? Which substance has the least negative impact on LC?
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Dear Christina Oettmeier although I'm absolutely no specialist in this field, I just came across the following potentially useful literature reference which might help you in your analysis:
Development and validation of a reverse phase-liquid chromatographic method for the estimation of butylated hydroxytoluene as antioxidant in paricalcitol hard gelatin capsule formulation dosage form
Fortunately this paper is freely available as public full text on RG. Also please have a look at the attached article in which the authors use a concentration of 0.01% BHT:
A comprehensive comparison of four methods for extracting lipids from Arabidopsis tissues
I hope this helps. Good luck with your research and please stay safe and healthy!
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Hi all,
I am trying to use Masslynx to run liquid chromatography. I have been able to configure the detector and pump, but I cannot find MS Tunes under the instrument tab. It is needed to inject my sample and then acquire data. Did anyone face such an issue?
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I used MassLynx about 4.5 years and that ended up two years ago, so bare with me here.
I guess that you have an LC/MSMS system, am I right? When you double click the MassLynx icon, the first window that opens the main window as shown in the first attachment.
Then, you must open two sub-windows within MassLynx to be able to control the instrument. They are MS Tune and MS Console.
From MS Tune window, you can turn On the MS detector, and from the MS Console you can turn On the LC flow and set your A and B percentages of flow as well as the flow rate.
You are saying that the MS Tune option can't be found in the Instrument tab. To me, this means that the communication between the LC and the MS is lost and you need to restart the communications.
First, there is a reset button on the MS that you can press through a hole using a paper clip or another narrow and long object. Before resetting the MS, exit all MassLynx applications and restart the computer. See if this solves the communications problem and brings back the MS Tune back to the Instrument tab.
One more thing you can do to check if the MS is online or not, is to access some MS functions in the MS Console window. Try to run IntelliStart for example. You can turn ON/OFF the nitrogen gas to the MS from this window. You can also fill up the infusion syringe with some liquid through the lines A and B. If these commands from the MS Console IntelliStart can control the MS, then your MS is online and communications aren't lost. In this case, try restarting MassLynx to see if this helps. If not, try uninstalling MassLynx and reinstalling it again.
Remember, always call the technical support line before doing something that you think it is going to ruin your instrument. Never take your chances with analytical instruments, they are so delicate and moody.
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I would like to analyse monocarboxylic acids higher than C20 (at least up to C30), but I cannot find a company for buying them. I prefer a mixture of more acids in an organic solvent but it can be solid standards. Thank you.
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Hi Kamil,
You may make an inquiry at Alfa Chemistry, they offer kinds of good-quality chemicals.
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During Elution of protein (say an Antibody) from Protein A column using Elution buffer which is at low pH (3 to 4) and immediately adding Neutralization buffer to the collected fractions to prevent the negative effects due to this extreme low pH. I'ld like to know during this process will the protein remains in native form only (or) It will first unfold during low pH elution fallowed by re folding during neutralization ?
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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.
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I am trying all the protocol to extract the clean phospholipase A2 from the inflammatory bowel disease patient's stool samples. All the time I am heading to the dead-end.
I have tried Chroloform-methanol extraction and then used the BCA assay to detect the concentration of the proteins. Then that sample used in gel electrophoresis to see the protein bands and could not see any protein bands. Can anyone help what went wrong, please?
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Wolfgang Schechinger Thank you for your answer.
Yes, I for the protein nice layer but don't know what happened the after steps because I couldn't see any proteins bands in the gel.
Of course, searching the " detection of PLA2 in stool" was my 1st thing.
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Hi all,
I will be quantifying amino acids from blood plasma via HPLC. I am planning to use leucine as internal standard. Is it already sufficient to profile all (20) amino acids or should I have more than one internal standard?
Thank you.
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As there are many HPLC methods for AA, it is rather important that you share with us which of the many procedures and methods you will use. For the very commonly used OPA/FMOC procedure, many choices for ISTD exist. Examples would include: sarcosine and/or norvaline.
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I am using Rostek RP C18 column on Shimadzu series 10 HPLC to quantify the compounds. There is a baseline falling down recently. Can you please suggest the reason? Mobile phase 5% acetic acid-methanol-acetonitrile=70:15:15
See attached chromatogram 1st compound comes at retention time 4.7 min and  2nd compound expected to come at 11 min but has not observed.
Thank you.
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Water probably absorbs more than solvent if you use a water-to-organic solvent gradient.
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Is the chromatographic peak a valid measure of a compounds` (relative) abundance in LC-MS data? We have recorded three MS/MS spectra; their retention time falls into a specific chromatographic peak (see attached document). Is this peak area an accurate measurement of abundance for the compound described by the three MS/MS spectra? If not, what would be a more accurate measure?
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No, we do not rely on MS signal alone for concentration. So called "abundance" is related to how well the various samples ionized under the specific HPLC method AND the MS parameters you selected (VERY SUBJECTIVE). Change the settings and you get different results. Some samples exist in high concentrations, but ionize poorly. Some in low concentration, but ionize strongly. Some compounds exist, are never seen. Hundreds of variation in signal detection lie between as this is a complex topic, esp for a web forum. 'Areas' or 'ion abundance' counts only have meaning when properly developed methods are used with formal standards. After that, complete calibration tables are created for quantitation (best done using a non-MS detector as MS is poor at quant). Isotopically labeled standards are best, but true "standards" in some form would be needed.
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I would like to get information regarding LOD and LOQ for my standard curve based on the software's calculations. Is there a way to show that and include in reports?
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there is a signal to noise ratio which can be calculated by masshunter automatically. You can calculate your LOD and LOQ based on that.
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Hi RG reserchers, recently our lab purchased a liquid chromatography coupled with mass spectrum brand “LC/MS-2020 shimadzu, Nexera XR/ LC 20AD”. When we operated it using phenolic extracts, we discover that the resulted picks appear without mentioning the name of the pick compounds or even a proposed name of the potential compound (like GC/MS). The mass spectrum fragmentations appear but without proposing a name of the compound. So, I am wondering if some of you have before used this apparatus and have faced this issue? also, does this apparatus need NIST or Wily bibliographies to identify the compounds? If so, where we can bay them?
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You have not done your homework to understand the basic fundamentals of how an HPLC or LC-MS system operate yet (or maybe have been watching too many of those TV shows or Movies where the person inserts a sample into the system and out comes the structure and name on screen!). There is no similar library ability with HPLC-MS as their is with electron impact detectors (hard ionization) used in GC-MS. When a proper GC method is coupled to a GC based MS (EI) detector, then one or more applicable libraries may be used, along with actual standards, to make qualitative assignments. For HPLC, there are far too many variables involved in the methods themselves (which determine selectivity for the compounds), the detectors themselves and the settings used (esp. for Mass detectors which have many critical setting that must be optimized, for each sample) which can create, change or make data disappear. And, as each instrument is different, similar methods may generate different data too.
  • LC Mass detectors are NOT universal detectors.
  • LC Mass detectors do not detect everything (compounds that do not ion well or at all, are unseen).
  • HPLC analysis and LC Mass detectors take many years of training just to achieve a basic proficiency in use. A well developed HPLC method must first be developed for use with the detector and sample. Changing the energy levesl used (gas, temperature, flow rates, LC method) changes the results obtained so a lot of training is needed to optimize the settings and interpret the data. Any one can use these tools to generate "peaks", but those peaks (and the data obtained) may not be real or scientifically valid.
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I have three dataset of quantitative and qualitative liquid chromatography, GC-MS and DNA barcoding of few samples.
What would be the best way to discriminate and visualize the data?
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Principal Component Analysis (PCA) is normally able to gather similar data and discriminate between different sets. We successfully did it with coffee aroma analysis using GC-MS.
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Does anyone have any tips on clearing blocked nanoViper (20 uM ID) tubing in a Dionex 3000 for LC-MS/MS? We're having recurring high pressure errors in our loading pump, and we've isolated it to a specific piece of nanoViper tubing between the loading pump and autosampler. The chemical identity of the clog is unknown, only that it is from a sample and is hydrophobic. Obviously identifying the source of the contaminant is important, we are investigating that as well. The pressure is excessively high but the flow is not completely blocked.
Currently we've connected the tubing directly to the loading pump, with the opposite end open. We have run first water and now 75% ACN through it, in both directions, but with little success in dissolving the block and reducing the pressure so far. Would a stronger/different solvent be useful?
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ACN is a poor choice as a 'wash solvent' for most methods. You need to use a wash solution (and perhaps a mobile phase too) that will fully (fully = 100%) dissolve the types of compounds you are running through this instrument. *This is a basic fundamental of training in the use of the system.
As you stated, FIND the cause of the clog first. Maybe you are overloading the system with sample? Maybe it is not fully soluble in the solution or when it mixes with mobile phase, it falls out of solution. This is clearly a training issue. In the meantime, replace the clogged tubing with fresh new tubing (tubing is cheap vs the time and solvents spent trying to clean it). Dissolve all samples in an appropriate mobile phase (one that they dissolve it). FILTER all samples with a chemically compatible filter before injecting. Insure that your HPLC method and mobile phase choices are appropriate for the samples (do they dissolve fully in the mobile phase?). Make sure you include a separate column Wash "step" after each run (this should be programmed as a 'Wash method" to be run after your analysis method). The "Column Wash" should utilize solvent mixtures that are fully compatible with your column and detector AND which are stronger than your mobile phase so they wash any potential late eluters or matrix off the column and out of the HPLC system and detector BEFORE the next analysis. Again, ACN is a very poor choice. Select solvents which will dissolve your materials and use a gradient during the wash.
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Right before my lab shut down, I ran several samples using Empower 3 and started analyzing the data remotely from home afterwards. Without realizing what I was doing, I deleted the sample set of data. While the project is intact, the sample set (raw data and results) are gone.
How this is possible is beyond me as most softwares will have safety mechanisms or the ability to still access raw data. Can anyone possibly help me in recovering this or let me know where it might be possible to access this data? I would greatly appreciate any advice! Tried to reach out to Waters but we don’t have a service contract so they refused to help at all...
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How did you delete the data? Did you delete it from within Empower, or delete the files from the drive?
If you deleted it from the local drive:
  1. See if your IT department has a back-up that can be used to restore the files
  2. Try one of the data recovery programs. As long as the disk sectors containing your data haven't been overwritten, it is likely that only the directory was wiped. I'm surprised
Some of the HPLC software I know of uses some kind of relational database to keep track of the data, so deleting it from within Empower may have deleted the files and the way to access them via their user interface. I don't have many suggestions in this case as they tend to lock those databases down to maintain data integrity.
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Column chromatography with Silica Gel columns with inbuilt UV detector.
Compound structure is mainly poly unsaturated fatty acids and analogs without UV handles.
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You have answered your own question. Proper chromatography methods require detection methods which are applicable to the samples under analysis. You may want to review the types of instruments, detectors, columns and materials available at your school before deciding on a final approach. Have someone in the school's analytical instrument lab assist you.
For HPLC samples with weak or no chromaphore, you will need to use a detector that does not rely on wavelength based light absorbance such as an RID, ELSD, CAD or MS, to name a few. Put a little time into researching this basic question so you will better understand the uses and limitations of these methods and detectors. I often suggest my students start with a keyword search on the web (GOOGLE or BING) to find examples and articles to review. This is one of the best ways to learn. Once you have familiarized yourself with the basics, be sure to ask your teacher for help in setting up and running some example methods.
BTW: You may also be able to derivatize some of your samples and use GC/FID or GC/MSD as an alternative to HPLC (only if the samples are appropriate).
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For a while, I have been experimenting with improving the linearity of my method. I have noticed that increasing my deuterium labeled (3x) internal standard to a concentration 2.5 times the ULOQ concentration, improved linearity a lot. However, I do not understand why. Does anyone have an explanation for this? (I am using LC-MS/MS)
The range I am working with is 10 - 20,000 ng/mL, which is quite a wide range to aim for. Adding 10 µL 50,000 ng/mL internal standard to 100 µL of my calibration standards, gives a near perfect linearity.
From the previous questions I have asked, I now assume that the most likely reason for saturation (in my case) is the formation of dimers/trimers etc. for the higher concentration standards. That can be the reason why, when I use isotopologues, the linearity does not improve at all.
How is it possible that increasing the SIL-IS, that has the same retention time as the analyte, improved linearity? I would think that it would actually induce more multimer formation, but this is obviously not the case..
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Mr. Asali,
LC-MS can be used to quantify highly reliably analytees in mixture without internal standard. Please consider, the following discussion, and the references therein, together with the references to the corresponding literature mentioned. This can be carried out by means of our innovative quantitative model formulast developed more recently. The method improves significantly the method performances of MS up to r = 0.99995 studying steroids in mixture, for example.
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Can anyone help me with peer reviewed papers that explain whether or not ions are formed in the ESI source? Is this what they call 'right-way-round' principle?
Then how come that some for some analytes the eluent pH does not affect ionization efficiency much? Can this be explained by the 'wrong-way-round' principle?
Im having a hard time understanding these two principles because they both contradict each other as they explain different theories for the ionization. Is one of the two an exception rule?
Furthermore, how can one predict what eluent pH you should choose as a starting point? Or is there no other way than trial and error?
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Noel W Davies Thank you again. This makes so much more sense now.
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I got a fraction contains of two or more compound by vacuum liquid chromatography with ethyl acetate:ethanol:water (100:26:10). The fraction was evaporate and i got chrystal mixed with brown semi solid. I want to get the crystal, what is the best solvent for recrystallization? Thanks.
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Dear Alik Kandhita Febriani,
I think you could recrystallized from ethanol or water. Also, maybe you could perform a sublimation for your coumarin, you will get a very pure crystal.
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For some unknown reasons, the software (Empower 2) doesn't provide mass distribution values for my samples. It integrates the chromatogram and detects all the peaks/retention times very well but my results table is just empty on these parameters (Mn, Mw, Mp, Mz, etc.). It gives me the raw measurements of peak height, area, and %area as well. Is there any way I can use these measurements to do my calculations? 
The methodology was as below:
Molecular weight characterization of the polymers were determined using a GPC unit (Waters 2695) equipped with a RI detector (Waters 2414), and a PDA (Waters 2998). The samples were characterized by passage through a guard column, followed by two Styragel 5 μm, HR 4E 7.8 × 300 mm column in series. THF was used as the eluent at a flow rate of 1.2 mL·min−1. Narrow poly(styrene) standards (580-156000 Da) were used to calibrate the GPC.
The chromatogram was obtained at 230nm wavelength and some of my peaks are within the range of standards.
Any comments, suggestions?
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Hi,
Did you finally manage to solve this question? I am struggling with the same problem.
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I would like to analyse monocarboxylic acids higher than C20 (at least up to C30), but I cannot find a company for buying them. I prefer a mixture of more acids in an organic solvent but it can be solid standards. Thank you
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Hi,
You may make an inquiry at Alfa Chemistry, they offer kinds of chemicals for research use.
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Hi everyone,
I am looking for the manual of the Spot Liquid Chromatography Flash from Armen Instrument: we have one of them in our laboratory but it did not run for a while. I never used it before so I would like to check if all pieces are here and follow the recommended start protocol. Does any of you have a copy ?
Thank you in advance !
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Isam, that is a sales brochure, not a manual.
Let us try and help people by showing them how to solve problems (a process of learning) rather than trying to do their work for them and give them answers.
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Can 2D-LC system be built by connecting two orthogonal columns with the use of two pumps and a switching valve? I want to build a tandem system myself. Is this practical?
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Yes. Take a look on literaturw. Your concept is ok, but remember that you need to "inject" continously dractions from 1st D to 2nd D, thus the valve between the columns must have a loop (s) (read about modulation/modulators dor 2D LC)
Regards
GB
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Dry Column Vacuum Chromatography (DCVC) and Vacuum Liquid Chromatography (VLC) are same OR diffrent techniques?
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I have read in some papers that nano liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) is more sensitive than conventional LC-MS/MS. But they never really mentioned why. I'm wondering how nano flowrate can achieve higher sensitivity.
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The detector response is based on concentration C=m/V if you reduce de volume the relative concentrtion increases and thus sensitivity.
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Hello, we have a Thermo Accela LC and we're having trouble with retention time shifts. Over repetitions the chromatograms look pretty reproducible, except drifting to longer retention times. It's also curious that not much elutes until 5 minutes, they're metabolite extractions so there should be polar compounds.
I think there's a problem with the mobile phase mixing. The LC is plumbed so the dynamic mixer is bypassed. Could it be a problem with the proportioning valves?
Also, we have four solvent lines, but we typically use two at a time. Could I plug the two unused lines at the quaternary inlet module (right before mixing) without causing pump problems?
Many thanks - there's so little on the internet about the Accela system. :(
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And you still have not provided any information about the method or answered the most basic questions...
Yes, some mixing is done whenever two lines intersect, as in the proportioning module (which has four valves and acts as a gate, allowing different proportions in at a time or inside the pump head), but most HPLC systems have an addition mixer. *For the Accela, they have in internal inlet mixer plus the pulse dampener also acts as a mixer too. Optionally (or std on some), a dynamic mixer can be installed too. However, as noted earlier increasing retention times are NOT an indication of a mixing problem.
Using only two of the four inlet lines can indeed result in problems. The two unused channels dry up inside and the valves can leak (resulting in cross contamination). Do not plug the unused channels. Instead, double up on each solvent line and place each of the unused lines into the complementary MP bottles (same as you use). Flush each one with MP to purge them fully. Then go back to using your two lines. Periodically re-flush each of those lines (~ 2 days) to maintain the degassed liquid inside them.
If you would be so kind to share your actual running conditions, column dimensions (basic HPLC info) and also provide numbers (Rt's) of some of the significant peaks for say, 6 runs in a row, then we could provide some free insight.
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When using the LC-MS/MS system to dertermine CPs compounds, I observed a problem that when the standard is prepared in Methanol:Water 1:1 solution, the system gives no result of analyte. However, when I prepared the standard in only water, the separation is quite good and peaks are observed clearly. Please explain
My mobile phase: MeOH:Water
Standard: Phenol mix 604
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A typical effect of to high elution power of the injection solvent. Try a lower MeOH ratio - e.g. by diluting your sample until your analytes are bound to the column. good luck
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Hello everyone!
You see, I was cleaning my Size Exclusion Superdex 200 prep grade column and everything went just fine.
I "reverse-flowed" NaOH 0.5M at a very slow pace
"Direct-flowed" Phosphate Buffer Saline (PBS) 0.01M, pH 7, NaCl 0.5M in order to clean up the sodium hydroxide, at 1 mL/min flow for 2 hours (my column volume is approx. 60 mL)
I got an awkward oscillating conductivity curve.
First, I checked if there was any bleeding in the column using a flashlight. It was OK!
Then I suspected the column was "overcompressed", so I loosen the adaptor at the top of the column. The problem was still there.
Then, I tried a slower flow rate of 0.2 mL/min. It worked! Both the conductivity and absorbance line were beautifully steady.
The column was sparklingly clean, right?
Albumin Test
Then I tried injecting 100 μL of my albumin 10mg/mL in phosphate saline buffer pH 7, supplemented with 0.2% NaN3, to test the column performance.
The conditions:
Flow rate: 0.5 mL/min, PBS 0.01M, pH 7, NaCl 0.5M.
The absorbance curve showed me that the column was clean indeed! We can see the albumin peak clearly and it was steady.
The conductivity line, on the other hand, wasn't steady at all. In fact, it was steady in an awkward way. It was steadily oscillating.
My Sample
I assumed that it was bad luck and moved on to my sample. It's a hydrolase in PBS 0.01M, pH 7, NaCl 0.25M. Sample volume: 5 mL.
I got the same result. Please, take a look in the attached picture.
Does anyone know what might be happening?
Thanks in advance.
Kind regards,
Guilherme
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The profile it similar to coluomns full of air for which you obtain strong oscillation when bubbles come out or in case of high pressure coloumns with out the flow restriction after the coloumn.
Which supedex 200 is it? 10/30? 16/10? the just to pass a lot of water at the flow reccomended on the mamual for at least 5-10 CV and see if it return normal.
Just as a practical suggestion. you can routinelly wash your coloumn just by injecting some ml of naoh concentrated and i think is it better wash out it with water than buffers.
good luck
Manuele
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I finished my masters in analytical chemistry, but there is so much more to learn. My main interest is the hard core fundamentals of chromatography and mass spectrometry. What positions in the working field/post academic courses should I apply for that further expands my knowledge? I am not done learning!
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Best advice I can provide (having hired and trained many in the field over the past decades): Get a job in industry to learn on the jobs (hands-on training) using a variety of different brands, models and types of modes. This is the only true way to gain practical experience with real-world compounds. Courses are great for learning the fundamentals and concepts (something that few spend the needed time to learn), but you will never gain any practical experience working at school, reading books or just taking more classes.
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I am struggling with Peak purity analysis, if anyone can help with that please. I am working on Dionex-Ultimate 3000 uHPLC.
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First, "Peak Purity Determination By HPLC" (specifically by use of a diode array detector) is a null test at best. It can only show possible impurities which may be present, but can NOT show true purity. As used by most HPLC instrument vendors, it is subject to many limitation and assumptions (such as all possible compounds absorb light with similar extinction coefficients, which is of course not realistic).
I teach the use of this feature on several software platforms and perhaps the two most important aspects of using it are: (1) That you have a great deal of experience and training in HPLC analysis and operation of the HPLC system and (2) that the analysis method used has been fully optimized and reviewed that is in compliance with following good chromatography fundamentals (e.g. Proper K prime's for peaks). This software feature is an advanced feature and can be easily manipulated to show any purity value you want. It requires formal training to use. Good documentation of the method settings and proof the method is selective for the given compound is required before use.
For more information on this topic, here is a link to a free, short article I wrote on the topic from my classes. Perhaps it will assist you with your questions.
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Which one do you suggest for quantitative analysis of cannabinoids: HPLC or GC? Why? What about neutral cannabinoids like THC and CBD? What about acidic form of cannabinoids like THCA and CBDA?
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Can I connect the column to a peristaltic pump operated at a low flow rate and leave the other end open to elute the contaminants out of the column? Or will it disrupt the column packing??
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Hi. Yes, you can use a peristaltic pump, as long as you respect the flow rate suggested by the manufacturer. Calibrate your pump before connecting it to the column and you should be fine. Watch for bubbles.
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I know that most (really almost all published) shotgun proteomics is performed using nanoflow liquid chromatography and nano ESI. Is it possible to get coverage of proteins in a complex sample for a shotgun proteomics experiment using microflow liquid chromatography? That is all our university has.
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I think that is difficult to say and will require some trial and error. It partially depends on your method, both from an LC perspective (what kind of gradient are you running over what time frame at which flow rate?), as well as from an MS (acquisition) perspective.
For what it's worth, a rough guess would be in the few micrograms range.
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What is the trends in liquid chromatography considering the development of increasingly selective HPLC detectors, such as tandem mass spectrometry? Could chromatography (separation of analytes using analytical columns) disappear in the future?
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Disappear? Not likely.
This question comes up frequently and progress has been slow. The technique of HPLC is mature (~ 53 years old) with few major advances in the past years. So called "UHPLC" is nothing more than HPLC, by a different marketing name. It is not new. We first starting using small particles packed in narrow diameter commercial HPLC columns coupled to low delay volume HPLC systems in the early to mid 1980's (e.g. Back then, I used one of the early HP 1090M HPLC systems with a DAD and DR5 pumps with great success that many modern HPLC systems have only recently equaled and surpassed). Low, solvent saving flow rates were used with super high-efficiency columns (1.0, 2.1 and 3.0 mm ID columns with 3u particles) to produce some great separations, but the market was just not ready for it. It was also in the 1980's that many of the HPLC systems became more reliable and added fully computerized automation for data acquisition and data analysis which were huge improvements over what we had before (strip chart records and cutting peaks out with scissors is not missed by me). In the 1990's, many said CE and CZE would replace all or most HPLC methods! That was nonsense. A decade later in the 2000's, SFC was advertised to eclipse HPLC as the most widely used technique. Another misleading prediction. None of these came true and SFC and CE are still niche techniques which are not likely to replace HPLC at all, but instead find their own niche applications (*Variants of CE found a great application area in DNA analysis).
Perhaps the most significant advance seen over the past decade has been in increased column peak capacity using columns packed with smaller particles, though this was developed and marketed back in the late 1980's (and few scientists were interested back then. It took a re-boot of the idea many decades later, when better instrument reliability was achieved and esp higher quality column packing techniques were developed to improve reproducibility, a challenge we still face).
In my view, as a professional scientific consultant who teaches scientists to use the latest techniques and methods, detectors and detection techniques for chromatography have not significantly changed. So much misleading detector sales literature and marketing would have us believe much has changed in the last 5 years, but really, little has. Detectors advertised with super high-speed data collection rates often far exceed the calculated need to collect accurate data by 10, 100 or 1000 fold. No recent order-of-magnitude changes (though the sales bias product literature from the instrument manufacturer's often claims each new system they introduce is orders of magnitude more sensitive than the one it replaced, LOL!). Sensitivity for some detection methods has certainly improved, but many of the improvements have been from other aspects of the technique or method (column choice, flow stability, better sample prep), not just the detector (e.g. longer path length flow cells provide significant signal improvement, but not 10x). Bench-top MS systems coupled to DAD units took time to perfect and now they are more commonly used. This is still an area where we will see improvements in both the technology and also in cost reduction and shrinking of size.
3D printing of columns and even instruments, "Lab on a Chip" solutions for specific application areas and the miniaturization of instruments are all areas where I think we can expect to see change in the coming years. Improvements have already been made and given time, hold promise.
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We've recently started using a single quad LC/MS system (Shimadzu LC/MS-2020, for what it's worth) for mouse plasma AA analysis (the 20 common EAA and NEAA). Pre-column derivatization seems to be ideal for this purpose, as we've gotten greatly increased sensitivity when using the Phenomenex EZfaast kit, but ideally I'd like to be free from kit prison for my AA analysis. When looking online, I've found a variety of AA derivatization protocols but not one that stands out as the standard. Is there any standard/common derivatization protocol for AA for use with LC/MS, or is it really as variable as it seems?
I'm quite new to using LC/MS, so any and all advice is appreciated.
EDIT: I am using ESI-MS, didn't even consider the remember importance of that until Karen mentioned it. Thank you both for the answers, they're extremely helpful!
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Dear Pszczolkowski,
I think attached papers are useful for your request.
Best wishes, Homayoon.
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The data that I have are of lcd format from liquid chromatography. Anybody knows how can be converted into matlab format? Any function for that?
Thank you!
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I see, Thanks Ludwig Gruber .
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I've observed that my procedure of mobile phase preparation affects the reproducibility of results. I used volumetric flasks, cylinders (for addition of second solvent) that is not so comfortable also for everyday use as flasks have limited number of volumes and Eppendorf pippettes for addition of organic modifiers. How to make the procedure more accurate?
1. I am thinking about bottle-top dispensette for measuring volumes of solvents:
2. I also used Glassco filtering system, hovewer after 2 months it started to leak and I could not see the reason of it (probably glass had some cracks). Nylon 0.2 um filters were used together with mentioned system.
Could you please share with your experience and advice devices (for measuring solvents volumes and vacuum filtration) that you usually use for LC mobile phases preparation?
I usually work with acetonitrile, methanol and TFA, formic, acetic acids, ammonium acetate and formate as modifiers.
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As the question is about the most "accurate" way of preparing the eluents, the only acceptable answer is to weigh all the constituents of your mobile phase. However, even with weighing in all the components make sure, volatile solvents are degassed before the final composition. For the preparation of an eluent, think about how to reliably and accurately prepare any liquid mixture. In other words: Do what we learned during the first practical lessons of our lab education.
Please allow me to talk the obvious: In gradient LC it is a very practical hint never to mix 100% pure solvents. Always have a small portion of the second eluent (in case of a binary gradient) present. It avoids any unwanted salt precipitations on the column or any significant volumetric contractions (or expansions) due to the mixing.
Now, did I always use the weighing method? Nope. I only weighed in the constituents, for ultimate performance. For most of our daily work, the faster volumetric approach is more than acceptable.
What made me stumble - several times in the past - was the imprecise description of eluent preparation in articles, posters, etc. I wished that people would accurately write what they did. For example instead of "5% (v/v)", one could write "add 50 mL of solvent A to 950 mL of solvent B". A couple of letters more, and everyone can reproduce the recipe...
My two cents.
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My lab is new to metabolomics analysis and for the past few months, we have been searching for a protocol to prepare our human fecal samples for LC-MS analysis, done by a metabolomics lab on campus. We ran a few validation samples using the attached protocol from the Sumner lab. However, I've seen other literature using 1) different ratios of methanol to water (50:50, 20:80, 1:1) and 2) cold methanol (anywhere from -20 to -80 °C).
I'm looking for information on how to decide what ratio of methanol:water is best and what temperature of the methanol you'd recommend? Also, if you have suggestions for other/better extraction solvents, that would be great! If anyone has done something similar, I'd love to hear your rationale behind your choices.
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Depends on the target compounds
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Dear all,
recently I was confronted with the statement that "rare sugars" can be used to identify food fraud or the use of enzymatic food treatment.
Initially, I was surprised to learn, that the term "rare sugars" is a known terminus technicus. Literature tells about some analytical methods - mostly based on liquid chromatography - to analyze some (psicose, tagatose to name the two most commonly mentioned).
What I would like to know is the following:
Is there any additional indication for an increased interest in such components?
Any hint and suggestion would be highly welcome.
Thank you very much in advance,
Detlef.
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Honey adulteration is a relatively hot area in food chemistry. As you point out, there are multiple published methods using NMR. I have done some work using HPLC-MS/MS, which I find to be vastly superior to the NMR methods (but slower, obviously) both in adulterated honey as well as adulterated aloe vera products, mostly concentrating on mannose. I can definitely see the advantage of looking for the rare sugars as adulterant markers (lower concentrations indicates adulteration, for example).
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We are having trouble with the injector clogging using our extracted plasma and tissue samples so need to add a filtration step in our method. We are thinking of filtering the sample after dry down and reconstitution, prior to injecting into the LC-MS. We only reconstitute in 250 ul
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Solid phase extraction
You clean and preconcentrate
It is the best way to clean dirty samples
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I am developing the method for simultaneous detection of gallic acid, pyrogallol, ethyl gallate, catechin,epicatechin and quercetin in a plant extract through Reverse Phase Ultra Flow Liquid Chromatography with isocratic mode. Except gallic acid and pyrogallol all the other compound peaks are getting separated clearly ..The retention time of the gallic acid and pyrogallol are very close..I have gone through the literatures and tried all the combinations of Acetonitrile:Water:Orthophosphoric acid (Buffer) and Acetonitrile:Methanol:Glacial acetic acid (Buffer), but I could not separate these two peaks. Can any one get me the solutions for this please...???
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Check the AOAC. They had HPLC methods.
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Separating sugars on a Hypercarb porous graphitic carbon column, retention time shift between runs is unacceptable. (Using MeOH/H2O+HCOOH eluent)
Any suggestion for a good washing procedure?
Thanks a lot!
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Two features of Hypercarb must be considered when using it for applications involving carbohydrates and anionic analytes. First, Hypercarb contains weak anion exchange sites on its surface. This aspect is primarily responsible for Hypercarb carbohydrate retention and selectivity. Second, these anion exchange sites are sensitive to oxidizing and reducing agents. The symptoms suggest the latter may be your problem. Try treating your column with ascorbic acid to restore the native oxidation state of Hypercarb.
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I am analysing isolated heme from blood using HPLC (Mobile phase: Methanol:H2O: Acetic acid) with identification at two different wavelengths. The chromatograms at 405 and 500 nm are shown. I can't understand the irregular slope-like curve at 405 nm.
Any suggestions?
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The retention time is very low, I would suggest to lower the methanol content to 30-40 % in the mobile phase as well as to lower the acetic acid concentration to 0,1% and compare the results.
regards,
G
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Have been trying to get metformin to elute later than wave/solvent front on a Poroshell 120 C18 2.1x50x2.7. Tried with mobile phases, 0.1% formic acid with ACN + 0.1%FA, 0.1% FA with ACN and 10mM ammonium acetate with ACN. Tried isocratic and gradient elution. Thanks
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I thank you should used buffer solution less than 4( 0.02 MKH2PO4) with 1% trethylamine , such as mobile phase Methanol: buffer: triethylamine :( 55:44:1) increase the retention time of metformin .
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Respected Sir,
I am doing method development.pKa of drug is 10.24. Column I am using YMC Pack Pro C18 250 mm in length and 3 micrometer in size. on one run of 100 ml of mobile phase pressure is 190kgf, second run of 100 ml of measuring cylinder of mobile phase pressure is 150 kgf, third run 120 kgf. This cylces appears as such. Same is the case with retention time sometimes 5, sometimes 5.5 sometimes 4.3. Mobile phase is 0.1 %formic acid and methanol 35 : 65.
These trials I tried in schimazdu HPLC PDA as well as schimazdu HPLC UV.
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First of all, you have to equilibrate for 15 min for every run.
Second, do you use gradient or isocratic system. In gradient system fluctuation of pressure is normal.
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Has anyone done LCMS on condition media for protein quantification?
I have this media (after the exposure of different flow conditions) that i would like to use LCMS for protein quantification because I want to test for any potential proteins that were exocytotically released during the conditioning. Any potential papers or suggestions would be greatly appreciated.
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If you don't use serum-free media as Alexey indicates, serum proteins and serum-derived exosomes overwhelm the amount of protein coming from your cells. Rather than using acetone precipitation, we concentrated protein by filter centrifugation using low MWCO Amicon concentrators. Using either method, you should calculate protein concentration in the media prior to concentrating, and in the case of filter centrifugation, after concentrating as well. If you're comparing across conditions or samples, you should compare based on the protein from equivalent initial volume of media, not equivalent amount of protein when running MS analysis.
Keep in mind that the amount of protein you obtain will be extremely low. In our studies, 48 hrs of cell culture in serum-free conditions yielded low ng per mL concentrations of protein (15-40 ng/mL, depending on experimental condition), a range that clearly requires concentration prior to MS analysis.
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Have been facing problem related to the column pressure from quite some time. Started the column clean-up procedure using 0.5M NaOH. Firstly, washed it twice the column volume with milliQ. Then, washed it with 0.5M NaOH. However, during this step, the pressure started increasing with time and reached to 1MPa at flow rate of 0.05ml/min. Before the washing, the column pressure of 1MPa used to be at 0.3ml/min. But, during normal course of work, after sample injection of the protein that I work upon (protein is involved in aggregation diseases), it used to follow the same pattern of high pressure even at low flow rate. Now, the column has been washed using 0.5M NaOH 1.5 times the column volume and now is being washed with milliQ. However, the column pressure is 1MPa at a flow rate of 0.05ml/min. Any suggestions on the issue are more than welcome.
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You could repack the column. Get the gel out of the column, give it a good washing, remove any fines. Make sure the filters and lines are clean. Then pack the gel into the column again, following manufacturers instruction (https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma-Aldrich/General_Information/1/ge-gel-filtration.pdf). This will take a whole day and require a gel reservoire that matches the column.
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Hey Experts,
I am planning to buy LC detector for lipid analysis. Can you please suggest me which LC detector would be best for lipid analysis? I am using SCIEX 5500 QTRAP and Shimadzu LC.
Thank you in advance
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*Mohamed's comment above is misleading and inaccurate.
There is no such thing as a true Universal Detector for HPLC (If there was one, RID would not be it.). RID is close and sometimes you will find misinformation about RID being "universal" (especially in vendor product literature) because it only relies on one physical property for detection (refractive index). However, as your sample's RI approaches the RI of the mobile phase, the sensitivity gets lower until it is finally zero. So that is not a Universal detector at all.
For lipid analysis, there are thousands of papers and articles showing the use of RID, ELSD, CAD and MS units for detection. ELSD is by far the most popular and RID is the simplest to use, least expensive and most limiting (very poor sensitivity).
Try a web search for example application notes and papers.
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Can someone please share the information or experience on flushing the anion exchange column from the arsenic speciation kit by ESI? The manufacturer doesn't provide any information about this with the product, nor any info on characteristics of the column, what stationary phase and what functional groups are. Is there possibility of reversing the flow of the eluent without damaging the column?
I suspect that the column is blocked be particulate matter. The kit is connected to ICP-MS equipped with SC-DX FAST system. The analytical sequence is set according to the instructions provided with the kit, 20 µL of sample, 120s of buffer 1 (ca. 2 mmol ammonium phosphate pH=9.7) followed by 660s of buffer 2 (ca. 40 mmol ammonium phosphate pH=8.6). The samples are calibration solutions of As species (As3, As5, MMA, DMA).
I observe problems with nebulization which is interrupted in and irregular manner. For a fraction of second nebulization stops then starts again like something is blocking the flow for a moment, which is also simultaneously visible as the brief brightening and dimming of the plasma. In the chromatogram one can see sudden drop of baseline to the zero followed by peak much higher than the baseline (see attached jpeg). This phenomena occurs more often while aspiring more diluted buffer 1 and after switching to buffer 2 with higher concentration it usually happen less frequently. Sometimes there is no drop, only very sharp and high peak, even when the sample is milliq water. When I run only milliq water, instead of buffers, this occurs very often, every few seconds throughout the analysis which takes over a dozen of minutes. I didn’t yet analyzed any sample besides the calibration solutions in water (max conc. 50 ppb).
There is no reply from manufacturer so maybe anyone of you fellow researchers have any recommendations in this situation?
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Try to change the prefilter of the column. It is on the inlet of the column.
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recently after few sets of sample and standard injection, the sequence stops randomly with the error message stating power failure on DAD icon with still power supply from ups. 
if anyone can provide some information from where we can begin troubleshooting. we are stuck in the middle of the analysis.
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we used different HPLC machine for the whole project. Started everything from scratch and completed the project successfully . Thank you all for sharing your expertice
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Hello,
I'm trying to upgrade to Analyst 1.6.3 so I can get off Windows XP and onto Windows 7. However, DCMS link version 2.1 is not working. It loads initially but then is not showing up in Analyst after we log out for the first time. Our Thermo rep tells me they haven't upgraded it for Windows 7. Does anyone have a work around?
Our MS is a Sciex 4000Qtrap and UHPLC is Dionex RSLC300.
Thanks,
JM
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So Sciex tells me that DCMS link only works on 32bit platforms! Incredible
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Curious about your research experiences. Ideally this is for ESI-QTOF electrospray ionisation. This will differ based on type of columns and equilibration as per dimensions, flow rate and size of particles. Can this be performed on a 250 mm column or up to 300 mm?. Does peak capacity increase in LCMS compared to HPLC UV?
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HILIC is not a column (though some vendors may place a HILIC label on it, they are only doing so to fool you), it is a mode of chromatography.
Column length alone has nothing to do with suitability for LC-MS analysis. First, develop the HPLC method and make sure it is compatible with the detector type and ion mode desired (e.g. ESI pos/neg; highest purity volatile additives and mobile phase ). For most types of MS systems, highest sensitivity will be obtained with the lowest flow rates. If sensitivity or LOD are not critical, then even 1.00 ml/min rates will be fine, otherwise, low flow rates (depending on the exact MS type) are preferred so the use of narrow ID columns helps to achieve this by maintaining linear velocity (e.g. for conventional methods) without loss of efficiency. You should get some help selecting the column when you first start to develop the actual method used. If LC-MS (ESI) is the eventual goal and sensitivity is important, then start with a column type and dimension which result in the needed separation (Have someone experienced in LC-MS method development help you). You will not know what the ideal column dimensions are until AFTER you start to develop the method. Work out the method on an HPLC system, then scale it ) to the LC-MS system so it works well (change column dimensions, flow rate, gradient conditions, inj volume and so on, as needed).
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Anal Chim Acta. 2017 May 15;967:12-32. doi: 10.1016/j.aca.2017.01.060. Epub 2017 Feb 7.
Recent advances in stationary phases and understanding of retention in hydrophilic interaction chromatography. A review.
Jandera P1, Janás P2.
I was looking for efficiency of gaurd column on its own for various compounds and for linking up of gaurd columns.
The fact is that e.g. phenyl column coupled to C18 column improved resolution (related to void volume in capacity factor) defied my understanding of  mixed mode chromatography.
The journal: J Chromatogr B Analyt Technol Biomed Life Sci. 2013 Dec 15;941:116-22. doi: 10.1016/j.jchromb.2013.10.005. Epub 2013 Oct 16.
Liquid chromatographic mass spectrometric method for simultaneous determination of smallorganic acids potentially contributing to acidosis in severe malaria.
Sriboonvorakul N1, Leepipatpiboon N, Dondorp AM, Pouplin T, White NJ, Tarning J, Lindegardh N.
serially links ZIC HILIC Gaurd column to ZIC HILIC which is common research for polar analytes. 
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Which methods are the fastest and easiest to do to save time. The LC-MS is broken for now and awaiting repair otherwise I would fragment the impurity with different m/z ionisation. 
In the meantime as I wait for the response from the manufacturer what can I do in the meantime for analysing this impurity in the small molecule. How can I remove this impurity before the To void volume marker, or elute it well off past the total analysis time?. Any suggestions and experiences would be helpful. Can surfactants remove certain impurities or dilute them?. Are any of surfactants compatible with HPLC whether they are cationic, neutral or anionic?. 
I want to centrifuge the solution and try to analyse it if there is a difference of nearly 100g/mol in molecular weight of the standard and the possible impurity. 
I do not have smaller filter paper available to try and refilter a stock solution of that standard below 0.45 microns used for buffer salts filtration. 
Also to switch on the HPLC visible lamp to analyse the molecule in the Vis-UV region to ensure that I know what peaks the molecule contains in both regions and to know how to change the elution of the impurity to prevent coelution with other analytes. 
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It's a solid in crystalline form that can be dissolved in highly organic solvent.  I was thinking to do a melting point analysis on just the solid.  
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Is there a simple and less expensive protocol for measuring the total antoxidant content of plants by HPLC
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 There is no need to measure total antioxidants content by HPLC. There are a number pf simple photometric tests, e.g. DPPH and etc. Why you want to use HPLC?  
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Hello,
Can anyone advise me where to find an example(s) of impurities profiling data (.raw files) acquired by liquid chromatography/mass spectrometry? Ideally, I would like to have several stages and a few replicates. Maybe there is a repository, where community uploads such data, that I am not aware of, or someone having them would be so kind to share them with me.
Thanks!
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Hello,
I am sure you are aware of MaConda: http://www.maconda.bham.ac.uk/downloads.php and the data downloadable in .csv etc. formats for specific contaminants  and the paper: https://academic.oup.com/bioinformatics/article/28/21/2856/236679/MaConDa-a-publicly-accessible-mass-spectrometry
Would be good to write to Ralf Weber for the source files.
Unsure if people put such data on DBs as .raw files but then you can go to the "Blank" files in submitted large scale metabolomics studies at MetaboLights:http://www.ebi.ac.uk/metabolights/  or MetabolomicsWorkBench:http://www.metabolomicsworkbench.org/  for example: http://www.ebi.ac.uk/metabolights/MTBLS218#protocols and so on.....
Because the "Blank" or "Solvent runs" in such untargeted data sets would constitute of impurities of various sources during sample preparation, chromatography and mass-spec runs.
Thanks,
Biswa
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My peaks have a slight peak tailing so half height or half width is appropriate but do I use 
R = (RT1 - RT2) / [0.5 * (W1 + W2)],  
OR
Half-width method (Resolution used in Performance Report):
page 249 of the report. That method used for agilent 1200 chemstation calculation of resolution so do I use this or the above. I do not know much about HPLC statistics or QC so make the answer simple for me to understand please. 
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My printer does not work in the laboratory. Converting min to mm does provide a number only
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I want to try basic conditions for LCMS in negative mode to try to satisfy the different pKa values of my 5 analytes which gear towards acidic and basic. pH5 is fine but the basic analytes can resolve better if I try to ionise them to see if I can sacrifice the performance of my resolution or acidic analytes. What column material have you tried in HILIC mode chromatography. I wanted to try charged phases but will only do cation or anion exchange. neutral phases not applicable. I am worried if that pH will degrade the material. Such as pH 6.5-8. Thank you for your support. 
I read that HILIC columns are not so sensitive to pH change but the production HILIC materials in columns is more difficult to attach the hydrophilic ligands. 
J Chromatogr Sci. 2013 Aug;51(7):684-93. doi: 10.1093/chromsci/bmt015. Epub 2013 Mar 13.
Main interactions and influences of the chromatographic parameters in HILIC separations.
Greco G1, Letzel T.
I am also wondering which pH range is most suitable for acidic analyte +ve mode ionisation and which pH range for negative mode ionisation for electrospray ionisation and atmospheric pressure APCI ionisation. 
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High pH (>pH 9) mobile phases have proven useful for efficient retention and resolution of ionizable compounds especially in combination with negative MS. I suggest you look up studies by
David Watson et al. at Strathclyde, UK.
Journal of Chromatography A, 1362 (2014) 168–179
You may also look at the study by Lucy et al.
Journal of Chromatography A, 1458 (2016) 82–89
and Teleki and co-workers
Analytical Biochemistry 475 (2015) 4–13
You will need to work with a basematerial that is pH stable at high pH and here is the polymeric phases recommended (keep in mind though that a polymeric particle is more sensitive to over-pressure and you should make sure not to work at pressures over 200 bar). You should also keep in mind that charged phases are pH dependent and they will alter their behavior with pH, thus not only your ionizable analytes. As a recommendation for buffer salt, a carbonate system will provide better buffer capacity than using only ammonia/ammonium hydroxide but both have been used in the literature.
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I am currently studying Individual and joint effect of PAHs and surfactants on CYP 1A enzymes in earthworms, looking at the EROD activity using the UPLC. My current mobile phase is K-phosphate/methanol (55:45) pH 2.5, though the method works, K-phosphate is not compatible with the column because of its crystallisation. I have not seen any other literature that has used a different mobile phase yet, so my first question would be: why is K-phosphate used as a constant mobile phase, and what other mobile phase can I use, bearing in mind that tris and hepes are no good either because they interfere with the mass spec. 
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Be sure both your mobile phases and samples are ultrafiltered (~0.1 um).
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Are all the peaks read of from Liquid chromatography–Mass spectrometry (LC-MS) always relevant. If yes, how will you analyse each of them assuming they appeared in 100's?
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Dear Victor, I assume from your question you are asking about mass peaks, and not chromatographic peaks? While not all peaks are relevant for both types of peaks (as mentioned by Hiten), the reason differs quite notably depending upon the peak which you are examining. I want to unpack Hiten's answer a little, assuming in all cases you are using ESI.
As Hiten noted, not all mass peaks are relevant, especially when running in full-scan mode. Often, peaks corresponding to plasticizers, column packing material, solvent adducts, previous injections etc. may be present in addition to the peak representing your analyte of interest. It is often good to have an idea about what to expect before starting - for example: do you expect to see a protonated or deprotonated ion? A sodiated or potassiated adduct? Calculate out the mass of your intended analyte, and then determine what the mass should be. If the observed mass deviates from the calculated mass, then reassess.
Similarly, mass peaks observable before the elution of the dead volume can be categorically disregarded.
Chromatographic peaks (often as a total Ion chromatogram or TIC) can similarly be disregarded where the mass spectra and retention times deviate from expected. Matrix may shift the retention time slightly, however, major shifts in retention or observed the mass (on MS instruments) or transition ratios/Qual/Quant ratios (MS/MS) will invalidate the peak, or hint at other issues in the LC/MS system.
I hope this helps, please feel free to ask any further questions.
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Analytes to be prepared in 85% ACN with 15% deionised water. pH 5 ammonium acetate. Mobile phase buffered same way. I tried amm. formate at same pH. I tried formic acid liquid buffering medium but split peaks. 
The running mobile phase equilibrating the column is 95% Amm acetate pH5. This is an amm. salt and I would expect precipitatation if 95% mobile phase prepared sample and running an 85% ACN mobile phase that is buffered because the analytes will become less soluble. This is for a HILIC setup and I am trying to set up a mini-gradient within the isocratic mode to allow the water layer to not be so pushed towards the material chemistry. So more polar interactions in the beginning to try to retain some analyte before the others. Then after 7 minutes the other analytes can become retained later in the spectrum. Moving the analytes past the To marker. 
Thank you for the experience you provide. I have tried optimising other parameters in HPLC approximately and this is priority for me, on the agenda now before I try higher temperature. 
I buffered the mobile phase with acetic acid of same molarity concentration at 10mM since this would be a contributing factor for the HILIC water layer to dissolve the buffer salts. I am using sonication after degassing once. 
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You've done a really nice job so far, but you missed one very easy step. Take your sample and pipet 10 uL into a mL or so of the eluant (simulate your injection) in a clear vial and see if you get a precipitate. 
I like the concept of how you've set up your RPLC run (HILIC runs typically start with very high solvent concentrations and drop down). You want to minimize your injection to eluant volume as much as possible to avoid the perturbation from the high %solvent sample to the low %solvent eluant, because at the 95% water even the small amount of ACN that you are injecting will cause some perturbations.
Keep up the good work!
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How are they related to anthraquinone, anthracine. 
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pKa values around 3 are in general associated to carboxylic groups. Nonetheless pKa values depend also on the neighboring atoms and molecule structure. In the case of pyocyanine the pKa of 4.9 is for a nitrogen atom and it is clearly low from general values associated to amines, which ranged from 9 to 10. 
At pH values above 4.9 pyocyanine will be a zwitterion. 
Water solubility is largely dictated by symmetry considerations as well as molecule charge. To determine if a phenazine behaves as polar or nonpolar you need to analyze the particular molecule. 
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Hello Folks,
I am planning to measure the endogenous compounds (e.g.retinoids) in liver tissue with LC-MS/MS. But it is very hard to get a control matrix sample for method development and validation purpose. What is the best way to quantitate such endogenous compounds? 
Many many thanks in advance.
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See if there is a C13 isotope, which would have a mass of +1 more than your native retinol (retinoids). Now you can do recovery and spiking.  This is the ability if you have an MS/MS system. You can probably use one retinoid labeled C13 compound for over all recovery of all your retinoids of interest  because the MW are so close.  The C13 standard is expensive but it is worthwhile.  There is a lot of retinol in liver.  See if NIST has a liver reference material.  Good luck.
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Hi All,
Am trying to quantify benzalkonium chloride by LCMSMS. I have optimised the method got the parent and daughter ion. Have injected the standard, peak was eluting. After that injected blank sample found some peaks in the same mass with the same response. Tried with different mobile phase but found the same peak with same mass in blank sample. checked for injector port carryover but injecting  directly to loop. but found the same peak eluting. Can anyone help me resolving the same.
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If I remember BAC is 'very' hard to clean.  See attached (they may have an idea on cleaning);
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I have a new molecule for which I have initially developed an isocratic LC-MS/MS method (Synergi Fusion column, H2O/ACN 20/80 (+0.1% HCOOH)). Analysis of this method gives limited sensitivity because of broad peak shape (but it was sufficient for the time being), and with no traces of carry-over.
Later on I needed a more sensitive method, so I switched to a gradient method to sharpen up the peak. Now I have great sensitivity but a carry-over of 50%!
Gradient:
A: H2O+0.1% HCOOH
B: ACN + 0.1% HCOOH
t        %A      %B
0       90        10
3       90        10
3.1    20        80
5       20        80
5.1    10        90
8       10        90
Rinse solvent: ACN
Injection of a standard solution followed by a series of blanks showed that the peaks decrease somewhat evenly by about half, after each injection. It will be impractical to therefore injection 10 blanks after each standard solution and sample.
Injection of a completely fresh blank vial (injected as a 'sample) after injection of a standard gave a peak, suggesting that the analyte does not stick to the outside of the needle. Use of an external wash vial showed no improvement in the carry-over. So the problem must be somewhere in the system.
I tried a number of things already, none of which had any effect:
- Tried a different column (Synergi Polar instead of Synergi Fusion)
- raise the gradient to 100% ACN and keep it for an extra minute
- rinse solvent: ACN/H2O 50/50, and MeOH/H2O 50/50 + 1% acetic acid
- used an external wash vial with ACN/THF 90/10
I then went back to using the isocratic method, and no carry-over was observed. But it is not sensitive enough, so I really need this gradient method to work.
What other rinse solution combinations could work? Perhaps something more similar to the mobile phase?
- H2O/ACN 20/80 + 0.1% HCOOH ??
- H2O: ACN: MeOH: Isopropanol (25: 25: 25: 25 V/V) ??
The LC-MS/MS system is an Agilent Series 1290 Pump and Autosampler, with an API 5500 MS
Any help would be appreciated!
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A couple of things. One: have you reduced the injection volume or better yet diluted the sample? Two: I have an Agilent QTOF in which I experienced the sample attaching itself to the plastic lines.... Lastly have you varied the gradient to have more organic in the initial steps?
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The column that I need to replace is a Luna C8 (150 x 4,6 mm) 5 µm. In the USP Column comparison web page appears as equivalent an XbridgeC18. The problem is that I don't know if the United States Pharmacopoeia allows the change in the stationary phase!, I mean replacing a C8 Column for a C18. Does anyone can give me an advise based on a regulatory knowledge?
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As per USP general chapter <621> you can change the dimension of the HPLC column within given range in chapter, but you cannot change the stationary phase. For changing the stationary phase you need to fully validate your method and prove its equivalency.
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