Liquid Chromatography - Science method
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Questions related to Liquid Chromatography
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?
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?
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
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.
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.
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)
- 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?
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?
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?
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?
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.
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 ?
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?
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?
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.
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?
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?
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?
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?
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?
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...
Column chromatography with Silica Gel columns with inbuilt UV detector.
Compound structure is mainly poly unsaturated fatty acids and analogs without UV handles.
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..
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?
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.
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?
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
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 !
Dry Column Vacuum Chromatography (DCVC) and Vacuum Liquid Chromatography (VLC) are same OR diffrent techniques?
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.
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. :(
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
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?
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.
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.
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.
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!
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?
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??
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.
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?
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!
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.
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.
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,
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
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...???
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!
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.
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
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.
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.
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.
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?
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.
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.
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?
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.
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.
Is there a simple and less expensive protocol for measuring the total antoxidant content of plants by HPLC
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.
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)],
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.
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.
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.
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?
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.
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.
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.
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%!
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!
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?