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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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Currently, I use pure water as my eluate. I wanted to use NaN3, but our company won't allow me to use that due to safety issues, so I wonder if there is something else I can use to prevent algae growth?
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How long do you need to keep the mobile phase? If the chemicals are not expensive, make new solutions of the MP in clean bottles every 2 weeks. You may be able to use sodium benzoate. If you are concerned about algae, keep the MP in the dark.
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Dear all,
what are the possible instruments to use to obtain the purity of a powder (NaCl sold in a pellet form) (has 99% purity or above)?
the cheapest instrument to the expensive one if possible according to your experience
Thank you in advance
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Purification of chemicals is a large branch of chemical technology, with a large number of non-universal methods. In the case of NaCl, it is sufficient to simply recrystallize it from an aqueous solution.
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Im curious as to whether the software for LCMS (Labsolutions from Shimadzu) already has a quantification feature or do I have to compute and compare the AUCs to the standards manually? If so, what are the steps in the computation/quantification of compounds?
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  1. In your LabSolutions main window, go to Postrun then open the Browser.
  2. In the Browser's main tab, press on Quant Browser, then open the files/samples you want to quantify.
Things to consider: quantifying requires a quantification method, where you assign m/z, retention time, etc. to your analytes/compounds, so they got recognized by the software. In this case, you will get peak areas (or area ratio in case of using an internal standard), and this can be called qualitative analysis. For quantitative analysis, you need to add the concentration of each calibration curve standard in order to quantify.
You can follow LabSolution training or watch tutorial videos on how to quantify using LabSolutions software.
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Hi I am confused as to how are solvent prepared for HPLC analysis. This is the protocol I got, I am not sure what it meant by the ranges 5-15% B (0-10min) etc.
A mobile phase of 5% (v/v) aqueous formic acid (A) and methanol (B) will be used with a discontinuous gradient: 5–15% B (0–10 min.), 15–30% B (10–15 min.), 30–35%B (15–25 min.), 35–50% B (25–35min.) and 50–80% B (35–40 min.), followed by an isocratic elution for 20min., at a flow rate of 1 ml/min.
Can someone explain to me? thank you so much
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Means you should have a gradient pump. So, set the pump to 5% and increase to 15% mobile phase B in a period of 10 minutes (by default mobile phase A is reduced from 95 to 85%). Increase B again from 15 to 30% in 5 minutes (10-15min- again A by default decreases 85-70%). Again increase B 30-35% in 5 min and so on. B is the strong solvent, used to remove better retained components.
min A B
0 95 5
10 85 15
15 70 30
25 65 35
35 50 50
40 20 80
60 20 80
after that you will have to equilibrated at 95%A 5%B for a long time <20 min before you run another sample.
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Hi, I'm a student so please bear with me as much as possible. I am measuring plasma acetaminophen levels using HPLC. My samples were in a -20 freezer which went down. They were discovered at +2C and were thawed at this point. They were placed in another freezer. We are using HPLC to measure the plasma AAP levels, so these samples will be thawed again prior to extraction and analysis. Is this going to cause a significant problem?
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Thank you kindly.
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I used sodium bicarbonate as the mobile phase and did a test sequence with water and mobile phase. In the chromatograph, I found a negative peak in the water sample but not in the mobile phase sample. Hence, I believed that the RID picked the signal from water, so I used a more diluted mobile phase. However, the peak is still there but smaller. Is there any way to eliminate the peak?
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Bruce Neagle Thank you.
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Can anyone suggest the HPLC method to quantify spheroidenone carotenoid with a DAD detector and an Agilent zorbax SB-C18 column?
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The bigger problem is perhaps the high price for the reference substance spheroidenone. Among others 10mg offered by Toronto Research Chemicals may become the best choice. You can test the purity the same time you prepare for your concentration-response correlation curve. The mass spectrum in the attached file has been made by GC-MS analysis. Isn't it to prefer before struggling with HPLC-MS?? It depends of course on your application and type of sample.
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Is there an effective way to detect oil/lubricant residuals dissolved in an organic solvent. Both qualitative and quantitative measurements are fine. I am looking for more of a bench top method, quick and easy. I know NMR or HPLC are good for detection.
Is it possible to use UV/VIS spectrophotometer or contact angle measurement??
Any other idea would be highly appreciated.
Thanks
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Hi everyone,
can anyone suggest me a method for hplc analysis of organometall complexes based on Zeise salts complexes?
Using a ordinary C18 column results in one peak, that comes from the free ligand.
However, studying the stability in solution shows promising results.
I need a reliable method to distinguish between free ligand and complex. Maybe a specific column that can be used for charged complexes? The platinum complex is negatively charged, its counterion is potassium.
It is now the analytical issue to solve this problem.
Any suggestions?
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What about HPLC-MS coupling? This may answer your question which compound is "in your peak".
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Dear colleagues!
There is a description of calibration mix preparation for GC method of milk triglycerides adulteration determination.
Prepare about 1 g of a mixture of the fat standards — containing at least the saturated TGs, C24, C30, C36, C42, C48 and C54, as well as cholesterol; plus, preferably, C50 and C52 — by weighing to the nearest 1 mg and recording the mass to 0,1 mg to obtain a relative TG composition similar to milk fat.
Analyse repeatedly a solution of the fat standards mixture in n-hexane or n-heptane in accordance with GC method. In the same sequence, analyse repeatedly milk fat of typical composition.
Determine the TG response factors from the fat standards mixture. Calculate the intermediate response factors of TGs not present in the mixture (please, see below) by mathematical interpolation. Apply the response factors obtained to the milk fat in order to obtain a standardized composition
Not present in mixture triglycerides C26, C28,
C32, C34,
C44, C46,
Could you, please explain how to calculate response factors of triglycerides not present in calibration mix?
Thank you in advance!
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Plot the response factors of TGs vs carbon number. Fit a regression line through the data. Use the equation of the line to calculate the response factors for the TGs of interest.
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My procedure which did not work was:
1. Cell lysis with 0.2% Triton X-100
2. Protein precipitation with TCA
3. SPE (I have tried without this step but results were the same)
4. Filtration through a 0.2 um membrane
5. HPLC analysis
I am trying to analyse the lysate for itaconic acid. The chromatogram did not have any major peaks exept one giant asimetrical peak (which can be triton x-100 judging by the size but not the uv absorbtion spectrum).
I am trying to find out which other cell lysis protocols do scientists use for HPLC? Thank you in advance!
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@Grant Simpson Thank you for replying! I have found that for metabolite extraction cold methanol works best. Also hplc with pda detector is not ideal for detecting metabolites in a cell lysate therefore i am trying to incorporate gc-ms for metabolite quantitation.
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There are certain drugs exist in liquid form available in the Sigma-Aldrich website, where they have mentioned the concentration of those drugs in "ml" rather than "mg" or "g". How one will find the concentration of such drugs?
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Actually, reading the question with very strict meanings, he might be looking for a conversion to grams. If the density is known, multiply the volume by the density to get the weight. The concentration of a pure liquid compound is 100%.
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I am working with carotenoids mainly Lutein and Zeaxanthin. For this I have purchased the standards from Sigma Aldrich which were very expensive. there are all in powder form and I would like to know what is the best solvent for dilution and long-term storage before HPLC analysis. I went through different papers and there was no consistency as there was mention of methanol, hexane, etc...
I would appreciate any technical and straightforward suggestion. Thanks
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Pure ethanol is best for dissolving lutein and zeaxanthin.
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Conducting HPLC analysis for carotenoid content on vegetables is not an issue, but what about carotenoid content incorporated in a high fat diet ?
My lab usually performs double hexane extraction for common fruit and vegetables but what would be an appropriate extraction protocol for fat food (45% butter, 5% tomato powder) ?
I've heard two possibilities : extract as usual or sample saponification prior to analyses.
As carotenoids are lipophilic, wouldn't saponification damage these compounds ?
What would be an appropriate extraction protocol in such a case ?
Thanks for your help !
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Hi Thomas,
The saponification works very well. Some research articles will do this in the cold so as to reduce the degradation of the carotenoids. Otherwise, you can also add BHT to the saponification reagent.
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Suppose in HPLC if Pump A and Pump B were running and the inlet tubing lines are un labelled, So how to identify which Pump's inlet tubing line is dipped in which buffer. I'd like to know what are the different possible ways that we can trace/identify respective inlet tubing lines of Pump A and B while they were in running condition.
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3/7/22
Dear Vikram,
This should not be difficult to do. What kind of HPLC do you have, and what are your mobile phases? We have an Agilent HPLC Series 1200 w/ a quaternary pump that can handle 4 mobile phases (A, B, C & D). The easiest way to trace the tubing to the pump head. That should tell you. If that doesn't work, disconnect the tubing as it leaves the pump but before it goes to the column, then go to the instrument control screen and turn on each pump separately (A or B) and let it run at 3-4 mL./min for a while and see in which reservoir the mobile phase level drops. Alternatively, you can test the mobile phase as it exits the tube, e.g., if they are buffers w/ different pH's, check the pH w/ pH paper.
I hope this information helps you.
Bill Colonna Iowa State University, Ames, Iowa, USA wcolonna@iastate.edu
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We intend to separate the mixture of three glycosilated flavonoids having two sugars units, each, according to their LCMS profiles.
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Depending on the gradient you used for your scouting run, they may be well-resolved for preparative LC.
You didn't mention the solvents used for your analytical run, but, assuming acetonitrile, try methanol or tetrahydrofuran. You didn't mention solvent modifiers, but if you didn't use any, try TFA to keep the phenolic groups protonated, and less polar to allow more resolution from the glycosylated compounds.
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I have 6 plant extracts prepared in ethanol. HPLC analysis of all is showing the retention time between 2.2 to 2.5 min but their mAU is different. GC-MS analysis showed different compounds in these 6 ethanol extracts. Is this okay or there is anything wrong
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Mrs/Miss Saxena,
By means of chromatography, there is unable to be determined tautomers and different protonated forms; if any. This can be done only by means of mass spectrometry; furthermore, exactly in terms of both quantitative and 3D molecular structural analyses.
Please, consider the following work. It deals, namely, with this issue:
The shown method is applicable to a set of different MS ionization and fragmentation MS methods. Please, consider the reference section, therein.
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How to check the contents(protein)present in a crude crop like flax in hplc and which solvent will be best to dissolve it for the hplc run?
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I do agree with Prof Dr Medhat Elsahookie.
I suppose that flax contains fixed oil than proteins unless exposed to stress. At that time, you can follow procedure to extract protein material. So, this can inject into HPLC device. Using standards, you can record a goal. Regardingly, check column diameter, solvent, proteins properties and impurities.
Cheers
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In other words, without a standard sample, can HPLC analyze the quality of a solution?
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Dear William Letter thank you very much. Your information created a new and useful insight for me.
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Hi all,
I'm trying to separate Alpha, Beta and Gamma cyclodextrins from each other in a mixed aqueous solution. Now I know that there are slight differences in polarity of the different cyclodextrin molecules so my idea was to try and separate them using SPE.
The components have excellent separation on a silver-ion HPLC column, which suggests to me that SPE could work.
However after a few attempts I have noticed that the CDs have barely any retention on the SPE column.
I would love any suggestions on how to improve separation :)
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I want to analyze Dopamine in the rat striatum with the HPLC method.
I used perchloric acid to the homogenization of rat striatum. but I don't know what should I use as a blank solution in this method.
Can anyone help me?
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Hi Sadegh,
I cannot help with this one. Big question is do you really need a blank for this type of analysis? My answer was based on (U)HPLC analysis coupled to electrochemical detection (we develop and sell EC detectors and (U)HPLC-ECD analysis solutions). This technique is mostly used in Neuroscience (analysis of catecholamines and acidic metabolites in brain dialysates or homogenates) due to its superb sensitivity and selectivity. Blank samples are not used in this case. In principle a chromatogram of your analysis of the work-up brain homogenate will give you a pretty clear idea about possible interferences eluting close to dopamine. By spiking (std. addition) your sample with dopamine and assessing the peak shape and response you will get more info. In case you have interferences you need to tune you separation conditions (see my previous answers).
Regards, Hendrik
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Wavelength is not mentioned anywhere.
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please helpI believe I have a clogged up HPLC (Agilent 1260 infinity II) due to there are salt precipitating as a result of using a mobile phase with a high concentration of phosphate, immediately after that the pressure increased and then Pump stop while running. Its not in the column, I believe, but I think it's clogged in a line somewhere before the column. If I try to flush the lines at only 0.1mL/minute, the pressure is getting so high the entire instrument is stopping.
I made several solutions, including cleaning the purge valve frit as well as removing the column, but the pressure is still high, and the pump is closed immediately after the pressure has risen.
Even when the purge is open, the pressure rises directly and the pump shuts off, What is the best way to determine where this clog is....removing line by line to see if the flow will start again?
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I used reverse-phase for the first time, and as you could probably notice the amount of water is already very high! Although, most of the compounds come earlier during the first 5 min!
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Hi, I'm going to separate PSSs STD mixture(PSS MW 16500,8890,5580,1690) with HPLC SEC column(jaigel gs310).
I prepared the STD mix by mixing each of the standard substances at about 10 mg/L in a 1:1:1:1 ratio.
but, Looking at the results, it seems that there was no separation.
1) What is the cause and what is the solution?
method
buffer : DI 40℃ 0.5ml/min 2.7MPa run time 80min sample : STD mixture
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I'm trying to pack an XK16/70 Column with a Toyopearl hw-50s, but I have some questions.
Question 1. I am not sure how many mL of % sludge should be prepared when packing the column.
ID is 16mm and length height is 70cm. I calculated that it is about π*(1.6cm/2)^2*70cm = 140.7ml, and the manual says that the bed volume of XK16/70 is 105-130 and the bed height is 52.5-65cm. do.
When I checked the Toyopearl manual, they told me to prepare 30-50% slurry as 1.1 x the column volume, so I am going to prepare about 1.1*130ml=143ml of 40% slurry (resin 40% and water 60%). Is it right to prepare like this?
Question 2. I don't know how much pressure and flow rate to give when packing the column. The manual says that 0.5 to 3 bar is recommended, so I am thinking of reducing the pressure to 2 bar based on shimadzu HPLC, but I am not sure how much to set the flow rate. When I asked how to do toyopearl packing on Youtube, it was set to 45ml/min at 2bar, but I'm not sure if I can set it like this too.
Question 3. If there is not enough resin when packing the column, can I fill it up once and open the upper part again to pack more?
Question 4. It would be appreciated if you could provide additional advice on column packing.
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I'm a beginner learning to apply HPLC. Although I understand the principle of HPLC, I'm unable to optimize the method for eluting Pregnenolone. I'm looking forward to discussing parameters that should be taken into consideration while establishing a method for eluting Pregnenolone.
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In the early days of high performance liquid chromatography (HPLC), the selection of column formats (particle size, type, and column diameters) was rather limited and thus, optimization often was done by adjusting operational variables such as eluent velocity, column temperature, and operating pressure.
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P.S: I wash the column with water and methanol when I open and close the HPLC system.
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Is MS or NMR recommended for further elucidation? Or, is there any standard library available and accessible?
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If you are looking for a specific metabolite, you can use the various papers that they have been published on HPLC (which that depend on the peak absorption range of the desired compound in different concentrations of the standard sample and using the calibration curve produced). However, if you are looking for a different range of various metabolites, it is necessary to prepare/purchase the standard of each samples, separately and it is prepare the calibration curve for each compound in different concentrations. Finally, it must be compared the unidentified peak made of output of (HPLC) and analyzed with standard of each of them, accurately. Then, it could be expected that the identity of the unknown/unidentified absorption peak is identified.
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I am looking filter that help to remove oil and petroleum jelly.
Diluent is 50% ethanol with water. I have ointment which has oil, petroleum jelly. I want to remove oil and petroleum jelly using syringe filter for my HPLC analysis. Other wise it go in to column . Any advise for sample preparation.
Add 5 gram of ointment. add 10mL ethanol. Extract active. add water mix well.
Try many filter to remove oil but come with sample
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What is the formula (the mix of ingredients)? Is it solely lipophylic as a real ointment, or does it contain some water?
You could try mixing it with methanol, instead ethanol and try to separate the constituents by centrifugation.
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I'm interested in analytical protocols for measuring exposure to methylphenidate in mice, especially HPLC-based methods. What are the possibilities regarding detectors and sample preparation procedures? Also, considering limited volume of blood can be obtained from mice (and sampling in more time-points probably affects the obtained results) - what would be the best option in the context of the minimal volume of sample needed for the analysis? What about enantiomers (e.g. 10.1002/bmc.3312). I'd like to find/establish a protocol for clinically relevant chronic oral dosing of methylphenidate in mice that reflects what we see in humans (https://www.researchgate.net/post/Protocols_for_clinically_relevant_chronic_oral_dosing_of_methylphenidate_in_mice)
Any info is greatly appreciated.
Jan
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I am using acetonitrile: water (40:60) as a mobile phase for making the dilution for HPLC analysis. The solution has quick degrade by taking HPLC analysis. Please suggest me how to reduce degradation rate of berberine chloride in mobile phase ?
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Berberine chloride is photosensitive and thermosensitive. So try to prepare the solution in dark, also keep the temperature below 25'C before and during use.
Hope this may help you for future.
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To have the chance to find new structures from plants, we need to collect all the fractions and isolate even the most minor compounds.
But, the problem is that we recapitulate at the end only a very small mg!
Is this kind of work still significant? or not worth!
Could we publish in high journal with only elucidation of new structures from plants?
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I realized that fumaric acid had not been tested in my lab with other organic acids compounds. Malic acid, citric acid, acetic acid, succinic acid, lactic acid, and tartaric acid are all tested in most methods. Is there a reason why this compound is always left out? Despite the fact that the HPLC column is capable of separating it.
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Dear Bongisiwe Zozo many thanks for asking this interesting technical question. To the best of my knowledge, fumaric acid does not differ significantly from the other acids mentioned in your question as far as HPLC analysis is concerned. In this context please have a look at the following potentially useful article in which fumaric acid is determined by HPLC besides various other acids:
Quantification of the Organic Acids in Hawthorn Wine: A Comparison of Two HPLC Methods
This article has been published Open Access and is freely available on the internet (please see attached pdf file).
Good luck with your work and best wishes!
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which column is best for XOS analysis or how much the run time?
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Generally HPLC, we can use it for qualitative and quantitative analysis.
What is the main difference while using it with PDA or with MS detector?
What are the advantages of MS to PDA and vis-versa?
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A photodiode array detector, or PDA, is a type of absorbance detector (such as a UV detector) that is able to detect light-absorbing compounds at the PG level. However, PDA detects an entire spectrum simultaneously. UV and VIS detectors visualize the obtained result in two dimensions ( optical density and time), but PDA adds the third dimension (wavelength). The analyte or related impurities peak can be determined simultaneously during separation at all wavelengths, which is convenient for choosing the most suitable wavelength and determining the purity of the analyte or related impurities.
But you must inject a standard to make a standard curve for qualitative and quantitative analysis (without a standard curve, we can not identify the analyte)
MS Mass Spectrometer
The analytes are detected based on their MW (Mass: charge). The obtained information is especially useful for compound structure and identification (based on the Mass: charge ratio (without standard injection)). By constructing a standard curve, a very low detection limit of analyte can be quantified.
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I am working in hexa valent chromium bioremedation by bacteria that belongs to pesudomonas and i want to measure chromium degradation products by HPLC and can you tell me if there ara any technique more suitable than it
thanks
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What you probably want to do is to determine the valency of the Chromium ions HPLC or CE would be the most logical choices. The issue is detection. In CE you can use an indirect UV/VIS colorimetric method based on inorganic ion displacement of ionized dyes in the mobile phase (see attached). Imidizole has also been used for this method in CE. You might be also be able to use a redox detector (in HPLC), but this requires careful choices for the mobile phase and sample preparation to eliminate cross-reactive species and will be unable to detect the highest or lowest oxidation state of the ion.
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I have a problem with my internal standard, it seems that there is a peak has the same retention time of my internal standard, this peak appeared in human plasma chromatogram although I made a derivatization step and SPE and my internal standard has no appearance in human body as the reference article saying ...
There are two photos ... one illustrates how the peak of my internal overlapped with the other peak which I don't know ... and the other photo shows the derivative of my internal and it's a cyclic derivative ...
I tired playing with mobile phase ratios but no change not even a small change ... they move with each other and they are totally attached to each other ... I'm thinking of using external standard method to solve the problem but is that logical while working with biological samples ....
My internal standard is 2-methyl cysteine and I'm using % acetonitrile in % acetic acid as mobile phase
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When removing the outlet check valve from the Thermo Horizon LC pump head about 1 mm of the tip stayed in the LC pumpe head. It sits very tight due to the peak seal sticking the fragment to the pump head. Any ideas on how to remove this?
Thanks in advance.
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Thanks, Alexey! I got it now with a tiny drill. Silly thing though! Take care!
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Are mobile phases used in chiral separation using HPLC and SFC the same?
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For experienced chromatographers using HPLC or SFC for chiral separations, here is a link to a free training article on the use of different alcohols in chiral method development. You may find the tips useful when developing methods.
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I'm looking for a low molecular weight PEG ladder (400 Da - 2KDa) that I can use as a mixed retention time marker for HPLC analysis of various PEG compounds. Does anyone know if such a thing exists and, if so, where I can buy it?
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Several chemical suppliers come to mind for PEG (PEO) standards. How about American Polymer Standards Corporation ? They sell lots of low Mw stds in the range you are looking for. Companies such as SIGMA will also have linear stds too, but far fewer in the low Mw range. I have used them often for GPC work.
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My mobile phase is 40 mL water ( with 0,112 g sls and 0,64 microliter of orthophosphoric acid), 56 mL acetonitrile and 4,8 mL methanole. A C18 column is used for the analysis. Detection is at 233 nm with UV. HPLC system is thermo finnigan surveyor. The standard solutions are prepared in mobile phase without sls. After analysing 3 repetitions of same standards with good similar results, peaks were smaller or disappeared in fourth repetition. What could be the possible reason for these results?
P.S: I prepared new standards and mobile phase but the problem persists?
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Thank you all for your answers. I washed needle and syringe about 10 times and now the problem seems solved. I think there was a problem with needle. Trapped bubble or sth ?
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I am having baseline issue when following the method outlined in a application note from Agilent. The application note references a method that utilizes a gradient in the attached image.
The whole application note can be found by searching for 5994-3791EN.
I do have a binary pump instead of the quaternary pump, but otherwise there is very little difference between my instrument's config and the reference method that would significantly effect afaik. If Agilent's newer software corrects for the baseline issues seen maybe that could be the answer.
I also have the older Agilent DAD from the 1100 series if that is relevant.
What could possibly be the root cause of the difference in baseline seen here?
The transition dip is seen at other gradients of these same two mobile phases.
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I see some researchers doing some isolation of already known and available compounds!
Does this isolation still meaningful?
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Dear Alexander, I would never do a mee too job. If you go commercial one has to invest substantial sums and somebody has to provide and invest the required funds. Nobody will invest if he can't get a reasonable return on money invested. On the other hand on everything we do we can add value so that all project participants can earn some money in various forms. To add value one requires intelligence and trustworthy partners. Unfortunately, this topic is never discussed in Universities. In a competitive world value creation is the most important aspect for everybody that wants to survive in research. What is important is also important is to create circular economies to create sustainability and multilateral trade to spread the wealth. This issue is particularly important in emerging economies.
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I'm looking for the isolation of polymethoxyflavones from my crude extract.
Which TLC plate is better, normal or reverse phase? And which kind of solvents giving a best separation?
Anyone worked already in this kind of polyphenols!
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Hello! How to view the UV-spectrum on the shimadzu SPD 20A device? My HPLC does not have a PDA detector. We have a HPLC in the laboratory with PDA detector and on this chromatograph it is very easy to see the UV spectrum. I cannot find the tab where the UV spectrum is displayed. This is about offline mode.
Thanks in advance
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Jack Silver, yes it's shimazu. SPD-20A. It turns out that only on the matrix detector you can see the range from 200 to 800 nm?
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I am using a chiral CD (cyclodextrin) column to analyze 1,1'-Binaphthyl-2,2'-diyl hydrogen phosphate (BNP) enantiomers with the mobile phase composed of ammonium formate buffered water and acetonitrile. The retention time of both enantiomers was consistent over the last several weeks.
After my colleague ran his dye samples (Rhodamine B) through another column last week, since he couldn't see the rhodamine B peak in the chromatogram, I suspect at least a small amount of rhodamine B has got into the chiral CD column because my sequence was queued right after his.
From then on, the retention time of the BNP enantiomers is very inconsistent while the other initial injection volume peaks are always consistent. For example, let's say I run 30 samples, the elution time moves downward for the first 5-10 samples and then suddenly moves upward for 5-10 samples and then again move downward. SO, I'd say the elution time change is not completely random but keep moving back and forth. I tried regenerating the column and also using reverse flow to remove any particulates for 2-3 days, but I couldn't fix this problem.
Here are some reasons that I suspect may be causing this issue:
1. Intermittent interaction of BNP with rhodamine B dye remaining inside the column
2. Sample matrix interference or impurity in my samples (not likely because it never happened before)
3. Build-up of the sample matrix over the sequence (not likely because pressure doesn't increase or change. and no noisy baseline and ghost peaks found)
4. Inconsistent pressure or mobile phase composition due to the instrument malfunction (not likely because the initial injection peaks except the BNP enantiomer peaks stay the same)
Could you provide your opinion on this and maybe the most suspicious reason?
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The answer will be found through proper troubleshooting of the complete LC method and system. Here is a link to a free article which addresses your question;
Most likely, the column is fouled/damaged (note: "pressure changes" are not a requirement to indicate this). Cyclodextrin columns are fragile. However, there are many other commons reasons for your observations too. Please have someone local who is experienced in using your specific HPLC and has many years of method development experience assist you through the troubleshooting steps to find the problem(s).
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The question is solved.
The problem was the lifetime of the column due to extensive sample load.
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it looks like You are working with ans isocratic separation? if tru try a gradient one. - it might be You are accumulating something on your collumn.
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I would like to resolve peak tailing in the hplc analysis of one drug. Mobile phase used is 100% Methanol. The sample injected after saponification. The sample pKa value is 16.5 before saponification. C18 column ,250 mmx4.6 mm, 5 um was used.
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If using 100 percent methanol as the mobile phase for a C18 column causes peak issues, use some water or a buffer in the mobile phase.
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GPC is a widely known technique to calculate molecular weight and PI. However, it is highly selective in terms of columns used. Please suggest some other methods.
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You're welcome, Sa Yao.
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Is is legal to use phosphate buffer saline as surrogate matrix for bioanalytical method validation?
P.S. I want something amino acid free and have the same matrix of plasma to do the validation.
I tried bovine albumin serum but it has amino acids in it and peaks appeared
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I hope its meet your needs
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I want to do validation but I need the same matrix of human plasma cleared from amino acids and other additives.
I actually I thought of using buffer, what do think? Is it legal to use buffer for validation of biological samples?
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I can't synthesize standards for BM adducts since they need a toxic reducing agent (NaBH4).
To my knowledge (forgive the undergrad me), without the standards, I wouldn't be able to know what the retention time of the adducts are. (There may be literatures stating their RT but the HPLC conditions may differ.)
Now, how would I determine that certain peaks on the chromatogram of the extracts correspond to the BM adducts (extension units) without the standards' RTs? Henceforth, calculate mDP.
Can someone clarify these things?
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you can use the relative retention time
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I will be analyzing condensed tannin extracts for their mDP, PD/PC ratio, and cis/trans ratio using the method of Gea et al. (2011).
For the calculation of mDP, the extension units will be identified as BM adducts (see photo).
Do I need to synthesize standards of these BM adducts? They used NaBH4 in doing so and our lab doesn't have that chemical.
Is there any alternative for NaBH4 or is there another way rather than to synthesize them?
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Lithium aluminium hydride (LiAlH4) is an active alternative to sodium borohydride (NaBH4) in reducing carbonyl compounds. It is more active because the Al-H bond is more polar and places more electron density on the hydrogen than the B-H bond.
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I need the requirements and needs of performing HPLC for Leutalyze which is used for estrus synchronization in cattle and the HPLC which we use is LC-AD 20. So can can anyone suggest the buffers required to perform this activity?
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Dear Shashidhar,
I found this article (attached), which treat the separation and quantification of cloprostenol via HPLC, i hope their approach will help you.
Best wishes
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Hello,
I'm designing a generic HILIC gradient to separate a complex mixture containing an unknown metabolite of interest for structure elucidation. The properties of the metabolite are unknown beyond its molecular weight and when it elutes on a C18 reverse-phase column (it is a polar molecule). I'm having difficulty finding online what a good buffer and pH is for a generic HILIC gradient, does anyone have any suggestions? I'll be using ACN and H2O as my solvents. My column is a Poroshell 120.
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10 mM ammonium acetate pH'd with acetic acid is a pretty common HILIC buffer system for LC/MS. You've mentioned a loss in sensitivity (assuming that's what you meant by the MS being worse) - you might just try negative mode on the MS just to see if you get better signal (go up to pH 6.5 buffer with negative mode, which is about the highest your column will handle). The change in sensitivity may be an indication that you need a more acidic buffer like the 10 mM ammonium formate with formic acid mentioned earlier. Again, without pKa info, you will need to do this through trial and error. If you are preparing 10 mM ammonium acetate, you shouldn't need much acid to bring the pH down, though more will be needed in the acetonitrile phase. Also keep in mind that your column may become charged as you go from low pH 3 to higher pH's, which can help or hurt depending on the unknown metabolite's chemistry. Also, make sure you always have a little water in the mobile phase (not 100% ACN) and that you condition the column well (see datasheet) before injecting. Best to try a few pH conditions in positive and negative mode and see what works best.
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I Tried HPLC of the sample with the following set of parameters set:
Instrument: LC AD 20
Flow rate: 1ml/min
Temp: 30 degree
Sample Injection: 30 microliter
Protein analyte conc: 5mcg/ml, 10mcg/ml, 20mcg/ml, 30mcg/ml.
Time for running: 15min
Pressure: 4293 Psi
Solvent used: 30% Acetonytrile(ACN:D.H2O)
Prior to the use I washed the column with solvent for 1 hour and found that the peaks were not clear and same upon repetetion. Can anyone suggest how to perform HPLC for a short aminoacid sequence(11 aminoacids) structure.
Thankyou
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As seen in the picture, the peak appeared in several retention times which is very weird (it has no fixed retention time) !!
Also, it appearedis later at 7 min and disappeard from 5 min !!
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  • Rawaah Y. Al-sharrab wrote: "I just put an empty vial in a certain position and name it as blank and run it between sample runs."
An empty VIAL is NOT a proper HPLC BLANK !
Please obtain some training and assistance in using the HPLC system. Incorrect conclusions will be reached without years of formal training and experience. Data gathered will be invalid. Time, materials and money wasted.
Please inject a real blank (inject the same volume of mobile phase as you normally inject for the sample, but without the sample inside, just 100% mobile phase). At identical wavelengths, compare the "blank" run to a real sample analysis chromatogram (overlay the two chromatograms). If you still see the "peak", then you have a contaminated flow path (column fouling or carryover, as noted earlier).
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I have a question regarding the communication between a HPLC 2695 Alliance and the PC/Empower software. I know that it works with a BUS/LACe card.
Is it possible to use a GPIB-USB-B connector from National Instruments instead of a BUS/LACe card ?
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We bought a Waters Bus Lace Card PCI on ebay
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During Hplc analysis two peaks are inter mixed. Anyone tell me how I get separated, sharp peaks at suitable retention times respectively. The mobile is phosphate buffer pH-3 and acetonitrile (80:20). C18 column.
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Dear Muhammad, thank you for asking this interesting question. This is a rather common problem which has been frequently discussed on RG and other scientific platforms. Thus a look at the answers given to some closely related questions might be helpful in your analysis. For example, please see
How can I separate two merging peaks in HPLC?
(14 answers)
and
Overlapping peak problem, help!
(12 answers)
Good luck with your work!
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Most researchers studying the taste active components of seafood perform specific analyses for classes of molecules they think will be taste active since previous research found them to be, such as amino acids, nucleotides, organic bases/acids, sugars, and minerals. But what if other molecules are taste active and aren't being found because the chromatography conditions or pre-treatment don't allow them to reach the detector?
Example papers:
thanks :)
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Yes, perhaps because it may be attributed to the instability of the reactive substances under certain conditions, where the suitability of the method depends on the type of product, method of processing and storage, and the degree to which the particular method relates to sensory analysis.
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I analyzed the internal standard "2-methyl-L-cysteine hydrochloride" using HPLC and the chromatogram shows three peaks instead of one which is weird!!
I repeated the preparation process two times to check if there is a contamination problem but the chromatogram still showing three peaks. So what do you suggest?! Is there a possibility that the internal standard is converting to other compounds?!
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Dear Rawaah Y. Al-sharrab,
Thank you so much for your interest. Actually, it is very difficult to solve the problem of internal standard. That is why, most of the researcher are now developing their method using external standard and to use matrix matched standard for avoiding the use of internal standard. Anyway, if you interested to work with internal standard, try to use the solvent as same as mobile phase, it may helps to minimize the problem. I read the answer made by
William Letter, he described many things, you may follow his advice. Thank you.
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Acetaminophen has a λmax of 243 nm. Why is detection λ of HPLC set at 254 nm?
Environmental Earth Sciences (2020) 79:457
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Dear https://www.researchgate.net/profile/Aarif-Shah-2 , Just ensure that DAD detector is working on VW ( Variable Wavelength ) priciple but UV detector is working on Lambert Beers law principle entirely different . You are again requested to calculate manually % RSD between two wavelengths ( not more than 2 % ) .Please go through the deatail - https://www.ssi.shimadzu.com/products/liquid-chromatography/knowledge-base/hplc-basics/uv-vs-pda-detectors.html
Further manual calculation of % RSD are suggested since two instruments are on defferent platform .
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Hello everyone,
i have a question about the peak of my pollutant, it doesn't appear in the HPLC analysis, on the contrary the intermediate's peak appears with high intensity. knowing that, the pollutant is dissolved in a water of a conductivity equal to 7, without being exposed to light, because it's photosensitive. i wondering if the conductivity of water can influence on the detection! thank you in advance.
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Please check using other column with DAD detector, may be it detected.
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It is HPLC analysis. Can also be done with GC-MS.
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At Tanzania Food and Nutrition Center laboratory we can do that test, it is done by HPLC with the help of an Amino acid score kit.
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Isolation of new compounds from plants and natural resources is a considerable target to find something new and relevant.
But, what is the main intention of isolating compounds already known and isolated?
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Alexander Sinko Good question!
In natural products research, we usually perform Bioactivity-guided isolation of compounds from natural sources like plants. Here, at first, we screen for bioactivity in a plant and if found potential then look for the individual compounds responsible for the activity and here comes the necessity of isolation of compounds. Till this point we don't know which compound is showing the bioactivity, we might only have idea about possible compounds from the literature of the previous works. In order to identify & characterize a compound, we first need to isolate it without knowing if it's known or novel.
Furthermore, someone may find a compound inactive for a particular activity, while another may find it highly potent for other biological role.
A sufficient literature review before selecting a plant for research usually leads to some novel compounds or at least known compounds with novel activities.
Hope you got what you were looking for.
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Purely asking out of curiosity - we have another protocol that seems successful, so I'm just curious chemically speaking why this might've happened with our first protocol. We initially used a mobile phase of pH 3.0 (MDTM Type II Mobile Phase), and a 2-channel analytical cell set to -100mV and +250mV, where peaks are quantified from the +250mV electrode chromatograph. Despite being the same concentration, HVA peaks are always CONSIDERABLY shorter than all of the other monoamines and metabolites, and consequently detection cuts off at a concentration about 10-fold greater than the other neurotransmitters.
Given the pKa of homovanillic acid (4.4), compared to NE(9.5), DOPAC(3.6), DA(8.93), 5-HIAA(4.22), and 5-HT(9.31), they should all be fully protonated at a pH of 3.0 when suspended in our mobile phase, so I'm not sure why HVA shows such unique differences from the others? Our recording electrode is set to a positive cell potential, so it should be oxidizing (taking electrons from) the sample - could it be that HVA has less 'available' electrons? If so, why is that?
We have supplies coming to switch to a different mobile phase, install a conditioning cell and tackle this problem, but I'm curious to hear why mechanistically this may be happening? Also, again out of curiosity, does anyone know what about a conditioning cell (installed between the column and analytical cell) makes it capable of increasing sensitivity?
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Hi Michael, did this answer your question? Regards, Hendrik
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The sample which I am working on requires filtration using 0.45 µm nylon membrane filter for HPLC analysis. However, it is costly. Could someone provide an alternative for this? Or can I skip the membrane filtration (I will be quantifying L-DOPA)
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Hendrik-Jan Brouwer sir, Thank you for your response:
This is the protocol that I will be following:
The sample is a plant. I will be using methanol and HCl as the solvent followed by centrifugation. The final step demands nylon membrane filtration(0.45 microns)
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Dear Colleagues,
In brief, I have a method for protein resolution using a C18 column but it was developed in a 250 * 4.6 mm column, at the moment I have a C18 but short in length 150 * 4.6 mm. Would you recommend shortening the time of 40% (as it is 100 mm shorter) and keep the same gradients between solvent A (polar phase) and B (non-polar phase) or playing with the gradients too?
Thanks for your help.
Best,
Giovanni
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For an identical, but shorter in length column running isocratic analysis by HPLC, the flow rate and composition would be the same as before (THE column ID is most important). A shorter column will have less load capacity so at the same conc, the inj volume should be reduced.
For a gradient analysis conversion, you need to specify the exact gradient composition program (in detail) for anyone to suggest an answer. Since only the length of the column is changing, try to initially keep the steepness of the gradient the same, then re-optimize the composition as needed (always checking for proper retention and resolution).
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Hello,
I recently had an issue with my HPLC. I restarted using it after some time, and as first thing I let it run with isopropanol, with no errors. Then, I wanted to let it run with the two mobile phases I usually use (water/MeOH) in order to start back analyzing samples. As I switch method (and phases), all the parts (sampler, DAD, column ecc.) go to error, showing a shutdown info message in addition to a leak error message. I tried looking for leaks from the pump to the column to the outlet of DAD but I can't see any leaks, and everything looks tight enough. I treid to redo a wash only with isopropanol but now I keep getting again this error. What could be the problem?
Thanks
Maurizio
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Actually, it is very difficult to say that for which specific reason it was happened. You may check all the system again and try to start one more time. If the same phenomena is occurred, I think, The best option for solving this problem, to communicate the respective instrument suppliers.
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Dear all,
Hope you are doing well.
I'd kindly like to ask if anyone may have any available internships/apprenticeships with regards to the field of research. I am a volunteer with IAESTE Malta and am currently in my second year completing a Bachelors of Science (Honours) with Chemical Technology where I place as one of the highest students in my class. I am looking for a 1-3 month period that focus' on spectroscopic technics or anything mycology and/or chemistry related.
I am asking here since I would like to have an experience outside of my country that would be both educational and enjoyable.
Anyone that may know of any offer and/or is offering one please leave a comment or email me through my student email: shaun.attard.e21254@mcast.edu.mt.
Thank you for your time and considerations.
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Hi Shaun! I'm not sure where you are located, but I would recommend researching Research Experience for Undergraduates (REUs) in the U.S. Many schools here offer a summer internship program for students from another school. I am unsure of their stance on international students though.
If you are a U.S. citizen, then I highly recommend looking into internships with NASA if they align with your interests. You can learn more about those opportunities at intern.nasa.gov.
You might look up some companies that you are interested and reach out to them specifically. The easiest way to do that might be to find employees of the company on LinkedIn and reach out to them. They might be able to point you in the right direction on how to get an internship with their company.
Good luck and I hope this helps!
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Hi everyone,
can anyone tell me which is the best way to quantify Melatonin? I've read articles that refer hplc-dad and also hplc-fluorescence. I'm interested in analyze melatonin in raw milk, making the extraction following ISO14156.
Do you have any sugestions ?
Thank you for your help!
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Hendrik-Jan Brouwer thank you for your answer. It was very helpful.
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My study involves establishing chronic nicotine addiction in mice and I would to check the cotinine level in mice urine using HPLC but currently protocols I found are done using human urine. Bioassays and GC are expensive, and I would like to use urine as the sample. Thus, how do I modify the HPLC protocol for mice urine?
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I have no practical experience, however, I hope the provided link will be very much helpful for you. Please have a look on the following link: