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Questions related to HPLC Analysis
My question is in the context of testing and choosing the best weighted linear regression for a calibration model (calibration curves using an internal standard). How to explain the use of the weighting factor 1/y^2, for example, when working on bioanalytical/analytical method validation? What practical factors lead to a verification that the use of 1/y^2 or 1/y, rather than weights related to "x" (1/x or 1/x^2), better describes the data from calibration curves analyzed on an LC/MS-MS system? Signal saturation? Contamination? Accumulation of biological matrix debris? A slight trend in the data that favors 1/y^2?
In addition to the above, if incorporating the weighting factor 1/x^2 (where x reflects the nominal concentrations of the analyte) or 1/y^2 (where y reflects the response of the equipment) into the model results in similar performances in terms of the sum of the absolute percentage relative error (%RE) values, which one would be the most appropriate to be assumed in the calibration model? What conclusions can be drawn from the choice of one or the other with regard to the behavior of the data (and the nature of this behavior)?
I must say that I don't have a strong background in Statistics, so I apologize if I've expressed anything incorrectly.
Thank you
Hi everyone,
Reaching out today to see if you can help me with my research project.
Lately, we're focusing on enzymatic synthesis. But I don't have a biologist background, rather analytical chemist one, so I am struggling with one question that came out a few months ago. We would like to find a way to first, detect and then, quantify the enzymes in the final product, to know if there are traces of enzymes in it.
The idea was then to try and dissolve the powder-form enzymes used as "catalyst" and developed a HPLC-UV and/or HPLC-MS (ToF) from this solution, before analyzing the final product. However, it was not conclusive (no peak in the chromatogram; and when there's a peak, m/z ratio are hard to exploit = is it the enzyme, a degraded version of it, something else that keeps the enzyme in powder form...?).
I am pretty sure I am not having the correct way of thinking this problem through.
So, shoud you have any experience in this field, I could really use some research data (articles, technical notes...) or any tips on how to proceed for this specific subject.
Thanks in advance for your time and help!
Célia
When I have modified gradient program in HPLC analysis, same sample shows different area response at different RT.
Welcome
I need help with solving a certain problem. Maybe someone had the same problem as mine and have a tip.
I have to prepare a calibration curves and make pharmacokinetic analize piperidine derivatives, which were synthesied. Purity all this compounds at least is 98%. They are free basis, acids or their hydrochlorid salts. During the preparation of the calibration curves compounds as a free form basis/acids, I noticed the appearance of two peaks (new in the dead volume and second in appropriate elution time). Peaks area of new peak increase with injected volume.
All hydrochlorid salts eluted as a one peak in adequate elution time.
Concentration probes: less than 0,2 mg/ml.
Solvent used: for salts: water or water/ACN 1:1; for free: MeOH (they don’t disolve in water or water/MeOH mix and water/ACN mix)
Injection volume: 1-10 µl.
Column: Kinetex 2.6µm PS C18 100A, 100×2,1 mm
Solvent A: water with 0,2% TFA (initial TFA concentration was 0,1%)
Solvent B: ACN with 0,2% TFA (initial TFA concentration was 0,1%)
Flow rate: 0,5 ml/min
Also i tried on other column: Accucore RP-MS 100×2,1 mm (changing column helps a little, but many of free forms still have two peaks in chromatogram)
Solvent A: 5% water/ACN with 0,2% TFA
Solvent B: ACN with 0,2% TFA
Flow rate: 0,5 ml/min
Probabilities:
- add buffer
- solvent is so strong
- altering the colmun but I don’t know which one will be better.
For me, the most interesting thing is when the same compound as a hydrochlorid salt elute as one peak, and as a free basic/acid elute in two peaks. Why some molecules, of the same compound have retention and interact with stationary phase and other moleculse say: "I dont like you, i'm out". Column isn't overloaded. I thought that additions in firts column have impact on retention and are saturating, but when i changed a column on "typical" C18 I observed two peaks.
Please help.
I am trying to analyze short peptides containing hydrophobic aromatic amino acids. The samples are insoluble in water or any organic solvent but only soluble in a very acidic solution such as water with more than 30% acetic acid.
So, in this case, I made a 20% acetic acid in DI water, which has a very low pH (1.8). I used it to dissolve my sample, resulting in a 0.1 mg/ml concentration. The HPLC mobile phase, however, is just an isocratic flow of 20% acetonitrile in water. I use a C18-column that tolerates pH in the range of 2-8 and the injection volume is 10 uL.
- Does the C18 column get hydrolyzed when my sample solvent has a pH that is outside the lower limit of the column pH range?
- In the case of me using a completely different mobile phase and sample solvent, how does it affect the peak shape? I expect to first see an "acetic acid" peak at the front" and then a compound peak and so on.
- Also, what happens if my compound is soluble in the sample solvent but much less soluble or insoluble in the mobile phase?
My amino acid condensation reaction (the reagents do not have protection groups) utilizes K2CO3 as a base. K2CO3, the resulting peptide product and unreacted amino acids all have decent solubility in water but poor solubility in organic solvents including methanol and acetone. I need to analyze my reaction mixture using HPLC, which uses aqueous buffer mobile phase and C18 column. I saw that the column will be destroyed if K2CO3 is not removed as it can trigger blockage. Is there anyway I can remove K2CO3?
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)
how to differentiate between the retention time of all these degraded products using the HPLC analysis. the column being C18
Dear Researchers,
I am currently conducting research on the presence of microplastics in fish tissues and sediment samples. I would greatly appreciate it if anyone could share a detailed protocol for performing HPLC analysis for this purpose. Specifically, I am interested in the steps for sample preparation, extraction, and the optimal HPLC settings for identifying and quantifying polymer types or plastic additives.
Your insights and shared experiences would be incredibly valuable for advancing my work.
Thank you in advance for your help!
Hello,
Why do i see this baseline drift when i compare my blank (black) to the sample (blue)?
Any suggestions as to why this happened?
Thank you!

After condensation of amino acids (they do not contain any protection groups), I believe that I have produced leucine peptides such as di-leucine, tri-leucine, leucine diketopiperazine, tetra-leucine and also unreacted L-leucine. I am using a C18 column, 250x4.6 mm with 3um particle size. Is there an aqueous mobile phase that can ion-pairing to separate oligomers with different length?
Hello,
I have a methanol extract that I have let stand for 3 days, three times. I have evaporated the methanol and, just to be sure, mixed it with water and lyophilized it. Before performing HPLC, I want to remove nonpolar impurities, chlorophyll, and pigments from my extract using liquid-liquid extraction to prevent damage to the C18 column. My first question is: should I use ethyl acetate, chloroform, or hexane for this purpose?
For one of my samples, I washed it three times with ethyl acetate and even left it shaking overnight. For another sample, I sonicated it. The sonicated sample changed color, and the water volume of all my samples increased by about 10-15%. How can I remove the ethyl acetate, and would using the extract in this state without removal damage the C18 column during HPLC analysis?
Thank you.
Hello everyone.
I have a problem with HPLC. A few days ago, to create a calibration curve with HPLC, I injected solutions with known concentrations into the device, which gave good peaks in the expected time. But now I inject the solution with the unknown concentration into the device with the same HPLC conditions as before and the device does not show a peak.
I have checked all the connections for leaks and flushed the column with water and methanol, the system is free of air, the flow path is completely open and the waste is coming out, but I still did not see the peak in the expected time.
I noticed some problem with hplc. As you can see in the photographs, negative peaks and strange chromatograms appear. There is no change in pressure during the run. What do you think could happen? The system is Thermo Scientific Ultimate 3000




In literature, they separate the simpler form of the mentioned compounds, Glycerol and Glycerol Carbonate by GC. I tried GC but I could not separate them so I decided to try HPLC. However, I have not found excessive information regarding the separation I want to perform.
Currently working on a USP assay method for HPLC that requires water-saturated butyl chloride as a mobile phase and water-saturated chloroform as a diluent.
What is the whole point of using water-saturated solutions like this?
Would there be alternative ways to substitute these solutions?
Hello everybody. Our instrument (i.e., ion chromatography) must stop working for more than 4 months because of some maintenance. My question is, during 4 months or more, does it need anything done for preservation? However, we have already removed the columns and are storing them in the refrigerator.
Thanks a lot
Our JASCO-PU-2080 HPLC pump is giving an error message reading : "TROUBLE UNDER PRES !!" and there is no liquid coming out of the outflow. The pump was left running at 1mL/ minute and was working normally to run HPLC samples in previous weeks. The error was noticed after 4 days where the pump was left to run with mobile phase on recycling. To get rid of the error message, we have turned the pump off and on but when we set our flow rate the pressure remains at 0 and the mobile phase is not being pumped through.
If anyone has a manual with a trouble shooting guide for this specific pump (ideally with the location of the specific components of the pump) or could give advice about how to fix the error message it would be much appreciated. Many thanks.

I will use retinoic acid (Sigma/R2625) and as I know is sensitive to air and light. After dissolving the chemical, I want to store my aliquots in an inert environment, but I do not have suitable vials or screw caps for this purpose. I wonder if anyone does this in a different way.
Thank you very much.
Currently, I am running an HPLC analysis to quantify 4 active compounds in my natural product extract. However, due to a limited budget, I am only able to run 1 concentration of each of the 4 standard compounds. May I know, if I can still quantify the concentration of the 4 active compounds in my extract? As such, I am planning to ratio the peak surface area of the standard compounds with the peak surface area detected from my extract.
Dear researchers,
I would like to ask you whether the retention of analytes of interest on the C18 column can be slightly affected by buffer concentration.
I have analyzed a mixture containing sulpiride (weak base) and diclofenac (weak acid) at 250 ng/mL on the C18 column with mobile phase A: ammonium formate and mobile phase B: methanol.
I have observed that when the concentration level of ammonium formate increased from 2mM, 5mM, to 10mM, the retention time of sulpiride slightly decreased from 6.72 min, 6.52 min to 6.46 min respectively, whereas that of diclofenac slightly increased from 10.04 min, 10.17 min to 10.28 min respectively.
As far as I am concerned, for a basic compound such as sulpiride, an increase in buffer concentration (ammonium formate) can result in a decrease in silanol activity so a positively charged compound such as sulpiride can have a slight loss of retention due to the ion exchange interaction between the compound and silanol group during a loading step. Consequently, the retention time of sulpiride was slightly decreased with an increase in buffer concentration.
However, for an acidic compound such as diclofenac, it is quite difficult to explain this phenomenon. In my opinion, it seems that when the concentration level of ammonium formate increases, the pH of the mobile phase slightly increases so the charged state degree of diclofenac slightly increases either. As a result, the energy configuration of diclofenac diffusing inside the pore of the C18 column is lower at a higher concentration level of ammonium formate (10mM) so it will have more interaction surface area with the C18 column, leading to more retention. However, this explanation seems not to be convincible because the higher charged state degree of diclofenac is, the more soluble is and the less retention is.
However, for the retention behavior of sulpiride and diclofenac, the above explanations are just my own opinion. Of course, I am not sure whether they are right or wrong.
If someone here can help me clear the retention behavior of sulpiride and diclofenac, I am really happy to listen to your valuable suggestions.
Thank you so much in advance,
Hello,
When working with methanol, I noticed that I could never take the exact volume of methanol with micropipette.
Is there any other tool or method that could solve this problem?
Many thanks for your concern,
Hi,
Can someone please suggest me a good diluent for the MCC (microcrystalline cellulose)? Further, I am looking for your valuable input, either based on personal experience or backed by strong literature references, about the HPLC analysis of hypromellose (HPMC), microcrystalline cellulose (MCC) and croscarmellose sodium (CCS). I have access to different types of detectors for this purpose.
Thank you
Anybody working on the HPLC analysis of CBD and THC from Hemp oil?
Hello fellow researchers, i started to run several HPLC methods to quantify an enzyme activity. I incubate my Enzyme (different mutants) with my substrate (futalosine, quite hydrophobic). To seperate the molecules i run a RP-HPLC with a LC-18 column (analytical) and use a Water-Ethanol gradient to elute my molecules. Few months ago everything seemed fine, i could always identify the peak corresponding to my substrate and a product peak as well. Since we only have one HPLC a coworker was using the device as well but for his experiments he used acetonitrile instead of ethanol. After i started to use the device again i washed the column with 15 CL water and ethanol and started my experiments again. But since then my peaks have horrible shapes and sometimes there is literally no signal at the expected RT. Now there are also very high intesities at the end of the run (~2000 mAu) which was never the case. Therefore i used a standard to see if the effect is due to my sample preps. I attached 2 files of my standard (HPX) in which one it is visible that the same molecule and prep shows a different signal intensity. Setup and method for this runs are identical. HPX1 is my standard i used shortly after i started with the HPLC again and HPX2 is the same standard run few days ago, after i notice the very high intensities at the end of my runs. Does anyone had similiar problems in the past or could help me out with tipps, why my runs got wrose day by day?
I am looking forward to your response
Hello, inexperienced grad student here. I've been processing samples via HPLC/LCMS (tissue extractions from plants) over the past year or so. We have two mobile phases that we use in a shared HPLC machine: Phase B: Acetonitrile with 0.1%Formic Acid, and Phase A: Water with 0.1%Formic Acid. I recently put on a run and was rushing a bit, didn't fully check the labels of the solvents before leaving and when I came back I realized that the Water that was attached to mobile phase A tubes had 0.1% ammonium acetate added, not FA.
Can I search for the compounds by mass and adjust that way, or are there other considerations I haven't taken into account? We use a 10.5 min run time per sample, and all of the compounds elute within 6 min when formic acid is used.
Our samples are extracted and then suspended in methanol.
Do i have to revalidate the method using some blank matrix or this matrix effect is acceptable?
I performed matrix base calibration(linearity) for validation.
All the parameters are well within acceptable limits.
I am just not so sure about matrix effect
Please guide.
I am trying to detect citrate in LC-MS. I ran the samples in both negative and positive modes. I performed direct injection (without a column) using an ESI ionization source. Thank you very much
In order to analyze small-MW thiol metabolites in plasma, I typically use a method with a mobile phase made of acetate buffer 0.1M (pH 5.25; 96.5%) and methanol (3.5%) running at 0.6 ml/min on a C18 column. The method works very well when I use a single pump with a pre-mixed mobile phase.
However, if I put the acetate buffer in pump A and methanol in pump B and I try to use my quaternary pump to mix the mobile phase, the separation is really poor and the retention times change a lot from a run to another in an apparently random way.
I tried to use different mixers (from 100ul up to 675ul) without obtaining significant benefits.
The HPLC system specialist told me that the problem is the composition of the mobile phase, which is "not compatible" with a low-pressure mixing system. Is it possible? Does anyone have similar experiences? There is some other test that i can do to check my system?
Thank You all in advance
I want to check if the PET, PVC, and pe have degraded in my liquid samples by using HPLC.
I am currently developing my SOP for sample injection into our soon-to-arrive Biorad Aminex HPX-42A column. The plan is to use HPLC grade water at 85°C for eluent. Based on the "Use and Care" guidelines provided for the columns, the HPX-42A has an acceptable pH range of 6-8. However, we intend to inject samples with a pH as low as 2. I would like to carry out neutralisation on the samples, but want to ensure that I am not causing any issues with my neutralising agent selection.
Are their limitations for neutralising agent use in samples to be injected into Aminex columns?
I am aware that there's a potential problem with counter ion replacement in these ligand exchange columns - if the cation that could exchange with the silver is in salt form, does this still pose a risk?
With a refractive index detector, there's a potential for eluent density fluctuation with salts that remain in the solution. Should I therefore use a neutralising agent whose salts precipitate? (Barium Hydroxide?)
Any suggestions or comments would be appreciated.
I am aware of the phenomena of counter ion loss in ligand exchange columns within HPLC. Due to this phenomena, contamination by anions, cations and salts can be problematic for a column. We use guard columns to prevent this counter ion loss.
My question is this:
What is the mechanism by which cations in a sample exchange with the counter ions in a HPLC ligand exchange column? Does this exchange occur even with salt forms of the cations?
I am doing hplc analysis and recently purchased a new column because the old one has degraded. Can one simply install the new column and start straight away with running standards or is there a procedure to do first before I start using the new column.
I am using a C18 150mm x 4.6mm, 5um Fortis hplc column.
I am attempting to replicate a HPLC method for the determination of water-soluble vitamins and the mobile phase consists of 0.1M Phosphate buffer (pH 7).
The paper states that they made up the buffer by "the addition of 0.1 mol/L potassium dihydrogen phosphate and 0.1 mol/L potassium hydroxide"
However, I would usually make up this buffer by adding potassium phosphate dibasic and potassium phosphate monobasic (in the correct quantities) to water.
Are these two methods the same and would the resultant buffer be the same?
Thank you!
Can anybody HPLC protocol for lactic acid isolation from bacterial culture?
Hello,
I need the help of a chemistry researcher who will be able to identify chemical compounds through HPLC-UV-MS/MS mass spectra in the context of a collaboration (he will be mentioned as one of the authors of the work).
Thank you very much in advance
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?
Long story short--the Chromeleon software I am currently using is old (and on an old computer), which is making it buggy and difficult to actually analyze data and generate attractive chromatograph overlays.
I would like to use OriginLab; however, I don't really see any easy way to do this. I figured out how to bulk data input; however, it seems that I would have to write a bunch of very long IF() functions to incorporate information from fit equations of standard curves and identify/label peaks based on retention time windows.
Is there a more reasonable way to do this such as built in app or standardized workflow similar to that included in Chromeleon?
What are the Secondary Metabolites that present? Is it necessary to do the tests to prove them in the lab by color changing chemicals?
hello, I've been developing a method on a c8 fortis column. the 4 peaks had excellent peak shape (tail=1 sym=0.9) then when it had an injection from an empty vial multiple times it started to have an increasing tailing for all peaks but having the same retention, resolution, pressure and even the plate number. tried to reverse the column and wash vigorously but it made it worse . Right now the tailing had reached 1.85 ... what should i do ?
The column is nonpolar on the other hand the vitamin b12 is polar, should I always use this method or not
Hi
I am searching for suitable HPLC column for the analysis of beta sitosterol, gamma oryzanol, sesamin, sesamolin, tocopherol and tocotrienols from edible oils such as sesame oil and rice bran oil. I have found articles that used PFP columns and C18 columns. I already got one C18 column (Kinetex 2.6um EVO C18 100A (100 mm x 2.1 mm x 2.6 um) column) but I do not find any publications that used the same column, but there are articles that used C18 columns of other brands.
Few researchers have used Kinetex PFP column (250 mm× 4.6 mm, 5 μm) for the analysis of same compounds from edible oils. So I am wondering whether I will be able to use the C18 column that I already have or not? If not, do I need to use PFP column or can you suggest me any suitable columns for this purpose.
Thanks in advance.
Hello everyone, I have a crude extract and I did MIC for it, it gave a minimum inhibitory concentration of 1 mg/ml. Later, I did HPLC for that crude Extract and the concentration of it inside the vial was around 500mg/ml, and I got 9 peaks from it.
The LC condition that was used:
Mobile phase:
- 0.1% formic acid in 1000ml of water.
- 0.1% formic acid in 1000ml acetonitrile.
Column: C8
The flow rate: 0.7ml/min, with an injection volume of 100ul. I used the isocratic method of 95 (water)/5 (acetonitrile).
The peaks were collected in different tubes, and I want to evaporate the acetonitrile and formic acid and then do the MIC for each individual peak that I collected, and I am not sure how to evaporate acetonitrile and formic acid, and once I evaporate them how many ml or ul of water should I dissolve my peaks with?
Your suggestions are highly appreciated, Thank you.
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
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?
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
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?
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
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?
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?
Can anyone suggest the HPLC method to quantify spheroidenone carotenoid with a DAD detector and an Agilent zorbax SB-C18 column?
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
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?
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!
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!
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?
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
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 !
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.


We intend to separate the mixture of three glycosilated flavonoids having two sugars units, each, according to their LCMS profiles.
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
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?
In other words, without a standard sample, can HPLC analyze the quality of a solution?
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 :)
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?
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!
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


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.




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.