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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
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Maybe, the paper attached is helpful
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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
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You may follow the activity alternatively...what is the primary substrate for your enzyme?... Lots of ways to do but, but it would be hard to quantify enzymes using native MS or bottom-up proteomic if the trace amount will be monitored.
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When I have modified gradient program in HPLC analysis, same sample shows different area response at different RT.
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Hello, the by changing gradient flow you changing mobile phase composition which definitely will act on RT and shape of picks.
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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.
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The appearance of two peaks for piperidine derivatives in their free base/acid forms may be due to differential ionization or interaction with the stationary phase; consider adjusting the mobile phase pH, adding buffers, or using a different column chemistry. Implementing gradient elution and optimizing injection volumes may also help improve separation and resolve the peaks.
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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?
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Yes, the pH of the sample solvent does indeed matter in HPLC. It affects the retention time and peak shape
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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?
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9-12-24
Dear Zongze,
You have already received some excellent answers/suggestions from several responders.
If you are still having difficulty removing the K2CO3, you can try adding some CaCl2 to your reaction mixture. The calcium will react w/ CO3 to form CaCO3 which is extremely insoluble in water.
I hope this information helps you.
Bill Colonna Dept. Food Science & Human Nutrition, Iowa State University, Ames, Iowa USA wcolonna@iastate.edu
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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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Use a silicone filter
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how to differentiate between the retention time of all these degraded products using the HPLC analysis. the column being C18
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While I don't fully understand your question, possible answers could be:
- You differentiate the degradation products by their retention times.
- As these are essentially more hydrophobic compounds compared with glucose, they should show up at higher retention times when using a C18 column (where glucose is basically not retained at all).
- To separate different degradation products, a suitable separation method has to be developed.
- Solely the retention time doesn't tell you which compound it is.
I hope, this helps somehow.
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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!
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Solution of KOH or H2O2 can dissolve the tissue leaving out the microplastics. Solvents such as TFA, chloroform and phenol can help to dissolve common variants of microplastics. These extractives can be analyzed using C18 column. However, depending on the composition of microplastics using only one type of solvent will not work and furthermore strong organic solvents will damage HPLC column.
A better strategy would be deconstructing the plastics and dissolving the monomers in benign solvents. For example, PET (a common microplastic) can be hydrolyzed in HCL or NaOH media and its monomer (TPA) can be dissolved completely in DMSO. It can be filtered and injected into the c18 column for analysis. A recommended HPLC condition for this analysis is gradient flow of 0.1% or 1% formic acid and acetonitrile at 0.6 m/L and elevated column temp. A calibration plot using external std concentrations is needed for quantification. Details can be found in the following source: http://dx.doi.org/10.1021/acssuschemeng.3c08286
Lastly, other techniques such GPC-MS, Py-GC-MS, and FTIR can help augment results from HPLC analysis.
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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!
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Not temperature related. You provided no HPLC method or sample information at all. *Based on the chromatogram showing the injection spike (notice the HUGE spike at T0), you overloaded the column in one example resulting in a different baseline response (normal). Use the same injection solution for ALL samples. Use mobile phase as the injection solution. Most importantly of all, as you appear to be using HPLC for the first time, please contact someone local who is experienced in HPLC to assist you.
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please answer it quickly.
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To calculate the conversion and selectivity from the area of percentage values with respect to retention time (RT) in HPLC data, what is the formula?
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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?
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What have you tried already, and what were the results? You might try an AQ type column using TFA as a solvent modifier. I suspect the compounds are very polar and retain poorly on regular C18
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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.
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Generally speaking,to remove nonpolar impurities from a methanol extract, you would typically use a solvent that is immiscible with methanol and has a higher affinity for the nonpolar compounds. One common solvent for this purpose is a nonpolar organic solvent like diethyl ether or hexane...Remember to handle all solvents with care and follow proper safety precautions, including working in a well-ventilated area and avoiding contact with skin or inhalation of vapors. Additionally, ensure that the selected solvent is compatible with your compounds of interest and that it does not introduce any unwanted impurities.
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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.
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Hello spike your sample with know concentration of analyte and see the results with the use calibration curve that you made.
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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
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Another possibility is a failing deuterium lamp, but I would investigate the degasser and check for leaks first.
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thanks
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High-Performance Liquid Chromatography (HPLC) is a widely used technique for the analysis of various compounds, including those present in plant extracts. Here's a general guide on how to perform HPLC analysis of methanolic extracts of plant leaves:
1. Sample Preparation:
a. Plant Material:
- Collect fresh plant leaves and clean them to remove any dirt or contaminants.
- Dry the leaves and grind them into a fine powder.
b. Extraction:
- Use methanol or a mixture of methanol and water to extract the phytochemicals from the plant material.
- Allow the extraction to proceed for an adequate amount of time (hours to overnight).
- Filter the extract to remove solid particles.
c. Concentration:
- Concentrate the methanolic extract using a rotary evaporator or other suitable techniques.
2. HPLC Instrumentation:
a. Column Selection:
- Choose an appropriate HPLC column based on the type of compounds you are analyzing.
b. Mobile Phase:
- Optimize the mobile phase composition, which may include a mixture of water, acetonitrile, and/or methanol.
- Add modifiers or buffer solutions if necessary.
c. Injection:
- Inject a small volume (typically microliters) of the concentrated extract into the HPLC system using an autosampler.
d. Detection:
- Select a suitable detector based on the compounds of interest (e.g., UV-Vis, fluorescence).
- Set the detector wavelength according to the absorption or fluorescence properties of the compounds.
3. HPLC Method Development:
a. Gradient or Isocratic Elution:
- Develop a gradient or isocratic elution program to separate the target compounds.
b. Flow Rate:
- Optimize the flow rate for the separation of compounds while maintaining system suitability.
c. Retention Time:
- Identify and optimize the retention times of the compounds of interest.
4. Calibration and Quantification:
a. Standard Solutions:
- Prepare standard solutions of known concentrations for the compounds of interest.
b. Calibration Curve:
Inject the standard solutions into the HPLC system to generate a calibration curve.
Quantification:
- Use the calibration curve to quantify the amount of each compound in the plant extract.
5. Data Analysis and Interpretation:
a. Chromatogram Analysis:
- Analyze the chromatograms to identify and quantify peaks corresponding to individual compounds.
b. Peak Integration:
- Integrate the peaks to determine the area under each peak.
c. Reporting Results:
- Express the results in terms ofthe concentration of each compound in the plant extract.
6. Validation:
a. System Suitability:
- Regularly check the system suitability parameters to ensure the reliability of the HPLC method.
b. Validation Parameters:
- Validate the method according to regulatory guidelines, including accuracy, precision, specificity, and robustnesss
7. Documentation:
a. Record Keeping:
- Maintain detailed records of the HPLC method, sample preparation, and analysis conditions.
b. Reporting:
- Clearly report the methodology, results, and any deviations from the standard procedures.
Always consult relevant literature, pharmacopeias, or method validation guidelines for specific details related to your target compounds and regulatory requirements. Additionally, it's advisable to consult with experienced analytical chemists or specialists for assistance in method development and optimization.
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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.
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Glycerol does not adsorb UV, therefore you need a RI (refractive index) detector, which requires an isocratic elution (it is not possible to use two pomps or a system with valves and one pump >>> no stable baseline), thus use a premixed solvent as eluent.
Retention of glycerol will be low on a C18 column, I expect that diglycerol dicarbonate shall have a much higher retention. Eluent composition water/acetonitril range 100 to 95/5. Start with the latter one and decrease the ACN concentration if there is insufficient retention of glycerol.
Alternative is to use a Pb-carbohydrate column at 80°C. The eluent is than 100% water in combination with RI-detection. You can add UV-detection for selective detection of the diglycerol dicarbonate,
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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?
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Using water-saturated butyl chloride as a mobile phase and water-saturated chloroform as a diluent in HPLC maybe to enhance sensitivity, aid in phase separation, or reduce ionization. The specific reason for using such aqueous form of solution could differ based the type of assay and the APIs or analyte being analyzed. Alternatives can be considered, but method validation (still dependent on the analyte involved) is essential to ensure the reliability of results.
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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
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Hello!
You can read the washing method and the conditions of storage for its manual, which you can download from the homepage of the manufacturer. For columns and suppressors, the conditions vary by type. The column and the suppressor (electrolytically regenerated) must be removed and stored in the specified solvent. If you follow the instructions you should have no problem.
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due to hplc analysis
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You have observed one of the most fundamental aspects of HPLC operation. It relates to both injector operation AND Column Void Volume.
Two common reasons usually account for the early change in amplitude observed near the start of an analysis (positive and negative, "blip").
(1) In all cases: At or around the Tzero time a pressure pulse should be observed in the HPLC chromatogram. This "peak" occurs from the sudden change in pressure when the injector valve switches from the "inline" to "inject" position (pressure drops then rises). This is normal and very useful as we sometime use the presence of the alternating "peak" to help estimate the Tzero time and/or confirm the injector switched.
(2) A related peak may be seen at or near the Tzero time due to the different chemical and/or physical properties of the injection solution vs the mobile phase (a positive, negative or if overlaid with the pressure peak, positive/negative "blip"). Ideally, your injection solution should always BE the mobile phase. With no change in the solution composition, no change would be expected or observed by most detectors (e.g. UV/VIS) from the introduction of this new solution to the flow path. Only the pressure "blip" would be observed. However, even tiny changes in temperature, small differences in solvent composition, refractive index changes etc may be detected by the system and a "blip" or "peak" seen at this same moment.
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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.
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Encountered the same problem on PU-980 which was left unattended for 2 years. The possible problem that something (salt sediments from buffer, for example) blocked pressure sensor.
Min pressure was set on 5 and actual was between -1 and 0. We disconnected the column prior the pump activation. The pump was working and liquid phase was pumping in the regular manner (1mL/min as it was set). After 2 minutes pump was stopped and the same "Under Pressure" message appears. We were pressing red Cancel button and restarting pump for around 1 hour while pumping DI water. After 1 hour pressure increase to 5-6 and problem was gone.
Then we started restoring the column but this is a separate story.
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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.
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Hi Gülşah,
I've not worked with retinoic acid, but we've got a similar problem with vitamin D which is also sensitive to light and air.
You can keep your aliquots safer by aliquoting in standard Eppendorfs, and then wrapping the tubes in tinfoil to block the light.
Air should be displaced from the tubes before storage. You can buy argon gas for this from some scientific suppliers, however it's very expensive. Argon gas is also sold as "wine preservation gas" and this is a lot cheaper.
All the best!
Sam
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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.
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A way to do this is standard addition. IF you know the standard concentration accurately, and it is relatively close to that in present endogenously in the samples, THEN you can run the samples with and without a spiked sample and infer the endogenous concentration from peak area differences. Note, it is better to do multiple additions and multiple runs, but not essential. You will, however, not have any idea of your LOD's and LOQ's.
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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,
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Back to your original question (and I am done with this thread)... You observed a small (tiny) difference in Rt from small changes you made to the mobile phase composition. No worries, this is normal because no two columns are the same and each may interact to a different degree (or none at all) when you make small changes to the mobile phase composition. Try 20 different C18 columns and you may find some show no change at all, some do. Pick and choose the one that you want for the application (this is what we do in analytical laboratories). The reason for the observed change (change, not "drift") is due to multiple interactions of the solute on the surface of the support. Hard to say if it is electronic or ionization in nature, but it is so small, practically, it does not matter. All chromatographers are familiar with this (no need to 'explain it'). Run enough samples and you will see it. In fact, the one thing that they might question is why you would use a mobile phase of just ammonium formate without an acid (e.g.formic). Why not use a buffer? For LC-MS applications, most would benefit from such a solution to promote ionization and maybe change the separation factor too (depends on samples).
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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,
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  • Michal, I am pleased that the eVOL works so well for you. It is a device I developed with my team at SGE. It is disappointing that it has apparently been discontinued but the same team that developed the eVOL have since developed at ePrep a far more capable automatic syringe which I mentioned above. https://www.digivol.com.au
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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
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You are welcome!
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Anybody working on the HPLC analysis of CBD and THC from Hemp oil?
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what exactly is the question? for general HPLC CBD analysis, you can refer to the following articles:
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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
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It appears that the analyses are incompatible with one another. Thus, it is more likely that the same column is being used for both analyses. Get a 'new' column and dedicate it to your analysis alone. Remember, other 'crap' is being injected on the column than just your molecule of interest.
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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.
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Hi Zoe,
So, your water (mobile phase A) contains 0.1 % acetonitrile and no formic acid? And does your acetonitrile (mobile phase B) contain 0.1 % formic acid, or did you also add acetonitrile to that?
I don't expect the 0.1 % acetonitrile will affect your chromatography, I'm more worried about the absence of formic acid, as this is both an ion pairing agent impacting chromatographic resolution as well as an acid that aids ionization of your analytes that is required for proper mass spec analysis.
So, if something is impacted by this little mix up, I think it will be your mass spec data, not your chromatography.
Hope this helps!
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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.
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Hello,
To validate a method, you have to demonstrate that your blank is really blank (no compound detected).
You have to find a real blank matrix that is not contaminated or in case this is not possible use other methods of quantification as standard addition.
Regards,
Andreu
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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
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I work with standard, the instrument is xevo g2-xs QTof.
I will ask my technician.
Thanks again.
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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
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As noted earlier, (1) Verify proper pump operation (check/clean and/or replace: seals, pistons, inlet/outlet valves, pickup frits, lines etc); (2) Verify proper multi-channel valve operation to insure no cross-flow leaks; (3) PRIME the system properly with FRESH solutions and operate at appropriate pressures. These basic steps will identify where the problem is.
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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.
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Thank you very much Bruce Neagle
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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?
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In HPLC ligand exchange columns, the counter ion loss phenomenon occurs because the ligand on the column has a higher affinity for the analyte ions than for the counter ions in the mobile phase. As a result, the analyte ions can displace the counter ions on the column and bind to the ligand.
The mechanism of cation exchange in a ligand exchange column involves the competition between the counter ions in the mobile phase and the analyte cations for binding to the ligand on the column. When the analyte cations encounter the ligand on the column, they can replace the counter ions bound to the ligand through a process known as ion exchange. This process occurs because the analyte cations have a stronger affinity for the ligand than the counter ions.
It is important to note that cation exchange in ligand exchange columns can occur even with salt forms of the cations. This is because the salt form of a cation contains the cation itself and its corresponding counter ion. When the salt form of a cation is injected into the HPLC system, the cation can bind to the ligand on the column and displace the counter ions, even if the counter ion is the same as the one already on the column.
To prevent counter ion loss and subsequent column degradation, guard columns can be used to remove contaminants and protect the analytical column. Guard columns are typically packed with the same stationary phase material as the analytical column and are placed before the analytical column in the HPLC system. Guard columns can be replaced or cleaned more frequently than analytical columns, which can help extend the life of the analytical column.
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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.
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Dear Damola,
Any column manufacturer provides column certificate with column efficiency report. check storage solvent given on the certificate and first give the flushing with the same solvent alongwith water in the ration 90:10. Second step is to check column performance. Refer CoA and perform the test. Compare the result of performance test with certificate results for Theoretical Plate, Asymmetry and resolution. If all parameters are matching closely with column manufacturer certificate results then you can take column for use.
before initializing analysis give the flushing with mobile phase for good baseline then you can initiate analysis.
If query feel free to whatsapp to me +91 9833618555 Vijay Bagul
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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!
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Can anybody HPLC protocol for lactic acid isolation from bacterial culture?
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Hello, before injection of a small volume into HPLC just add 4N sodium acetate 1/10 volume and freeze the sample to precipitate the proteins, centrifuge the sample at 10000g, and then inject supernatant.
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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
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You can contact me
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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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Sodium azide is active even in very low concentration, so you don't need to handle a large amount in total. But if you company restrict you to use it, try benzalconium chloride, thus assuming that the compound doesn't interfere with the other chemicals in the mobile phase.
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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?
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Update chromelon software or get originlab are your two best choices it seems to me David Booth
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What are the Secondary Metabolites that present? Is it necessary to do the tests to prove them in the lab by color changing chemicals?
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Olive bark is a term used to describe the outer layer of the bark from an olive tree. It is composed of layers of thick, dark-colored cork cells, which protect the inner bark from weather and pests. Olive bark is harvested for use in various products, including homeopathic medicines, cosmetics, and dyes.
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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 ?
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Phenomenex suggest washing a reverse phase column at 1/5 to 1/2 typical flow rate as follows: (V = pi x r^2 x L, length and radius are in cm and volume is in mL).
10 column columns of 95% water and 5% acetonitrile - to remove buffer
10 column columns of THF
10 column columns of 5% water and 95% acetonitrile
10 column columns of mobile phase
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The column is nonpolar on the other hand the vitamin b12 is polar, should I always use this method or not
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Vitamin B12 analysis in tablets using HPLC is certainly possible because concentrations are relatively high and the matrix is not compicated. However, determination in food products can be very difficult, because at very low concentrations it is "sticky". Difficult to liberate from the matrix and it also tends to stick to components of your HPLC system (e.g. tubing and column)
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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.
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We routinely use YMC c30 carotenoids column (https://www.ymc.co.jp/en/columns/ymc_carotenoid/)
for the simultaneous analysis of tocols (tocopherols and tocotrienols), carotenoids, phytosterols, and many other lipophilic compounds.
It can resolve all 4 tocopherols (α-, β-, γ- and δ) and 4 tocotrienols (α-, β-, γ- and δ).
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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:
  1. 0.1% formic acid in 1000ml of water.
  2. 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.
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You could use a freeze dryer or a rotary evaporator, depending on what equipment you have in your laboratory.
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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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Victor Alexandrovich Nikolaev thank you very much. This information was very helpful.
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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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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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I think recrystallization of NaCl from aqueous solution may be sufficient. because the purity of the chemicals is usually determined using the physical properties of those chemicals
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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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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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