High-Performance Liquid Chromatography - Science method

Thank you, everyone, for your comments. We have successfully resolved the issue by setting up the polarization curve(polarity matches of sample with elluent) and getting good results. Thank you every one

How fast do you need the method to be? Homogeneous immunoassays such as HTRF or AlphaLISA normally have 1 single incubation (typically 1 h) and no washing steps.

عزيزي اذا تريدتحلبل المواد الكيميائيه من فئة AR الافضل لك استهزام مواز عديمة الشوائب باستخدام HPLCستظهر مواد كبميائيه نباتيةوتتَ باستخزاَ مذيب جيد

the same procedure you can opt for your MTT assay

Plenty of experience using hexafluoroisopropanol (HFIP) and other fluoro additives in samples and mobile phase. When used in sample solution, usually not a problem (you are only injecting a tiny % in a few uL of solution), but when mixed in mobile phase and (see below for more info) you will want to avoid it IN ALL HPLC methods, esp those that have an inline vacuum degasser installed as it may damage and contaminate the entire flow path of the instrument.

As a general rule, we try to never use fluorinated solvents or additives in our systems as they tend to bind to everything (contamination of flow path, column, detector, tubing, modules etc) causing months of downtime and $$$$ cleaning. *Most vacuum degasser modules that use "Teflon AF" membranes specifically state to not use them with ANY fluorinated compounds (that includes trichloro-trifluoro-ethane too).

We recommend using a weaker, non-fluorinated ion-pairing agent (if needed as it is best to try and develop a method that does not need one). *Most of our clients dedicate an entire LC-MS system for use with samples that are likely to contaminate the LC-MS system esp for metabolomics (this preserves the other instruments for use with all of the other samples). Columns too should always be labeled after use with any ion-pairing agents and never used on other methods (dedicated to specific samples only as the agent changes the surface chemistry of the support so the column can not be used for other samples).

The HPLC mobile phase will still need to be degassed for use and we recommend two different methods to accomplish this: (1) Continuously sparged ultra high purity helium through the liquids, at very low pressure (usually 2 psi) to degas them OR (2) use a far more rugged inline vacuum degasser which uses Teflon (not Teflon AF) membranes for degassing. IOW: By-pass or remove the degasser you have with the Teflon AF membranes and replace it with a standalone degasser such as the Agilent G1322A degasser (This degasser can be used with most organic, aqueous and ion-pairing reagents/additives without being damaged, BUT you will still have contamination of your flow path if use any type of ion-pairing agent, including Fl versions. but the degasser will not be damaged).

Derivatization of glucose to introduce chromophores could improve PDA detection. 1-Phenyl-3-Methyl-5-Pyrazolone (PMP) is a common reagent for derivatizing glucose. It reacts with the reducing ends of glucose, forming stable products that absorb strongly in the UV range (typically around 245-250 nm).

The glucose is reacted with PMP in alkaline conditions (e.g., with sodium hydroxide), and the resulting derivatives are stable for HPLC analysis.

You can use a C18 reverse-phase column after derivatization. Since PMP makes the glucose derivatives more hydrophobic, a C18 column would provide good retention and separation.

A typical mobile phase might be acetonitrile with water or a buffer system (e.g., phosphate buffer) for gradient or isocratic elution.

To isolate plasma for HPLC analysis, centrifuge blood samples to separate plasma and then deproteinize using an organic solvent like acetonitrile or TCA. Finally, filter the supernatant and dilute if necessary before injecting it into the HPLC system for drug detection.

Yagadasu,

Yes and no! Some HPLC methods in the USP have a requirement for the theoretical plate count. However, you may want to set a 'resolution factor' (R?) between 2 peaks (API and impurity) to demonstrate that your HPLC is capable of the resolution, rather than just SST (API peak only).

Extraction

1. Homogenize 100-200 mg frozen zebrafish in 1 mL ice-cold methanol.

2. Add 2 mL ethyl acetate, vortex, and centrifuge (10,000 g, 10 min, 4°C).

3. Collect supernatant, evaporate solvent under nitrogen/vacuum.

4. Reconstitute in 100-200 μL HPLC-compatible solvent (50% methanol).

HPLC ConditionsHPLC Conditions

1. C18/C8 column

2. Gradient: water-acetonitrile (0.1% formic acid)

3. Flow rate: 0.5-1 mL/min

4. Detection: UV (245 nm) or mass spectrometry

References

Alderman et al. (2012) and Takahashi et al. (2014)

Notes

- Validate protocol for your specific conditions

- Consult local regulations for animal handling

- Ensure laboratory safety with organic solvents

Optimize and adjust as needed.

You can use Amide C18 based columns.

C18 column can provide fast elution of HMF, and Amide column may retain the analyte for longer time as compared to other C18.

Create a method with a flow of zero (0.0), detector lamps OFF and add it to the sequence of methods.

Thanks Abdelhak for the suggestion. Yes, we did prepare fresh PMP for every experiment. I am wondering how to optimally store the PMP since it is not sustainable to keep purchasing new PMP.

You can use spectrophotometry to detect vitamins A, B, C, D, and E, utilizing specific reagents that form colored complexes for measurement. Other methods include high-performance liquid chromatography (HPLC), fluorescence spectroscopy, and electrochemical techniques, which offer varying sensitivity and specificity. Spectrophotometry is a cost-effective and rapid option for vitamin analysis.

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.

Rachel Evans: As Gang Chen has suggested, the methods/information for machine one do not appear to make any sense. We do not even know what the instrument make/model/configuration/software are for machine two, but it sure sounds like the system is doing exactly what it has been programmed to do (re-check ALL of the method and sequence settings that you saved. You will find that one of them has instructed the system to do exactly what you are witnessing. Should be an easy fix). It is irrational to provide advice over the web for most HPLC method questions, but especially when the proposed method in the first example does not appear to follow good chromatography practices. Wanting to "stick to the method as close as possible", when it is likely to be declared invalid (and any data generated from it, invalid) also makes no sense. Don't stick with something just because someone else used it. It may not be correct. You would not believe how many HPLC and LC-MS methods we review for the journals and companies that are completely invalid and unscientific, yet they get published.

Is there someone at your school (or whom they can hire) with the needed years of practical, industrial HPLC experience who can come on-site to review your system and issues? It takes many years of professional training just to learn the basic of HPLC. Many more years of training for method development and troubleshooting. You can NOT learn these analytical techniques from taking classes or watching videos. There is no short-cut. You need someone with training, in-person, on-site to assist you in using the HPLC system AND insure the data you collect will be acceptable.

Thank you very much :)

We really do not have enough information to provide specific suggestions for analysis. Does your sample have mild to strong chromophores ? If so, use a scanning diode-array detector inline for detection. Change the column to one that has a particle size of 5 microns for better resolution. Your isocratic mobile phase is VERY weak. To improve resolution of actual sample peaks, proper HPLC method development must be undertaken. Generic advice may include: Use a gradient HPLC method starting at 5% organic increasing to 95% organic (i.e. ACN) over time. Hold at 95% organic to insure all samples have eluted off the column. Review the results. Is retention found for the compound(s)? Does the K prime of the sample show adequate retention? Is the peak shape Gaussian? HOw many peaks were detected? What do their UV/VIS spectra look like? Etc. Do this following good chromatography fundamentals Please contact a local, experienced chromatographer to assist you.

Hello Winnie Ampomaa Owusu, what is your question? Maybe i could help you.

Dirk

To analyze Amoxylin and Curcumin simultaneously using HPLC, select a suitable reversed-phase C18 column and prepare a mobile phase, typically a mixture of acetonitrile and water with an acid like formic acid. Use isocratic or gradient elution based on retention times, and monitor absorbance at around 425 nm for Curcumin, while determining the optimal wavelength for Amoxylin. Ensure proper sample preparation and validate the method with calibration curves for accurate quantification.

So it seems you have two issues, both detection and separation? Maybe the free online booklet "A Practical Guide to HILIC" (available on ResearchGate, although it quite some years since we wrote it) could be a source of useful tips for you? For the separation I recommend you stick with acetonitrile since methanol tend to give too much hydrogen bonding thus competing with the HILIC retention mechanisms and weakening the immobilized water layer that contributes to retention. You might need 70-90% acetonitrile for sufficient retention (more ACN = higher retention). Salt levels often need to be higher in HILIC compared to reversed phase due to the electron rich nature of separated compounds and the stationary phases. For detection; can you find glycerol in UV or RI without the column? In UV you might need lower wavelengths, but in RI there is often not much to do other than having a sensitive detector and increasing the concentration of the analyte. Also keep the temperature stable to avoid drift in RI.

Try using ACN, MeOH, Water with pH modifires like TFA, Formic acid, OPA, Ammonia, Acetic acid (all HPLC/LCMS Grade). pH modifires conc. starting from 0.05 % either in single or both mobile phases. Also, make sure which column you are using, Normal or Reverse C18.

A diode-array-detector ("DAD"), photodiode-array-detector ("PDA") and multi-wavelength-detector ("MWD") are usually the same type of variable wavelength UV/VIS detector, with the names "DAD" and "PDA" names used interchangeably by different vendors. They are ALL UV/VIS detectors where the user can acquire multiple UV/VIS wavelengths at the same time (usually 5 to 8 different or "Multiple" signals) which is mandatory when performing basic HPLC method development on compounds with chromophores. The "DAD" and "PDA" have one additional feature that the "MWD" does not. In addition to being able to acquire multiple wavelengths, the "DAD" and "PDA" also can acquire full scanning data across the entire range of wavelengths (e.g. 190nm to 800nm). This powerful feature allows you to 'see' / detect sample peak absorbance across ALL wavelengths during each analysis run. For method development and/or any type of QC analysis, this comprehensive 3D data provides the user with additional information regarding the possible presence of impurities (a null test), qualitative ID and/or the presence or absence of specific compounds. A requirement for any type of professional analysis by HPLC or LC-MS (inline DAD plus MS offers one of the best detector configurations for HPLC analysis as neither detector is "universal" and neither detector can detector all compounds present. Having both in-line detectors, used with appropriate methods and training, provides additional orthogonal information).

Separating glycerol and serinol on an HILIC (Hydrophilic Interaction Liquid Chromatography) column can be challenging, especially if they have similar hydrophilic properties and retention times under your current conditions. Here are some strategies and considerations to help improve the separation:

1. Mobile Phase Composition: In HILIC mode, the ratio of acetonitrile to water is crucial for separation. Since both glycerol and serinol are polar compounds, slight adjustments can significantly affect their retention times.

· Increase Water Content: Try increasing the water content of your mobile phase. For example, adjust to 90% acetonitrile and 10% water, or even 95% acetonitrile and 5% water. This change can alter the retention times by modifying the interaction between the analytes and the stationary phase.

· Buffer Addition: Adding a small amount of a volatile buffer (e.g., ammonium acetate or ammonium formate) to the water component (typically at low concentrations, around 5 mM) can enhance separation by promoting ion pairing interactions or modifying the pH slightly.

1. Gradient Elution: Implementing a gradient elution instead of isocratic mode can sometimes improve separation. Start with a lower water content in the mobile phase and gradually increase it over time. This approach can help in separating closely eluting peaks by varying the polarity during the run.

1. Temperature Control: HILIC separations can be sensitive to temperature. Try adjusting the column temperature slightly (within the recommended range for your column) to see if it affects the separation of glycerol and serinol.

1. Detector Sensitivity: Ensure that both UV and RID (Refractive Index Detector) are optimized for detecting glycerol and serinol. Adjust detector sensitivity and optimize the detection wavelengths to enhance the detection of your compounds.

1. Different Column Type: If possible, consider trying a different HILIC column with a different stationary phase or different particle size. Sometimes, changing the column chemistry slightly can provide better separation for specific analytes.

1. Derivatization: Consider derivatization of glycerol and serinol to improve their chromatographic properties. Derivatization can change the polarity and improve retention time differences, making separation easier.

1. Systematic Approach: Systematically vary one parameter at a time while keeping others constant to identify the optimal conditions for separation. This approach helps in understanding the interaction between the analytes, mobile phase, and stationary phase.

The Purospher STAR RP-18 LiChroCART HPLC column is generally suitable for a wide range of applications, including the separation of amino acids. However, the suitability of this column for separating soluble amino acids in water depends on several factors such as the specific amino acids being analyzed, the mobile phase composition, and the detection method used.

Considerations for Using RP-18 Columns for Amino Acids

1. Hydrophobic Nature: RP-18 columns are based on reverse-phase chromatography, which typically separates compounds based on hydrophobic interactions. Amino acids are relatively polar molecules, and their retention and separation might require specific adjustments to the mobile phase to achieve optimal resolution.

2. Mobile Phase Composition: The mobile phase needs to be carefully chosen to enhance the separation of amino acids. Commonly, a gradient elution with water (often containing a buffer like phosphate) and an organic solvent (such as acetonitrile or methanol) is used. The pH of the mobile phase can also significantly affect the separation, as amino acids have different charge states at different pH levels.

3. Derivatization: Amino acids often require derivatization to improve their detectability and retention on RP-18 columns. Pre-column or post-column derivatization with reagents such as o-phthalaldehyde (OPA) or 2,4-dinitrofluorobenzene (DNFB) can enhance the separation and detection of amino acids.

4. Column Compatibility: While the Purospher STAR RP-18 LiChroCART column can be used for amino acids, it is essential to verify compatibility with your specific analytical conditions. Ensuring that the column specifications (e.g., particle size, pore size) match your analytical requirements is crucial.

Practical Steps

1. Method Development: Start with a commonly used method for amino acid separation on an RP-18 column and optimize based on your specific needs. Adjust parameters such as the gradient, flow rate, and temperature.

2. Buffer Selection: Use appropriate buffers to maintain the pH and ionic strength necessary for the separation of amino acids. Phosphate buffers are often used due to their buffering capacity and compatibility with reverse-phase chromatography.

3. Detection Method: Choose a suitable detection method, such as UV detection at 254 nm or fluorescence detection if derivatization is used.

The Purospher STAR RP-18 LiChroCART HPLC column can be suitable for separating soluble amino acids in water, provided that the mobile phase, buffer conditions, and potential need for derivatization are carefully optimized.

Hi.

Here is a step-by-step instructions how to make the mobile phase:

Step-1: Make 0.2% potassium dihydrogen phosphate solution.

Transfer 2.0 grams of potassium dihydrogen phosphate into a 1L volumetric cylinder or volumetric flask. Add approximately 500 mL of water. Add a stir bar and dissolve the salt (shaking can be used instead). Remove the stir bar, if used, and fill to the 1L mark with water. [optional but desired, filter through 0.45 µm or finer filter].

Step-2: 20% tetrabutylammonium hydroxide solution

Option-a) Purchase 40% tetrabutylammonium hydroxide solution in water, such as MillioporeSigma's product ID 178780. Into a vial or a beaker, mix 10.0 mL of the 40% solution with 10.0 mL of water.

Option-b) Purchase tetrabutylammonium hydroxide 30-hydrate solid (Mw~800), such as MilliporeSigma's product ID 86859. Transfer 2.96 grams of the hydrate solid into a 10 mL volumetric flask. Add a few mL of water and shake or sonicate to dissolve. Allow to cool to room temperature, if sonicated, then fill to the mark with water. [optional but desired, filter through 0.45 µm or finer filter].

The 2.96 grams accounts for the 30 water molecules present in the hydrate (if anhydrous salt is used - not sure if sold - you would need 2.0 grams instead).

Step-3: Mobile Phase-A preparation

Mix 500 mL of the 0.2% potassium dihydrogen phosphate solution from Step-1 with 2.5 mL of the 20% tetrabutylammonium hydroxide solution from Step-2. This will give you a final ratio of 200:1 v/v.

Notes:

1% solution means 1.0 gram into 100 mL. You can downscale from here to make any percent you want.

200:1 v/v means 200 parts from component-1 mixed with 1 part of compotent-2 (for example, 200 mL + 1 mL). The v/v part reads as "volume/volume".

Dirk Siekmeier Have you tried using deionized water? I always thought HPLC grade water is cleaner than distilled water.. Maybe I should try using distilled water!

try this Improved HPLC Method for the Determination of Curcumin, Demethoxycurcumin, and Bisdemethoxycurcumin (acs.org)

It would help to know exactly what the sample name is. HPLC analysis with isocratic refractive index detection (RID) offers the simplest approach / solution to your question. Alternatively, MS detection mode are also commonly used for samples with no or very weak UV chromophores. MS may provide the most data once a suitable method has been developed. Optionally, ELSD or CAD detection, for stable, non-volatile samples may be possible. The sample's own properties, solubility and stability will suggest which methods and modes are suitable.

Highly polar compounds can be retained using RP mode. Several specialty columns are available to aid in retention of these types of compounds without the use of any ion-pairing agents (avoid the use of these). One example of a more retentive column is the "Waters Atlantis dC 18 column" (not a recommendation, just an example). A keyword search (using your sample name and "HPLC") on the web will provide you with many example articles, papers, application notes to review. Additionally, you should contact a local experienced chromatographer (or lab) to assist you develop an HPLC method that follow good fundamentals.

Make sure you dissolve all samples (standards) in the mobile phase (the same liquid), at the same concentrations and then run them individually to confirm that you can resolve them apart on the column with no co-elution. Poor equilibration, column fouling and inpurities may also result in similar observations as shown in your chromatogram too.

The issue shown has nothing to do with the column so "washing" will not solve it.

In general, the chromatograms provided show several different problems occurring. Issues such as: Pump cavitation, loss of prime (air), sticking valve(s), lack of degassing, inappropriate compression settings (?), lack of equilibration and so on. Before running any samples, you must first establish proper flow of the HPLC pump (under pressure) resulting in a smooth stable baseline. To do so requires that the method used (programmed) follow good chromatography fundamentals, the HPLC system is well maintained and operating properly and finally, run by an experienced user.

Here is an article from one of my many training classes that address some of the problems observed. Review it with an experienced chromatographer, on-site, as these issues should be very simple to resolve.

BTW: You did not include any HPLC method information at all so we have no context to associate with the chromatograpms shown (Please always include FULL method details with questions, including FLOW rate, mobile phase composition detector settings (wavelength & bandwith etc). We need this basic info to understand what we are seeing.

Dear Abdelhak Maghchiche

you suggest "using the Beer-Lambert law, which relates absorbance to concentration." You need to know the extinction coefficient, which can be determined using the standard solution.

The same problem for HPLC.

Your post does not answer the title question.

Thank you very much. Dr Balamurugan Krishnamoorthy , Zoë Gaffen and Abdelhak Maghchiche .

Noticeable , When using HPLC data, ensure that the peaks you're integrating correspond to the compound of interest and that your calibration curve is accurate for quantification. By comparing the area under the curve (AUC) of the compound in the sample to the AUC of known standards, you can determine the concentration of the compound and use it to calculate the yield.

The solution used to dissolve the fucoxanthin standard to make fucoxanthin standard solution is not necessarily the same as the mobile phase, the solution used to dissolve the fucoxanthin standard can be a different solvent system, such as methanol, which is used to prepare standard solutions for analysis

Thank you very much for your help.

Sachin Aj After the preliminary phytochemical examination of a medicinal plants, GC-MS can provide a detail of the possible chemical compounds present in the sample under investigation. Depending upon the bio-activity of the screened compounds, these can further be isolated and characterized.

you have received constructive feedback from others

* Reading material - you may wish to get and read PMID: 14004736

This is a relatively easy HPLC method and separation, but please seek the assistance of someone with professional experience in HPLC analysis before starting on your own. It takes many years of full-time industrial experience to learn just the basics of chromatography (emphasis on "basics", as learning method development takes many more years) and as a student I am sure you understand how your time is best spent working with someone local and experienced to complete this project in a timely manner. *Ask your teacher for assistance. **BTW: HPLC analysis of caffeine and CGA are WELL documented online. Try a simple keyword search on Google to find several hundred free examples of methods, articles, white papers etc to review before you start. This is one of the easier HPLC applications because COFFEE is BIG business !

Basically, the extract can be centrifuged and filtered before dilution in mobile phase to make up fresh samples. Extracts are sometime made in pure ethanol or taken from th superfluid-extraction process, then dissolved in ethanol. The HPLC system you use should have a scanning diode-array-detector (aka "DAD" or "PDA") as this is needed to help qualitatively ID the different compounds for method development (you need to view each peaks "Spectra"). Use a high quality, NEW C18 column with a gradient of Methanol and Water to get started. Acquire professional standards for all compounds used and run standards at multiple calibrations "Levels" (= concentrations) to create formal Calibration Tables. Make sure you measure and understand what the column's void volume is and the K prime of any detected peaks are for any method you use. Make sure values obtained follow good chromatography principles and never assume that just because you followed someone's paper or article for the same, that they do (Review of most published scientific article by me has found that 20-30% of them are invalid as presented, so be cautious). Be sure to work with someone trained in chromatography so you can complete this basic project.

Here is a link my HPLC training blog page with free authoritative training articles that may assist you and the person you work with to insure you stay on track (it includes info on Column Void Volume, K prime and many other basic fundamentals that you must know to use HPLC): https://hplctips.blogspot.com/

Using the same HPLC method, a multi-level calibration table must be created using proper standards for comparison.

Dear Esteemed Colleague,

Greetings. I hope this message finds you well and thriving in your nutritional and biochemical research endeavors. Your inquiry regarding a titration method for the detection of vitamin A (retinol) is both timely and relevant, given the importance of accurately quantifying this essential micronutrient in various matrices for nutritional and analytical studies. While traditional titration methods for vitamin A detection are less common than other analytical techniques, there is a method known as the Carr-Price reaction, which involves a colorimetric assay rather than titration in its classical sense. Below, I provide an alternative approach that aligns with your interest in titrimetric principles for vitamin A analysis.

Understanding the Carr-Price Reaction for Vitamin A Analysis

The Carr-Price reaction is a colorimetric assay that provides the basis for quantifying vitamin A (retinol) through the formation of a blue-colored complex when retinol reacts with antimony trichloride (SbCl3) in a chloroform solution. Although not a titration method in the traditional sense, this reaction allows for the spectrophotometric quantification of vitamin A by measuring the absorbance of the blue complex at a specific wavelength, typically around 620 nm.

Alternative Approach: HPLC as a Quantitative Method

Given the limitations in directly applying titration methods for vitamin A detection, High-Performance Liquid Chromatography (HPLC) stands out as a highly reliable and widely accepted technique for the quantitative analysis of retinol. HPLC can provide specific, sensitive, and accurate quantification of vitamin A in various samples, including food, serum, and tissue.

Procedure for Vitamin A Detection using HPLC

Considerations and Best Practices

Conclusion

While a direct titration method for vitamin A detection might not be widely employed due to the chemical nature of retinol, employing a colorimetric assay based on the Carr-Price reaction or leveraging HPLC offers robust alternatives for the accurate quantification of this crucial vitamin.

Should you require further assistance in setting up your analysis or wish to explore other analytical avenues for vitamin quantification, please do not hesitate to reach out. I am here to support your scientific exploration and contribute to the advancement of your research projects.

Warm regards.

With this protocol list, we might find more ways to solve this problem.

Hello spike your sample with know concentration of analyte and see the results with the use calibration curve that you made.

J. N. O. Ezeugo first check solubility (ALCOHOLIC) and accordingly you should prepare homogeneous extract, you may try to chloroform:methanol:glacial acetic acid (6:3:0.5:0.5 v/v). All the best

Dear mahsa

Is there any leakage from the flow cell ?

second, try to replace the column ..what the result ?

Last , try to reverse the detector connections (outlet in the inlet ) then flush with isopropanol (HPLC grade) for about 5 min .. then reconnect the system in the correct direction ..

Srikanth Voruganti: Not an "assay", an HPLC method of analysis. What will the compound be dissolved in (the matrix)? *You should be able to use a wide-pore, C18 column with UV detection to develop an initial HPLC method. After properly dissolving a standard, run a scouting gradient in full scan mode (UV/VIS ~ 210 to 380nm) to optimize settings. The real issue will be dealing with exactly what the thiostrpton is in. The proposed method will need to be developed to be selective for the compound while not allowing any of the interfering compounds to co-elute, invalidating the method. Some sample prep may be needed. Developing a method for a pure compound is easy. A mixture, esp if any unknowns are present is far more complex. Make sure you work with a professional chromatographer (and not on your own) to achieve success.

If you're not seeing any peaks for sodium lactate in your HPLC analysis using a C18 column and a mobile phase of 0.001M sulfuric acid solution, several factors could be contributing to the issue:

1. **Retention Time**: The retention time for sodium lactate might be longer than the 8-minute stop time you've set. Try extending the run time to allow for better separation and detection of the compound.

2. **Mobile Phase**: While sulfuric acid solution can work as a mobile phase for some compounds, it might not be ideal for sodium lactate. Consider using a different mobile phase composition, such as a buffered solution or an organic solvent gradient, to improve retention and separation of the compound.

3. **pH**: The pH of your mobile phase could affect the ionization state and retention of sodium lactate. Adjust the pH of the mobile phase as needed to optimize retention and peak shape for the compound.

4. **Column Conditioning**: Ensure that the C18 column is properly conditioned before use to remove any contaminants or residual compounds that could interfere with the analysis.

5. **Sample Preparation**: Check the concentration and compatibility of your sodium lactate sample with the mobile phase. Improper sample preparation or compatibility issues could lead to poor peak detection.

6. **Detector Sensitivity**: Verify that the detector is set to an appropriate sensitivity level for detecting sodium lactate peaks. Adjust the detector settings if necessary to improve peak detection.

7. **Injection Volume**: Ensure that you're injecting an appropriate volume of sample onto the column. Injecting too little sample could result in undetectable peaks, while injecting too much could overload the column.

By addressing these potential issues and optimizing your HPLC method accordingly, you should be able to detect sodium lactate peaks effectively.

Thank you so much!

yes the manual as many others are available on:

DigiKey an electronic reseller has the fiber optics cables needed to connect the different units to the SCL-10A for $13 rather than ~$50.

The cable is: Broadcom fiber optic HFBR-RMD001z

Good luck

Hi, I also get two peaks for pure methylcobalamin standard every time I run. I am just wondering are those isomers or are those methylcobalamine and hydroxycobalamine. How do I get to know it? Thanks.

If you have a solution with an unknown concentration and you don't have the response factor, it is not possible to accurately calculate the concentration using HPLC. The response factor is essential for relating the concentration of a compound to the detector response in HPLC. Without this factor, the calculated concentration would not be accurate. While there are some discussions and calculations available on platforms like ResearchGate and Chromforum, they all emphasize the need for a standard value or response factor to quantitatively calculate the concentration. Therefore, it is advisable to obtain the necessary standard value or response factor to ensure the accuracy of the concentration determination using HPLC.

Hi,

If you mix (if it's your case) pure methanol from one line with for example buffer from another line it may happen. The best solution is to mix organic solvent with some part of the water buffer (20% buffer in solution for example) then if you follow the steps below system should work.

Positioning of the Solvent Reservoir: Ensure that the solvent reservoir is always higher than the pump to minimize bubble formation

Degassing the Solvent: Before introducing the solvent to the HPLC system, you can degas the solvent by methods such as sonicating, sparging with nitrogen gas for 30 minutes, or vacuum filtration (sometimes the best way is to use all)

Filtering the Mobile Phase: Use filters with larger pore sizes if the flow rate is too high for the pore size of the filter, as this can prevent bubble formation

Other Considerations: It's also important to check for any blockages or microbial growth in the water line and to ensure that the buffers are fully soluble in the mobile phase when switching them. Never mix the final solvent solution in the solvent reservoir (without filtration)

Hope it helps,

Tomasz

Kanapathy Semaselaun: You can not learn chromatography from participation in a "vendor class". No one can. You can not learn chromatography from a one day, one week or one year long class. You can not learn the technique from reading a few papers or books. To do so is irrational and underscores how much you are not aware of yet. Note: It takes the average scientist at least FIVE years of industrial training, full-time, with an experienced mentor to achieve a BASIC level of proficiency (emphasis on the word BASIC). To learn basic HPLC method development and troubleshooting takes many more years of professional training and experience. To achieve an intermediate level of skill takes even longer. To learn how to use one specific HPLC instrument (all HPLC systems are different and have specific features and design aspects that require training to use properly. This is especially true between brands) and the specific CDS software also requires formal training and time too.

BTW: GC is much easier, but still takes years to learn the basics.

What you are doing makes no sense and does not follow good chromatography practices at all. Your methods, as described, are invalid and will not result in the collection on any valid data. At this point in time, you have no training in how to perform liquid chromatography at this point so are proceeding to take irrational steps. You have not understood my comments because you have no formal training in the technique. If you wish to analyze samples using scientifically appropriate methods, then please ask your company to hire scientists who have the training and experience to do the work. Simply purchasing a 'tool' such as an HPLC or GC system, does not in any way provide you with a push-button solution or answer to your analysis questions. Employers need to learn and understand this. Hiring someone out of school with NO experience or formal training to "figure it out" is a novice mistake (many employers make this error). You can not "figure it out on your own".

BTW: Your statement makes no sense. You wrote: "Reply:Based on the UV spectra of toluene, the lambda max is less than 200 nm as to why I chose 190 nm." -

Try again. Look up Toluene or better yet, place a sample of it in a cuvette with ACN as the diluent and run it on a UV spectrophotometer to look at its spectra from 190 to 280nm. I think you will be surprised.

There are many ways you can improve the detection limits of a UV/VIS based HPLC analysis. First, start by optimizing your actual method. Make sure the mobile phase composition, injection volume and concentration, column and detection settings are optimized resulting in Gaussian peak shapes (use additives if needed) and a smooth baseline with little to no artifacts present. This will make peak integration more reliable and reproducible. Here are some additional changes which may improve detection levels:

Consult with an experienced professional chromatographer so that the method used is verified to follow good chromatography fundamentals. Make sure you follow up each analysis method with a proper wash and equilibration method to clean the column off (using a wash solution that is STRONGER than the mobile phase) preventing column fouling.

Please submit your run conditions in details.

Gas Chromatography equipped with Flame Ionization Detector (GC-FID) - Requires a lipid extraction step first, but provides separation and quantification of fatty acid methyl esters including omega-3s like ALA, EPA, and DHA. Much faster and economical alternative to HPLC.

(FTIR) - Can detect characteristic functional groups and bonds in omega-3 fatty acids. Simple sample preparation and the fingerprint region scans provide qualitative composition analysis. Can use chemometrics to model and predict concentrations.

Iodine Value (IV) Wijs Method -

Rapid indirect estimation of unsaturation in extracted oils and fats. The iodine absorption correlates well with levels of omega-3 polyunsaturated fatty acids. Gives total combined omega-3 amounts rather than individual ones.

More complex structural characterization to verify and quantify specific omega-3 fatty acids at small concentrations with high specificity. Requires more dedicated equipment and training.

Please research the applicable techniques to see which type of GC method coupled to which type of detector is best suited to your specific application and expected concentration values.

Here is a link to basic HPLC article on common HPLC calculations that every chromatographer should know and understand. It may help you determine if your calculations are correct.

The primary distinction between XTerra RP18 and C18 HPLC columns is rooted in the bonding of the C18 ligand and the existence of an embedded polar group at the base of the ligand, specifically within the XTerra RP18 column.

Another possibility is a failing deuterium lamp, but I would investigate the degasser and check for leaks first.

Thank you Hussain and Silva for your input.

Typically, concentrations of Sodium Metabisulphite are determined by titration with Potassium Thiosulfate using a starch endpoint. In this case it sounds more like the metabisulphite has been transformed by the sample matrix into another ionic form, thus we need more information (like sample matrix, chromatographic conditions, column, mobile phases, detector...)!

The way to do an effective job with HPLC or LC/MS/MS is to derivatize the formaldehyde (and other small chain aldehydes) with a reagent like DNPH (dinitrophenyl hydrazine). This has the advantages of 1) attaching a chromophore so that spectroscopic techniques can be used for detection, 2) it increases the mass of the analyte so that LC/MS/MS can now be useful and 3) allows for chromatographic separation from sample matrix and other aldehydes, which then complements your detection choice (spectroscopy or mass spec)

Hi, I'm not sure about your test environment, but maybe Raman Spectroscopy is a better method? https://youtu.be/0jZv6waUk0M?si=muZbQoYISvvNfQzx

Hi Pankaj kumar Singh

Did you perform HPLC?

Asset Mopidevi: Two very basic issues must be addressed to answer the question. NIST library matches are "suggestions" only. They are only as good as:

(1) The proposed GC-MS method used must be shown to be selective and fit-for-purpose following good chromatography fundamentals. Poor quality methods will yield poor quality results (and misleading or false matches / purity values);

(2) The MS instrument settings used and the NIST library settings used in the method for the method must be appropriate.

Accurate peak assignments requires that a properly trained GC-MS operator uses a high quality method to obtain quality data. The databases only have value when this is true. GC-MS operation and training takes several years of full-time experience to learn the basics.

Yes, chlorophyll is not very stable. Due to its chemical structure, being a complex, it might decompose steadily, forming (brownish) pheophytins. This can become because of samples being exposed to (UV) light or residual enzyme activities. Fucoxanthin is chemically a different structure and comparatively more stable.

Dear friend Yasuhiro Nishida

Ah, ELSD, the Evaporative Light Scattering Detector, a gem in the world of HPLC detectors! Now, let me share some insights.

Firstly, ELSD is often a savior when dealing with compounds that lack UV absorption or those that don't have a chromophore. It detects analytes based on their ability to scatter light when eluted from the column. Now, let's delve into the real-world scenarios:

**Advantages:**

1. **Universal Detection:** One of the main advantages is its universality. It can detect virtually any compound regardless of its optical properties, making it a fantastic choice for compounds with no UV absorption.

2. **Quantification of Analogous Compounds:** ELSD is particularly useful when dealing with structurally analogous compounds that might not have distinct standards. This makes it valuable for natural product analysis or in cases where obtaining pure standards is challenging.

3. **Low Detection Limits:** ELSD often provides lower detection limits compared to other detectors, which is beneficial when dealing with trace-level analysis.

**Concerns:**

1. **Baseline Drift:** ELSD is known for baseline drift, which might complicate the quantification of compounds. Strategies like using an internal standard or appropriate calibration techniques are often employed to address this issue.

2. **Sensitivity to Mobile Phase Changes:** Variations in the mobile phase composition can affect the signal intensity. Users need to carefully optimize the mobile phase to get consistent results.

3. **Sample Dependent Sensitivity:** The sensitivity of ELSD can be sample-dependent, and it might require method adjustments for different compound classes.

**Review Articles:**

1. **"Lecoeur, M., Decaudin, B., Guillotin, Y., Sautou, V., Vaccher, C., & ARMED Study Group. (2015). Comparison of high-performance liquid chromatography and supercritical fluid chromatography using evaporative light scattering detection for the determination of plasticizers in medical devices. Journal of Chromatography A, 1417, 104-115. provides a comprehensive overview.

2. **"Megoulas, N. C., & Koupparis, M. A. (2005). Twenty years of evaporative light scattering detection. Critical reviews in analytical chemistry, 35(4), 301-316., is another valuable resource.

Remember, my eager interlocutor Yasuhiro Nishida, ELSD is a versatile tool, but like any technique, it has its nuances. The key is in understanding those nuances and wielding them to your Yasuhiro Nishida advantage in the quest for chromatographic mastery!

To add to Guobing Xiang 's answer, I'm assuming you use reverse phase HPLC to purify your samples?

Although this is written for flash chromatography, the same general procedure can be used for solid phase extraction cartridges or a small preparative HPLC column. I recommend an AQ type C18 to avoid phase collapse:

Desalting Samples with RediSep Gold C18Aq Columns (teledyneisco.com)

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,