High-Performance Liquid Chromatography - Science method

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

To calculate the concentration (mg/kg) of a pesticide residue in a sample using the area of the peak obtained from an HPLC analysis, you'll need the following information:

1. Calibration Curve: First, you need to establish a calibration curve for the pesticide of interest. This involves analyzing standard solutions of known concentrations of the pesticide using the same HPLC method. From this, you can plot a calibration curve relating peak area to concentration.

2.Peak Area: During the analysis of your sample, you'll obtain a peak corresponding to the pesticide residue. The area of this peak can be obtained from the chromatogram.

3.Sample Preparation Details: You need to know the details of your sample preparation, such as the volume of the sample extracted and any dilutions performed.

Once you have this information, you can use the following formula to calculate the concentration of the pesticide residue in your sample:

Concentration (mg/kg)=Peak area of standard×Sample volume (L)Peak area of sample×Concentration of standard (mg/L)​×Dilution factor

Where:

- Peak area of sample: Area of the peak corresponding to the pesticide residue in your sample.

- Concentration of standard (mg/L): Concentration of the pesticide in the standard solution used to generate the calibration curve.

- Peak area of standard: Area of the peak corresponding to the pesticide residue in the standard solution.

- Sample volume (L): Volume of the sample extracted for analysis.

- Dilution factor: If any dilutions were performed on the sample before analysis, you need to account for this by including the dilution factor in the calculation.

By plugging in the appropriate values, you can calculate the concentration of the pesticide residue in your sample in mg/kg. Ensure that all units are consistent throughout the calculation.

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/

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

Not that I recall. But there was a colorimetric method in the AOAC.

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,

I haven't verified that it can be analyzed correctly, but it seems to be possible with Elisa as well.

Similar kits are available from other manufacturers.A number of melatonin antibodies themselves are also available.

The kit targets human-derived samples, and the sample type is limited, but melatonin itself is a small molecule. Yeast (cells or culture supernatant?) might require pretreatment, but just for reference.

Hi,

Too vague, what kind of drugs? I would use Google it first to see if it was done before.

Eef Dirksen For 1L of 1X there is 60g Na₂HPO₄, 30g KH₂PO₄, 5g NaCl, and 10g NH₄Cl. Thanks for your help!

Drospirenone has a couple of strained 3-member rings. Look at your process to see if there are conditions (heat?) that opens one of the rings.

A Google search shows many options for me, including MilliporeSigma (as vendor, they supply it from someone else). See links below based on

CAS# 2483832-10-4 (also existing as 128-23-4):

You can find this information very quickly with a simple web search. The manufacturer's example for monosaccharides separation on the column you are using does not show sucrose in the chromatogram, so I suspect you will not be able to separate it from maltose and you will need to use a different column. A different column chemistry such as propyl amine is common for carbohydrate separations. If you want to stay with the ligand exchange column type you will need one with a different cation.

Hi Andrew, I am currently not doing PCD, but I am happy to be part of the discussion.

Regards,

Danijela

You have a seal wash leak and your mobile phase is leaking through the worn seal(s). Please have your HPLC pump professionally serviced. Regular maintenance and cleaning are required for operation.

Note: For any future posts on "HPLC" please provide the exact make. model and type of HPLC module, the mobile phase used in it, the flow rate and other basic information to help users put your observations into context. Help is most likely to occur when you share basic information in your initial posts (or requests). Please contact some at your school who has professional experience in HPLC to assist you with these types of questions (and/or hire the local instrument service tech to perform the needed services on your instrument.

I just heard back from the instrument manufacturer; the symptoms indicate a lamp failure.

Kevin

Observing a "linear relation" as you say does not in fact show any indication that you have a valid HPLC method of analysis at all. When no chromatography takes place (as may be the case in your example when the sample(s) elute at or near the column void volume or with low K prime values), the area counts often show near perfect r2 values. *Adding unknown peak areas together that you do not understand is not the approach that should be taken.

To put into context what your chromatogram may imply, the actual LC chromatography method information must be shared (required). The details of the HPLC method should be provided. These include all of the common parameters and settings which describe the method used (e.g. Column name, type, dimensions, particle size; flow rate, detailed mobile phase composition (and if applicable, gradient time table); temperature, injection volume, injection solution, detector type and settings used (e,g, for UV/VIS this would include wavelength and bandwidth), sample concentration etc.

For the most practical assistance with this project, please contact someone at your school who has practical experience using HPLC for direct help. Learning the basics of HPLC takes many years of full-time, hands-on work so working with an experienced local chromatographer will improve your chances of collecting valid data.

Hello.

You can get structural information on PCs by comparing retention times and UV spectra with the literature. Please check the following books:

Campos, M.G., & Markham, K. R. (2007). Structure information from HPLC and on-line measured absorption spectra: flavones, flavonols and phenolic acids. Imprensa da Universidade de Coimbra/Coimbra University Press.

Mabry, T., Markham, K. R., & Thomas, M. B. (2012). The systematic identification of flavonoids. Springer-Verlag.

Regards.

Dear Agnė Keselytė,

First of all, I would like to inform you that the best method for DMSO analysis is Gas Chromatograph (GC) because having solvent this technique is best suitable.

Gene@rally, DMSO is not analysis by HPLC because the traces can be remain in system and DMSO will cause corrosion.

It depends on HPLC procedure. Use ethanol solvent to extract hisperdin

I believe that calcium citrate is prepared from the reaction of modifying citric acid with calcium hydroxide

No its should not change significantly like 2-3 fold.

Dear Dr Vijay Arjun Bagul

Since my lab have no OPA and the order is still in process, thus, I came up with the idea to find another possibility to analyze SMM without OPA.

I found several references that analyzed SMM without OPA, I followed those papers but I'm still failed.

Hi Jimson,

I hope the attached article will help you.

file link: (PDF) Determination of chlorophyll in plant samples by liquid chromatography using zinc-phthalocyanine as an internal standard (researchgate.net)

With warm regards

Anup

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.

No, zebrafishes can't use chromatography. In general, fishes are not qualified as operators of scientific instruments since it is necessary to have hands for that.

OK, now for a serious answer: if you want to analyze zebrafish tissue with chromatography, you need to separate the completely insoluble parts of the tissue first, otherwise you will just jam the column in both cases.

For the analysis of the insoluble parts, you will have to do some sort of chemical cleavage procedure to make them soluble.

Follow the curcuminoid extraction and analysis from turmeric...

Similar molecular structures will yield similar analysis accuracy with minimum modification...Use as template methodology.

Your questions suggest you really need to contact an experienced chromatogtrapher at your school so the instrument is not damaged. It takes many years of full-time experience to learn the basics of GC-MS analysis and we can not teach the technique in a forum. Please contact a local expert to help you use the system, and plan your methods so valid data is collected and the experiments follow good chromatography principles. Now, a few comments which address your questions...

"GC-MS Grade" solvents are of very high purity usually containing far less residual materials than may be found in HPLC grade solvents. HPLC analysis (and the detectors used) have different requirements than Mass Spec systems (or even FID etc detectors). *Without additional information, for GC-MS analysis, use GC-MS grade solvents where possible (this is good advice and protects the instrument and column). BTW: Specific solvent grades are usually offered to minimize certain potentially interfering compounds (e.g. Pesticide residue grade; MS grade, less particulate, less water, lower UV abs etc ...). The REAL differences, if any, will be found when you read the actual solvent analysis specification sheet provided by the manufacturer. It is on this sheet that THEY will specify which purification procedures were used and what test results were found for the batch. Different manufacturers often have different specifications and/or different definitions of "purity" so you must COMPARE the different specifications. Depending on your specific application(s) and instrumentation, you can select the solvents which are best.

Regarding methylene chloride (HPLC or GC-MS grade) for GC-MS (I assume injection solution), well that depends on the EXACT method used (of which their are millions...). I can think of a few where it could damage the system so please speak with a professional chromatographer before moving forward.

"It has to do with an air permit stating we can only use HPLC";

* Again, more information is needed to understand your real question....however...

There are scientific and unscientific ways of doing things.One needs to find a solution that makes sense. Send the samples to an outside lab that CAN perform the test correctly.

Absolutely the result will not match because, first need to understand the basic principle of both techniques. Separation takes place in HPLC hence all components get separated and individual results shall be obtained whereas colorimetry is measurement of solution based on absorbance and hence reading is sum of all compounds. Hence both the results will not be matched.

Have you tried mass spectrometry with electrospray ionization, possibly in combination with ion mobility like TIMS or TWIMS?

Thanks a lot for your help and valuable advice.

Best regards,

Jose

Agree with William Letter,

You need to isolate the target chromatographically (do not need to separate -S and -R) and use CD to elucidate the racemic composition...

Under constant conditions, a deviation in the g-factor for a sample with respect to the value for a pure enantiomer indicates the presence of a chiral impurity.

Here are the links for clarification;

Actually, this may be very easy to do. Two methods have worked well for me in the past. Go slowly and do not "dig" into the column head with any metal parts.

(1) Using a pair of pliers (with internally serrated/grooved tips for gripping, not smooth), grip the PEEK tubing right up against the broken tip of the fitting in the column head (so the pliers are 90 degrees to the tubing, parallel to the head of the column). Now try to turn the tubing like a screw to un-thread the ftting (turn anti-clockwise). The broken plastic tip should be swaged onto the tubing allowing it to spin out. You may also be able to back it out enough to grab onto the tip with the pliers and spin it out.

(2) You can use a pair of pliers to grab onto the PEEK TUBING stuck in the broken fitting and using leverage against the column head, slowly pull the tubing out of the broken fitting tip. Once the tubing has been removed, use a small sheet metal screw (one that fits tight, just a few threads) to thread into the remaining PEEK fitting tip inside the column head. It should grip onto the plastic. You should be able to twist it or just yank it out once the screw has a grip onto the plastic (the broken part is soft plastic so you will not damage the SS threads of the column).

The solubility of ergosterol is very high in chloroform. So you can use chloroform

This RG thread might answer your question.

What specific data are you looking for? General precautions should be followed with all HPLC columns. e.g. Do not overload or foul the column, Maintain pH under 8 and above 2.5, operate under 5K psi max, wash with a solution that is "stronger" than the mobile phase after use etc.

On the web I found this:

"Table 2. Characteristics of 12 μm Sephasil Peptide media.

Peptide C18

Base matrix: silica

Bead form: spherical

Nominal particle size (um): 12

Mean pore diameter (Å): 100

Spec. pore volume (ml/g) 0.7

Spec. surface (m2 /g) 300

Bonded phase: C18

Carbon content: 16%

pH working range: 2-8 ".

Q: Does your column have the 12u particles inside (the label should show the particle size)? If so, this looks like the correct data. When Pharmacia sold columns, they often rebranded other vendors generic columns (re-labeled). It appears to be a very old type of C18 (maybe type A or possibly B) and I would not recommend using it for method development (because you will never be able to reproduce it since the column no longer exists). The pore size is shown as 100A (OK for low Mw samples and/or very small peptides). If you are just using the column as a student to gain experience with HPLC and run some samples then it should be fine for generic use. Columns are very inexpensive consumables used with HPLC systems. *Start with a brand new column for anything of value.

Once you have specified the EXACT compounds you wish to analyze, contact the local column suppliers and review their application notes for suggestions. *Make sure you work with an experienced chromatographer first, to develop HPLC methods that follow good fundamentals.

In case you are experiencing "carry-over", here is a link to an article that may assist you in troubleshooting and solving it.

8/17/23

Dear Dick Dick,

In our lab, we frequently measure the components of wine, which contains small amounts of acetic acid (HAc). We do this by running a series (5 or 6) of HAc standards on our HPLC, along with our unknown samples. I can't recall the column we use, but it may be similar or identical to yours, as we use 5 mM H2SO4 as the mobile phase. After the run, we call up chromatographic profiles of the standards and integrate the HAc peak areas, then plot this vs. the HAc concentrations of the standards to get a std curve. R2 is usually ~0.98 or higher. We also integrate the HAc peak areas of the unknowns. After that, it is a matter of getting the HPLC's computer to calculate the concentration of HAc in the unknowns.

We use an Agilent HPLC. Usually, the device automatically integrates the peaks, but occasionally, depending on the baseline, we have to go back and manually integrate them. The computer software will do the calculation for you after the std curve is plotted. If your HPLC can't do this, you can also prepare a standard curve in Excel. Both ways work.

I hope this information helps you.

Bill Colonna Iowa State University, Ames, Iowa, USA [email protected]

Did you have your broken HPLC degasser professionally serviced or replaced?

The culture is too expensive - and you have HPLC capabilties?

This is easy (I think though,it's been >50 years!). We had an HPLC method for Citric Acid in fruit juices. If I remember it was 15% cold meta phosphoric acid (not the more common ortho!) in Methanol, PDA/DAD at 248 nm, and a common C18 ubondapak (Waters) HPLC column.

I also found 0.05 seconds decrease in retention time with 10 fold decrease in concentration of one phytochemical