High-Performance Liquid Chromatography - Science method

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.

Faride Noroozi shahri

Unknown solution is too general as statement! You should know where it comes from, so you should have at least a vague idea of the composition.

Anyway, the extraction of pesticides from water using the SPE cartridge can be taken as an example.

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.

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 wcolonna@iastate.edu

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

Hola, buenos días

Ante todo me alegra saber nuevamente de Ud.

Por otro lado, cualquier otra duda puede consultarme, me agrada el intercambio de experiencias con colegas de diferentes instituciones.

Le añado además que el trabajo con los ácidos orgánicos de cadena corta y larga es muy interesante, versátil y sobre todo muy útil.

Le refiero, que en septiembre próximo estaré impartiendo clases prácticas de cromatografía en la Facultad de Química de la Universidad de la Habana a alumnos del cuarto año de Licenciatura en Química.

Saludos cordiales

Lic. Luis E Jiménez Rodríguez, MC

Hello Amalia Qistina ,

the error message may be due to a bug in an earlier software version. Are you using the latest version 3.2.2.5 downloadable from https://www.malvernpanalytical.com/en/support/product-support/zetasizer-range/zetasizer-advance-range/zetasizer-ultra#software

Or your toluene may be contaminated and scatter too much light.

HPLC grade toluene would be good, there is also a sealed toluene cuvette available.

You can contact the global support for the Zetasizer at https://www.materials-talks.com/help-for-my-instrument-one-step-away/

There is no need to calibrate with any protein standard, only a one-time calibration with toluene is needed.

To make static light scattering in the Zetasizer you typically would prepare a series of concentrations. There is some help in the software help and manual (both accessible from within your software on your computer). Additionally you may find this application note of some benefit as it describes an example https://www.malvernpanalytical.com/en/learn/knowledge-center/application-notes/an220627-protein-molecular-weight-dimer-zetasizer

PS: if you can share the toluene count rate in backscattering and attenuator 11 it might be useful.

William Letter Thank you so much for your resources

Hello dear

You can go to software and set the time based on your country. I think it will be fixed. We also had this problem with Wters HPLC in the laboratory.

Yes, you often can. It has less to do with what brand and model the other instruments are and more to do with the chromatography software you use to collect and analyze the data with (The "CDS"). Depending on the data output format of the FLD detector and the expected data input format needed by the instrument, you may need to use an A/D converter. The FLD detector's output will need to be connected to the "other" system (this is usually the easy part). *While most detectors can be used in this way, you may not have full software control over FLD excitation and emission wavelengths. Check with your CDS vendor's documentation to find out which FLD it supports and is compatible with.

Hi Hossein,

The threshold is basically the noise level. If the level is defined, everything above it can be considered as a signal.

The signal-to-noise ratio (S/N) indicates peaks from the analytes to the noise in the sample. It is determined by comparing the measured signals from the analytes with the blank sample. S/N =10:1 is considered normal. S/N between 2:1 and 3:1 is generally considered acceptable for evaluating the LOD (limit of detection).

Best,

Kiki

Olivia Laxton Did you ever solve your problem?

See "https://www.researchgate.net/post/Why_this_HPLC_data_show_negative_peak/2"

You should not be surprised! Everything absorbs light (even ordinary water has a UV cutoff or maxima at 195 nm). Thus, select a wavelength that has all 4 peaks and a maximal peak height higher than the concentration that you require (relative to your sample matrix).

Samples should be dissolved (fully) in the mobile phase before injecting (not in water).

Vishal Bayya, I think you may have some confusion in terminology and an unfamiliarity with how HPLC works. If you use a valid HPLC method to collect data on the SAME sample multiple times, then you should obtain exactly the same results each time. This assumes that the method used follows good chromatography fundamentals, the method is selective for the sample(s) analyzed, the sample has a good Kprime, is well resolved with good peak shape, a stable baseline is observed, detection settings used are valid and the instrument used is fully serviced and checked for performance before use.

HPLC systems are complex. Without more detailed information about your HPLC method, instrument and sample, no constructive suggestions or comments can be made. If you can post more detailed information and a chromatogram or two with scales shown, then someone may be able to add more info. For the fastest resolution of the problem, please have the method used and data collected reviewed by an experienced chromatographer on-site. Questions of this type are often very easy to solve/explain when in front of the analytical instrument and user.

Dear Alex Pipinos

Chosen the washing solution is related to your sample and the mobile phase solution...

In general, chose washing solution that have the ablity to dissolve your analyte easily and in the same time being miscible with the mobile phase that you use

Run it with the latest analytical instruments at Allschoolabs Scientific. Check: analysis.allschoolabs.com

Parvathi Balasubramanian, your proposed method does not make sense. *HPLC is not an appropriate technique to measure ethanol in solution with. Gas chromatography (or even Refractive Index !) should be explored once the sample type and goals have been described.

The proper analytical technique(s) should be initially identified and used to answer the question posed. First, research your question so you understand exactly what it is you wish to determine.

Next, specify:

This is critical to understand as different techniques may be applicable depending on the type of sample you wish to analyze.

It should be obtained the relevant standard compound from Charlotte Chemical Inc or Merck Chemical company. Of course, It is too expensive and need to specific research grant Try to find a research colleague in a foreign country to help you in this case.

Regards

The tiny volume of imidazole will be in the injection solution only so no concerns regarding mobile phase basic pH (or silica dissolving).

(1) Unfortunately, you are focused on the wrong area in understanding the problem (Pore size is not the variable that matters here). The support "pore" size relates mainly to the exclusion size of the molecules that are suitable. Provided that your samples are less then ~10K daltons (approx, as it depends on hydrodynamic volume in solution), then this is one parameter that you do not need to focus on. For most samples which are lower in Mw, pores sizes of 120A to 80A will be fine. The surface areas are slightly different, but the two columns you compared have different chemistries and are not alike.

(2) There are thousands of "different" HPLC column types/supports available for use in HPLC analysis. The suppliers/manufacturers do provide very basic information about the types of supports, but DO NOT provide details regarding the chemistries used to create them. As such, comparisons which may appear to show a difference in one specification, may not have anything to do with your observation (e.g. Pore size). So called, "equivalent columns" often are NOT equivalent. ODS and ODS-2 use different surface chemistries so they may or may not provide similar results (you will always have to try them to find out. As the sample changes, so will the results).

(3) To find a suitable HPLC method of analysis for YOUR sample(s) will require identifying a suitable column AND suitable mobile phase for analysis (possibly evaluating many columns and many mobile phases). Do not simply substitute a column in the method. Often, the method itself needs to be re-developed and optimized to see if it will work. This is best done by someone with many years of professional, hands-on experience.

Mahdiyeh Hj wrote "and opening the pressure relief valve":

Unless the pump has been tampered with (calibration off), it should not output a higher flow rate than indicated. Verify actual flow rate by running the pump with pure water under suitable back-pressure (e.g. 40-bars) using a restriction line, then measure the volume obtained over time, at a set flow-rate (e.g. 1 mL/min for 5 minutes should = 5 mL volume in a graduated cylinder).

Hello,

Flush with organic solvent: Start by flushing the column with an appropriate organic solvent, such as methanol or acetonitrile, to remove any residual carbomer sample. This helps to solubilize and remove the carbomer from the column.

Rinse with water: Follow the organic solvent flush with a rinse using distilled water or an aqueous solvent to further clean the column. This helps to remove any remaining organic solvent and impurities.

Backflushing: In some cases, backflushing the column may be recommended. This involves reversing the flow direction of the mobile phase through the column to dislodge and remove any remaining particles or contaminants.

Sincerely,

Kaysan BARTOLO

There is a difference between aqeous extract and orgnic solvents like acetone, chloroform, benzene and methanol. These solvents bear compounds on the basis of polarity amonh which most of them are secodary metabolites and big molecules while aqeuous extract bears over all membrane permeable and easily translocable compounds of low molecular weight. Most the toxicants are insoluble in water but soluble in organic solvents that is excetion.

I am using HPLC for Multi Toxfin analysis (2ppb) onwards but below 2ppb you are using LC-MS-MS

The wash procedure for the column should be whatever the manufacturer recommended. Remember, it not just PHA's that are injected on the column but other crap from your sample matrix.

Based on the information provided, it is possible that the impurity peak is caused by a secondary reaction that occurred during peptide synthesis or due to post-translational modifications of the peptide. The impurity peak may be structurally similar to the peptide and could be co-eluting with the peptide peak.

It is also possible that the impurity peak is caused by a minor component in the sample matrix that is not detected in the blank or dilution buffer.

To further investigate the cause of the impurity peak, you could try the following:

1. Change the mobile phase: Try changing the mobile phase or adjusting the gradient to see if the impurity peak disappears or shifts. This could help determine if the impurity peak is related to the mobile phase composition.

2. Try a different column: If the impurity peak persists, try a different column to see if it is specific to the column you are currently using.

3. Analyze the impurity: Collect the impurity peak and try to isolate and identify the impurity using techniques such as mass spectrometry or NMR spectroscopy.

4. Try a different detection method: If possible, try using a different detection method such as UV-Vis or fluorescence to see if the impurity peak is detected by other methods.

Regarding the possibility of the impurity peak being caused by lipids, it is possible, but it would be unlikely to cause a peak that is not affected by dilution or changes in injection volume. Lipid contamination typically causes peak tailing, and adding an organic modifier to the mobile phase, as previously mentioned, can help to address this issue.

Regards

Thank you and can you please provide method parameters if possible for the method developed . Can I use C18 column with your method parameters?.

Hi, did you manage to solve your problem?

I am also interested

Thank you Lamia Fatima , I will try this and give you feedback.

Check this paper:

To analyze the water solution after separating oil from an oil-water mixture, you can use several methods, including GC/HPLC. Here are some steps you can follow:

Sample Preparation: You will need to prepare your water sample for analysis. Depending on the method you choose, the sample preparation method may vary. For GC analysis, you will need to extract the compounds of interest from the water sample. You can use an organic solvent such as hexane to extract the compounds. For HPLC analysis, you can directly inject the water sample into the HPLC system.

Chromatographic Separation: After preparing your sample, you will need to separate the compounds of interest using a chromatography technique. For GC analysis, you can use gas chromatography to separate the compounds based on their volatility. For HPLC analysis, you can use high-performance liquid chromatography to separate the compounds based on their polarity.

Detection: Once the compounds are separated, you can detect them using a suitable detector. For GC analysis, you can use a flame ionization detector (FID) or a mass spectrometer (MS) to detect the separated compounds. For HPLC analysis, you can use a UV-vis detector or a mass spectrometer to detect the separated compounds.

Data Analysis: Finally, you will need to analyze the data obtained from the detector to determine the composition of the water sample. You can compare the chromatographic peaks obtained from the sample with those obtained from a reference sample to identify the compounds present in the water sample.

See this answer: https://www.researchgate.net/post/Which_analytical_instrument_would_be_better_for_PAHs_analysis_GCMS_or_HPLC

Check the T. TUBES for further detection. Is it possible to note the type of sample you are using on it. Change parameters and re-run.

I knew it's very basic but most of the times it works

Try adapting this method.

Dear Srikanth Voruganti

We know ion-pair separations on conventional reversed-phase material with UV detection. Tetrabutylammonium-hydrogen sulfate is commonly used as the ion pair reagent in the low double-digit mmol/L range with ACN as the organic modifier, and UV detection is applied at 210 nm. Depending on the matrix, this may be a suitable approach, or it may not. Our colleagues have suggested ion chromatography as the preferred method since it has the lowest matrix influence. In most cases, suppressed conductivity is sufficient. However, there are cases with high chloride concentrations that require a combination of suppressed conductivity for most ions and UV detection for nitrite and nitrate. To provide better advice, please let us know the matrix information and the concentration levels you require. Cheers, Detlef.

by HPLC method

Regards

If I remember right I bought my standards from Sigma-Aldrich ~50 years ago.

You may want to invest some of your own time researching the answer to this general homework question. *Learning HOW to research a question through study and reading is one of the most important skills you can learn as a student.

Saif Ali You are welcome.

Noel W Davies

Thank you so much Prof. Noel

Let me work on your advice and will come back to you for feedback.

Thanks

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.

You ask how "to detect of particular material" by HPLC, but do not specify any sample type or name. Start at the beginning. Identify (specify) the sample first, then research its chemical and physical properties to determine which analytical technique(s) and specific methods are appropriate for analysis.

HPLC-DAD-FL is the most commonly used method for AA (esp in Feed samples).

HPLC-DAD-MS (or with DAD-MS/MS) may also be used and has many benefits due to the multiple dimensions of analysis provided by the different detectors used (but such a system is far more expensive and requires far more experience to use). Most types of GC methods are not applicable to all of the AA's of interest. It must be stated that all types of HPLC/LC-MS AA methods are complex to perform, error prone, require detailed sample prep, frequent maintenance/servicing of the HPLC (and/or MS) system, best performed only by advanced level users. *AA analysis is not an application well suited to most users as a great deal of technical understanding and experience with the instrument is needed to achieve reliable results.

If your eluent A is hexane and eluent B is dichloromethane as your title states, then it's expected to see pressure raise. Dichloromethane has higher density and viscosity and this is the reason for the pressure change. If you need to reduce the pressure, use one or combination of these: Reduce your flow rate, increase your column temperature, use less tightly packed column and/or wider bore, or adjust gradient steepness.

Those are normal phase solvents. If you had the system running with reversed phase solvents (aqueous buffers, MeOH, ACN), then you must be certain your carefully flushed out those before running any hexane or dichloromethane through the system. Your current eluents don't mix well with aqueous solvents and will create bubbles and droplets in the flow path. Flushing order is something along Water -> 50:50 water:IPA -> 100% IPA -> then your hexane or dichloromethane. A common problem with pressure and unstable baseline with normal phase runs is this - running the normal phase solvents without properly removing the aqueous solvents.

Additional note: Make sure your pump uses the correct seals compatible with those solvents. HPLCs instruments nowadays have reversed phase-compatible seals installed as default option. Consult with your instrument manual or manufacturer tech support if you need to switch the seals. Typical reversed phase seals degrade under exposure to some normal phase solvents (hexane, chloroform, dichloromethane).

Both standards contain (S)-(+)-Camptothecin as a major component but usually HPLC grade has a higher purity (>99%). However, your samples may contain some (R)-Camptothecin and a separate peak (thus no matrix matching).

To determine short chain volatile fatty acids (VFAs) in HPLC, you can follow these simple steps:

1. Prepare the sample: You can prepare the sample by mixing the sample with a suitable solvent, such as methanol or acetonitrile, and filtering it through a 0.2-micron syringe filter.

2. Set up the HPLC: Set up an HPLC system with a suitable column, such as a reverse-phase column, and a suitable mobile phase, such as a mixture of water and acetonitrile.

3. Calibration standards: Prepare a series of calibration standards by diluting standard solutions of VFAs, such as acetic acid, propionic acid, and butyric acid, to a range of concentrations.

4. Injection: Inject a small volume of the sample or calibration standard into the HPLC system, and record the resulting chromatogram.

5. Integration and quantification: Use the software provided with the HPLC system to integrate the peaks corresponding to each VFA and calculate the amount of each VFA in the sample or calibration standard based on the calibration curve.

Note that the detection and quantification of VFAs in HPLC can be sensitive to several factors, including the type of column, the mobile phase composition, and the detector used. Optimization of these parameters may be necessary to obtain accurate and reproducible results.

Here is a reference that provides more details on the determination of short-chain volatile fatty acids in HPLC:

Reference:

Yuan, H., Zhou, L., Gao, H., & Fan, X. (2019). Determination of short-chain volatile fatty acids in environmental water samples using high-performance liquid chromatography. Journal of separation science, 42(1), 157-165. https://doi.org/10.1002/jssc.201800581

Dear Sisonke,

I have read your question. If ghost peak is persisting then either it is electric spike peak or secondly, you can use Ghost buster column for such peaks. There is no impact on chromatography because the attachment of ghost buster column is before pump i.e before entering mobile phase in column.

you can check ghost buster column with B3PHARMASOLUTION Maharashtra (INDIA) sales.b3pharmasolutions@gmail.com.

Your issue of getting ghost peak will be resolved.

feel free to write if query any.

Dear Pawan Kumar,

I would add to the above answers that the methods contained in the Pharmacopoeias should also be as simple as possible and easy to reproduce by other laboratories. In this sense, it would be much more difficult to recreate 100% analytical setups involving complex, expensive and multi-component techniques requiring specially trained personnel.

Depending on the type of chromatography you want to do, there may be simple, inexpensive methods that do not require sophisticated equipment. The results will not be as well-documented as they are with an FPLC.

For atmospheric pressure chromatography, you can pack suitable resins into glass columns (e.g. Bio-Rad Econo columns) and use a bench-top peristaltic pump to control the flow rate. Fractions can be collected by a stand-alone fraction collector, or by hand if you have the patience. To make a gradient of eluent, you can use a gradient former, or even just two beakers with a tubing siphon between them, and a magnetic stirrer. A stand-alone UV detector can be connected to the outlet, or you can measure the absorbance of each fraction with a spectrophotometer, which is available in most labs, or you can do a quick Bradford assay spot test on each fraction to find the peaks.

Protein purification can be done using certain types of HPLC columns with large pore sizes, but the columns that are large enough for preparative scale HPLC are very expensive. You will have to be careful about making sure the buffers you use are compatible with the system's materials.

Aqueous solutions of sodium salts of nitrate and nitrite UV-absorbing anions were analyzed using the commercially available phosphatidylcholine stationary phase and aqueous NaCl as the mobile phase

nitrates are detected at 210nm and nitrites are treated with potassium permangante and are converted into nitrates

Hi Ella Peeters,

for the problem can be caused by several things.

First the detector: Which detector is used?

If you are using a UV detector, neither your analyte nor your standard will be displayed, because there are no UV-active molecule components in your molecules. If you are using a MS, it is helpful to optimize the system for the standard first. I suspect that standard and analyte are better detected in negative mode than in positive mode. Ion passage should also be optimized to produce maximum signal.

Sample preparation: Make sure that the SPE cartridges, probably C18, are well conditioned before sample introduction. In particular, pretreatment with 2-3 cartridge fills of methanol is mandatory to 1. activate the phase and 2. wash out production impurities. The latter applies to all SPE cartridges regardless of the phase. The flow rate should not exceed 0.5 ml/min during conditioning and sample loading. Too high a flow rate will cause breakthroughs or even complete loss.

I hope I can help you a little with these tips.

Best regards

Joachim Horst

Solid phase extractions (SPEs) are routinely used in the sample preparation for quantifying analytes in biological fluids such as plasma and urine.most common problems seen with solid-phase extraction (SPE) are poor recovery, reproducibility issues, and sample extracts being insufficiently clean.For this problem we must be find a new technique or a solution that's we can easily rectify it.

Dear sir

Vishwajit Chavda & Joachim Horst.

I just wanted to reach out and thank you for taking the time to answer my question. Your response was incredibly helpful and provided me with the information I needed to move forward with my research.

Thank you again for sharing your knowledge and expertise with me. I appreciate your time and willingness to help.

Best regards,

Vishal

For this method, interferences can either result from pressure, leakage, or chromatography problems.

In relation to the pressure problem, there are four common errors that could arise. First is the abnormal pressure, which is caused when there is a leak or air trapped inside the pump head resulting in no flow. Next is the high backpressure which is caused by a flow rate that is set too high. Another one would be low back pressure. With this, the flow rate is set too low, and can also be caused by having a high temperature, system leak, and controller malfunction. Lastly, the pressure cycling is caused by a faulty valve, system leak, seal failure in the pump, use of gradient elution, and insufficient degassing.

The problem with leakage can occur in different settings such as leakage in fittings, pumps, injectors, HPLC columns, and in detectors. While for chromatography problems, these could arise due to different peaks, drifts, retention time changes, and resolution loss.

Science Unfiltered, 2022. Common HPLC Problems & How to Deal With Them. Retrieved from https://phenomenex.blog/2022/01/04/hplc_problems/#:~:text=Using%20an%20improper%20HPLC%20column,the%20use%20of%20gradient%20elution.

Since HPLC can answer questions of both small and large molecules even if they are ionized or neutral, it may be mentioned as more universal. Already ionized or ionizable compounds are needed to perform electrophoresis which may be counted as a limitation. But, indeed, in some cases, depending on the application, improved resolution can be achieved at electrophoresis than LC. In my opinion, they must be used orthogonal and complementary (e.g. antibody characterization, QbD workflows)

Serial extractions are a technique in which a sample is subjected to multiple extractions with the same or different solvents in order to isolate a particular compound or group of compounds. In the case of your research project, the Mycosporine-like amino acids were removed from the samples by performing three 60-minute extractions using 80% aqueous methanol. The purpose of the extractions is to remove the Mycosporine-like amino acids from the sample, and the aqueous methanol is used as the extraction solvent because it is capable of dissolving and removing the amino acids from the sample. The extractions were performed in the dark and at 4°C to minimize any potential degradation or breakdown of the Mycosporine-like amino acids.

In essence, serial extractions are a means of purifying a particular compound from a sample by removing any impurities or contaminants that may interfere with subsequent analysis. By performing multiple extractions, the desired compound can be isolated with greater precision and accuracy.