Method Development - Science topic

Review the websites:

For those arriving at this question: to properly answer this question you have to measure the solid-liquid phase diagram. It will tell you the maximum solubility of choline chloride in 1,2-propanediol at a certain temperature (e.g. 30 ºC). Probably a choline chloride mole fraction of 0.5 (1:1) is higher than the maximum composition at the temperatures you work.

Adding water increases the solubility (lowers the melting point) and can be a solution. Of course also within the thermodynamic limits.

Kindly check...

In my opinion try to use Methanol instead of Acetonitrile.....

Search first for analytical method for cholesterol to analyze the sample through LC-MS.

Dear Dr Siti Noor Hajar Zamrus,

I found an article that might interest you and the approach used for the quantification of sodium dodecyl benzene sulfonate seem interesting.

Best wishes,

Sabri

I suggest Mendeley and Endnote. But Mendeley is user friendly software.

I think looking at the overlay spectrum photodegradation is taking place rather than photoisomerization. You may check the fluorescence behavior of cis and trans isomers in isolation if available with you by following their kinetics.

Dear Bhimanna Kuppast,

Thanks for your interest. I am working with LC- MS since 2011. My research area is: development and validation of analytical methods for pesticide residue determination in different agricultural commodities. For the development of method, first of all, you have to follow the international guidelines for which compound, you are interested to develop and validate the method. For example, for the development and validation of LCMS method for pesticides and veterinary drugs, you may follow the EU guidelines: SANTE/12682/2019. Additionally, you may read several scientifically sound articles published by a very good quality journals. If you want , you may visit the following LCMS based articles:

DOI 10.1002/jsfa.8716

DOI 10.1007/s12161-016-0537-z

DOI 10.1007/s12161-014-9898-3

I have no practical experience, however, I hope the provided link will be very much helpful for you. Please have a look on the following link:

In addition to SEC methods (some have poor resolution), many methods using a C18 or C8 column with wide pores can also be used to retain and resolve many of the key peptides too. When trying to work with clinical Amyloid Beta peptides, the matrix and "other" compounds which are not of interest will guide your separation goals. Try several different modes of chromatography to survey what you have, then let the data point you in the right direction for what to try next.

Dear Alexander , Thank you so much for the article.

May I ask, I read some articles about positive controls drugs (tereparatide injection) test with RAW Blue cell line. Will those drugs stimulate cytokines expression or any Optical density in the assay?

My thought, is these drugs should inhibit cytokines release and OD should match with blank? Am I right?. Or high concentration of drugs can kill the Raw Blue cells?

Kindly share your thought....

Dear @ Calvin D Leech

Thanks a lot for your interest to work with GC-MS. I have some experience to work with GC-MS for the quantification of pesticides residues. I think, this variations related to the preparation of standard and the sample as well, it is not related to the instrument. I suggest you to prepare the standard properly, and I advice you , please do not prepare the standard following serial dilutions, prepare the standard from one working standards solution. The another important issue is matrix. If you are using matrix matched standard, then you have to be very careful to mix with the matrix properly, otherwise a large variations may come. Thank you for your interest.

According to my findings, the temperature does not have to be more than 20 C.

This might be to late but since you are doing HILIC, and the strong solvent is water in HILIC, you have to change your needle wash to contain much more water. Just make sure you have as little water as possible “stored” in the needle for the next injection.

Like all empirical formula, the CN method for calculating lag time should only be used within the parameter range used to derive it. Having said that, when compared to other methods for calculating lag time (all of them empirical), the CN method has a better physical basis - in that it is based on the time of travel of water along various flow paths. So, there appears to be no compelling reason why it could not be used for larger basins. I found that the CN method is very reliable and used it for basins as large as 20 km2.

Following

Dear Girdhari,

published isoelectric point of liraglutide is approximately 4.9.

You can also calculate this (https://pepcalc.com/) from it's primary sequence (HAEGTFTSDVSSYLEGQAAKEEFIIAWLVKGRG).

It is should be soluble in neutral water. Solubility decreases dramatically below pH 7. Double-check if the final solution really is neutral.

Adjusting pH to above 10 or below 2 should significantly increase solubility.

You could also use liraglutide prescription as a guide:

3ml solution containing 18mg of liraglutide, 1.42mg disodium hydrogen phosphate dihydrate, 14mg propylene glycol, 5.5mg phenol, dissolved in water for injection, pH 8.15

European patent EP 3 295 952 A1 (p. 4)

You can also add an organic solvent to improve solubility (e.g. 30 % ACN).

But make sure the elution strength of your sample solvent does not exceed the starting conditions of your LC-MS method, as this will lead to irregular peak shapes.

Kind regards,

Lorin

I do not know

Dear Mateo,

I suggest you to see links and attached files on topic.

Sameera -

I'm guessing that for your GLM software to do something other than OLS, you have to instruct it to do so.

Have you tried WLS? You might consider this for regression of form y = y* + e:

https://www.researchgate.net/project/OLS-Regression-Should-Not-Be-a-Default-for-WLS-Regression

There is this, with references noted in it: https://www.researchgate.net/publication/333659087_Tool_for_estimating_coefficient_of_heteroscedasticityxlsx

The WLS regression coefficients are often very close to those for OLS, but the prediction intervals associated with smaller predictions would be shorter, and the prediction intervals associated with larger predictions would be longer, often much longer.

If the sample size is small, the regression coefficients would be more likely to differ more substantially from the OLS ones, but then we would be less sure of the regression weights. But with smaller samples there is more uncertainty, so that is to be expected. Choosing OLS when that is obviously not correct would not help. Sometimes it can be reasonable though. Model and data issues may cause that to be the case.

Cheers - Jim

You might need to look at whether your lab needs a Micro or Ultra Micro Balance. Mettler Toledo do ones that measure down to 0.1ug.

This is outlined in the method......

https://www.epa.gov/sites/production/files/2017-04/documents/method_8260d_update_vi_final_03-13-2017.pdf

Try this "free" program that you can play what if in mobile phase method development. It is a good tools that I did not have when I was in school 35 years ago.

Dear Dr. Ramachandran,

In general, the type of measurement scale does not significantly influence the procedure of psychometric assessment. The assessment of psychometric properties involves the check of reliability (internal consistency, test-retest) and validity (construct validity, criterion validity) of the results obtained with the measure. The type of measurement scale may change statistical procedures that you apply to obtain necessary evidence of reliability and validity, but will not change the procedure of scale development.

Best regards,

Kirill

good answer Adam Zmysłowski

These forums do not provide a means to address such a broad question as posed. The basics of HPLC as a formal analytical technique and method validation basics fill volumes of books and journals. Read the books to research the basics. As HPLC method development and HPLC method validation take many years in the workplace to acquire a basic level of proficiency, I suggest that after researching the topic, you hire a contract laboratory to perform the work for you OR work with a local laboratory whom has experienced liquid chromatographers with a demonstrated history of doing the same for compounds of interest.

Additionally, you may wish to take some time and begin to research the basic of liquid chromatography [e.g. The text, "Introduction to Modern Liquid Chromatography" by: Lloyd R. Snyder, Joseph J. Kirkland, John W. Dolan is excellent] , HPLC methods, the basic concepts and fundamentals of analysis. BTW: Avoid sales oriented HPLC websites such as chromforum or chromacademy which have many inexperienced users who may misdirect you. Additionally, if you wish to be involved in the actual analysis work, then you will need to obtain formal training on the specific HPLC system and software that will be used for the work (Many types of HPLC systems and each one requires training in how to use it).

I am an investigation methodology teacher and yes there are established methods for devoloping workshops and for the evaluation procedure. To develop a workshop, the methodology is as follows: Fist of all you must define the objective, after that, take into account what are the activities that all persons involved in the workshop are going to develop and for the workshop evaluation, we apply the observation methodology of all the activities developed by the participants, following an observation guide.

In my personal oppinion, once the activities you devoloped are finished, you can apply a survey or interviews, to be able to prove the effect or result obtanied according to the planned objective.

I might suggest you consider using the Covaris G-tubes for plasmid DNA fragmentation. However, I agree with that there is no need for plasmid linearisation prior the library preparation for Nanopore MinION sequencing.

Hello, why you need to split peaks in LCMS, you do not have differences in m/z ?

Hi Diane,

Could you shed more light on the presenting sorting you used? I'll also wish to know if there are freely available online sorting websites I could use for postgraduate research.

Thanks for posting the new question.

I agree with A-B Lauren on both points. I also recommend you include any data you suspect might not be respresentative in a sensitivity analysis to let the reader understand your results better.

It is always up to you (the LCA practitioner) to use your expert judgment on what data can be used and to what extent it is representative. Processes modeled on data from other locations are however often better than no processes at all.

To have recommendations on how to deal with impact methods you would have to give more background info, both on the system, aim and scope, as well as the impacts you want to asses.

Dear Asal Asali,

calibration functions with quadratic fits are allowed according to USP 1736:

"A calibration curve is created by plotting analyte-to-internal standard peak area ratios versus concentration for the reference standards analyzed. For the somewhat narrow calibration range required for Category I and Category II measurements, a simple (nonweighted) linear fit is sufficient to define this response versus concentration function. However, 1/x or 1/x2 weighting also can be used, as well as nonlinear (e.g., quadratic) functions, as supported by appropriate and provided validation data."

Due to my experience, most of the people familiar with HPLC-UV sticks to linear calibration functions due to Lambert-Beer law. However, this law does not apply for LC-MS, since LC-MS is not based on (UV)-light absorbtion.

Best regards

Andre

Hi Omer,

Target compounds are inositol phosphates. We have selectd reverse phase anion exchange Chromatography .

You could do qualitative interviews with experts in the relevant area. You might also consider a "delphi" design where you collect rounds of interviews with experts via email. Here is a brief introduction to delphi techniques:

Please look at the following below links which may help you in your analysis:

http://www.plantphysiol.org/content/plantphysiol/96/1/262.full.pdf

https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1399-3054.1989.tb02065.x

Thanks

Hello, serial dilution is widely used in HPLC method development and validation. Only thing use the rage with is corresponds the range of analyte in future analyzing samples.

The method was invalid as shown. The consequences of using instrumentation and techniques without proper training often lead to erroneous data being collected and incorrect conclusions or quantitative results being reported. In your case, there are two reasons for this:

(1) The DAD reference wavelength feature on your Agilent 1260 DAD was turned 'ON'. This must be turned 'OFF', not 'ON'. To learn why, please read the attached article below.

(2) Your signal acquisition wavelength is very close to your Reference Wavelength. This guarantees that your data will be invalid as even the peak of interest will fall under the Reference range you specified (360,10), invalidating the method and data obtained.

Those who use HPLC for the first time and/or who have not had any formal training in liquid chromatography (and especially in the use of a diode array detector (aka: PDA)) often makes these mistakes. Clients often use systems setup just like this one and are unaware of the problems caused until a negative peak shows up. The appearance of the new, negative peak is this hint that all of your previous data may be invalid and the only reason you discovered it now is because this time the system had to subtract-out so much data from your main peak that it ended up being negative!

Nano technology based sensors for different antibiotics may be created....good idea but you have to point out specific group of antibiotics....

I agree with William.

aCompCor within the CONN toolbox does a great job!

Interesting discussion.

You are quite welcome!

1. PRESUMED IVIVR:

Keeping Product concept in mind Define Target In vitro & in vivo specification (based upon solubility & permeability profile of drug)

2. RETROSPECTIVE IVIVR:

Prototype Formulation Development with level of variation in release rate & its selection by extensive in vitro dissolution testing in MULTI media, Pilot PK study (cross over study on 6 -12 subjects)

3. PROSPECTIVE IVIVR

Define formulation meets in vivo performance (with empirical basis of formulation related rate controlling variables), Define BIO RELEVANT & DISCRIMINATORY media, Confirmatory IVIVR PK study & Define LEVEL

4. FORMULATION OPTIMIZATION & PROCESS VALIDATION Formulation/Process optimization, Scale-up/ Pivotal batch manufacture with Process Validation & Pivotal PK studies (on at least 12 subjects)

5. REGULATORY SUBMISSION PRODUCT REGISTRATION/ REGULATORY APPROVAL SCALE UP & POST APPROVAL CHANGES (SUPAC)

Dear Paul,

I think there is a misunderstood here!

At the current time, I am not a PhD student but I am searching and looking for a PhD study opportunity and I thought you have a project and need a PhD student to supervise and work with.

Regards

Ruqaya

Since tobramycin lacks chromophore group its estimation is difficult using uv detection unless derivatization is done which is again a tedious process. Its better to go for LC MS for better results.

We have differentiated THP-1 cells (cultured in RPMI) using the following differentiation media composition:

RPMI media supplemented with 10 % (v/v) foetal bovine serum (FBS), 25 ng/mL recombinant human sRANKL (Gibco, USA), 200 ng/mL PMA (phorbol 12-myristate 13-acetate

Differentiation time 8-10 days

REFERENCE:

The above differentiation media pushes the monocyte non adherent cells towards osteoclastic adherant phenotype. Slowly you can acclimatize the cells with (50:50 RPMI: DMEM)

For osteogenic differentiation of BMSCs, 10mM Beta-glycerophosphate, 100nM dexamethasome, 0.2mM ascorbic acid in DMEM supplemented with 10% (v/v) FBS.

Differentiation time 14-21 days

For co-culture:

Predifferentiated (day-5) osteoclasts (from THP-1) and predifferentiated (day-7) BMSCs (towards osteogenic lineage) can then be co-cultured with 50:50 RPMI:DMEM (with the osteoclast and osteogenic supplements) till day-21.

Hope this helps.

I mean for Titanium oxide

Dear Sir. Concerning your issue about the Temperature and Emissivity Separation (TES) method. TES algorithm hybridizes three established algorithms, first estimating the normalized emissivities and then calculating emissivity band ratios. An empirical relationship predicts the minimum emissivity from the spectral contrast of the ratioed values, permitting recovery of the emissivity spectrum. TES uses an iterative approach to remove reflected sky irradiance. Based on numerical simulation, TES should be able to recover temperatures within about /spl plusmn/1.5 K and emissivities within about /spl plusmn/0.015. Validation using airborne simulator images taken over playas and ponds in central Nevada demonstrates that, with proper atmospheric compensation, it is possible to meet the theoretical expectations. The main sources of uncertainty in the output temperature and emissivity images are the empirical relationship between emissivity values and spectral contrast, compensation for reflected sky irradiance, and ASTER's precision, calibration, and atmospheric compensation. I think the following below links and the attached file may help you in your analysis:

http://ieeexplore.ieee.org/document/700995/

http://www.tandfonline.com/doi/abs/10.1080/0143116031000115274?journalCode=tres20

https://unit.aist.go.jp/igg/rs-rg/ASTERSciWeb_AIST/en/documnts/pdf/2b0304.pdf

Thanks

It depends on what your compounds are?

Amar,

1. For gradient work use a 5 cm column as 25 cm column is too 'sluggish' in responding to mobile phase changes. In addition never use 100% water in a mobile phase (your mobile phase A). It can cause column collapse (I still believe in 'old' wives tales). Add about 5% ACN.

2. Even put 5% water (Mobile Phase A) in your Mobile Phase B. This will reduce gradient peaks or spikes due to refractive index differences.

3. Inset a sample chromatogram so we all can see it. Use a PDA (scan 200-400 nm) instead of 1 wavelength. Your impurity (related substance) may not absorb at 254 nm.

Thanks Harrie and Isam,

I am aware of the SIL and the different metabolomic apporaches... maybe I did not pose my question well enough. I was just looking a chemically defined Std mixture to validate my LC-MSMS methods, that is, if I inject a 'known' standard, how good is my chromtography and the MS/bioninformatics methods to detect them before I move into more complex biological samples.

I have received an answer directly through a message of another RG member (thank you Biswapriya Biswavas Misra), which I will reproduce below, which I think best addresses my question, just in case someone else is going through this process:

Hello Alejandro,

Hope you are doing well!

Saw your question on metabolomics standards for your laboratory/ core facility I suppose.

To be very honest, as suggested by a couple of people (not from metabolomics background but analytical chemistry), stable isotope labelled standards (SILs) are truly expensive and only used by top notch core facilities and rarely by research groups. Also, they do not solve any problem- except a better quantification- but by no means reliable!

[1] If you can afford and have got some funds, I would highly recommend the MSML Library

MSMLS Sigma Mass Spectrometry Metabolite Library: https://www.sigmaaldrich.com/catalog/product/sigma/msmls?lang=en&region=US&cm_sp=Insite-_-prodRecCold_xviews-_-prodRecCold10-5which is Supplied by IROA Technologieshttp://iroatech.com/page/Mass%20Spectrometry%20Metabolite%20Library%20of%20Standards and if you get the quote (and can bargain!) would be lot cheaper than that from Sigma. This is MUST have for any facility doing LC-MS (and even GC-MS) based routine metabolomics. With 600+ standards one can easily create a library of all generic pathway wide metabolites. All are VERY RELEVANT to all sample types, starting from sugars amino acids, TCA cycle to phosphates and metabolic intermediates. And currently, there is not other company that provides such a huge list. Do not go for their SIL standards as that would be too expensive and needs further software and specific applications. Just the unlabeled 600+ standards in micro well plates would do the job!

So, there are a few Sigma Kits available that I must point to, and they are grouped by what you will looking into in a typical untargeted/ targeted analysis.

[2] ML0010 Sigma TCA Cycle Metabolite Library : https://www.sigmaaldrich.com/catalog/product/sigma/ml0010?lang=en&region=US&cm_sp=Insite-_-prodRecCold_xviews-_-prodRecCold10-1

[3] CAR10 Sigma Carbohydrates Kit :https://www.sigmaaldrich.com/catalog/product/sigma/car10?lang=en&region=US&cm_sp=Insite-_-prodRecCold_xviews-_-prodRecCold10-2

[4] V1 Sigma Vitamins Kit ~98% (Components, TLC) :https://www.sigmaaldrich.com/catalog/product/sigma/v1?lang=en&region=US&cm_sp=Insite-_-prodRecCold_xviews-_-prodRecCold10-4

[5]47264 Supelco Organic Acids Kit (individually packaged), analytical standard : https://www.sigmaaldrich.com/catalog/product/supelco/47264?lang=en&region=US&cm_sp=Insite-_-prodRecCold_xviews-_-prodRecCold10-4

[6] 47266 Supelco Sugar Alcohol Kit (individually packaged in quantities indicated), analytical standard : https://www.sigmaaldrich.com/catalog/product/supelco/47266?lang=en&region=US&cm_sp=Insite-_-prodRecCold_xviews-_-prodRecCold10-3

[7]A9906 Sigma-Aldrich Amino acid standards, physiological analytical standard, acidics, neutrals, and basics : https://www.sigmaaldrich.com/catalog/product/sial/a9906?lang=en&region=US

[8] If you plan to get/ use a lot of Human plasma or such samples, then better to have and compare NIST's human plasma pools available from Sigma: Metabolites in human plasma NIST® SRM® 1950 : https://www.sigmaaldrich.com/catalog/product/sial/nist1950?lang=en&region=US

I mean the choice is yours and depending on the sample types you are expecting. But, I would always go for the IROA kit and se what is missing and then compensate it with the Sigma kits, and for sure have the NIST plasma as QC run always.

Hope the above lists help you focus on a few and go for them. If you got more questions please do not hesitate to ask again.

Thanks and regards, Biswa

Hello: Your question is far too broad in scope and required time to answer. Are you aware of the fact that operating a LC/MS or LC/MS/MS system is very different than using a pH meter? It takes many years of professional experience and practical hands-on use to learn how to properly operate this type of complex analytical instrumentation and develop methods properly. Additionally, each brand and type of system is different, and this requires even more specialized training.

While some aspects of your question are very basic in nature and easy to solve ("how to create the calibration curve"; This involves developing a proper analytical method ad defining the detection settings, then analyzing many standards and plotting them using the software), this is absolutely NOT something that can be taught in a class or offered in a few paragraphs on a website.

Websites such as Researchgate are best for very specific scientific questions and are not a substitute for long-term professional training. Please seek out the assistance of someone local in your organization (e.g. The scientist in charge of operating and maintaining the LC/MS system) or at a professional laboratory to to assist you with theses matters. You will learn much faster in this way.

Dear colleague,

I agree with M. Farooq Wahab

This may be in left field - although not dealing with Metal composition - we are trying to create a measurement tool around Crisis Intervention in the Worplace..... We have developed a validated took..... but don't know how to gain any control data... We would just be utilizing the tool a certain number of days after the incident.... does this relate and have you come across a method that works where you can only gain data one time?

check out my publications on researchgate. Hypercarb column is old tech and needs to inject blank matrix to prime it. I used mixed-mode column (Acclaim Q1) for many polar pesticides (glyphosate, paraquat, ethephon, fosetyl Al, ETU, PTU, Chlormequat and more.

Hi,

You may be interested by this paper related to the Fama French model:

Creative Destruction and Asset Prices.

By: Grammig, Joachim; Jank, Stephan. Journal of Financial & Quantitative Analysis. Dec 2016, Vol. 51 Issue 6, p1739-1768. 30p. DOI: 10.1017/S0022109016000557.

Sujets: Rate of return; Investment risk; Small capitalization stocks; Large capitalization stocks; Assets (Accounting) -- Sales & prices; Creative destruction

Best regards

I would have to say that it is probably contamination in the injector. If you are describing an issue with a mass spec then you could also look to the inlet of the mass spec.

Chiral method development is an advanced technique best suited to chromatographer's with many years of practical HPLC experience. Your method development skills should be strong as should your understanding of the fundamentals of chromatography (as with all chromatography, understanding Kprime, T0, R and S are very important). The most important key is to first identify the correct column for the sample which will retain it with good peak shape (finding the correct mobile phase is the second step).

Chiral method development by HPLC and/or SFC requires the use of many different chiral columns and mobile phases to find a suitable separation.

If you would like to learn about practical Chiral HPLC (and SFC) screening methods for pharmaceutical drugs (for Racemates and Enantiomers), then I would suggest you consult the following articles :

"Chiral HPLC and SFC Column Screening Strategies for Method Development:" http://hplctips.blogspot.com/2011/06/chiral-hplc-and-sfc-column-screening.html

"CHIRAL HPLC / SFC METHOD DEVELOPMENT (Alcohols):" http://hplctips.blogspot.com/2011/04/chiral-hplc-sfc-method-development.html

"Some Stereochemistry Terms (Chirality)" http://hplctips.blogspot.com/2011/06/some-stereochemistry-terms-chirality.html

"Modern HPLC Method Development Tips (PART I):" http://hplctips.blogspot.com/2016/07/modern-hplc-method-development-tips.html

Thank you for the answer, I will ask the tech support. 

Thanks

thank you!

This is too general a question for a web forum, because there are dozens of possible directions to go depending on the available information. For example: Is the sample a "known" or "unknown" compound? Do you know its structure and/or which detection techniques are applicable? Is it a pure sample or mixture? The more information you have, the better the initial screening will be. So first try and obtain information about the sample before using HPLC.

Some initial guidelines for unknowns that I teach professionally would include (these suggestions try and utilize inexpensive and common types of columns and solvents found in most HPLC labs):

These are very general guidelines and follow the 'keep it simple' protocol which relies on running a few simple initial experiments to obtain data, then  add your observations and experience to plan the next steps .

BTW: A two-part HPLC article series on "Modern HPLC Method Development Tips:" (Part 1) can be found at the link below. 

Please email me, Deepak. Would like to talk there and exchange info. Could you also mail the model and make. Email: bbmisraccb@gmail.com

Thanks,

Biswa

Dear Sir, Thanks for  replying. but the papers you sent I already had. And my need is still the same, that is, said numbers of methods in AACC (2003) and AACC (1990).

Sir

Thank you very much for your precised Answer

Thank you very much

Hello,

Which programming language are you using?

You can use MATLAB built in function graycomatrix if your are using MATLAB as your implementation language.

Details can be found in the following link:

If you are using python, you can first install scipy, numpy, and skimage and then use skimage.feature.greycomatrix and skimage.feature.greycoprops.  

Let me know if you have any specific question about the detailed implementation. 

Thanks.

Dear Sir. Regarding your issue about the method to quantify the oxidation of Menadiol into Menadione other than HPLC-MS/MS. Different analytical methods (spectrophotometric or spectrofluorimetric) have been proposed. The following below links may help you in your analysis:

Thanks

Dear Sir. Regarding your issue about how to detect ddNTP by HPLC-ESI-MS The following below links which may help you in your analysis:

Thanks

At small scale the precipitation might not be visible clearly. vigorous vortexing and sonication may also help to solubilize the solute.

Precipitation can be avoided by

1. using solvent in which compound is very soluble (it may not be applicable in your case as you might be working with biological system and organic solvent not allowed)

2. use DMSO (you have already tried but you might not be allowed to use higher percentage of DMSO for the experiment)

3. use Cosolvent / low amount of surfactant/ b-cyclodextrin (this might helpful)

below is attached link of the article.

Microbial population and its activities other than fungi is generally low. So organic matter present in acid soils are not fully humified and N content is low in these soils though the organic matter is high in the the soil.

I don't know that there is a perfect answer unfortunately. I grapple with this a lot myself. It's hard to take ratings during the exertion part of interval training, but I would say immediately after the effort bout, as soon as the rest period begins would be best. Its not perfect but it will give you a sense of how RPE develeps during the session. You could also include session RPE as well, it may even be interesting to see how that compares to ratings taken during the actual effort bout.  Good luck :)

As our friends mentioned above the best place is hand book of chromatography, or better hand book of stationary phase. Obviously you are trying to separate the components of a sample, and you have chosen a correct method, because the polarity of the analyte must be matched with the stationary phase, if not the retention times would be too short. 

Hello: I teach HPLC method development and am familiar with most of the commercially sold as well as 'free' chromatography method development application products available.

The software products mentioned above, by previous posters, is not free.

Free software is available. Example: DryLab is one of the most popular chromatography simulator software application available.

NO known SOFTWARE application can perform truly automated HPLC method development. All require high quality input in the form of data and a skilled chromatographer to function. All of these applications rely, to different degrees, on the input from a very skilled chromatographer to provide initial values for the software to use. Even some of the more advanced software applications such as ChromSword require a great deal of skill on the operator's part to define the types and dimensions of columns used, mobile phase combinations tried and specific methods used for the screening protocols (best for only the most technically savvy chromatography users only). The applications can only fine-tune methods or scout for methods based on a user's input OR the data input from pre-run chromatograms (simulated or real). They are a poor choice for teaching people how to develop methods by HPLC. These programs are just a tool, not a solution. In the hands of a skilled operator, they can be useful. For most users, they are serious overkill and I do not recommend them (BTW: I personally use and like some of the programs, but they are neither free nor the best applications for most users).  For basic and intermediate levels users (99% of all chromatographers) I do recommend the simpler programs such as the basic version of the DryLab application to help teach HPLC fundamentals. This is the area where most chromatographers are deficient and if used with real experiments, these types of programs can aid users in understanding the relationships and concepts needed to develop proper HPLC methods (such as learning about T zero, K prime, Resolution, Solvent % effects and so on). This is a great way to fine tune methods based on just a few initial runs. Most people can start using the software with minimal training and it encourages lots of "what - if" experiments and simulations which I like to see.

More info on Dry Lab can be found at this link: http://molnar-institute.com/drylab/