Increase Confidence in Your Results with the ACQUITY QDa Detector

Increase Confidence in Your Results with the ACQUITY QDa Detector

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Hello, everyone. Thank you for joining today's webinar. It's my pleasure to welcome you to the Material Science Session, as part of Waters Asia Pacific LC Symposium 2021 for accelerating materials innovation with modern separation technologies.

My name is Jen. I'm the Assistant Marketing Manager for Waters LC and MS in Asia Pacific, and I will be the host for today's event. For today's event, we will begin with my colleague Charlotte's presentation to introduce Waters Liquid Chromatographic portfolio. Then we will have our full feature topics from different speakers to talk about the needs, the challenges, and adaptive technology in the different application areas, such as polychemicals and polymers. Before we begin, I would like to draw your attention to one housekeeping item.

On your screen, you can see the main presentation screen. If you hover your cursor to the CC button, you may select the appropriate language subtitle according to your preference. And please take a few minutes to complete the survey at the end of today's event. Now, I would like to pass over to the first speaker for today, Charlotte, who will introduce the current Waters Liquid Chromatography Portfolio. Hello, and thank you for joining us today.

My name is Charlotte Yu, and I am a Separation Business Development Manager at Waters Asia Pacific. As many of you here already know, Waters has launched several new products in recent years with innovative technologies. To start today's session of the LC Symposium, I'd like to start by giving you a short overview of the current Waters LC portfolio. As a scientist, we have concerns every day. Do my samples contain toxic substances? Is our product safe? Is my data accurate? Our goal is to help you, our customers, competently make critical decisions to move your business forwards. From fundamental research, to method development, to routine testing, our integrated robust offering of instruments, chemistry, informatics, and support, addresses your research complexity.

Our chromatographic portfolio provides you with abilities. First one is useability. Our platform support a consistent user experience, whether you are using an LC optical detection for a routine analysis or an LC with mass spectrometry for trace-level analysis. Scalability, our portfolio allows you to scale to meet your needs, regardless of chromatographic form, column, or solvent. For many applications, AC configurations are paired with control models and informatics, minimizing the time being spent from a sample to [INAUDIBLE]..

Transferability, our model [INAUDIBLE] methods would have run from instrument to instrument and left the lab producing the exact same data. As you develop or transfer your methods, you can rely on Waters to keep them fit for purpose as they move from development to routine use. Dependability, our application scientists and global technical support teams answer your questions, have develop methods, and problem solve every step of the way. We are here to maximize your research and the innovations that result. There are many examples of the innovations in LC that we have brought to market in our history as a leader in separation science. The Alliance LC has been the backbone of [INAUDIBLE] for over 20 years.

But there may be no better example of how Waters has enabled scientists to gain competitive advantage than the advent of the Acquity UPLC in 2004. Since 2004, it's addition to the Acquity family has been designed to allow a broader range of scientists to benefit from this game-changing family of products, without compromise what they do. And with this industry following Waters lead, the phase of chromatography has changed. We have also added new specialty separations technologies, such as convergence chromatography with UPC squared, Polymer characterization with the Acquity AP system, and process monitoring with Patrol.

Finally, we introduced several products in recent years to meet customers' needs, and to solve another challenge. The Waters LC portfolio can be broken down in many different ways. So I would like to introduce our portfolio in five overlapping groups.

The first one is analytical LC systems. These systems allow you to conduct your routine analysis with confidence. This group includes the original Acquity UPLC, the [INAUDIBLE],, the [INAUDIBLE],, and the Acquity Premium. These products provide the flexibility for these method development, reliable method transport, with uncomplicated routine analysis.

Next one is LC for MS. High performance-based technology delivered in optimized configurations, the power of the sensitivity, reproducibility, robustness of your meta-analysis. Here, I would include systems such as the Acquity I-Class Plus, the Acquity M-Class, and new Acquity Premier system. Next, we have system solutions. These [INAUDIBLE] proper solutions, like the Patrol system or the Acquity APC system, were designed to address the application of specific workflow requirements, complex metrics analysis, or production environment operating constraints.

We also have super separations. These systems are ideal for those new to separation science. These core technologies, such as Alliance HPLC, or the [INAUDIBLE],, or for affordable, highly reliable, lower complexity systems for routine analysis. And, finally, purification systems, proliferative scale HPLC plays a critical role in applications where compounds must be synthesized, identified, isolated, purified, and characterized.

These systems enable substantial increases in throughput with an added bonus of system robustness. But the chromatographic separation itself is only one piece of the puzzle. We also need to collect, analyze, report, and secure our data to be able to make critical decisions with confidence. Our informatics solutions capture, manage, integrate, and analyze your LC and MS data, improving discovery and manufacturing quality and integrity, and reducing time to market, easily and seamlessly conduct scientific searches, integrate networks, manage compliance, and streamline operations.

You invest a significant capital in your LC system. Why not use the very best consumables? Waters is committed to developing, producing, and manufacturing innovative and consistent products for solving your most difficult challenges. As you can see here, our enabling technologies meet your best standards for performance, reproducibility, and quality, giving you confidence in your analysis. As I mentioned before, Waters has added two new systems to our portfolio in the last nine months, the ARC-HPLC and the Acquity Premier. So, lastly, let's take a look at the benefits of these products.

The ARC-HPLC was released in the summer of 2020. It is built on what is proven technology. Matter transfer could be transferred 14 different labs for different vendor instruments, or different separations platforms.

The ARC-HPLC ideally designed to show equivalent separations between different platforms, not only for Water's instrument, but also other vendors HPLC systems. We can use features such as gradients smart start to facilitate this process, saving time, leading to more productivity and better asset utilization. If you want to improve your productivity and efficiency, this can be performed on an Arc HPLC as well, because of its higher air pressure tolerance compared to the existing Waters HPLC. We can scale the method moving to our 3.5 micron column,

and all performance can be improved. And additional benefits include lower solvent purchase and disposal costs. As the next product, in February 2021, Waters will define separation science once again with the introduction of the Acquity Premier. This system solution integrates consumables, column chemistries. And there is a system to solve some of the most challenging separations, analytical chemistry, with mass spec technology. What are the challenges of separations today? Analyte loss is one of the most troublesome challenges in chromatography that labs experience today.

This problem manifests itself in many ways, poor sensitivity, poor reproducablility, poor recovery, or even missing analyte. Mass spec high performance are [INAUDIBLE] technology that we are applying to the LC system, the column, and the consumables used in the previous solution are designed to improve the sensitivity, increase analyte recovery, improve resolution, and minimize those interactions which lead to sample losses. Finally, as your new products, we are going to introduce another innovative new product based on this premium solution platform. This new system is coming this year, and I'm very excited.

This system will also bring innovation for your success, again. Today, I give you a brief overview of the current Waters LC portfolio. Thank you very much for your time and attention. Please contact your Waters representative at any time if you have any chromatography questions. Waters has a system solution to help you answer the questions with confidence.

Thank you again for listening. Thank you, Charlotte, for the great overview of our current LC program portfolio. Next, I would like to welcome the next speaker, Mr. Shanmukh Patil. Mr. Shanmuk Patil is the Senior Manager in the Analytical Department in Rallis, India.

He has over 28 years of experience in analytical area with GLP/GMP/ISO platform. He is the [INAUDIBLE] not only method development development in HPLC, LC, and MSMS, GC, GCMS, [INAUDIBLE],, but also has a wealth of experience for standardizing analytical method and improvised QC procedures as per ISO norms. He's widely active both in India and overseas in the small molecular field focus on agriculture chemicals and pharmaceuticals. I will now hand over to Mr. Sanmukh Patil for his presentation.

Hello, everybody. Good morning. My name is Shanmukh K Patil.

I am heading the analytical department RICH-Rallis India Limited, R&D, Bangalore. Let me thank the Waters [INAUDIBLE] for giving me an opportunity to present pesticides perspective from a regulatory aspect. Coming to this pesticide understanding from a regulatory perspective, I'll briefly cover about the general definitions of pesticides formulations, and how it is being taken in a regulatory perspective. A few talks on that pesticide residues, and what are the important factors in considering that pesticide is reduced. The model fixation kind of regulated terms. So let us move on to next slide.

Here, pesticides broadly classified as technical and formulations. Technicals are the one which have more concentrated, like more than 90% safe. Formulations are the pesticide which are formulated using technical material. So as a definition, technical pesticide are bulk pesticide manufactured in the concentrated form, and its purity and impurities as per the registration requirement.

Because it has its own requirement to be called as technical. And as far as formulation is concerned, it is a stable combination of various ingredients designed to render the product useful and effective for the purpose claimed, the form of pesticide as purchased by the users. So the formulation is upon the [INAUDIBLE],, like various formulations are available, like WPEC, I will call it in the next couple of slides. So those are the stable combinations. That means the active ingredient present in the formulation should work as is required to be used.

So that is formulations. And the active ingredient is a term, is a biologically active part of a pesticide present to the formulation. So this is a main element which controls the pests. So how the formulating a technical pesticides helps, Because now it is like increasing population we have been witnessing, we have to feed increasing population with [INAUDIBLE],, which have to be sufficiently produced. This is the biggest challenge every country is facing.

So unless we use a scientific usage of pesticides, we can not feed the complete population with food. That is inevitable. A pesticide, you say, is inevitable, but judicial, you see, is the one which is challenging. Because the crop production itself has a lot of constraints, among which, major threat from pests, which completely devastate the crop production thereby it reduces the food production. So pesticides comes in the picture, pesticide [INAUDIBLE] formulations, which scientifically control pests attack on the crops, thereby serving mankind. So this formulation of a technical pesticide helps to optimize the amount of active ingredient at target site.

Because it's directly technical. [INAUDIBLE] use directly to the crop. So it is a diluted one, which is because the active ingredient to very small quantity sufficient to kill the pest. So that it has to be formulated in such a way that only targeted pests are affected. And this also helps to maintain the physical and chemical properties during storage, dilution, and use.

Step one, once formulated, pesticide which would keep its vigor throughout the period of storage. That was the biggest challenge during the pesticide formation. And enhance the biological performance, so formulation which we use, that has to enhance the biological performance, reduce the effects on the environment and non-target organisms.

The target is to kill the pest. So non-target crops are not affected by [INAUDIBLE] formulation. Like when we spray on the pesticide, it should kill off only pests. It should not affect the crops or beneficial organisms.

And one more and the most important aspect is that the production is safe to handle. Because usage of pesticides by large is a farmer. So farmers are not well educated to handle, I think, most of the farmers. So it should be, we have to make a formulation in such a way that should be safe to handle, safe to maintain.

So in formulating such pesticides or formuating a regulated product, having those advantages requires a lot of study and many factors, which influence the formulation type. Not all formulation types are used for all pesticides or all crops. There is a difference between this age of one kind of formulation and the other. So these factors influencing formulation types are physico-chemical properties of technical.

So sometimes, purity matters like that physico-chemical properties, like its density, liquid, [INAUDIBLE]. It is a crystal nature. It is a boiling point, [INAUDIBLE].. So many factors are there, which governs the formulation type. And biological activity and mode of action, how exactly it affects surface, of in the surface, or some-- on the field, on the crop, on the air.

I guess so many mode of actions also. It depends. Method of application, that you are spraying, you are spreading, or granulation, and mixing with the seeds, so the method of application also determines. Safety and the cost, of course, this will be priority basis. We have to consider it. Then market preference, then comes the regulatory requirements.

So once everything is OK, but a regulated requirement is not met in this, it will not be marketed. So regulatory requirements are very stringent, and we have to comply with that at all time. So these are the conventional formulations that I have just listed here. . And that's called DP or D, it is a code. And means the package which you see the product note, in that these are codes [INAUDIBLE] granules, GR or G; soluble concentrate, SL or WSC; soluble powder, SP; emulsified concentrate, ED; wettable powder, WP or WDP.

And the seed treatment, also many kinds of, [INAUDIBLE] DS, LS, FS, WS. Several things which are moving forward, many new formulations are being water soluble granules, water dispersal granules, capsule suspension, CS. These are the new formulations, very new generation formulations, which are very popular nowadays, and ecofriendly, and water-soluble. So many advantages to be, say that content like CS, ZC, and ZE, all these things, those require really meticulous formulation and usage of the active ingredients at a very, very, very calculated.

Suspo emulsion, micro emulsion, oil dispersion. I'm not going to detail it everything, but just to have what the formulation calls, and new [INAUDIBLE] formulation in the market I just listed here. These are now some more list, ultra-low volume tablet, emusifiable gel, and also, [INAUDIBLE]..

So coming to the main aspect, like how we are living with the pesticide, and how it is playing a role in helping mankind. So by and large, pesticides mainly used on crop to target or to kill the pest. Pest could be insect. Pest could be disease.

So pest is [INAUDIBLE]. So it will target on pest. However, the application of the pesticide on the crop or food, or wherever it is essentially used, it will not work completely. There is a residue.

There is a residue of pesticide present on the crop, on the soil, on the water, so and so forth. So ultimately, what happens after this stage, the crop will be harvested and the grains are separated, that grains maybe containing pesticides, water, soil, everything is contaminated. Ultimately, it will be a part of pesticide [INAUDIBLE] that man or the person, all who are using food, maybe intake the pesticides. So that's [INAUDIBLE] residue. By and large, they may be contaminated in the food material.

So that residue, the term residue itself, we have to define, what residue? And how much there should be for the presence of pesticide itself is not a big thing, but how much is present in that it is important. And what is the safety limit of residue, and its management. Because when the consent is that pesticide has to be used to survive, like I told you, just I mentioned that to feed an increasing population, we need to have a pesticide. This is inevitable. So in that sense, how can manage? How we can lower the residue level in the food? That is very important. So for that, many regulatory things are [INAUDIBLE]..

So there are things which are how they are evaluating the toxicity of pesticides, just I'll give you an example how they go by the severity. If the LC50 is-- it means those is less than 50 ppm, parts per million. A very highly toxic pesticide is characterized. [INAUDIBLE] 30's. And 50 to 500 is highly toxic.

500 to 1,000 ppm, moderately toxic pesticide. To 5,000, it is highly toxic pesticide. If it is more than 5,000 ppm residue, then it is practically non-toxic pesticides.

Similarly, it varies with the species, also. In mammal is less than 10 ppm is very, very highly toxic. [INAUDIBLE] is practically non-toxic. So these I want to category, because each pesticide is behaving differently. Not all pesticide behave equally. So there are so many chemistries are involved for certain how the pesticides are being evaluated.

So this is how the classification for the regulated, And coming to the residues, what exactly residues means. We have the concentration of any specified substance in food, the agricultural commodities or animal feed, resulting from the use of a particular pesticide. Due to usage of pesticide after the crop harvest, if there is a residue, that means the remaining remnant of that particular pesticide is called residue. The residue may not be only the parent compound.

It includes parent compounds, its the metabolites, derivatives, reaction products, impurities. So mainly the highlighted part is toxicological significance. The pesticide residue which is present is highly toxic. This [INAUDIBLE] significance governs whether it is highly toxic, or severe, or non-toxic.

For the parent component with it in the residue, metabolite would be the-- sometimes, metabolites will be toxic than the parent compound. [INAUDIBLE] is also possible. Sometimes due to the reaction. So different products are formed, which are toxic again. But these products are found due to this pesticide.

So this is how they're related. And impurities from the-- because if there is going to happen, particularly in the technical materials, that may cause the toxicological significance. So these are [INAUDIBLE] ascertain the residue, due to which and what. It has to be significantly calculated. Then comes the term maximum residue limit.

This is the regulated term, which is very much important in ascertaining whether your dosages or formulation product is safe or not. So by and large, maximum residue limit is the maximum concentration of a pesticide residue. Normally, we express it in the ppm or milligram per kg. They are legally permitted, that is important. Because zero MRL, there are new possibilities.

So legally permitted concentration, where in our food commodities, or animal feed, this has been said by CODEX or National Regulatory Authorities. So certain limits are being assigned. If you go to CODEX site, are a lot of pesticide list with the MRLs fixed with the particular crops, all the categories you can see.

So this is how they have fixed it over the period of time, based upon pesticide nature, based upon crop nature, based upon season. Various factors have been considered, and [INAUDIBLE] that, OK. This is the MRL for this crop [INAUDIBLE].. So maximum residue limit, it should not exceed.

So the models are derived from an assessment of residues found when crop is treated according to GAP. Not all, whatever the field some study that has been taken, and as I said before, the residue, pesticide residue and [INAUDIBLE] very high. That has to be considered. It is not like that. The good agricultural practice, under a good agricultural practice, if the crop has been raised and used as per the dose requirement pesticide, then analyzed. So based upon that, MRL will be fixed.

Consideration of various dead residue intake estimates, and determination of comparative acceptability in this called ADI, We will add a regulatory term to indicate that food complying with MRLs are safe for human consumption. So ultimately, human consumption happens for the food and the materials. So those should be compliant. That's called acceptable daily intake. If a particular intake of that particular food is considered by continuously taking your food, then the pesticide residue is by 0.1 ppm, then doesn't have any effect, and that they recorded it.

So ADI and MRLs are very important terms to the pesticide registration. So normally, MRLs are legal trading limits. These are set by regulatory authorities. I consider it to principle to ensure proper usage of the pesticide, and as a precaution to reduce potentially harmful effects. So actually on the term MRLs are not safety levels, because different countries have different trading levels based on agronomic needs, good habits, usages, climate, so many things.

Many factors go with it. But generally do not have different safety standards. MRLs are not derived from ADI. ADI [INAUDIBLE] term used for the human safety, but MRLs are not derived from API. MRLs are not toxicology, MRLs are not toxicological threshold concentrations at which if they are exceeded, toxic effects must automatically be accepted, theoretical. So coming to ADI, this is the limit of your daily intake of a pesticide that can be ingested over the lifetime of an individual without appreciable health risk based on the facts known at the time, expressed in ppm.

The result milligram per kg body weight per day, is how the ADI is. [INAUDIBLE] So exposure to public residues in food cannot exceed ADI. This is very important. Then TMDI predicted maximum daily intake, theoretical, of pesticide residue based on assumptions of MRL levels of residues in the food, and average daily food consumption per person. They are expressed in the milligrams of residue per person.

Then PHI, that is first harvest thing in this. Number of days which should lapse before harvest after the last treatment of the crops in the field with the pesticide. That means pesticide was sprayed, crop harvest has to be done-- like say if the crop has to be harvested 14 days after last spray of the pesticide, then the amount to be received will be less, like that. A pesticide application and harvest of the crop, very important to ascertain the MRLs.

So actually speaking, MRL and ADI, no direct relations, based on the separate evaluation of a different data. So MRLs are proposed only after establishing off [INAUDIBLE] ADI. So here comes the importance of our analytical tools. Because whenever we say a residue MRL, all these things are based upon the data generated by the analysis.

So the analytical methods to be regulated and validated, those methods which results are accepted. So the reliability of data that is, under which circumstances of this datas are generated, wheteher a GLP, GFP, GAP, like that. So that matters, actually. Then the raw agricultural commodities, also, which [INAUDIBLE]. Processing how the [INAUDIBLE] the storage of the samples.

And a number of field trials is not one or two. So there is [INAUDIBLE],, dosages. Agro-climatic zones also important. And which variety of crop is used for the particular establishment or the MRL? And the part of commodity, that whether it is a grain part, or stem part, or root part, those matters for MRL clarifications. So as we see here, there are limits.

When it was early, it was 0.1 ppm limit of detection. Below it, there [INAUDIBLE] to detect as years increasing. [INAUDIBLE] '60s, 70's, 80's, and 2000. ppb level, we can see now. ppt level also we can-- some ppt level also we can analyze.

So that is how the analytical tools or techniques are developed over the years. So MRLs are very important. So they will all regulatory authorities continuously monitor the MRL levels of particular crop or pesticide based on fresh data, because climate in zones changes sometimes. Sometimes variety changes. So many actors.

So there's a continuous monitoring process, so fresh data is required. Registered users and new users, and analytical methods, earlier conventional methods are there to fix the residue level. Now we have advanced tools so that now it will be affecting the MRL levels. So very, very stringent measures were taken, and fresh information on metabolism toxicity.

There are also figures to review their model storage stability of the component in the commodities. So because pesticide productions with-- they're not the same as, old as 1960s or '70s. So many changes happen, so technical materials are produced in different sources, different reactions. So that also affects the [INAUDIBLE] of the particular pesticide, even though they are the same thing. But that also gives us MRLW, information and processing this also. In India, so almost all registered pesticides have been established for an MRL.

All new pesticides has to get an all registration granted without MRL. MRL is a must. So even all label expansion, label expansion is once the product is released for some crop, we want to ensure that the same product for different crops or different seasons.

So label expansions, that also require MRL fixation. So how do we fix the models? Information on chemistry of the component, which you are going to give; information on short and long-term toxicity, then NOAEL, that's called No Observable Adverse Effects Level, and ADI, the acceptable daily intake. This has to be established information on environmental behavior, metabolism, including toxicity of metabolites.

[INAUDIBLE] All of the original parameters considered [INAUDIBLE] for the metabolites. And it is our recovery studies, it is our field studies location to say so many factors which are important. Number of treatment, seasons, other practices, multi-locational and multi-seasonal residue data. This is very important regulatory agreement. Regulatory authorities keep asking on this, and we have to supply this data.

Trial design protocols for a treatment of a sample, transport, storage, pre-harvest interval, all that are even-- and whenever data have to be submitted. Storage stability of residues in the samples. So any factors are the which are data required for the [AUDIO OUT]. So when it comes to the validation of the parameters, we just briefly know how they classify as whole analytical tools are being used. I believe pesticides are-- most of the pesticides that are volatile and were not in [INAUDIBLE] houses. So the client classification based upon [INAUDIBLE] unstable, [INAUDIBLE] stable.

[INAUDIBLE] for the liquid chromatograph. [INAUDIBLE] is a governing factor, then this general classification GC [INAUDIBLE] are the most commonly used. Again, advanced technologies improving the HPLCs performance, like UPLCs. And GCs are the [INAUDIBLE] mass spectrometer. So those are governing factors in establishing their model. So in our R&D, we use-- we have a lot of HPLCs.

And our Waters ARC-HPLC and we LC-MSMS [INAUDIBLE],, which are being used to assess the pesticide content in the matrices are more reliable and rugged in such a way that it improves the confidence level on the analytical results delivered from these systems. So we are very much happy to use such systems which are giving us a confidence in the level results. Those sensitivity and reproducibility are very important parameters in getting accurate results. So those are complied [INAUDIBLE].. So coming to the registration in India, so overview, I'm just giving overview. So identification or screening of the new product, the product being registered, then product will be selected.

Then it has to be added to the schedule. The schedule comes until this one. Then we have to take a permit for the import cases, and we have to do the general data registration. Then quality [INAUDIBLE]. Then online data submission is here. Now, it is all data submitted online.

Those datas are screened by [INAUDIBLE] and RC, and reviewed by scientific experts. If they need any clarification, they write back with a query. Then we have to do the explanation on the queries, and, again, submission of deficiency answers for applicant. Then before that, [INAUDIBLE],, along with the data, we have to submit a sample.

And one of them is product, and analytical report, also. So [INAUDIBLE] also analyze the samples, that products for its active ingredient. Then a review of [INAUDIBLE] happens, a committee will be formed. That final assessment of inclusion of that product and approval as I said, we have too fixation of the MRL, the grant registration, [INAUDIBLE],, then launch of the commercialization of the product. So in between, finally, we have to take the [INAUDIBLE] clearance and the production of the product. And a pesticide comes [INAUDIBLE] modification.

[INAUDIBLE] for a regulatory body, which is fixing the MRL. So this is how it goes in the process. Because the data was to be submitted for one product, So a a lot of toxical data, plus crop production data has to be given.

So it takes several years, sometimes more than 5 years to 15 years. Some products are registered even after 7 years, 10 years. So that depends on the pesticide, what we have [INAUDIBLE]. So briefly, so what we can say is we in RICH-R&D is what is the systems are here, excellent confidence in releasing the results, and thus getting no QC query from the regulatory authorities on submitted results. We're extremely happy to have Waters HPLC LC-MSMS in our laboratory. Residues are inevitable byproduct of pesticide use.

The fact that they are found at all is only due to the significant advances in analytical chemistry. Because analytical tool which specifies this is the schedule of this particular pesticide. This is a particular pesticide metabolite.

So that is very important. The tests are now so sensitive now that the detection level that can be reached and is equal to detecting one teaspoon of salt in 1 million gallons of water. So this is considered to have been achieved here with the various companies analytical tools. Levels even lower than that can be detected. The mere presence of a trace amount of a pesticide does not mean a product, or water, or food, is unhealthy. So the toxic significance has been established to [INAUDIBLE] at a minimum level and maximum level, and so many parameters.

That determines whether it is safe or unsafe. So this is how the pesticide and pesticide residues are being highlighted in our present scenario. And thank you, once again, for giving me an opportunity to present on pesticide perspective on the regulatory.

I thank Waters and [INAUDIBLE]. Thank you, one and all. Thank you. Thank you, Mr. Shanmukh Putil for the informative talk

regarding the regulatory requirements in pesticide development and manufacturing. So our next presentation is entitled of expanding detention capability in SEC and APC. Online SEC-ICP/MS and APC-ICP/MS hyphenation. This talk combines SEC and the APC couple with [INAUDIBLE] in order to do polymer containing heteroatom characterisation. I'm happy to introduce the speaker, Dr. Miroslav Janco.

Dr. Janco is a Senior Research Scientist, and a polymer separation [INAUDIBLE] using the [INAUDIBLE] science team of the co-R&D organization at Dow Chemical. Currently his research is focused on the separation and [INAUDIBLE] characterization of polymers and of particle in the molecule level by HDC and LC, including SEC, HPLC, LC [INAUDIBLE] models, using conventional and enhanced detections, as well as a couple and the hyphenated tactics.

His latest contribution to the field of polymer separation and characterization is development of ultra-high pressure size [INAUDIBLE],, which is more commercialized as an advanced polymer chromatography system by Waters. I will now hand over to Dr. Janco for his presentation. Thank you for joining this talk.

The topic of my presentation today is enhancing detection capabilities inside the exclusion chromatography and advanced polymer chromatography, shortly SEC and APC. And more specifically, I will cover online hyphenation of this size-based separation technique, with inductively couple plasma mass spectrometry, shortly ICP/MS. Before I start, I would like to acknowledge my colleagues Betha Snow and Patrick Fryfogle for their contribution to this work.

In the course of this presentation, after a short introduction of polymer heterogeneity and liquid chromatography modes, I will cover APC as a way to address the continuous demand for short analysis times, better resolution, and improved precision. I will also demonstrate that increase, obtaining information about analyze sample when multiple conventional detectors are employed. Next, I will present the example of both SEC-ICP/MS, and APC-IPC/MS hyphenations, and demonstrate that these are effective approaches to determine not only molecular weight, molecular weight distribution, and dispersity of analyze polymers, but also to determine the amount of heteroatom containing monomer, and its distribution within molecular weight of analyze polymers, thus increasing information knowledge about analyze samples. Let me start with the statement that only mother nature can make uniform polymers.

Man-made polymers are dispersed materials distributed in more than one direction. In addition to a distribution in molecular weight, they show distribution in chemical composition, in groups, tacticity, and molecular architecture or topology. To process all these distribution with the single analytical technique is a very challenging task, if not impossible one. It's clear that the simple analytical method cannot provide all the required information.

Methods which do not involve any separation such as osmometry, light scattering, viscometry, or even NMR spectroscopy, generally yield only an average of the property to be determined, such as molecular weight, chemical composition, and functionality. When information about the distribution of these properties is required, a separation step has to be included. Liquid chromatography its different chromatography modes depicted in this image are often used. SEC and APC are chromatography techniques that are most often used to determine molecular weight distribution, while HPLC and UPLC are most often used to determine chemical composition distribution.

Liquid chromatography at critical assertion point is used to process end functionality and tacticity distributions, as well as molecular weight distribution or one block in block or polymers. To further enhance the richness of information on our analyzed samples, still within one chromatography graph, a coupling of two different chromatography modes is required. We refer to that as a 2D LC. This topic is out of the scope of this presentation, but it's covered by one of my colleagues presenters in this section. In my presentation, I will adhere to single chromatography mode, specifically SEC or APC, and the richness of the obtain information about analyzed sample will be achieved through the coupling of multiple conventional detectors, or employment of enhanced detectors. The representative list of detectors used in liquid chromatography that are divided into two arbitrary groups labeled as conventional and advances is presented on this slide.

Enhancing the richness of information about analyzed sample can be easily achieved by combining several conventional detector, such as RI, UV, light scattering, and viscometric detector. By using detector from advanced group, or by proper combination of detectors from both groups, as it will be demonstrated in the course of this presentation. It has already been several years since SEC scientific community is taking advantage of APC technology. APC is application technique for size-based separation of polymers using columns packed with sub 3 micron, rigid, high pore volume, hybrid particles, combined with fully optimized low dispersion Acquity LC system.

APC is superior to SEC in speed, resolution, precision, and last but not least, sustainability. Typical SEC run can take from half hour to one hour, while APC analysis can be finished as short as in several minutes. In terms of resolution, I believe that presented figures speak for themselves, demonstrating much higher resolution power of the APC. In terms of precision, RSD below 1% can be achieved using APC technology, while RSD as 5% to 10% is generally acceptable for SEC. Let me just say that at the early stages of APC development with only UV and LSD detection option available, APC technique deliver only a limited amount of information about analyzed samples. However, today, the detection capabilities in APC are equal to those in SEC.

And the information revealed about analyze sample is significantly improved, as it is demonstrated on the next slide. The informational richness improves when we start to couple two or more conventional LC detectors together, as depicted in these setups on the left, where APC is combined with the set of four detectors, RI, UV, light scattering, and viscometer. Delivered result is not only a relative molecular weight, but also absolute one.

Plus information about the hydrodynamic radius, radius of gyration, and the branching, or conformational information. And in some instances also, chemical composition distribution within molecular weight that is shown in those figures on the right. All that, with the significantly shorter analysis times due to the speed delivered by APC technology. While the obtained information about analyzed sample is quite impressive, in some instances, it still might not be sufficient.

So in the next part of the presentation, detector from enhanced group of detector, specifically ICP/MS, will be employed with attempt to further increase informational richness about analyze samples. Let me also make clear that ICP/MS detection is feasible for substances that contain elements. I will refer to them as a heteroatom in the course of a presentation, that are detectable by ICP/MS. These heteroatoms are highlighted

in this table. There is a vast number of small molecules, monomers, and corresponding polymers, and copolymers. Several of these structures are also shown on this slide that contain the heteroatoms detectable by ICP/MS. And there is also great interest to know the fate of these heteroatom containing moieties when incorporated into polymer chain, and in fully formulated products.

When the information about the total amount of the heteroatom is only required, spectroscopic techniques such as XRF and ICP/MS alone are most often used. However, the result is only average value. So when the distribution of the corresponding heteroatom containing moiety is needed, a separation step has to be included. Our current state of the art online SEC and APC ICP/MS set up is depicted in this slide. We utilize Waters ACQUITY APC separation model to achieve desired separations.

And the key component is the ICP/MS instrument as an online detector. In this particular case, Agilent's 7700 series ICP/MS instrument is shown, and utilized as a very sensitive and highly selective online detector, with a highly dynamic range capable of detecting all elements from lithium to uranium, as it was demonstrated in the table on previous slide. So far, we have demonstrated successful utilization of ICP/MS online detector in HDC, SEC, and HPLC separation modes, and most recently also in APC mode.

In terms of compatible solvent, both volatile aqueous buffers and a wide range of organic solvents were confirmed to be compatible with ICP/MS detection. On this slide, let me briefly describe ICP/MS detection. Column effluent, actually our sample, is continuously pumped into nebuliser, where it is converted into fine aerosol with argon gas.

The fine droplets of the aerosol are separated from that larger one in the spray chamber. The fine aerosol is transported into the plasma torch. In plasma, at high temperature, atomisation and ionization of the material take place. Subsequently, the ions are extracted into mass spectrometer, where the elemental composition of the material is determined. Now I would like to present a couple of examples of a combination of the resolving power and speed of size by separation techniques, both SEC and APC, with the high selectivity and sensitivity of ICP/MS, made significant contribution to the discovery of unknown information that helped more customer project forward.

First example is a separation and characterisation of heteroatom-containing polymer by SEC-RI-ICP/MS hyphenation. Effluent from SEC column is split and monitored by refractive index shown on this slide, and ICP/MS detector on the next slide. Using SEC charge of analyze samples, and the proper calibration curve, a relative molecular weight data, and fraction below 1,000 and 500 can be determined.

While the first 5 lot of analyzed materials are consistent in terms of molecular weight, lot number 6 depicted here in cyan color show lower molecular weight, and has been identified as an outlier. SEC charts of the analyzed samples as detected by ICP/MS detector are shown here. There are three distinct peaks on the chromatogram. Peak number 1 is assigned as heteroatom response in the polymer.

Peak number 2 is labeled as a residual, or unincorporated heteroatom-containing monomer. And peak number 3 is unintended heteroatom-containing byproduct. Using calibrational curve of heteroatom-containing monomer on the upper right, the total amount and the distribution of the heteroatom-containing monomer can be determined within polymer molecular weight, and is summarized in table below. It's clear that twice as much of heteroatom-containing monomer was charged in the sample 5. By combining information from both reflective index detector on previous slide and ICP/MS detector on this slide, two of six analyzed batches where identified as outliers.

Next example is even more challenging one. There is a need to characterize heteroatom-containing component in fully formulated system when component of interest shown here in the red trace, quite a lot with other formulation components, shown here as a black trace, and is the level in the formulation is low in the range couple of percent, as shown by the blue trace. In other words, this is an example when we are looking for a needle in a haystack. Shown here is a overlay of the SEC chart of the need heteroatom-containing component, blue chart; and the same heteroatom-containing component in the fully formulated product, red chart; as detected by ICP/MS detector.

Due to high sensitivity and selectivity of ICP/MS detector, a challenging task become quite a straightforward one. Broadening of molecular weight distribution of heteroatom-containing component is revealed with the high sensitivity. It is hypothesized that the heteroatom-containing component is undergoing chain scission and chain recombination during formulation process, resulting in the presence of a low molecular weight and high molecular weight species. A summary of our analysis of several lots of fully formulated products by SEC-RI-ICP/MS hyphenation is shown on this slide. While no difference can be revealed by refractive index detector, see insert in the top right corner.

The ICP/MS detector reveals significant differences in the level summarized in the table on the left in molecular weight and molecular distribution of the heteroatom-containing components, which correlated well this process variables. The disadvantage of that SEC ICP/MS approach is a long analysis time, usually 30 to 60 minutes. That become an issue when using solvents that char, or carbonize a lot, causing a drift in ICP/MS detector signal. ICP/MS detector drift can be minimized, and even completely eliminated by utilizing APC technology, which allows to achieve the size separation in the significantly shorter time. On this slide, four batches of heteroatom-containing polymer were analyzed and compared using APC, is the analysis time as short as 12 minutes. Another advantage of using APC is that the range of the applicable solvent is greatly increased due to [INAUDIBLE] column and liquid chromatography system back pressure limitations.

Due to a short analysis, time, the ICP/MS detector response stays linear over a wide range of concentration, as demonstrated by calibration curve on the left. And the amount and the distribution of the heteroatom-containing monomer can be easily determined and compared. Three batches from different windows are quite consistent in the amount of heteroatom-containing monomer, but one batch shows significantly lower amount.

APC ICP/MS results were validated by independent technique, XRF, and good agreement between APC ICP/MS as the result, and the result obtained by XRF is demonstrated. On this slide, I would like to address a potential concern of peak broadening caused by ICP/MS detector. Overlay of SEC chart of heteroatom-containing standard, covering a broad range of molecular weights, as detected by refractive index and ICP/MS detector is presented. Detector responses are high normalized and appear as a single chart, despite the fact that there are lines. One smooth one is for refractive index detector, and second more spiky one is for the ICP/MS, suggesting no, or minimal peak broadening caused by ICP/MS detector. With that, I would like to conclude that SEC and APC using conventional detectors, including RI, UV, MALS and VIS, are very robust and cost-effective analytical tools, but provides somewhat limited information on analyze samples.

We have demonstrated that SEC and APC hyphenated to ICP/MS detector deliver significantly richer information on analyze samples, but add to the complexity of experimental setup and data processing, resulting in a longer turnaround times and higher cost. Further improvements in both instrument and software technologies are required to minimize the complexity of the experiment using advanced detectors, so that SEC and APC hyphenated technique become routine analytical approaches for polymer characterisation in both academic and industrial settings. Last but not least, I would like to acknowledge the support from both Dow and Waters, and thank you for your attention. Thank you very much Dr. Janco for the great talk on hyphenated SEP-MS for SEC and APC. Next I would like to welcome Dr. Bastiaan

Staal to the next presentation in the title of Recycling Gradient in Polymer Chromatography, a New Approach in HPLC on Polymers? It will focus on interactive chromatography and how it could determine the chemical composition distribution of polymers. Dr. Staal is a polymer chemist who finished his PhD in 2005 in the University of Idaho in polymeritization by [INAUDIBLE]. Since 2006, he's a lab leader at BASF, responsible for GPC and HPLC of polymers for the Center for Research at BASF.

His main interest including developing alternative methods in data processing for both HPLC and GPC. Searching for stable conditions for performing proper GPC, unraveling multi-MS data of copolymers, and the hyphenating techniques such as GPC-IR, and pushing the boundary of 2D chromatography to it's limit. I will now hand over to Dr. Stall for his presentation. Good morning, or afternoon. My name is Bastiaan Staal, and today I would like to present you something about recycling gradients in polymer chromatography.

Is this a new approach, or is this just another curiosity? Before we're going to dive into all kinds of details, I would like to take you on a journey from a perspective, like what is it, why we are all doing this? And one of the main goals of characterization is understanding one of those fundamental relationships, which we always like, to the picture, the chemical, the physical, and of course, the application and how do they interact with each other. Forget about the picture on the left. Unfortunately, things are more complicated if you want to understand the overall application properties. It is not just linear combination of chemical and physical properties.

But first of all, we need to characterize our samples. And with the characterization methods we use for polymers, we are basically close to the chemical properties, which we can describe, whether it's NRM, or HPLC, or GPC. If you want to know more about the physical properties, we have to use other techniques. Nevertheless, in many cases, understanding why application is performance good or bad, it is simply defined by the difference between good and bad performance as samples.

And that is the main task of characterization, to see if it can distinguish between good and bad performance. So why is it then so difficult, and why are we talking so much about the characterization? So what type of do we have then? Well, there are many types of different characterization techniques we can use for characterizing polymers. SEC is just one of them, or GPC, but there are many more. The problem is that there is not a single technique who gives the full picture of a polymer and characterizes it at once.

So what is it then that it makes so hard to characterize polymers? The problem with polymers is that they are never single distributions. They are distributions and distributions. Because think about the length of polymers, they can be long or short. They can be different in chemical compositions. That is already a challenge. And they can be different in topology, for instance, branched or not branched.

And all those properties together form our final product, or a final polymer with its characteristic properties. So as you can imagine, it is impossible to separate all those distributions simultaneously. And especially for industrial polymers, this normally consist of many more different components. Today, I will only focus on the characterization methods where we separate polymers. Because only by separating, we might have a chance to understand those underlaying distributions. So what I are then the standard methods for polymer separation, because that's where we need to go to.

The GPC, separation to size is definitely one of them. Interactive chromatography is another very important. And there are many more, but those two are the main ones if you're talking about polymer separation.

And all those methods require that polymers can be dissolved. So I will not talk about polymers in a solid state. But we need to dissolve polymers, otherwise if they can't be dissolved, all those methods cannot be applied.

So that narrows down our methods which we can use, to some extent. And one of them already addressed is GPC, or SEC, to determine the volume-mass distribution, or actually it's the size which we measure. And the other one, interactive chromatography, is one of the systems where we can determine chemical composition, or n groups for all kinds of things.

And today, I would like to focus on the chemical composition distribution. So before we can continue, I need to make sure that you understand the principle of interactive liquid chromatography. I try to put a scheme here where green is the non-solvent and red is the solvent. Then at t0 we inject on top of our column our sample with precipitate, or absorbs there and doesn't move. And while the time increases, we move from a non-solvent to a solvent.

Then our peak starts to move, and elute in a particular order until everything is out of the column. It is based on the fact that we are weakening the interaction with the column, as we are moving to a much stronger solvent. In this sheet, we see an example of how separation according to chemical composition could work.

In this case, we use a gradient from acetontrile to THF, where we separate a copolymer SAN called styrene acrylonitrole. With the time we increase the solvent composition from, let's say, to a nitrile to more THF. And we see that [INAUDIBLE] from our system, it contains more acrylonitrile. This was measured offline. It's used as a collaboration curve. That's why you see those four red peaks in there.

The green sample was a commercial sample, which was used for comparison to see to what content this acrylonitrile was in there. So this shows the ID of how we can use interactive chromatography for separating according to chemical composition. But remember, this only works if there is no molar mass dependency.

And to prove that is what we see in the next slide, by doing two dimensional chromatography. If we combine our size exclusion methods with our interactive chromatography methods, we can actually see that this separation is not completely molar mass dependent. Especially in the low molar mass range, we see there is a strong molar mass dependency in the HPLC separation which we are doing. On the right, we still see the same picture as we see on the slide before. And, remember, the part which is marked in red where the green curve goes down, that is actually based to the molar mass dependency.

You can also see it directly in the two dimensional picture. Because if your molar mass should be independent on the chemical composition, it should not have a banana shape, but it should become horizontal. And they'll bend it down. So that means, if we go back to another example here for polyethylene glycols, which are lower molar mass, we see this effective even much more pronounced. What we see here is an HPLC separation, but from a homo-polymer.

On the left, we see the very low oligomer [INAUDIBLE] polyethylene glycol. And the three one to the right are standards with an increasing molar mass. So this is all nice and good, because the separation looks very nice, but this is actually not what we want if we want to separate for chemical composition. We want to diminish this molar mass effect. Actually, we want to don't have any molar mass effect at all, because it only jeopardize our way, how we have to look to our distribution.

So congratulations to the people who made it so far, because now we finally can define our aim. Can we, indeed, diminish those molar mass effect of an HPLC separation for polymers? That is a very good question, and it took me a long while to think what we can do about it. So here comes the ID.

If we use an infinitely long column in such a way that each polymer molecule have enough time to reach its equilibrium state within a gradient, we might receive [INAUDIBLE] reached our target, because we do not have a molar mass dependency anymore. Wow. That's really a mouthful. So how does this work then in practice, if it works at all? Let's first have a look to the experimental setup. What we see here is that we use two columns, column 1, and 2, ordinary GPC columns.

And they are switched in such a way that if we put a gradient in, we can recycle it. And I will show you on the next slide how that works, because it's a bit difficult to read it from such kind of pictures like. So let's start at the beginning where we are just doing an ordinary chromatography, in this case interactive liquid chromatography. On the left picture, we see column 1 is going to be filled with the gradient from our pump. Remember, green is the non-solvent, and red is the solvent.

Starting completely filled with ACN, and we see that column to the right is red, it's coming in our column. So our column 1 is going up in solvent composition. If you follow all those arrows, you will see that column 2 still remains green and is completely full with acetonitrile.

So that is what happens at the beginning, at t0. Then we move to the right, and then we see something that at the end of a certain time, nearly all the gradient which I've put in at the first column at the beginning is simply flushed through the first column and parked or moved to the second column. You can also say like, you simply moved the gradient from the first column with time to the second column. That is correct.

And we notice that our pump has moved back from the gradient from our good solvent to our non-solvent very rapidly. And that's why we see this gradually changing of color very rapidly from red to green in our first column. So in this way, we have parked our gradient into the second column. Note that there is a UV detector between column 1 and column 2.

If you follow the lines, you will find it out. But the summary here is, and that is very important, that we parked our gradient from column 1 into column 2. So and now the fun can start. Because what we are doing now is we are switching our valve from position A to position B. And what then actually happens is that the gradient, which is now in column 2, is starting to enter again the beginning of column 1.

If we look to the picture on the right, we can see that after a certain time, the entire gradient from column 2 has moved back to column 1. And as you can imagine, this can be repeated all over, and all over again. But before we can continue this, we have to put back our valve, of course, in position a. And then it starts where we were two slides ago. In this way, we can repeat our gradients, parking it from column 1 to column 2, and visa versa. Remember that the time, which is needed, is what we later will call the cycle time.

And another aspect we should not forget is we need to move our gradients from, in this case 100% A to 100% B, within the column volume of the column we are using. So let's have a look at some results, because maybe it's a bit difficult to imagine. Every time you switch, it reminds me about a 2D chromatography. And that is actually how those data, the raw data, these looks like.

On the left, we see the UV detector, which was in between those columns, if you remember. And that just picks up, in this case, some signal of some polystyrene standards. On the horizontal axis, we see the time for the number of cycles, if you like. And every time there is such a black line, we are switching the valves from one position to the other. Down to the right, you see a magnified part where you can see the gradient showing you how stable this gradient works. Another way of looking at those kind of data is to plot it in a 2D plot.

And that might help to visualize, actually, if peaks are stable or not. If we are looking to this dataset as a 2D dataset, then it looks like this. And the first time when I saw those results, I got very disappointed. And the reason why I got very disappointed is because I was expecting that all those molar masses would migrate to a single point. Remember, I wanted to create a method which could measure molar mass independent.

And even with those columns and very long run times, lower molar masses still seems to move, didn't became constant, and those higher molar masses never reach a single point. So besides the fact that those measurements took an ugly long time-- I mean, 250 minutes. That's by far too long for industry standards-- it still did not merge to a single point. So I should came to the conclusion here that this method failed.

So that triggers the question, what did I do wrong, or what is it that I need to change? Well the previous column we were measuring on a silica column. That is probably the wrong type of chromatography for those kind of things. So we changed to a C18 column. Using a bit of smaller dimensions, then the measurement would not take so long, and therefore we could do a gradient a bit faster, in three minutes instead of 12 minutes. Again, down, we see the same picture as what we saw before. Every black line is simply the switching valve moving from one position to the other position.

Now we see our control plot, our two dimensional chromatography plot. And one of the things immediately should get your attention is the low molar mass pieces to the left, the molar mass of 2,800 grams per mole. With each cycle, it is moving to the right. And, indeed, after a certain number of cycles, all those individuals tend to line up in a single line. And that is exactly what we wanted to achieve here, the separation where we can measure molar mass independent. I think some of you might get a bit confused, and might wonder, I'm missing the whole picture, what was this all good for? So let's go back to our comparison, what we normally need to find those conditions.

In liquid chromatography, we have a name for methods which we use if we measure molar mass independent to so-called HPLC under critical conditions. And here we see an example of how those critical conditions are found from HPLC method where we are running isocritical conditions. That means, for instance, to the curve to the right, if we use 45% of acetonitrile on this type of columns, we see that the PEG 600 elutes just before the PEG 4,000, the red and the pink color. We also know if the higher molar mass elutes after the lower molar mass, we are clearly in the HPLC modus. If we didn't increase our content of acetronitrile, so we are increasing our solvent strength, because a non-solvent was there, water. We are moving back into the GPC modus, and that is here where the blue curves presents.

By doing trial and error, you can find that for this particular column, that at 45.8% acetonitrile, you have reached the critical conditions, hooray, hooray, indeed that 604,000 are eluting at exactly the same time. And the moment you achieve that, you can clearly say, OK, I found the critical conditions for my system at those temperature.

As you can imagine, it is laborious to find this critical point. And now comes the fun part. By using this recycling gradient methods, we end up exactly at the same critical conditions. The only thing what we need to do is recycle our gradients. So although we are still running a gradient, we will reach exactly the same critical point as what we do otherwise by trial and error offline. So this means that if I have to compare those methodologies, I know which one I will choose, the one to the right.

Because I don't need to look exactly for those conditions, because by cycling this gradient, it will find its critical conditions by itself. The only thing is, I don't know how long I need to cycle. And that brings my presentation to my conclusions. I've shown you today that a recycling gradient is a potential method for reducing the molar mass dependency in HPLC separations.

I've only show it for a homopolymer, And I cannot show it for a copolymer, but that's something I hope to in the future. Playing with a relatively simple setup as I've been show, it gained you a lot of insight into separation mechanisms. We could clearly see that the same separations on the silica column did not work, then we can also clea

2021-08-23 21:19

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