Online Webinar: Surface Area of Pharmaceutical Ingredients at Real World Conditions by IGC

Hello everyone and thank you for joining today's webinar with Surface Measurement Systems. My name is John Dowd and as the Head of Marketing for Surface Measurement Systems, I'll be the host of today's webinar. The topic we'll be discussing today is Surface Area of Pharmaceutical Ingredients at Real World Conditions by Inverse Gas Chromatography and we're very lucky to be joined today to discuss this topic by Dr. Daniel J. Burnett. Now, just to let everyone know, there is a live Q and A taking part with Dr. Burnett at the end of today's session and you can take part in this in one of two ways. The simplest way would be to submit questions in the Questions tab in the webinar control panel that you see to your right, but we understand that some questions may be multi part or be slightly too long to type up. So please do raise your hand which is another function of the control panel you can use. And when I see your hand is raised during the live Q and A, I will give you the opportunity to unmute your microphone and ask your question to Dr. Burnett directly. So once again you can either submit the question in writing in the Questions tab or simply raise your hand and I will let you know when you can unmute your microphone. So without further ado, I would like to introduce Dr. Daniel J. Burnett, the Vice President of surface measurement systems, NA and one of our resident experts in IGC. Dr. Burnett is located in the North American headquarters for SMS in Allentown, Pennsylvania. His team works on research studies and science applications for many prominent companies. He received his Bachelor's in Professional Chemistry from Eastern Michigan University in 97 and his PhD degree in chemical Engineering from the University of Michigan in 2001. Dr. Burnett has co authored and authored over 25 papers in peer reviewed journals and presented at numerous national and international conferences. So without further ado, I would like to pass over now to Dr. Vinat Tabidlik to begin today's presentation.

Thanks John, Appreciate the introduction and for the nice introductory video to Inverse Gas Chromatography and the igcsea. So I will very briefly talk about the technique you know how to measure surface area by inverse gc. But the bulk of the talk will be some case studies and why you might want to measure these at real world conditions or process and storage and control relevant conditions as opposed to cryogenic temperatures and vacuum conditions which is typically required for most traditional surface area methods. So we'll move forward and just to remind people of the principle for Inverse Gas chromatography as the the Video showed the sample is typically placed into a glass column and that is where the sample is prepared and packed. And then we elute a probe molecule with known properties and essentially measuring the retention time. So how long that probe molecule takes to elute through the column is related to the retention volume and then that is related to a wide range of physical chemical properties that can be determined of the solid phase. So inverse GC is a technique that is not new. It's been around since the 1960s. Here is just showing a histogram of some IGC publications as noted by Google Scholar. If you do an inverse gas chromatography search, you'll see over the last 20 to 25 years a relatively steep uptick in the number of publications and just marking some notes. Prior to roughly around 2,000, these were all home built systems. So converting a traditional GC into an IGC Surface Measurement Systems in 2000 came out with the first commercially available inverse GC instrument that was based off of GC platform. But then in 2009, sorry, my arrows must have shifted. They're not directly related, but the timeline shows that's when we came out with the current igcsea, which was the first purpose built from the ground up inverse GC instrument and just a flow path. And as the video showed, you load the samples, you load your solvents, and then the instrument does the injections and the measurements from there. From that point moving forward, a wide range of different powders or granules or fibers or films or different sample morphologies can be measured. And there's lots of options to do all sorts of different types of materials. So the inverse GC and the IGCSEA can measure a wide range of physical chemical properties. Excuse me. As the alluvial diagram shows here, these are interconnected very often with both surface modification, research and development, quality control, formulation and composites. So what we're going to focus on today is BET surface area. Although we can measure things like heat absorption, surface energies, acid base solubility parameters and whatnot, today we're going to strictly focus on measuring the BET surface area of pharmaceutical materials. So in order to measure the BET surface area of a material, you need to measure the adsorption isothermic. The BET method is typically these are the IUPAC classifications of isotherms on the graph on the left. In order to apply the BET model, you need to make sure that the isotherm is the right type. That's typically a type 2 or a type 4 isotherm, which is where the molecule will Form a monolayer and then grow layer by layer. So although the BET equation is widely used on a wide range of isotherms, it's very important that the BET model is only applied to the right isotherm shape and isotherm type. In theory and in practice, any gas or vapor can be used to determine the BET surface area. As you can see here. As long as it forms the type 2 or type 4 isotherm, you can use traditional adsorbates like nitrogen, argon and krypton. However, you certainly can use other molecules like water or vapors like hexane or octane or benzene. As long as you know the cross sectional area of the probe molecule, then you can determine the BET surface area once you measure the appropriate isothermic. So this is well established and part of ISO guidelines that it is not necessary to only use gas orbates like krypton or nitrogen or argon at cryogenic temperatures. You can do these experiments at room temperature or at or near room temperature using different vapor molecules as opposed to gas molecules at cryogenic temperatures. And just as a note, there is an international standard and all three volumetric techniques, gravimetric techniques and carrier gas techniques are applicable and able to be used according to the international standards. So most traditionally or most commonly used and probably most widely used is the volumetric nitrogen absorption technique that is done typically at liquid nitrogen or cryogenic temperatures. And you can measure using nitrogen or krypton or argon. For different applications, you can measure an isotherm. From the isotherm you can get the BET surface area. Likewise, you can do it gravimetrically using an instrument like the DBs that SMS also makes. And this is typically done at atmospheric conditions, so not under vacuum, and again typically at room or at or near room temperature. The carrier gas method is the inverse GC technique. This again can be done at room temperature. We typically can get away with a smaller sample amount, so it makes it more applicable to lower surface area materials and also materials we might not have a lot of sample. The degassing is done in situ, so you pack a column, you can outgass the sample in situ, you can measure it at controlled temperature and which I'll show later, a relative humidity condition. So I can not have to do these only at cryogenic temperatures and under vacuum. Just to show some general applicability of the different bethods. On the top, we have the scale going from low surface area materials, around 0.1 meters squared per gram up to higher surface area materials, you know, 1400 meters squared per gram and above. So the DVS does a very good job across a broad range. You know, maybe it could stretch this down to about a half a millimeter squared per gram. But you might challenge to get very low surface areas because you need a lot of sample and you can't get a lot of sample on the sample pan. Nitrogen absorption technique, if you're using nitrogen, typically around 1 meter squared per gram is where the RSD becomes a bit challenging. And they can be difficult to run. You can use krypton or argon. Those get very expensive to run as well. But they do have limitations. The IGC's kind of sweet spot is, let's say, between 0.1 meters squared per gram up to about 150 meters squared per gram. Above that point, it does become a little bit challenging by igc. So there are some limitations that won't work for all materials, at least in the pulse method that I'm talking about today. But certainly by frontal method, you could get to higher surface areas if necessary. But this is where IGC kind of fills in in a nice sweet spot of, you know, let's say 0.1 meters squared or below, up to about 150 meters squared per gram. So this fits in quite well for things like excipients and drugs and pharmaceutically relevant materials. Just as a kind of overview and summary, we can. The DVS can be done over a wide temperature range. We typically use organic solvents, and we do this under ambient, or we can measure it under vacuum conditions if necessary. The igcsea, again, a very broad temperature range for measuring surface areas. We typically use, again, organic solvents, organic vapors to measure the isotherm and then subsequently the BET surface area. And these are done in ambient conditions. So this is not under vacuum or. And not under pressure. The nitrogen absorption technique is done at cryogenic temperatures using either nitrogen or krypton. And these have to be done under high vacuum. So there's no way around exposing the sample to very, very low pressures. I'm not going to go through all the math and the derivations here, but essentially what you need to do in inverse GC to generate an isotherm is you do several pulses of different varying concentrations of your adsorbates, and then from that you can construct the isotherm. And then once you get the isotherm, you can apply the BET equation to get the BET surface area. And just as a way to validate the technique, you certainly can use BET standards. Excuse me. This is a CRM170 which is a 1.05 meter squared per gram bet standard. This was done with about 500mg. It was conditioned in situ for 100 degrees C for six hours. We then measure the octane isotherm. You can see it has the right shape, which means it has this hump and it's concave instead of convex. That would indicate a type 2 or monolayer type formation. And then I can apply the bet equation, as you can see on the right graph. And you can see we get a bet surface area of 1.01, which is in very good agreement with the 1.05 reference value for this material. Further details can be obtained in SMS application note 225. And to show this on a pharmaceutically relevant material, some paracetamol from Sigma Aldrich, multiple runs on multiple columns were done and you can see that the surface area values were between 0.2876 and 0.2788. R squareds were very good standard deviation, you know, between 0.27 and I mean 0.0027 and 0.006 meters squared per gram. So we can see, we can get excellent RSVs, you know, approaching 1% of total value, even for a very low surface area material, which many crystalline pharmaceutical materials will follow in this range and excipients as well. So some of the challenges with volumetric techniques, as I alluded to previously, if you're using nitrogen as your adsorbate in the volumetric techniques, typically below 1 meter squared per gram, is where the errors start to approach unacceptable values, maybe 10%. So typically we would recommend, or most people recommend that nitrogen absorption only down to about 1 meter squared per gram. If your sample has a lower surface area, you'd have to go to krypton, which is possible, becomes very expensive. But you can see for typical nitrogen absorption, the limit may be around 1 meter squared per gram. In addition, the cryogenic temperatures and the low pressures for some fragile in particular, maybe some amorphous materials or some spray dried materials or other things that might be relatively unstable. The low, excuse me, the low pressures and the thermal shock could cause phase changes, dehydration, particularly of a hydrate, which I'll show you later. You can see some of these conditions are artificial conditions. They're not relevant to where the materials are processed and used. They can artificially change the morphology of the material and then give you misleading or non atmospherically relevant surface area values. So again, you know, at room temperature, as opposed to, you know, liquid nitrogen and freeze drying There's a high thermal shock, you know, very quickly lowering the temperature by 200 degrees C. Again for organic fragile amorphous materials or partially hydrated materials, this can cause some challenges and again induce some unwanted changes to the sample just due to sample prep and the measurement technique. So to move on to some case studies by IGC of where these are used for pharmaceutical materials, some of this work was done by our group. This is a paper done by Dr. Williams Group at Imperial College using inverse GC to measure the specific surface area of freeze dried biologics. So the goal was to compare nitrogen absorption versus using octane absorption in inverse GC to determine the specific surface areas for some freeze dried biologics. The model materials were IgG, sucrose and influenza nantrogen that you can see here. For the nitrogen absorption technique, the gassing was done at room temperature under vacuum overnight. The experiment conditions were as necessary at 77 kelvin. On IGC we only used about 100 to 400 milligrams of sample. Preconditioning was 2 hours under a flow of helium at 30 degrees C or 3 or 3 kelvin. Then the measurement technique was done at 25 degrees C or 293 kelvin using octane as the probe molecule. And hopefully you can see the numbers on here. If we compare the different techniques, you can see that we get good agreements. On the top we have the IGC measurements. On the bottom are the nitrogen measurements. You can see here an average of 1.08, 1.26 very close sucrose 0.46, 0.9 and the antigen 0.55, 0.44. So the numbers in generally agree. However, you can see, excuse me, that the RSDS or the error margins in the repeats are much lower for the IGC technique. So these are relatively low surface area materials between, you know, 0.5 and 1 meter squared per gram. And the RSDs range from around 1 to about 3 and a half, whereas by the nitrogen absorption technique they range from around three all the way up to about seven and a half. So you can see that the RSDS or the accuracy in repeat measurements is much better on these low surface area materials measured by IGC compared to the nitrogen absorption technique. And just to highlight that or to show the comparison sample weights were about the same between 100 and 400mg RSDS 3 to 8 for nitrogen, 1 to 4 for IGC runtime, 4 to 6 hours where this was 5 to 8 plus the 24 hour preconditioning time temperature was at 25 degrees C as opposed to 77 kelvin. This was done at atmospheric pressure where these under vacuum and which I'll show later, not on the data that I showed previously from Dr. Williams paper. But you can do these over a range of relative humidities. Whereas nitrogen techniques, you're forced to do them at dry conditions. So to highlight or to show the impact of relative humidity on specific surface area, we'll first start with a relatively inert and robust material like this CRM171, which is an alpha alumina. It's not expected to go through any phase changes or be affected by water. You just have water coating the surface. So we measured the BET surface area. This is a reference column where the reference material is 2.95 meters squared per gram. We measure this at 30 degrees C and we get 2.95 meters squared per gram. So at 30 degrees C and 0% relative humidity, we get one to one agreement with the reference value. Now we did this same sample, here it is just repeating this at 0% relative humidity, 2.95 meters squared per gram. Then it was done at 50% RH. You see that the available surface area has dropped to 2.73. And then at 90% we can see it's at 1.45. This is basically just highlighting that as you increase relative humidity, the available surface area will decrease because water is coating the surface. So, so water isn't causing any phase changes to this material. It's just showing you what's actually available at these conditions. So again, I can get the specific surface area at environmentally relevant conditions on your material. Now moving on to a spray dried lactose. Spray dried lactose is very moisture sensitive and it's well known that it goes through a phase change at different relative humidities. If you're not familiar, we have several application notes that highlight this. But lactose will go through a moisture induced glass transition around slightly under 30% relative humidity. At 25 degrees C and then above 60% relative humidity, spray dried lactose, which is mostly amorphous, will spontaneously recrystallize to the crystalline form. And here we can see, we can track that and we can show that in the surface area value. So at 0% relative humidity, you can see we get a surface area value about 1.1. At 35% relative humidity, which is above the glass transition point, but below the point where it would crystallize, we can see about the same value. Maybe it's increased very slightly, but within the error margins. However, when you expose the sample to 70% relative humidity, you can see the surface area decreases dramatically by about a factor of 10. Not surprising, as the crystalline phase will have a much lower surface area than the amorphous phase. So we can do this all of this in situ on the same exact column. Pack the amorphous column, measure it at 0% relative humidity, then measure it at 35% RH, then measure it at 70% RH, and we can directly see how moisture is causing these different phase changes on the sample. On a different case, this is where actually the surface area goes up with increasing humidity. This is on a cotton sample, so maybe not as pharmaceutically relevant. But just to illustrate, we can see that as you increase the relative humidity, the surface area actually increases. And this is due to moisture causing the material to swell and expand, which can be confirmed by doing DVS experiments with our video options. So in this case, you can actually see an increase in surface area as the material swells and expands with increasing rh. Also, the drying or outgassing or conditions can dramatically affect the surface area. One material that's commonly used as a lubricant in pharmaceutical materials is magnesium serrate. And it is certainly affected by the relative humidity and what the apparent or available surface area is. So at 0% RH, we get a surface area around 4 meters squared per gram. At 35, around 3.3, at 70, around 3.16. However, if we bake this sample out to 100 degrees C and then measure it, we can see the surface area goes up. So we can see that the surface area decreases as humidity increases because the available amount of water on the surface is covering more of it. However, if we force all the water out, the surface area may go up. It may be just drying it at 0% relative humidity is not forcing out the bound water. You have to go to much higher temperatures to force that water out. And then you get a more dry surface and the surface area increases. Again, this is just highlighting that the outgassing and measurement conditions can dramatically affect the apparent and measured surface area of the material. So we talked about the effect of relative humidity. Now we're going to show some papers where they looked at. This is not work done by us, but this is work done by other groups where they looked at the effect of the vacuum or the thermal shock and the low temperature, how that might affect materials. And this is looking at the effective surface area on some pharmaceutical ingredients. And what the bottom line was from this paper is that the nitrogen absorption technique was not good enough to measure the low surface area. So Krypton could be employed. However, there was significant risk for structural damage from the low glass jettison temperature. And it determined that IGC using a flow technique at atmospheric conditions reduced the risk of altering the crystal form. Thus the pulse method by IGC also reduced the risk of potential crystal from the solvated form analysis. So just showing that if you look at the details of this paper that the vacuum and cryogenic conditions of nitrogen or Krypton had a high risk for altering the state of the material, which wasn't the case in the inverse gc. And further on to that, a nice paper published by a group at Genentech was looking at the use of IGC to measure pharmaceutical hydrates. And you can see here that you know, both comparing the anhydrous formed done by IGC and using Krypton, you can see you get very good agreement. But when you talk about the dihydrate, it's impossible to measure the dihydrate in the nitrogen system because by exposing it to a vacuum, you, you dehydrate the sample, you remove the water. So I can't measure the dihydrate form in the nitrogen system. So the only way to properly measure the dihydrate was to measure that in the inverse GC at different relative humidity. So you can see we controlled or not we this paper, these researchers measured it at 50% relative humidity. And then you can directly get the dihydrate. You can see here that it's much different than a Krypton because it's probably dehydrating during the experiment. I'm not actually measuring the hydrated form. And some of the conclusions from this paper is that since the IGC does not require outgassing step at cryogenic temperatures and low conditions and we can do them at different relative humidity conditions. It was the only way to maintain the hydrates in these surface area measurements. And finally the gas orbtion is not suitable for bet surface area measurements of certain types of pharmaceutical hydrates because going again to those cryogenic and low vacuum conditions and finally very similar study and conclusions were done using some nanocellulose cellulose is sometimes or can be and has been used as an excipient in the past. But this paper just highlights, it does compare gravimetric, volumetric and chromatographic techniques to measure surface areas and shows the risks of with these spray dried or freeze dried nanocellulife samples, when you expose them to vacuum and cryogenic temperatures, you do see some significant phase changes. So in conclusion, I hope that I've highlighted some aspects of where inverse GC might be applicable to measure surface areas of pharmaceutically relevant materials. And at the end of the talk I'd be happy to answer any questions that may come in the chat if John can help me out. But I would like to direct your attention to if you want to get any more details, I can direct you to the Sorption hub which houses SMS's relatively new Zorption Academy. If you are a user of our instruments you can request access to this and then it has a wealth of information of past webinars, other information that might be relevant to you. And excuse me, if you are a actual user of our instrument, you ever need technical support, you can also log on Adsorption Hub for any technical support or service tickets or whatnot. So please utilize these resources for both scientific support and technical support and we can help you out with our worldwide team in both the us, the uk, Europe, China and India, we have staff around the world that can help you with your challenges. So without further ado I will pause and hopefully there'll be some questions and John, I'll see what you can help me out with.

Thank you for a great presentation Dan. Yes, we have a few questions in coming in already but so please anyone in the audience who would like to have a question asked, please do submit it now. So our first question is I see you have chosen different PP0 value. How do you choose this range and how would you know what is the best one?

Okay, excuse me. The classic or the prescribed or recommended range is 0.05 to 0.35. So that's the definition of the BET surface area. For those of you familiar with inverse GC I guess maybe one of the downfalls is you don't typically dial in a specific p over p knot. It's based on the peak height and the resulting chromatograph. But you want to choose as broad a range between 0.05 to 0.35 as possible. If you only limit the data to let's say below 0.1 P over P0, typically you will artificially get a lower surface area value that is there. So I would recommend get as far as you can into that 0.2 to 0.35 range to get your best results.

Thank you. That person follows up with a question saying do you exclude any points when.

Calculating bet you can you would sometimes you might want to drop off the lower ends if they what what's often maybe a little bit more for microporous materials but it is acceptable to essentially look at the R squared and if you drop off the lower points to improve the R squared, that's recommended. We typically would not recommend the drop off the high points. So start with your all the values between 0.05 and 0.35. If some of the lower ones pull the line less linear, it's acceptable and common practice in surface area measurements to drop off the lower values to improve the R squared. I would not typically recommend to drop off the higher values because that can sometimes artificially give you a lower surface area value than is actually there.

Thank you. Question here. If the sample contains, for example, a 20% moisture, won't preconditioning dry the sample and change its nature?

Exactly correct. And that's the whole point of it. So the inverse GC you can precondition at any conditions you want. So I could condition the sample at 20% relative humidity or 40% relative humidity to maintain that moisture there. We only when we're doing comparisons of the different techniques and we know the materials are stable at 0% relative humidity, then we would only dry them. So I can precondition my sample at any conditions I want. That's the advantage of inverse GC as opposed to volumetric techniques where you're forced to precondition the sample under vacuum with no more moisture present. So you're absolutely correct. And we would only Recommend preconditioning that 0% RH if you know your sample is safe at those conditions.

Thank you. We've got a good few more questions. Don't worry everyone, we will get round to all questions asked because we have a few in the queue at the moment. But the next question is, will the results still be accurate if the specific surface area of the sample is less than 0.01 meters squared per gram? If yes, is there anything we need to pay attention to when measuring. When measuring a sample with such a small specific surface area.

The rate limiting or the limiting factor would be the retention time. So you may have to use more samples. So if your surface areas are below 0.01, you may have to use several grams of sample. So your limiting factor would be how much sample I can get in the column. Do I get good quote unquote, good retention, which means your probe molecules are separated from methane by at least 0.1 minutes. So we do offer larger column oven configurations that can take up to a 10 millimeter sample column. That's not our standard system, so if you already have one, it would be a different system. But yeah, the only Limiting factor would be how much sample I can put in there, because I just need enough surface area total in the, in the column such that I can get reasonable retention. So I've certainly measured and seen things down to 0.05, 0.01. Again, you might have to use, you know, 10 grams of sample to get something reliable. That would be. That would be the limiting factor on that.

Thank you. Is there a way the surface area can be measured on the IGC without having to enter an approximate value when setting up the sample information for the measurement?

Yeah, unfortunately we do require you when you're entering in. You can put in a nominal value. It doesn't need to be accurate. You can just put in a value of 1 just to run the experiment, or a value of 10. But that is something that our software at the moment does force you to do is to enter in a estimated or known surface area. So you still need to put something in there. So that's how the software calculates how much solvent to inject. But it doesn't have to be a real number. You can just put a nominal value. I typically recommend maybe a value of 10. If you don't know anything about your material, it's better to guess high than guess low because we want again, cover the monolayer coverage. That's slightly different from a surface energy experiment where you want to focus at the low end. A surface area experiment, you want a broad range of concentrations and a broad range of essentially coverages. So if you don't know anything about it, I would try to suggest putting the value of maybe 10 meters squared per gram just to ensure that I can do the full isotherm.

Thank you. Why or how did you choose octane for an adsorbent? Is this choice independent of the specific surface area of the material?

Great question, and it is something that I did miss. It's related to the fact that octane is nonpolar and will typically wet the surface and form a type 2 isotherm. So as I mentioned, the only caveat to using the bet equation is that you have to have a type 2 isotherm or type 4 isotherm, which means the molecule will wet, will form a monolayer before growing layer by layer. This is the reason not that water is typically an awful molecule to use for BET surface area because it doesn't typically wet, it'll clump and agglomerate and it might even swell into the sample. So we choose octane for most materials. It will form a type 2 isotherm. It doesn't mean it will form it for all materials. So there may be some method development. I might. If octane forms a type 3 isotherm, we would recommend choose a more hydrophilic molecule like ethanol or something like that, or methanol. So the one potential downfall is that the isotherm is a function of both the sample and the probe molecule. So although the volumetric technique nitrogen is inert, it'll form a monolayer on pretty much any surface. You might have to choose a different molecule depending on your sample. But we typically recommend to start with octane because it's nonpolar and typically will form the right shape isotherm. So great question.

Great, thank you. How do you determine the best parameters for generating an isotherm?

Typically, to generate the isotherm, what I do is if you're familiar with the software is you start with the lowest. You can use the software to determine what your lowest concentration injection would be and then what your highest concentration injection would be. And then I try to get maybe 10 to 15 points in between. So start with your low point and your high point and fill it in with maybe 10 to 15 points. You want to get as big isotherm as possible.

Great. Okay, this question here is. Hi, I'm doing this practically I've struggled to get a high enough PPO value, usually not getting past point 2. This is on a crystalline solid with a nitrogen BET value of 5 to 10 meters meters squared per gram specific surface area with a sample mass of 50 gram milligrams. Sorry. I've also tried with lactose and gotten about 10 times of civic surface area determined by BET.

Well, to answer the first part of your question, I would recommend when you enter in the column surface area, even though BET says it's 5, I would choose a higher value, maybe 10 to 20. That will force the instrument to inject more molecules than what it thinks it should. And then you can get the higher coverages. So if you, if you struggle with that, try a lighter alkene. So if you tried with octane and you're maxed out around 0.2, try with heptane. Heptane is a lighter molecule. It will inject more moles because the vapor pressure is higher. So it'll inject more moles to cover more part more of the isotherm. So if you, if you've already tried increasing the surface area that you enter, that will artificially. That'll train the software to inject more molecules. If that doesn't work, try a smaller alkene like heptane or hexane. And the second part of your question is, I mean, if it's crystalline lactose or is it morphous or spray dried lactose. Again, if it's crystal and lactose, I would expect very similar results between nitrogen and dbs, except lactose monohydrate, even alpha lactose monohydrate. If I expose that into a nitrogen system, you're probably removing the monohydrate. And so you're not actually measuring alpha lactose monohydrate in the nitrogen system. You're probably measuring anhydrous lactose because that water molecule will be easily removed under vacuum. So there could be a reason why you're getting differences. So they may be real and that's the fact that you know under vacuum you're going to pull out that monohydrate. Even if it's crystalline lactose, you could pull it out if it's amorphous or spray dried or freeze dried lactose, you certainly could cause phase changes by going to those conditions.

Thank you. Two questions here from the same person. The first part is, can we remove the moisture in situ? And the second is, why does the surface energy result not change when I input the actual BET value to recalculate the surface energy in the software?

Okay, great. So sorry about the background noise here, but yeah. So the first part of your question was. Let me ask the second part. The surface area is actually not used in the surface energy value. So if you're familiar with the Y axis which shows the surface energy, the surface area is not used at all. The surface area only is used to determine the fractional surface coverage. So your X axis might change if you're looking at that surface energy profile. So that is why it doesn't change the surface energy, although we forced you to put it in there. I mean the surface area, although we forced you to put it into the column information, that value is actually not used at all to determine Gamma D or gamma ab. It's only used to determine the fractional surface coverage. So that's why the actual gamma AB or Gamma D will not be changed at all. But the fractional coverage or the actual fractional coverage will change if I alter that surface area. And the first part of that question was, yes, I can remove the moisture in situ. You just choose what conditions you want to use. You can outgas or dry the sample at any temperature and any relative humidity you want prior to measurement.

Great, thank you. Last few questions here. What do you do as a conditioning step prior to Octane injection.

Again, it would depend on the sample. So if for most materials that should be safe, we would Recommend maybe a 2 to 3 hour conditioning at 0% relative humidity and room temperature. That would remove any surface water from the sample and provide you a clean surface. Now, of course, if you're doing a hydrate or you're doing something that can't be dried, we would condition it at whatever humidity is relevant to your process or your material. So usually a 2 to 4 hour conditioning at 25 degrees C would be sufficient because you're flowing through nitrogen or helium at those conditions that remove any surface water from the sample.

Does the packing of the sample have an influence on the bet obtained?

No. Just like in surface energy and all the things, we use methane as a dead volume. That will track and and correct for any packing differences between sample to sample or column to column. So no, the packing will not affect the service area results. It will affect the gross retention time, but it will not affect the net retention time or as that retention time changes. So no, that will not affect the results. But good question.

Thank you. And our last question here. Do you integrate the resulting peaks by hand, by their real borders, or by the automatic calculation using the whole spectrum as a range of integration? Sorry.

Typically I do it by hand or just to make sure that I'm capturing the entire peak. It's good practice to look at the peaks just to make sure you catch them. And if you had any impurities that maybe came out early, you don't want to include them. But if you want to do a quick screening, just go ahead and have it automatically select the peaks. Typically you're only injecting one molecule at a time, so you're only injecting octane or hexane or whatever it may be. So it's usually pretty easy to just use the software. But for me, I like to look at the injections, make sure the peaks make sense, make sure I capture the entire peak and therefore I can put in proper limits.

Thank you, Dan. That is the last of the questions for today, so thank you so much for being here to answer the audience's queries.

Well, thanks for your time and attention and I appreciate all the great questions. And you know, if you have any further things, you can contact me directly or email scienceurfacemeasurementsystems.com and I would be happy to follow up with anything you need.

Thank you, Dan. Yes, and I'd like to second that by thanking all of our audience for being here and engaging so enthusiastically in the Q and A. I hope you've learned something useful to take back to your research. Please remember that all our webinars are available on our website to view and in addition, you can also go to thezorptionhub.com sorry, that is just absorptionhub.com no the where you can sign up free for the Adoption Academy, which has our full catalog of webinars, application notes, and exclusive learning content devised specially for that platform. So once again, you can find all of that and sign up for free at www.ZorptionHub.com. thank you all again and we'll see you next time.

Thank you. Bye bye.