Originally broadcast on 2-Sep-21
Have you ever transferred glycerol or ethanol with a pipette? Most people KNOW that this can be challenging. But often, they do NOT KNOW which liquid handling system is best to use in this case.
While you may already realize that your liquid type and pipetting technique have the power to influence your analysis results, you might be wondering how you can improve your pipetting technique.
Eppendorf has spent the last 60 years researching the physics of pipetting. In this webinar, they will share this knowledge with you and show you how to optimize the handling of liquids using some simple tricks, ensuring that “the pipette” does not influence your analysis results.
In this webinar, you will discover:
– How different liquid types can influence pipetting accuracy;
– Different liquid handling techniques and the best approach to adopt for each liquid type;
– How to implement a good pipetting technique to improve the accuracy of your results.
Learn more about Eppendorf Germany privacy practices.
Boost your bioscience career with our upcoming webinars covering technical, vocational, and soft skills: https://events.bitesizebio.com/webinars
hello this is Adam Paulson welcoming you to this bsize bio webinar which today is sponsored by endorf endorf is a biotech company that develops produces and distributes systems for use in all spheres of life science the company’s broad range of high quality products and systems is used by researchers worldwide to carry out their work efficiently today’s presentation is titled reproducibility mastering the art of petting and is being presented by Dr Barbara cifka product marketing manager and Dr Ora gust application specialist manual liquid handling and pet calibration service both of whom are from endorf Barbara obtained her PhD and developmental biology in 20 2004 from the University of cologne where she researched the asexual reproduction of jellyfish using molecular biology techniques she went on to gain sales experience at peren Elma and Micron seek lab before joining EP andor’s product market manager and manual liquid handling certified trainer in 2014 Barbara has been product marketing manager for manual liquid handling Solutions in Europe since 2018 and is happily celebrating the 6 year anniversary of endorf Pistons stroke pets this year or specializes in applications of liquid Handling Systems and in teaching their correct use she provides guidance to users to solve difficult petting questions and has supported endorf pet Calibration Labs in the accreditation process Ora holds a PhD in biology from the University of tubingen she has a great deal of practical experience in both the correct use and liquid Handling Systems and their performance test as always we will have a question and answer session after the presentation so please type any questions that you have into the questions box which appears at the bottom of your screen and I’ll put them to Barbara and Ora at the end a link to view the recording of This webinar will be sent to you in due course so now over to you Barbara and Orica for the presentation thanks so much Adam uh and welcome everybody reproducibility mastering the art of pipetting that’s our topic today and we will guide you today through difficult liquids we will talk about functional principles and pipetting techniques but first do we have the right information around us we checked lot lots of pages uh uh on the web and nearly 60% of the found videos around correct pipeting were not fully correct this webinar should show you the the right handling of liquids and we want to eliminate some myths and we’ll show you where you can find the right information as mentioned we start with the liquid classes or with difficult liquids the most liquids in the life science research are unpro equa solutions however often we have also difficult liquids in the lab this could be volatile liquids viscous liquids forming liquids or some more but we will focus on these three ones today but how to handle them all what should we do with all these diff difficult or different liquids let’s have a short view into this video are you aware of this problem this is a um slightly colored liquid I will tell you later what it is or I hope you know what it is and I hope you can see the dripping out of the tip properly I have the first poll question for you what kind of liquid do you think this could be please choose only one answer could that be water could could that be oil could that be ethanol or maybe a detergent and I hope you all see now uh the popup window and you can vote what do you think could that be thanks for the results so lots of you are voting for ethanol and this is correct also some of you go for water or detergent let’s have a closer look into that volatile liquids are ethanol as shown in the video and most of you gave the correct answer it could be also acetone or chloroform and what is the problem here we have a high vapor pressure of the liquid and additionally we have a lower surface tension than water and the consequences here are that you as shown in the video that you have liquid drips out of the pipet tip and this leads to inaccuracy and also to a non-reproducible result and we have also contamination risk you have the evaporation into the piped cone as shown here the liquid evaporates into the air cussion of the piper tip higher pressure builds up here and the liquid drips out of the tip what we recommend for this type of liquid the best choice is a positive displacement system maybe you don’t know this name but you know the name may be a stepper or a dispenser we had endol call it multipad if you have no air cussion you have no problem and later on Ure will give you some more insights into that system another best choice is the visualized pip pipet manager system with the Explorer plus this system um have a liquids um stored as a program inside this um this system and you can choose uh between different liquids like here like ethanol and when you choose that there’s running a program for you so to say in that case for example pre-wetting or pre-saturation or working especially slowly or and or if you want to uh have more information about the visionar pipet manager please go on the link uh on our homepage and you find that in the chat now if you don’t have a positive displacement system or the piped manager we have some tricks for you if you want to use the air cussion pipet very important here when using ethanol or other volatile liquids pre-wet more than three times to decrease a frequency of Dripping you can very easily um uh try that out um with a non- preed tip with ethanol and a preed tip and then you see the difference very easily but we have to say this is not sufficient with very high vapor pressure chemicals like acetona chloroform the reverse pipetting technique um can also um be an idea to decrease the error but this will not avoid the dripping this will only reduce it a bit and you should adjust the piped temporarily to the liquid according to the user manual this is our application Noe 198 what we found on the web uh the recommendation of positive displacement is really rarely found on the internet and this is a bit astonishing for us because it is the best choice here I have the next video for you are you maybe aware of this problem in the web uh sorry in the lab um as you can see here um there are bubbles inside the tip or inside the liquid and another effect you will see while dispensing now here a huge residue is building up um inside the tip so we have remaining liquid inside the tip this is a viscous liquid and we would like to know from you how do you solve this problem in the lab so this time you can choose more than one answer you can aspirate or dispense extreme slowly or you can simply ignore the residue in the tip maybe you use the reverse pipeting technique maybe you cut the tip or you use a positive displacement device so I’m happy to or I’m very curious about your answers so let’s have a look into that soon here we are okay most of you are voting for aspirate and dispense extremely slowly this is really really good answer to ignore the residue is uh is rare and this is also so very good but also we see that some people are cutting the tip and uh let’s have a closer look into that how users solve the problem with cutting the tip some people are doing that and here we make an experiment uh to show the influence of cutting of tips on the pipeting error and what you can see here is a systematic error and the distance of the cut off above the orifice so orifice is the opening of the piper tip and shown here is pipeting water with a non- cutter tip and with a cutter tip of 2 cmet distance and the blue bar here uh the blue bar here is uh the normal error limit for this volume of th000 microl and you see with water and no cut a tip it’s a great result with cutting the tip even when you’re only um pipeting water you are much higher than the arror limits and this effect is higher with glycero when you non cut uh when you’re not cutting the tip um you have um you are inside the arrow limits but when you cut the tip to use glycero with 50 % then you are far away or far above uh the arrow limits so you see cutting the tip is not the best idea unfortunately this tip is really often recommended on the web so what are viscous liquids beside glycero it could be blood it could be mineral oil and mineral oil was liquid we used in the video before and the problem here is uh viscosity the fluid resistance you have a higher inner friction and these liquids have a higher density than water the consequences are um you have an inaccuracy because of a poor flow Behavior because of the risk of air bubbles and drop formation and you have leftovers inside the tip as you can see here and as you saw in the video and this leads to um really poorly reproducible results here we sh it uh show it in another way now we used or we Tred to pipet 50 microliters of 85% glyce Rule and this is a normal pipeting um technique forward technique with an air cussion pipet and as you can see here the different pading steps are very very diverse this these are not reproducible results when you’re doing the same with a reverse pipeting technique the results are a bit better I would say they are not so diverse but they are still not at the 50 microliters but when you use a multiped or in general a positive displacement system then the results are really really um reproducible as you can see here so as um hopefully obvious shown uh in the diagram before the best choice is again the positive displacement system I know I’m repeating myself but we have no air cussion then we have no problem and again the vision is pipot manager with the Explorer plus could be uh a very good choice beside the predefined liquid types in the pipot manager you have you can also Define your own liquid types here is viscous liquids and um you can enter these values you will always find everywhere um for your liquid um the dynamic viscosity and then you define your own liquid type if you want to um pipet viscous liquids with an air cussion pipet then as most of you uh answered really right work extremely slowly and this is not so easy with an um mechanical pipet so the recommendation is here to use an electronic air cushion pipet also the reverse pipetting decreases the error because we have a residue anyway in the tip but I have to point out it decreases the error but not eliminates it and again here it could be of uh good help if you adjust the pipit temporarily to the liquid according to the user manual I have no other video for you I’m sorry for that but I have another poll question for you foaming liquids how do you pipet foaming liquids and you can choose again more answers than one use a positive displacement device could be one choice aspirate dispense extreme slowly and or ignore the form in the tip what do you think y I like these answers very good so to ignore it is not the best idea that’s that’s correct use a positive displacement device it’s a very good idea and uh when you work with air cussion aspirate and dispense extreme slowly very good so with the forming liquids um first what are they these are liquids um contains uh detergents that could be in the lab mostly Protein Solutions with high concentrations for example PCR Master mixes or cell culture media the problem here is you have an increase of bubble and foam formation and you have additionally a change in the wetting behavior of the liquid sorry and you have additionally a stickiness to the surface and the consequences are the detergent sticks to the tip wall here in the picture you see a blue color detergent and you see hopefully very good um that this is it should be an empty uh pipet tip but uh the liquid sticks to the inner wall of the tip you have foam with in the pipet and the blowout um used normally while pip pading leads to Bubble or foam formation and splashes so again the best choice here is the positive displacement system and again I have to repeat myself no air cussion no problem and also here the visualized pipot manager um could help out you have an um uh another um predefined liquid type uh the enzymes and an antibodies and um here again as I mentioned before a program runs behind and in this case um working very slowly and without a blowout if you would like still to work with an air cion pipet as most of you mentioned work extremely slowly and again the recommendation is here you are not able to work such um such slowly uh with a mechanical pad uh please use an electronic pad and or will tell you a bit more about that um in the next slides avoid performing a blowout when you have forming liquids you can apply the reverse pipetting technique or with an electronic pip at the dispensing mode and you can uh use Piper tips with a low retention functionality there are different companies uh including EP of offers um low retention tips so these are our information about difficult liquids and if you would like to know more we have a liquid guide for you on our L uh on our homepage it’s under the Endor handling Solutions and there you find the liquid guide with lots of more information with videos diagrams and so on so please have a look there and you find the link now hopefully in the chat now I hand over to thank you so much Barbara for sharing um some practical um examples uh for yeah challenging liquids so by this we have seen that there are some liquids um that push the air cushion pipits to their limits and in such cases we have seen um that it is better better to use a different liquid handing system and Barbara already mentioned the uh positive displacement instrument so this is also an example um for um the fact that there are a varing uh requirements of of the different applications uh in the lab and uh that we need different tools um to really answer that accurately and for that reason there exists a large bouquet of options so think of the different instrument models and the different volume variants and and the tips uh with the different features like filters or certain Purity grades alone the Endor catalog shows about thousand Solutions the interesting question now is when to use which and in order to help you a little bit through this question it makes sense to step back and differentiate a little bit by the name by the main functional principles and these are the air cussion versus a positive displacement mechanic versus electronic and handheld versus automated let us start with the air cussion versus positive displacement instruments to understand when to use them we need to take a look to at the construction of these tools the air cion pipod basic basically consists of air okay there are also a piston and some mechanics enabling you to move the piston but in principle it is a piston moving air and when a tip is attached and immersed into water the system moves the liquid too it’s a very simple system unfortunately the air cussion is sensitive to influencing factors such as temperature holding angle or other liquids and water and as a rule of a thump the bigger the air cussion the bigger becomes the pipetting error induced by these influencing factors and this brings up a question wouldn’t it be good to have no air cushion at all indeed we have seen challenging liquids are transferred best with a so-called positive displacement instrument here the Piston is built into the tip not into the instrument and that has the great advantage that the liquid runs up to the piston and the air cushion is avoided with a little exception at of a small air bubble influencing factors on the air cussion do not come into play anymore and uh since the Piston wipes the tip clean even sticky liquids like blood or glycerol can be transferred accurately for these reasons a positive displacement instrument is a perfect solution when pipeting challenging liquids let us now take a look at the electronic pipets electronic pipets are more ergonomic uh than the mechanical ones because you do not have to move the piston the electronic pipet does this for you so it supports you in terms of ergonomics when you have to process an increased throughput Additionally the range of the functions is bigger they simply offer more modes um this is what you can see here on this uh little um yeah popup so there is a a number of the different modes of typically you know um programmed into electronic pipets and while with a mechanical pipit you can only PIP IT the electronic one also offers for example a dispensing option which is especially helpful when filling plates this mode enables an efficient working as you will be faster when filling the plate by the dispensing mode the third interest interesting aspect although rarely mentioned in the internet um is the improved reproducibility sorry the improved reproducibility um of electronic pipets this graph shows the result of a study focusing the random error of pipetting at different volumes as shown by the blue bars pipetting with mechanic pipets had a higher random error as pipetting with electronic pipets which is displayed by the green bars the stud was performed with skilled and well-trained lab Personnel the within run pris and the between run pris is better when the pipetting is done using an electronic pipet and this means the electronic pipet helps to increase the reproducibility of your results this phenomenon is caused by the motorization of the Piston H let us stay a little longer with this topic you can low the human error by applying good pipetting techniques Barbara is going to show or to give you more input on this um in a minute we have seen that electronic pipets further help in reducing this error but there is an option Beyond totally avoiding the human factor in pipetting and this is the automation here you do not hold the pipet anymore but the PIP pettings are done completely automated the reproducibility is top there are a number of advantages coming with an automation but there is one more factor I would like to highlight when you are pipeting tasks often become very complex and you need to concentrate very heavily for longer dur sorry for longer dur durations um automated systems be can be of enormous help an example is the normalization in the frame of NGS here every cavity of a plate is to be filled with a different Dent volume a task that can only be solved by a single Channel pipit as every well is to be filled with a different volume of water those uh you of you who are doing normalization will agree that it is exhausting to repeatedly search the table for the needed volume setting it on the pipet and pipetting the water into the correct well 96 times or even more it is so much easier to just upload your Excel file with the delusion calculation and press the start button on your automation so far about the three most important functional principles but there’s one more thing uh that needs to be mentioned and um I’m now having a question to you what do you think how much does a piper tip impact the pipetting result is it 10% or 25% or do you uh say or guess it’s 50% just give it a guess and maybe you are surprised okay so the um votings come in now thank you very much so most of you say it’s um 25% and um as I said you might be surprised um the iso 8655 which is something like the Bible um in liquid handling um um that uh yeah shows that the um that the pipeting error caused by the piper tip can be up to 50% and uh this is quite a bit and uh here I think it is important to understand that that the pipet and the tip really build the system and the tip although being a single used item uh may have a great influence on the pipetting result so it should be chosen with the same care as the pipet this is due to the fact um that pipets are adjusted to a certain tip or let’s being more precise um the pipet is adjusted to the shape of the tip the tip shape defines for example the air cussion SI size inside the tip on this picture you can see an end of th000 micr tip on the left on the right hand side there are some competitors watch out for the difference in length between these tips manufacturer wise an Endor th mic pipit will be adjusted to the Endor tip on the left when it is then used with one of the tips on the right it will show an increased error and needs to be adjusted to the air cussion size of this alternative tip this physical principle is true for all air cussion pipets in general now what can you do if you wish to use another manufacturer tip you should look out for the tip induced influencing factors such as tip fit shape of the tip or quality of the tip orifice so when you are introducing alternative tips we recommend doing a performance test of this new system to check its accuracy send the pipet together with the alternative tips to a calibration lab they will check and if needed adjust the pipet all right so this was an overview over the of the main functional principles next slide please and now it’s up to you uh to make the right decis for your application so when being in doubt about the right tool please feel free to apply our selection guide you can find the PDF in the chat now so it is time now to turn back to the air cussion pipets again the air cussion is sensitive to influencing factors and thus it is important to have a good pipetting technique barara is now sharing with you the most important aspects thanks yeah let’s have a closer look into the pipetting techniques as shown before using the right tool is one part of the metal but using it in the right way is something a bit different and because we showed you before that the most problems um caused by an air cion uh we will focus here on the air cussion pipets so this is uh where the problems arise here short overview about um all the things you have you should have in mind when you aspirate and dispense liquids to choose the right pad volume is important a small immersion depths the holding angle the pre-wetting or also the pre-saturation of the piper tip the slow and uniform piston movement and the delay before taking the tip out of the liquid these are all the things to have in mind when aspirating and when you dispense a liquid the holding angle um is different than while aspirating you should release a liquid against the wall of the vessel and only if you have small volumes you should release the liquid directly uh the liquid in the tip directly into the liquid of the vessel we will focus on these three ones the right pipetting volume the holding angle and the slow and uniform piston movement and if you would like to have more information on all the other topics you will receive the source uh of that um later on first I have again a poor question to you you want to pipe at 10 microl you have three pipets available with each of them you can pip at 10 microliters what would be your choice you have a pad of 0.5 to 10 microliters a pad of 2 to 20 microliters or a pipet of 10 to 100 microliters and I’m curious what do you think what could be the best choice let’s have a look into the results okay the very good again so 10 microliters and the 20 microliters or 20 microliters most voted um Pad but not the 100 and I’m happy to see that so we have an experienced audience here I will show you why you choose the right answer here is shown the systematic error while pipeting 10 20 or 100 microl uh with 10 20 and 100 microl pipets and pipetting 10 microliters the systematic error with the 10 microl pipet is one% with a 20 microliter it’s only a bit higher but when you see the result of the 100 microliters we have a um systematic error of 3% and this is a lot so our recommendation is choose a pipet always with the smallest possible air cussion as we shown before the air cushion causes problems so when as uh the smaller the air cushion is uh the more reproducible is your result or we can say the closer the aspirated volume um to the nominal volume is the more accurate the dothing is this factor is very rarely mentioned uh in on the web how important this is and I’m wondering about that because you have normally all these pipets in your lab so it’s easy for you to to select the right one but yeah you don’t find so much information on that let’s talk about the holding angle or let’s first have a video on uh to show that our second topic is the setting angle when aspirating liquids you need to hold the pet as vertically as possible I will show you why this is beneficial with a series of eight samples whoops there we go the more you tilt the pipet the more variations you will get in a series of samples since hydrostatic pressure in the tip changes with the angle the more you tilt the pet the more liquid you’ll dispense it’s difficult to control this effect since the variations are too small to see with a naked eye for best results just keep the pipet as vertically as possible so here Sean again the aspir uh while aspirating um you have to take care on the holding angle hold the pipet as vertical as possible during the aspiration ideally at an angle of 0% uh zero degrees sorry for that and again in the web there’s often mentioned it is okay um to have an holding angle up to 20% but why they recommend that you will aspirate more volume even it is small amount more and also it will not possible for you to have always an angle of 20% you have to estimate it so we would really recommend to have always the angle of um 0 degree and here’s also shown uh in a graph um the vertical holding angle and an angle of over 45% when you hold it like like that uh in the lab and I would say we see it often uh in in videos to have it like that um and these are result results when you do it like that we want to um pip at th000 microliters and here we have 106 microliters around 1006 or 1005 microliters while have a high um or have the wrong uh holding angle while vertical pipetting leads to a a really accurate and reproducible pipeting the third aspect of pipeting technique is working slowly today we have the problem we have to work fast and we are always in a hurry and um yeah it’s a higher pressure I would say in the lab and um this is not the best choice while pipetting because it’s a very precise technique and here we say slowly working is key and as shown here in the picture you have um you have the sea and you have the fog Over the Sea the same happens in your air cussion pipet you have Aerosoles above your liquid and the faster you are working um or pulling the button vigorously the liquid scatters into the PIP pad and you have um lots of Aerosoles in the pipet that leads to contamination on the one side and it leads to um uh inaccuracy on the other side so to work really slowly especially as shown before um with uh some difficult liquids please choose an electronic device um here is shown our Explorer Plus or the multiped e3x and here you can see we have um speed buttons and you should select for difficult liquids not always not when you work with water but for for problematic liquids you can choose um the slowest speed of one to pipe it very very slowly and this uh leads to very reproducible results also this is very rarely mentioned in the web how important slowly working is and why for sure um Barbara may I hook in here quickly yes please um there is one more aspect about the pipetting speed um and this is a factor that can be important when pipetting functional units um like proteins or genomic DNA or cells and this is a sheering and um in order to to understand the she GE Force um we need to understand two physical contexts firstly liquids move faster in narrow tubes than they do in tubes with a bigger diameter and secondly there is a velocity gradient from the wall to the middle so towards the wall of a tube or of a piper tip um the liquid speed is zero and in the middle it is fastest we know this phenomenon from for example from Rivers um and where where they flow very fast in the middle and um towards the sh Shore they are rather slow for yourselfs um the G genomic DNA or the proteins this velocity gradients um between the wall and the middle is quite important so the bigger the gradient the higher is the sheer force and this means that on the other hand that you have two options to decrease the shear Force the first option is the tips diameter and the second option is the pipetting speed and uh for pipetting liquids uh which contain functional units like DNA cells or let’s say genomic DNA cells um proteins Etc um we recommend applying an electronic pipit which is set to a low speed or if not the lowest speed um please note here that different manufacturer electronic pipets offer very different speeds um so I can of course only speak for the endol Explorer pipet here um you may now ask what or why not using a mechanical pipet and the answer is that all of us really tend to pipet too fast and an electronic pipet ensures pipetting with a very low speed with an even liquid movement and in a reproducible way and these are factors that are beneficial for lowering the risk of shearing and making the experiment itself reproducible now decreasing the pipeing speed pave the way for a safe transfer of most functional units however um if you’re working with very very sensitive molecules um or cells um even the lowest pip heading speed may be too high and in this case it can be meaningful to additionally apply a whiteboard tip we have performed a number of experiments about shearforce in liquid handing and uh its influence on the cell viability and there is an application Note coming in November showing the results and explaining this in more detail so for those of you who are working with such functional units please stay tuned and I saw Ora um that we have just uh came out the biion news appor biion news um with an Outlook uh on that topic that’s true but I have to ask you it should be talk about shortly also um about maintenance of pipets um because I would say it’s an important topic too oh yes it is yeah you’re completely right um uh so we have spoken about examples for challenging liquids now um about functional principles and we have um also talked about examples for good pipetting Tech techniques um but um as you said this uh the performance of the um liquid handling system itself um is also quite important and uh next slide please um sorry now it works yeah so let’s uh thank you very much so let’s have a look um at this graph um it shows the performance of a pipet in standard use and what you can see is that it Alters slightly um the performance over time so when applying a trend line here we can forecast that it will exceed the red marked error limits approximately in August 2022 so how can this be and why do pipets alter their performance that’s the next question I would like to explain to you so it’s because pipets contain wear out Parts um just as you know it from your car car or from your from your bicycle um so you know that a new tire has a good grip and a worn out one offers only a poor grip and in worst case the worn out one may even burst um and due to the abrasion and the aging process um tires are wear out parts and pipets also contain wear out parts for example the Piston seal that seals the air cushion another example is the O-rings on the cones of the same model of some model models um of pipets and uh factors like the frequency of the use or the aggressiveness of the transferred liquids influence also the lifetime of such wear outs parts so in order to keep the pipets at top performance and to avoid down times they should become maintained and checked on a regular basis the checks are called calibration um and the maintenance is as as well as a calibration can be done by a pipot calibration lab okay Barbara this brings us towards the end of this webinar um we have shown you today uh that the reproducibility of a pipetting result is influenced by the liquid the selection of the right tool or let’s say of the most suitable liquid handing system it is influenced also Al by your pipetting Technique and by the maintenance status of your pipet Barbara would you like to add something yeah only I have uh only a bit I have uh one or two last slides for you so if you want to know even more as Adam mentioned uh at the very beginning we celebrate 60 years of pipetting or of pipets now uh at endol so in uh 196 1 we um we brought the first um air cussion pip head um into the market and so we celebrate the 60 years with a with a very informative um ebook it’s called the science of Piper think to Perfection and you can um download it on our homepage but uh we would like to thank you uh to your participation into that this webinar and so you will receive this ebook as a PDF um later on so and there are all this information mentioned um today and much more information um so I hope you you like it and uh you will find it useful my last slide is only to summarize what we wanted to um tell you R summarized um the main topics but I would focus more on think about the source of your online information um so so-called experts um showing videos in the web are not always the right choice of information the best idea to search for for valuable um information is um or are knowledge databases either from manufacturers of pipets and tips or um also the calibration service companies they’re working the whole day um on on calibrating pipets and they are very uh very good sources for good information an example could be here an us-based company called artel this is a very trustful information base uh to have really really good information on all that so please don’t trust always the information in the web go to reliable sources to find the correct information that’s it from my S uh side and thanks so much um for your audience now you know the art of pipet well thank you um Barbara Nora that was an excellent presentation very interesting uh we have a number of questions from the audience and uh if anyone else has a question please feel free to post it in the the questions box which appears at the bottom of uh the screen so um coming to the first question uh to either of you Barbara Orica uh this question’s from Andy who wants to know what do you recommend for preting high concent ated sulfuric acid okay sulfuric acid um yeah so there are a number of of options um so first of all we have learned today that the best U um option always is the um positive displacement principle um again I would like just to highlight that uh the Piston is built into the tip um and this um has the advantage that um I would say it is more safe um yeah way to transfer an aggressive liquid like soric acid um so of course when we are talking about aggressive um assets we have also to talk about um chemical uh compatibility so um are the materials of of the liquid handing system um resistant to uh this chemicals and uh with the um multipad and comi tip system which is our um um positive displacement system um I can say that is okay um and uh if you are but it you could also um yeah do it with a with a um normal air cussion pipe head um although I would as said recommend the positive displacement system but you could also do it with an air cussion pipot and therefore we offer our customers on the website a a chemical uh resistance chart for our pipet so um you can check out um if you have also other aggressive uh chemicals um organic solvents or you know bases or assets um then you can check it um if if there’s a chemical comparability there um when we are talking about uh the air cussion pipets and soric acid please take into account that the density of this uh liquid is much higher than that of water um and that means that uh due to the air cussion um inside the air cussion pipet uh you will um have or you are going to um pipet a smaller volume uh than you set on your on your pipet so um again this speaks for the positive displacement principle because there’s no air cussion thank you or um so the next question Barbara this one is for you and this is from V who who asks uh do we have to pre-wet pets every time hey V and thanks for your question um uh I will answer it um it we yeah what is the answer it depends so um the the main question you have to ask yourself how correct um has to be your result if you have to work um in a very controlled um area and you need and be very very high precise results um then uh we would recommend for um the most liquids um to um to pre-wet but you don’t have to do it for example with equa Solutions in general because it’s not such necessary and on the other side for um foaming foaming liquids for example um this is not a good recommendation because you you um increase um the eror type so um for difficult liquids like ethanol um it is more than recommended for equa Solutions um I would say you you don’t need it um um if your sop is not recommending is so means uh your standard operation procedure um of your protocol um from your company you don’t have to do it all ways but you should have it in mind that this is in some cases very very um um helpful and and leads to more reproducible results yes I hope that question thank thank you very much uh or the next question is from you uh this is from Anna who Begins by saying hello and thank you for an extremely informative webinar and then she asks um I find petting small volumes of less than 10 microliters very problematic I after dispensing the liquid I often find residues in the tip but I’m not sure that the problem originates from the type with liquid since my labmates do not have the same problem when using the same reactors do you have any recommendations on how I can solve this problem for me it is very worrying because I’d like to improve my PCR performance yeah okay thank you Anna for this um uh for this question um yeah small volumes um can be quite tring I I totally agree um and the smaller the volume becomes the more you know the the greater the trouble is um I’m I don’t know what type of um pipot you are using or uh which volume variant you are using um so always keep with a very small air cushion if possible so um pip at the 10 mic with a 10 mic pipet um instead of using a 10 to 100 mic pipet for example um so that was would be the first thing the second um also check out for the tip quality um I don’t know whether you have different um Piper tips in your lab sometimes that happens um just uh make sure that you have tips with a good quality because um I have feedbacks from the market um where especially the pipetting of small um liquid volumes can be troubling um and uh yeah most important um is probably um really pip it slowly um and when you are doing the PIP petting um just do not only you know let lift up the thumb but then um wait a minute and leave the tip inside the liquid wait a minute um well not a minute but wait a short time let’s say count 20 21 22 23 and then take it out of the um out of the liquid and when you uh dispense the liquid also um be careful do it into a um pipot it into a um already you know placed liquid in that was previously PL placed in the in the tube or pipot towards the wall so do not do this freejet um pipeting um because that helps the liquid to really run out of the out of the tip and of course because you are mention in um PCR um I don’t know um whether you may use another you know PCR Master mix or things like that um so Master mixes are different and they can trouble because they contain detergents or um yeah tend to be slightly whiskers um and also here um there may be difference between the reagents really um and uh that that can also be um troubling so I mean if we are talking about pipeting 10 microliter I always recommend really to pipe it into liquid what that was already placed in in the tube or in the plate uh and then you can uh pipe it up and down to really make sure that everything runs out of the tip um in case you are working with for example a slightly wiscus liquid so that was a bunch of of options now and from my answer you already maybe get an idea um how difficult is it it sometimes is to solve customers problems because we do not know the background so we don’t know with which wume they are working which Piper tips they are using um which um liquids they’re really U actually pipetting and and and so there’s always a bunch of options or possibilities that can or may cause this trouble but I hope maybe with one of my answers um I I hit the core to help you otherwise please um yeah yeah get back in contact with uh with Endor um and then uh yeah um we can try to solve the your problem thank you thank you or uh Barbara a question for you this one from ANA meta who who asks uh should a mechanical pipe test be stored vertically and when stored should they be on the biggest volume to preserve the spring inside um to the first part um so the the most important thing is never lay down a piped with a piper tip and especially when a filed filed Piper tip so then you have to hold it and store it always vertical but you should not store it with a piper tip on it but um in general it’s often mentioned um but you can lay down your pipet this is not a problem um in general as I mentioned without any tip or liquid um but um I would recommend uh to to have sort it out more on the table to have it uh in a in a vertical option and um Adam can you um repeat the second part to have it on the thousand on the on the nominal volume position was it right uh that’s correct here on the biggest volume to to be honest I never heard about that um before for or do you have um more knowledge on that topic no no um but um there are a lot of M out there um so you can um if you put it in in in the pipet into the um pip pading stand you can just you know leave it you don’t have to set it to th microl when it is for example a thousand microl pipet we call it the Nom nominal volume by the way so the the volume you can the maximum volume you can um of of the volume range um printed on the pipot is called the nominal volume um but there is no need really to to store it at the nominal volume for me it was always new I never had that question but it’s it’s funny you can work for years with a topic and you have always new questions thanks for that so interesting question thank you Barbara uh or the next one’s uh for you and this is from uh John who asks um in calibration of of pets is this standard all over the world since many companies are involved in production okay um the calibration um I I don’t get the really the um I have problem to bring together the production and the calibration so um when you are um thinking about the um calibration in uh calibration service Labs um so there are um of course different uh quality standards let’s put it this way um uh and and if you have or if you have the need or you wish to um choose the highest quality standard then you should choose an accredited calibration lab um regarding the production um so our pivots I can only speak for for Endor um our pipets are manufactured in Hamburg in Germany so they are not different manufacturing sites um and of course the um the um production or in production um calibration and and checking and the the quality control uh is also done in Hamburg does it somewh somehow answer your question okay well I hope so thanks or um actually the next question is also for you um this is from CARiD who asks what is the lifetime of mechanical prepared yeah okay um yeah the lifetime is as long as you keep it maintained um so if when we speak about an mechanical pipot endov gives three years warranty um but uh to my knowledge um and I get feedbacks from a number from a lot of customers um also through these trainings um who say oh I um opened a drawer and there was an Old endol Piper that was more than 20 years old and it’s still working um yeah this is completely true so I mean it’s mechanics inside and these mechanics it’s like a you know like a watch um and and uh yeah it’s it’s working forever so to say um and the only thing you could you should take care of is that you maintain your pipets especially when you’re working with um you know for example um contaminating Solutions um you have to make sure that nothing you know sticks in the cone or yeah um disturbs um the the functionality of of of the piston for example um so clean them maintain them send them to calibration or do the calibration yourself if you can uh and then you will have yeah a very long lifetime of your pipet so there’s no end date thanks or uh this next question I think is very interesting and and this or is also for you I think um there are instan this one’s from CeeLo Marquez who who asks there are instances where Labs reuse Petes for various reasons what are your thoughts on this and how is reusing the tips um uh how does that affect low retention function of the tips yeah to reuse pads is completely is completely okay um as said just make sure when you take them over that um they are send them to a calib lab or make the maintenance yourself and the calibration just to make sure that the the everything’s okay with the with the device reusing of the tips is a different uh question so um the the piper tip is a single use item and um that does not mean that you yet that you are only allowed to make only one um pipetting with it um so we all know that for example if we do a dispensing of a plate uh we attach the piper tips um to a multi- channel pip and and uh use it for the full plate so that’s obviously more than one um pipetting step and this is fully okay what you should not do is you know wash the piper tips and reuse them again because there is a a contamination risk of course um how to get that clean really um and uh the second point is that um you have a you know there molecules um of the water um um going along the the hyped wall sorry the tips wall and um after I I don’t know we have I have no um real number in my head now but um after a high number of um um yeah movements of these molecules uh the surface of the of the tip may be uh um um yeah affected and um the um liquid May tend to retent a little bit more uh but this is nothing that you should make um yeah be really concerned about um as long as you you’re completely safe as long as you are um you know attaching your tips filling your plate and that’s it and then you throw it away okay um thanks Ora uh Barbara um this question’s for you from Laura who asks when you use the same petp multiple times the pet uh to pet the same volume what will be the influence on this on the petting error and uh when do you need to change tips hey Laura uh and thanks for your question um a good question we we had the topic um shortly before but but not in the in the presentation itself is a pre- weding um so there is always an influence and you find also diagrams on that uh on our um landing page in the in the handling Solutions um that when you pre-wet um your Piper tip um three four five times uh you come closer to the correct volume and when you pad then um I don’t know 10 times with the same pipet tip um you stay on the same volume but this is maybe for equa Solutions or more unpraised your Piper tip because the the problem with the forming increases always when you pip it again and again and again with the same Piper tip and so it again as we always have to point that out it depends totally on the on the liquid you’re using and on on your approach um or application so I would say with with with a unpro liqu bids uh you can use it I can’t tell you a number but you can use it more often the same tip with problematic liquids with ethanol for example there is a pre-wetting very important so um there is better you do it more than one time to pipet a pre-wet first and then you can do it U different times then the problem decreases with foaming it increases when you use a piper tip uh lots of times so um yeah it’s really the question of um of the liquid and if you want to know more please um give us a hint um directly and then we can talk about that thank you thanks Barbara actually staying with you there uh this question’s from John who asks uh what ways are there to test the performance of a pet in the lab good question thanks so much and um it’s really um recommended to do some tests uh in the lab a very easy thing is um the let you make a leak test or leakage test with your pipet and everybody can do it um on their own um to um yeah to to fit the tip um to the to the um to the pad and use water and hold the PIP head really vertically and wait one minute and if you see not only drops like in the video of the of the um of the ethanol um but um um also if you see only a very very small building up um of of a bubble then maybe um your pip pad is not um is not in a good um shape or the system is not good so pad or the pad tip fits not well to the pipet and this is a first test you can do and maybe it depends again how how accurate your um applications need to be um you can do it every day every week every month um this is very easy and with higher volumes if you have a um um um what is the wording uh balance um maybe with a thousand 100 microliter pipet you should um sometimes try to um pipet that on on your balance and and check out if the results are um the same as they should be um but this is not possible in the most cases for for small volumes and so we would recommend um like um I I saw another question from another person uh the the um the periods of calibration the isorm this is so to say our Bible um recommends uh one time a year a calibration and this then there will be checked everything really properly also the things you are not able to do in your lab but it depends on your uh standard operation procedure in the lab if you have to do it much more often maybe every six months or every three months but the leakage test and maybe to check with a balance with a with bigger volumes you can do on your own in your lab I hope that answer your question yes thank you Barbara um I think you also answered someone else’s question there at the same time so that’s very helpful thank you um or this question’s for you um and this is uh from LF theia uh who asks uh when reverse petting is it correct to do serial petting or do we have to use a different pet every time um yeah that’s a good question um so reverse pipetting means that you have uh that that you leave a certain amount of liquid in your tip um which is to be discarded then so uh the advantage is that most for example when doing or pipeting wiscus liquids that uh yeah the error will you know be in that um volume that you discard afterwards so from that side that is not that speaks nothing against uh doing a Serial dispensing um with the reverse pipetting to my eyes okay thanks and the the the same person actually also asked can we pit phenol with plastic tips yeah okay I just in parallel um I just uh checked out our um chemical compatibility um chart and um so I do not recommend in this case uh yeah to to go for the positive displacement uh principle surprisingly um so phenol is an organic solvent um so from that side the organ the the multipet or the positive displacement principle would be the right choice but um we have a inconsistence or let say we have a um in Advantage uh with the uh chemical compatibility with phenol so when you use it very very short it should it might be okay but you you should not um um have it in longer contact so um the other option the air cussion pip head um would be okay but also there’s a butt um so the polypropylene is resistant to phenol um but the um the seals in the pipit are not um so when you um wish to um PIP IT fenol with an air cussion PIP IT uh then you should take care to have a very short interaction time and take an uh or keep in mind that you um have a higher um maintenance fre frequency because this um piston seal will you know yeah wear out faster this is what I meant with aggressive liquids uh um in during the webinar so yeah phenol actually really is one of the liquids that uh are a little bit problematic yeah that’s true so go for positive displacement only if you have um smaller volumes and if you have um you know short very short contact times don’t leave it for longer um in that um in in the tip and if you go for the air cussion um then make sure that you have a smaller maintenance cycle okay thanks or staying with you actually um a question from uh neur Ria who who asks is the positive displacement pit available in the Philippines Ah that’s a very good question yeah um so the the um our positive positive displacement um instruments are called multipad M4 and multipad E3 or e3x which is the electronic version um and they are part of the catalog so they will be available also in the Philippines as they are part of the catalog okay uh thank you or Barbara uh this one is from Alicia who asks what type of tips uh should be prepared filtered or nonfiltered oh a good question um um in general uh for equa Solutions um it’s um absolutely fine to work um with normal tips without a filter but um when you have um yeah aggressive or um not problematic yeah I I guess the best word is aggressive uh liquids uh we recommend uh absolutely the filter tips um to um avoid the aerosole building inside the air cussion uh we had up off we have a dual filter tips so um it um sort out um not only um small liquid drops but also really the Aerosoles um and so you you save your um your pipet um with that so that you avoid in general the Aerosoles um um but um in you have to take care maybe that you have to work a bit slower you should always work very slow but um to work a bit slower um when you use a filter um tips because the liquid needs a bit more time to aspirate um or to to uh rinse into the piper tip so it really depends again this question is not easy to answer in general um on the liquid on your application and what do you need that’s great that’s great thank you actually staying with you there another question from V uh who asks uh What uh what one should do what should one do when some VI viscous liquid like enzymes stick to the outside of the pit tip while aspiration okay thanks for that question um I would say in the first line line uh we um didn’t point that out more in detail uh in the webinar but you will find that later on in the in the ebook um about the correct pipeting um to you can avoid that U mostly um because of the immersion depths so um a lot of people are not taking care on that and uh Deep dive with a pipet tip um yeah too deep into the liquid and then you have um liquid outside of the of the piper tip so the best thing is um to to really take care on the on the um depths to to go with a tip inside the liquid and so um how do you say that um um oh I don’t know sorry I’m not a native speaker I miss a word but um yeah so use uh a very uh little immersion depths um this avoids uh that and um I don’t know if I would recommend it you can very carefully when you but but it’s it’s it’s a bit dangerous to go um with a with a wipe outside um the pipet tip but then you can um cause a problem that you um it’s suck liquid out of the tip so it’s a bit it’s a bit dangerous if you are really a professional in pipeting you can do it maybe like that but the best choice is really to to take care on the immersion depths and and work really really um on the surface okay thanks uh Barbara uh just a few more questions uh staying with you again here froma who who well says thanks for a very informative webinar and I think you’ve covered calibration but uh this isn’t interesting question how often should we calibrate our pets and is there a way you could check this in the lab since I heard about weighing some drops uh weighing the drops and comparing uh them with the volume okay um so as I mentioned before shortly the recommendation not from us but from the isorm 8655 um two is that you should calibrate your pad in a calibration lab um at least one year uh one time a year but this is only a recommendation and um it totally depends on your on on so many things how many people uses a pad is it only you or are different users working with a pad what are the type of liquids um are they aggressive then you have to calibrate it more maybe more than one time a year what what uh recommends your sop if you have one one in your lab in in lots of lots of lab you have defines Sops who who Define that um that period of calibration um what else um R do you have more points uh to what what could be influence the um the aggressive liquids users so I would say um these are the most important topics so we cannot give you an answer you do you have to do it one time a year or every six months so it’s really depends again on the circumstances uh in your lab yeah Barbara this is completely correct um just a side note to that um the iso at 655 um as said this is so so I call it the Bible of liquid Hing um so that’s that’s a standard an international standard and um that recommends um a calibration at least once a year um and as Barbara said um it belongs very much on the use so it’s also the frequency of use how heavy is the pipot becoming to used how aggressive are the the liquids um how many users are there because everybody uses um the pipot a little bit different um so there is a bunch of different um yeah uh factors that come into play but there’s one thing uh which is really important and this is please focus on on your analysis so if you have a very sensitive analysis um it may be a good idea to um calibrate the piped more often um than when you are you know doing just pipeting 10 milliliters from A to B um so and there’s no um no requirement in in terms of the accuracy so focus on your analysis what does your analysis really need and from that side you can answer um this question how often a piper should be calibrated taking into account um how aggressive is your liquid how many or how heavily is the piper used okay thanks all right well we we thank you both uh we have so many questions and to the audience please be assured that uh you know that we will um uh Endeavor to answer these questions uh offline uh the questions will be sent to uh to the speakers um so that brings us to the end of of today’s webinar thanks again Barbara and or for a very Illuminating presentation and a great discussion I think and and uh thanks also to our sponsor endorf and uh finally thanks to you the audience for taking the time to attend and and listen uh so until next time uh good luck in your research and goodbye from all of us at endorf and bsize bio thank you