[Music] all right so today we're gonna be talking about calibration curves why do we need a calibration curve it's because when you inject a sample into an instrument like an HPLC you see an absorbance you don't know what it means a calibration curve it's a method by which you inject a known set of standards and you use that to compare against an unknown set of samples and then you can use the known set to extrapolate the values let's give you a real-life example say I have a 100 ppm standard and a 200 ppm standard and they give you roughly like a thousand absorbance units and and 2,000 absorbance students it could be whatever they could be area absorbance unit response units schmeckel units doesn't matter if we had a hundred and 200 ppm for the standards and it corresponded to a thousand and two thousand absorbance units and we had an unknown and the unknown was about 1,500 absorbance units we could be relatively confident that it's a it's about a hundred and 50 ppm sample today we're gonna be building our art Cal curve so let's see here we got some vitamins here all right so I would say the workable range for for this vitamin we want it to be about 100 ppm so so let's do a let's do a five point Cal curve that's pretty strong if our sample is 100 ppm we'd want to encompass the high point would be 200 150 25 and pen that that sounds like a like a pretty good Cal curve to me so how do we build a cow curve the first thing we want to start with our neat standard meat pretty much means pure a pure sample would be a million parts per million and you usually go to a stock solution our stock solution will probably be somewhere around mm mm ppm and from there it'll be really easy to dilute out to your 200 and 100 and la blah blah let's talk about materials what do we need to build a cow curve some neat standard 100 million metric flask this is gonna be for the stock solution a bunch of 10 mil volumetric blasts these are gonna be for the individual calibration points transfer pipette very handy smart spatula or dumb spatula whatever you want weigh boats balance pipette and pipette tips so those are all the items that you're gonna need so the first thing we're gonna do is we're gonna wait out roughly 200 Meg's of of meat if we go to hundred Meg's into a hundred mils that give us your 2,000 ppm stock solution right first tear the the way the weigh boat fancy that is there so we're gonna weigh up about a MIG trying to make this all right all right and we need a little bit a little bit too much so we'll take some out and just get rid of that so that should be good so that's about 200 rings from there we're gonna dump the neat standard into a volumetric flask that goes in and then we're gonna top it off with water doing the calculate can just trust the meniscus because the density of water is one gram per mil so it's gonna work but if you were using another solvent like say you were using methanol or something like that you're gonna have the factor the density in let's go ahead and pop that off so now we're just going to vortex this and then we should have our 2000 ppm stock solution so now that we have our stock solution what I like to do is rather than trying to get a pipette in here I like to take some of this out and put it in a simulation vial so it's just easier to do the pollutions and then I dump it in here and I'm just gonna label this properly from here we're gonna do our dilution so we're gonna do a temporal dilution for the 200 ppm so basically what we're gonna do is one mill of this into a 10 mil for the 200 half a mil into 10 mil so let's go ahead and get that done so let's get a pipette so let's do the first one so let's get a pipette and let's take one mil of this and let's put it in here and let's get a cream transfer pipette and then let's top it off all right so let's just for text this and then we're gonna have our first calibration point and then let's do that for the rest all right so we finished the volumetric calc curve I got my 200 ppm standard 100 ppm standard 50 ppm standard 25 ppm standard 10 ppm standard so we're gonna get all of this in two vials and then we're gonna inject it on the HPLC and then we'll see how we did so now that this is done we also want to do it gravimetric wait what does that mean that means we just weigh everything to do the Calcutta metrically we're gonna weigh every step so the first thing we're gonna want to do is we're gonna want to tear the volumetric glass let's put that in there okay so now that that's tired we're gonna add our stock solution in there again it's 1 mil into 10 mils so let's weigh the one though first let's grab our 1 mil pipette all right let's grab our 1 mil pipette and let's take our 1 mil of stock solution let's get into the volumetric and let's lay this and the next step so let's record that and let's top it off so when you do the Calcutta treat it doesn't necessarily need to be in a volumetric class it could be you know centrifuge tube it doesn't matter because you're recording every step of the way so let's try to finish up our Cal curve and get everything into vials we finished our two sets of Cal curves on this side we have the gravimetric we done Cal curve and on this side we have the volumetric Lee done how curve let's compare them and see how they turned out so we got them in the HPLC we ran both and as expected the gravimetric way of doing the cow curve was more accurate both were pretty spot-on the r-squared values for the volumetric with 998 and the r-squared values for the gravimetric for $9.99 so a slightly better result there both are very accurate but you know if you spend a little bit more time to do things gravimetric ly you're gonna get a better result because you're just weighing every step of the way