 Good morning. Good morning. Good morning. So welcome back to another video discussion that we will be having this time We will be continuing the last part of Your spectrophotometer So last time we discussed about your beer's law we discussed about what spectrophotometer is What spectrophotometer is all about compared to your photometric Instruments and we discussed what spectrophotometric Photometric instruments are all about and then we discussed all things about the different components and this time what we're going to do is to discuss your Quality control being done for your spectrophotometer. So again, I just want to welcome everybody back So enjoy learning and I hope that you'll be having a great time listening to this Video discussion and learning a lot with us I hope you will be having your pen with you your notes and also if you have your books on the side You can also do that so that you can go along with me as we discussed. So let's move on. So What we're talking about today will be your quality control for your spectrophotometer. Yes, your spectrophotometer again is very important because most of our clinical measurements done in the laboratory are actually being done using your spectrophotometer So let's dig in to what we have in store for today. So for these quality control for spectrophotometer I will be first discussing to you the two major and general type of your spectrophotometer We have the single beam spectrophotometer and the double beam spectrophotometer This video will actually be just short and I hope that all of you will be learning a lot Even we have a short time. On the other hand, we will also go through Quality control. So for the quality control of your spectrophotometer, we will be discussing here What are the different parameters that you check to be able to know whether or not your spectrophotometer is really Functioning well. On the other hand, what we will also be discussing Blanking technique. Yes, if in the gym you are doing the blanking here in Spectrophotometer, you will do the blanking and I will be discussing blanking later on and I'll also be providing you a Video, a link to the video that actually best explain how your blanking technique works Okay, let's move on. So for the types of spectrophotometer The first one are your single beam spectrophotometer. So these are the simplest type of absorption Spectrophotometer So these instruments are designed to make one measurement at a time at once specified Wavelength. So having said that your single beam spectrophotometer is actually the most common Type of spectrophotometer that we have in the laboratory, although there are some Specifications that are different from one spectrophotometer from the other Generally, most of the spectrophotometer that we are using are actually single beam spectrophotometer And before I go deeper to the single beam, maybe you're wondering how does it look like? So for you to be able to understand this discussion well, I will be showing it to you So this is actually a single beam spectrophotometer So I just don't have it in a linear form, but technically once you look at the single Beam spectrophotometer, you have your it just it's actually just a straight line a straight flow of the different parts So technically you have your light source here, and then it will pass through your Entran sleet going now to your monochromator and then your exit sleet and then once that the specific Wave length has been isolated It will now pass through your sample cell or your cubit and then the Transmitted light now will be detected by your photo detector. And again, what does your photo detector does it? Transmit or it. Yeah, it transmits your light energy your Photons and to its equivalent Electrical energy that will now be read by your reading Readout device and eventually you will be able to Know what is the measurement of your test so so in a nutshell what you're got what you're saying is that is straight or a Linear or a single beam spectrophotometer. So going back now Sir, how different is my single beam and my double beam? Maybe this is where everything will be Change or will where everything will be different? I want you to look at bullet number two It mentioned here that these instruments are designed to make one measurement at a time at one specified wavelength Okay, so what do we mean by that you big sabihin isa isa yung pag-measure mo nang iyong Analyte isa isa yung pag-measure mo noong iyong cubit or noong Analytical cell mo so what we're trying to say is that when you are using your single beam spectrophotometer What you're doing is that you actually measure one sample one sample cell one cubit at a time Yes, one at a time and one At only one specific wavelength, so how does it happen isn't it that later on I will be Discussing blanking technique and blanking technique is very important when it comes to your spectrophotometer before you proceed with your measurement in your in your Analyte you do the blanking technique first so most of the time it's actually part of the QC procedure of your spectrophotometer So going back now When we say single beam spectrophotometer you do it one at a time you first you do the blanking technique first and Then you do now the measurement of each of your samples Okay, each of your samples It is different compared to your double beam spectrophotometer because in the double beam spectrophotometer later on when we Discuss I'm actually jumping ahead of myself But I just want you to understand it clearly by this time so that we'll just have a smooth time when we go to your double beam Spectrophotometer, so technically what happened is that in your single beam spectrophotometer What you do is to measure it one at a time at a specific wave length on the other hand when you do the double beam spectrophotometer the blank is already inside The spectrophotometer and you only need to add up your Sample so I hope that's kind of giving you a Idea of how it looks like because later on when I go to the double beam. I'll gonna I'm gonna explain that further Okay, so going back to the single beam spectrophotometer the absorption Maximum of the analyte must be known in advance when a single beam Instrument is being used again It's very important for you to take note of the absorption maximum of the analyte for you to be able to use as Specific wavelength so again This is how your single beam spectrophotometer looks like and I hope by this time you actually have been Familiarizing yourself with the different components of your single beam spectrophotometer, which is very important Okay, so moving on now Do the double beam spec? So this is a much bigger picture of your Single beam spectrophotometer. So moving on. We have your double beam spectrophotometer So how does your single beam and your double beam spectrophotometer? Differentiate the first is that in your single beam. You only have one line of monochromatic light Okay, you only have one line of monochromatic light in the double beam spectrophotometer You try to split You try to split or you try to chop the monochromatic beam of radiation into two components, okay into two components Likewise what I have been mentioning a while back in your double beam You are actually measuring the Absorbance of your blank in the absorbance of your sample at the same time So that is what's happening here The reason why you have to split and you have to chop the monochromatic light Because one beam will be passing through the sample and the other beam will be passing through The reference solution or your blank. Okay, so Having said that now, this is how it looks like okay, so In your double beam spectrophotometer, we actually have two types the double beam in space and the double beam in Time and I will be explaining to you what the difference between in space and in time So in the double beam in space, this is the very Very important Very important difference that they have in the double beam in space. It's actually have two Photo detectors. Yes, two photo detectors. The two photo detectors is one For the sample and the other one is for the reference beam or for your blank. Okay, this is for your blank so sir, can you go us through about blank or Blanking technique how important is blanking technique again? Blanking technique is very important for you to be able to make sure that 100% of the light is actually being transmitted to the photo detector and Technically what the the blank actually looks like it's actually just at the ionized water It doesn't has it doesn't have these the analyte or the take for example the glucose the lipid the cholesterol that you want to Measure it actually contains nothing. Yes, it's just plain water. It's the ionized water Okay, so having said that sir, maybe you're asking Sir, what about the the ionized water? Why is it important in the blanking technique? It is important in the blanking technique for you to make sure that 100% of the light is actually passing through the quvet Okay, kasi di ba kapag if a Kaya sinasabi natin blank wala talaga siyang laman. So ang mga ngayon yan Yung laman ng blank mo is just the ionized water and you try to Let the light pass through that for you to make sure na walang light na na absorb or walang light na nakakatakas Or walang light na sa iba na pupunta. So that's very important for you to check So going back now to the double beam in space. You have food toe to photo detectors Okay, one for the sample and one for the blank or the reference for you to check the reference beam so here The correction will be made. Okay, the correction will be made if there are some Corrections to be made it will be done automatically and then you will be having your readout device reporting now your Reporting now your results as you can see here. You have here your mirror. The mirror is the one that That splits your light into two one going to your One going to your Blank and one going to your sample. Okay, so by the way, if you have any questions, feel free to comment down below Comment down in the YouTube video so that I will be able to answer you and we will be having a very meaningful interaction in Discussion on the comment section So please do leave your comments if you have any questions Clarifications or any comments with regards to this video. Thank you. So let's move on to your double beam in time Okay, so double beam in time. This is now the biggest Difference between the double beam in space and the double beam in time the double beam in space in time Uses only one photo detector. Yes, only one photo detector and Alternately passes the monochromatic radiation through the sample cubit and then going now to the Reference cubit using your chopper. So unlike this one you automatically split the He automatically split the light one going through the sample one going to the blank And then there is a specific photo detector that will be measuring Each of the light transmitted light when you do your beam in space They are actually waiting for their turn. That's how it looks like So one will go to your sample cubit first and then will be detected by your photo detector And then it will go to your reference cubit for the blank and then it will also be read by your photo detector And this is how it looks like. So I Hope you're actually seeing it clear. So this is where you're this is where your Double beam in time spectrophotometer. It looks like so here what you have here is that is actually the The light, okay. This is actually the light. This is the light source Okay, the light source passing through now your Entranslate so this thing here the one that you are seeing here is actually a monochromator this entire thing here Isn't it? This is the light source. You enter to the light and translate and then mirror For your diffraction grating your monochromator. This is actually is very have fold for you to isolate a specific Wave length and then your exit sleet. Okay, your exit sleet and then it will go out now. So first Okay, so first that would actually happen is this. Okay, it will first pass through your it will first pass through your Sample cell and then your sample cell going directly to your Detector and then the readout device the same time after the sample it will now be shifting its mirror Now going to your the light now will pass through your mirror and then again another mirror and then to your blank To your reference cell and then it will now be delivered to your photo detector That's actually how it looks like. It actually looks more complicated than your double beam in in space. Okay, but The same the two are actually the same So having said that now in the double beam in the single beam spectrophotometer The main difference is that in the single beam spectrophotometer You actually measure one sample or one cuvette or sample cell at a time and in the double beam You actually Have to load them at the same time and then the machine will take care of it already. So I hope that's clear Okay, so let's move on now to the quality control for your spec spectrophotometer. So again Quality control is very important to make sure the reliability the accuracy and the precision of your results in the laboratory And we have a couple of parameters to be checked to check the quality of your spectrophotometer. So let's move on so before I Move on. I have a question for everyone. So What are the three factors that affect? Okay, that affect your What are the three factors that affect your? degree of isolation so the degree of Isolation so what are the things that? The effects or that influences the degree of isolation so Um, please pause this video and then comment your answer down below Okay, and then we will be revealing the correct answer later on so that I will be able to see who are watching and who are actually Giving interactions in our comment back. So this is the question that I have for you what Okay, miss Our bear here is very much curious. What are the three factors that affect the wavelength of isolation? Okay, what is The factors that affect your wavelength isolation So leave your comments leave your answers on the comment box below So moving on to the different parameters that we have the first one is the wavelength or the photometric Accuracy, so what is the importance of your wavelength? This means that the wavelength Indicated in your dial is actually the the actual wavelength light that passes through your monochromator to take for example I set my Again review isn't it that your your wavelength? We have actually your visible spectrum from four hundred to seven hundred Denometer and your ultraviolet light less than four hundred and greater than 100 that is Greater than 700 rather is your infrared region So for you to take for example, I set the dial into 360 360 nanometer for me to be able to May I I need to be able to make sure that when I set 360 it actually is 360 nanometer that is Passing through my monochromator and how do I do make sure of that? I made I make use of your day Diedimium glass absorb which has an absorption peak of 600 nanometer and your volume oxide filter that has a sharp peak at 360 nanometer so this Material these are the one that we are using for us to check the wavelength or the photometric accuracy So again, how do we me? Why do we need to make sure of that because we need to make sure that the wavelength indicated in the dial or in the in the in the machine in the The the dial is actually the same wavelength that passes through the monochromator So that's very important. Okay on the other hand, we also have your absorbance check Okay absorbance check you make use of a glass filter or a solution with known absorbance for a specific wavelength And this is actually what your Blanking blanking also does. Okay, you're blanking Also do this. So what you want is that isn't it? Okay, isn't it in the absorbance check if you actually make use of a If you actually make use of the ionized water, you actually can expect that it is zero percent absorbance Zero percent absorbance. So none of the light coming from your light source are absorbed By the solution so meaning if it is zero percent absorbance, it only means that it is 100% transmittance Okay, zero percent yung na absorb kaya 100 percent yung Transmittance parang ganyto lang yun okay, so Take for example sa isang Sa sa sahod mo take for example sa sahod mo your absorbance take for example your absorbance is the tax and then your wallet yung Yung yung pinaka matatanggap mong pera is the transmittance So kung walang tax yung sahod mo take for example if your salary is one 100,000 pesos and it passed through zero percent absorbance So you will be receiving the entire 100,000 pesos But if some of the light or some of the salary had been affected by your tax take for example 20 per May 20,000 that there is 20,000 that had been taxed or had been absorbed So you will only be getting at a transmittance or a salary of 80,000 pesos So that is how that is actually the analogy when it comes to your absorbance and your transmittance They are inversely proportional okay, so What you want to do is to check the absorbance if it is really zero percent absorbance So what you do is to use Samples or solution with known absorbance Okay, you you actually know the absorbance and you want to hit that absorbance again You're checking the accuracy and the prestation. So let's move on to the fourth the third one We have your linearity. So When we say linearity it should form a linear relationship between the radiant power or the radiant Energy and the concentration it talks about more about the bear's law for both your slope and your intercept So, how does your linearity looks like? Okay, I want you to imagine a solution with different With different colors so a solution with different colors take for example, you have the Let's just go for for the red. Okay, if for example, you have the dark red You have the red and then you have the light red and then you have your Pink and then your powder pink and even a lighter color of pink. Okay, so take for example The color represents your concentration The more color the darker the color of the constant the solution the higher the concentration of the sample The higher the absorbance that it has are we clear? So Take for example What you're actually do do in your linearities that you actually measure a series of sample from the darkest to the lightest So if I'm gonna translate that to concentration from one with the highest Concentration and one with the lowest concentration and what you should expect that the absorbant should also be decreasing okay, the absorbance take for example, I have samples letter a samples letter b and Letter a letter b. Okay Take for example, I have sample letter a sample Let letter b Letter c and letter d. What actually what we're actually gonna do with that is that Okay, let me try to Let me try to give you an example by doing this one Example you have samples a b c and d Okay, the dark the dark red the red the light red So the pink and the light the light red. So or the light pink so to speak So those are samples of decreasing solution You also expect that their absorbance are also decreasing. So if the absorbance of letter a is 100 You should expect the absorbance of letter b is 80 The absorbance of your letter c is 60 and the absorbance of your letter d is 40 So what I'm trying to say is that the when we are talking about linearity if it if the Concentration is of decreasing absorbance or decreasing concentration. You should also be able to see that Okay, you'll also should be able to see that linearity. So the change in concentration Result in that straight line calibration curve of your beer slow. So that is for your linearity So last but not the least is for your stray light. So Again, what are stray light? So this your stray light So remember that when we discuss your entrance lead This is the number one job of your entrance lead to prevent your stray light because it causes your absorbance error So your stray light again, let me explain what is a stray light for you stray light or any wavelength in Any wavelength outside the bond transmitted to the Monochromator so Usually any light take for example, all I want is from the visible spectrum 400 to 700 nanometer. So any wavelength outside outside that is considered to be a Stray light. Yes, correct. It is considered to be your stray light and you want to prevent that Okay, so what are the usual causes of your stray light? What are the usual causes of your stray light number one is the reflection of light from your Scratches or mga gas gas, okay, so usually if you have scratches in your monochromator or scratches in your Your sample that could actually or your cubit rather that could actually be Causing stray light, okay, because the light will reflect from the scratches Okay, the other one another cause of your stray light are dust particle If the that's the reason why you also have preventive maintenance for your your machines to Clean them up from the not only from the outside, but specifically more importantly from the inside So that is the common causes of stray light against stray light. These are any light outside the band Okay outside the wavelength that you want So if you have stray light, well, what will it cause it will now cause absorbance error Okay, it will now cause absorbance error So how do you detect your stray light and your absorbance error you detect it using a sharp cut-off filters So you use your sharp cut-off filter for you to be able to identify or For you to be able to make sure that there is no stray light in your Spectrophotometric system. So in a nutshell, we have four parameters. We have your wavelength. We have your absorb We have your Absorbance check and we also have your linearity and your stray light and these are very important quality control parameters. So Now we go to your blanking technique, okay, and I'm actually linking a A video and you can see it here in the Cards and the in the cards that I am showing the link that will be showing up in the video right now Okay, so I'll please click on that afterwards I will also be leaving that in the description box so that you will be able to just click it there and proceed to the blanking technique so reagent blank Okay, what do you want to correct in the reagent blank as simple you want to correct the absorbance caused by the color of the reagent Okay, and on the sample blank What you want to correct is to measure the absorbance of the sample and the reagent in the absence of the end product Okay, so in the in the reagent block blank sometimes the color of the reagent would affect the absorbance of your Samples so you want to So you want to remove that, okay, so what you use is a reagent blank so this happens if your sample is a Colored sample, but if your sample is colorless you can actually just use a you can actually just use In the ionized water, okay the ionized water It's a transparent and then you will also be using a a colorless sample if your sample is colorless your blanking technique using The ionized water would do its job, but take for example, you have a color the agent take for example For your cholesterol, it's colored pink. So what you're going to do is to Pipet the reagent and then you will also be doing a blanking of that Okay, again, I'll be linking the video so you understand it better So you also have the sample blank you what you do in the sample blank is that you add up the sample and the reagent without the Without the end product first, okay Well without the end product first so The the definitions I have given you the reagent blank and the sample blank is very much the simplest Definition that we can have so that you will understand it better, and I hope it is helping you a lot. So I'm actually nearing the end. So these are the references that I'd made I use for this video So I hope that not you will not only be relying on the video or the Summarized notes, but you will also be looking into your books. Okay, so Again, we have a questions a while back So what are the three factors that affect the degree of? Wavelength ice wavelength that is wavelength wavelength isolation. So what are those three? I hope so this is your final chance to put your answers in the comment box section and the answer is it is your monochromator the weed of the entrance lead and the weed of your exit sleet, okay, so thank you very much for Thank you very much for Sticking with me throughout this video. So if you have any question, please feel free to comment down below And then I will be very much glad to answer your question. So Again, don't forget to hit like button share it to your friends and Don't forget to subscribe to my channel Jomar add-downs and For you to be able to be updated for the latest Academic videos that I will be posting here in my channel. So that would be all for today Thank you so much and have a great day Matt and like what I always say Enjoy learning because learning will get you far far in for your dream So, thank you very much and have a nice day ahead of you