Questions related to Spectrophotometry
Would you please let me know if you are expert in the determination of P and Si in ores by spectrophotometry methods? If you help me, I will add you as a co-author in one of my ISI papers.
I use spectrophotometer to detect surfactant SDS with different concentration. (wavelength 300-700) peak at 450nm.
However, when I add a RNA virus in my solution, the spectrophotometer can also detect virus at wavelength 650nm.
My question is: is the spectrophotometer detecting the proteins on virus ???
what type of molecule can be detected at wavelength 650nm?
I have searched this question myself but I am still confuse about it. In one article, I read that such quantitative analysis of ethylene glycol was done via HPLC. I am using ethylene glycol as carbon source to grow a bacterial strain. Now I wish to do spectrophotometry to measure its quantity in culture media at different intervals but I am not sure whether it is doable and what wavelength should be selected. I really need guidance on it from relevant expert.
Thanks in anticipation!
Hi, we have a trouble to use atomic absorption spectrophotometry method (PinAACle 900Z graphite furnace) to measure calcium, i.e., the detector was always saturated with Ca standard and samples. It will be highly appreciated if you could provide other alternative way to accurately measure total mitochondrial calcium.
Hi everyone, I have a question regarding the determination of dissolved aluminium by colorimetric method using pyrocatechol violet according to ISO 10566:1994. In the preparation of 'mixed reagent', we have to add 5 mL of the aluminum standard solution (10 mg/L) into 100 mL of this mixed reagent (total volume).
My question is:
- What is the role and function of the addition of standard solution for this method? In this standard it only says "accurate addition of the aluminum standard solution is essential in order to allow a linear calibration at low concentrations".
Note: if we calculate the concentration of this aluminium standard solution in the sample is about 15 µg/L.
Thank you for your answers.
I was determining flavonoid concentration using AlCl3 method from natural product extract (based on Estimation of Total Phenols and Flavonoids in Extracts of
Actaea spicata Roots and Antioxidant Activity Studies; by Madan, Banshal, Kumar, Sharma; 2011) and used Sodium acetate instead of Potassium acetate (Evaluation of Antioxidant Activity, Total Flavonoids, Tannins
and Phenolic Compounds in Psychotria Leaf Extracts; Formagio et al, 2014).
I have prepared a calibration curve (With R^2=0.9918); by preparing different concentration of Quercetin (SRL chemicals) yellow powder in methanol. I did not notice any 'perceivable' colour change after I add reagents into Quercetin aliquot. However I prepared a conventional calibration curve.
It gave me a strange result. Very high flavonoid content in the test extracts; even greater than Total phenol content (by FCR method) of the test extracts
Based on this ( https://chemistry.stackexchange.com/questions/35389/does-the-aluminium-chloride-assay-ordonez-et-al-2006-for-total-flavonoid-conte ) stackexchange post; I prepared a control set for the yellowish background correction (for each concentration of quercetin) where only water was added to quercetin aliquot (instead of same volume of AlCl3 reagent). This second experiment gave me weird result; the ODs increase with quercetin aliquot but OD(Reaction) - OD(Control) keeps fluctuating (positive and negative values at random).
Where I might be doing the mistake? Anybody else encountered similar problems?
Experimental bacterial growth curves with OD600 in the y-axis typically show rate of growth until a stationary/plateau phase is reached. Theoretical growth curves with the number of cells in the y-axis additionally show the death phase, where the number of cells declines.
Can the spectrophotometer distinguish between dead or living cells at the plateau phase, for example if they scatter light differently? or can this only be known from additional viability assays such as stains?
Sample = 0.5g .
After extraction process, 1ml of sample was mixed with anthrone and sulphuric acid.
Concentration of sample obtained from glucose standard curve equation =103 mg/l.
How to calculate total carbohydrate content in g/100g?
I am going to analyze the degradation of diclofenac carried out by an experimental reactor with UV-VIS spectrophotometry. However I am not sure what concentration I should use to observe changes in absorbance.
What problems could I have to observe the evolution of the absorbance if I use a concentration of the order of mg/L or μg/L?
Thank you for your time.
Spectrophotometry applications are useful to measure the absorbance, reflectance, and transmission of light by gases, liquids, and solids.
If spectrophotometry can be used to measure TSS, then what are the steps to calculate total soluble contents from the light absorbance or reflectance values?
Given the absorbance spectrum, how could one calculate concentration using the slope (tangent) of linear part of that spectrum (e.g. from 300 nm to 450 nm is linear)
I want to know, what's the cause that we are measuring the absorbance at different wavelength? why not the same? what would be if I used a same wavelength for all the assays related to it.
I am initiating a discussion on measurement of spectral transmissibility of ophthalmic lenses. I intend to investigate how these properties change over time with a view to understanding how much vision/protection ophthalmic lenses allow wearers over time and use.
A comprehensive way to find the concentration of random solutions would enhance benefits related with health, industry, technology and commercial aspects. Although beer lambert law is a solution, there are some cases where Epsilon is unknown (Example: A Coca-Cola drink or a cup of coffee). In this cases, proper alternative ways of determining concentration should be suggested.
We are interested in assessing the quality and integrity of our formulation (Solid Spray Dried Bovine Serum Albumin particles suspended in a polymer solution of Ethylcellulose in Dichloromethane). One measurement that we believe will be helpful is measuring the Turbidity of this suspension. Is there a method to measure turbidity of this suspension using UV Spectrophotometry? I have seen two different wavelengths that are commonly used in these measurements, 330nm and 600nm. Is one wavelength preferable to the other for proteins?
I am trying to assess the potential for a set of small molecules to bind to transition metals (Cu2+, Zn2+, Fe2+, etc.), and am wondering if there is an established protocol for assessing binding constants.
One of the closer examples I have is the following paper (https://pubs.rsc.org/en/content/articlepdf/2006/cc/b611031b), in which the researchers test the absorbance spectra of a small molecule probe against ATP using a standard spectrophotometer. Would I then construct a concentration-response curve from a selected wavelength that shifts with a titration of metal?
I appreciate any and all help! Why is it that Beer's Law is popping in my head?
In an experiment, data produced by uv-vis spectrophotometry for a solute is taken against solvent without any treatment we are checking it's conc. reduction so the initial conc.which is control Is measured against treated samples we found the reducing OD ,now is the direct result sufficient or it should undergo ANOVA ?
I am trying to isolate RNA from vesicles but despite the RNA concentration by spectrophotometry. I am concerned by the 260/230 ratio of these samples, all values around 0. What could be a reason for that?
For note, EVs were purified by sequential centrifugation. Then, the vesicular RNA was isolated using a commercial kit.
Thanks in advance!
I have tried nitrate adsorption by 2010 Hach spectrophotometry(based on cadmium reductin). But some results(adsorption capacity) have been negative.
I want to try cadmium reduction method by an usuall spectrophotometer. do yo know is it working?
i want to determin the chromium concentraion via spectrophotmetic method, but in this method the reagent reacts with the metal to form a pink-red complex, now the solution to be analysed is red to brown before even adding the reagent , i am afraid that it might interfere with the results !
any suggestions on how to remove the color of the solution before spectrophotometric analysis?
I'm looking for a recommendation for an alternative to the Roche assay for analysis of 2,3-diphosphoglycerate (2,3-DPG) in blood, since they apparently have production issues with expected delay of at least a year.
The Roche assay is based on extraction through perchloric acid and subsequent neutralisation by potassium bicarbonate, and using this kit, 10 148 334 001, for detection by spectrophotometry (detection range of the kit is 0.02–0.15 umol).
I have freeze-stored samples already prepared according to the described method, awaiting analysis, but I also have blood units which I haven’t sampled yet. Ideally, I’d like to find a substitute to the kit, but changing method is also an option for my future samples.
I’d appreciate any recommendations very much!
Kind regards, Linda
I'm currently working on examining the activities of mitochondrial respiratory chain complexes. I successfully measured complex I, II and III; but the assay doesn't seem to work for complex IV.
Its an assay on whole tissue lysate (mouse heart) and its measured by monitoring the absorbance at 550nm. I follow the protocol outlined in this paper.
The only advice I came across was to check that my cytochrome c is reduced. I tested that but it wasn't the issue.
If anyone has any ideas about what could be wrong with my assay, I would be grateful for any advice :)
Hi everyone. I've been working with a bacterial isolate that potential for IAA production (quantitavive method with spectrophotometer). Last January, the isolate produces IAA with relatively high consentration (the color is red). But last week, we're testing it again but it's not producing IAA anymore (the color didn't change). We tested it with LB and NB medium and nothing change.
Is there any factors that affect IAA production since IAA was their secondary metabolite? Or do you guys have the same problem and how to solve this? Thank you.
Is there a method to determine GSH and GSSG in plant material using spectrophotometry but without using GR nor NADPH?
I use the Noctor and Foyer (1998) method but it needs expensive reactives such as NADPH and GR
I have hydrolyzed a protein source (like meat) and produced protein hydrolysate.
The resulting compound is a mixture of amino acids, small and large peptides. I am looking for a way to measure the amount of total amino acids and proteins (peptides) separately. Because amino acid assay methods may also identify total peptides.
Due to the large number of treatments, I wanted a method that is fast and simple, such as spectrophotometry.
Thank you for your help.
I understand that it is possible to quantify total protease enzyme activity via spectrophotometer by using casein as a substrate and measuring the reaction of tyrosine with Folin & Ciocalteau's Reagent. Is it possible to read the absorbance of specific protease inhibitors such as aprotinin (serine protease inhibitor), pepstatin A (aspartic protease inhibitor), and leupeptin (cysteine, threonine, and serine protease inhibitor) by simulating such a reaction, and could this be accomplished using a spectrophotometer? So far, my literature search leads me to believe that the activities of these protease inhibitors can only be quantified through SDS-PAGE. Any help is greatly appreciated.
I will be performing a study on how well anatase nanoparticles are attached on the fibers. As such, I would like to have a measure of how the amount/concentration of TiO2 NPs on the fibers is decreasing upon application of agitation. UV-Vis was suggested to me as the technique of choice since it's facile and the response of TiO2 to UV-Vis excitation is already well-documented in literature.
Should I just be examining how the relative intensity (or absorbance) is decreasing and correlate it with the decrease of TiO2 NPs present on the fibers? Thank you for your answers!
- Is There Any Feasible Method To Test The Efficiency Of Fluorescent Compounds Other Than UV Spectrometers ? Suggestions Would Be Appreciated !
We are looking for a buffer system for acetonitrile (pure, no water addition) with pHS around 16+ for comlexometric measurements with UV-Vis technique.
I work with circulating miRNA in plasma, so detection of hemolysis is extremely important to me.
I'm trying to measure it with the Nanodrop 2000c and I'm using either the A(414nm )/A(376nm) (DOI: doi:10.3791/56326) or the HS (A(414nm)-A(386nm)+0.16*A(386nm)) (DOI: 10.4155/bio.13.344) and sometimes these two techniques give me contradictory results, can anyone tell me which one is more reliable? And can you tell me if you use other techniques that are based on spectrophotometry?
Thank you so much!
I'm currently using Bradford method for a protein assay. And I am measuring the proteolytic activity in roots of certain plants, as many studies found plants have in situ proteases to aid the nitrogen uptake. To determine the proteolytic activity, I will incubate the roots in a known ovalbumin standard solution. Then, after a period of time, the Bradford Assay will be conducted on the incubated solution to look for a decrease in the concentration. I am stilldetermining what concentration to use. In case you don't know, the dye itself without proteins has a maximum absorbance at 495nm, with proteins, it will have a maximum absorbance at 595nm. The dye also has a protein detecting range of 200 - 1500 μg/mL, which is the case for albumins (often BSA is used, but I was restricted to use ovalbumin). When I tested the ovalbumin solution with a concentration of 1500 μg/mL, no color change could be seen, the peak from spectrophotometric analysis also remained at 495nm. I then tested with a range of ovalbumin solutions, then realising with a concentration at or above 50mg/mL, a color change can be seen. The expected results are, there should be a color change & a peak at 495nm when the ovalbumin concentration is at 1500 μg/mL, but there wasn't, and it also requires a high concentration to detect protein concentration, which is odd for a sensitive assay like the Bradford method. Do you have any insights or ideas of what is causing this problem? My current idea is, there is something abnormal about the dye (this is the product I'm using from Bio-rad), or the ovalbumin powder that I used to make the solution has impurities. The impurities idea might be incorrect though, as the powder I'm using is lab grade, and the impurities shouldn't be too significant to this extent.
I am currently conducting a protein assay with the Bio-Rad Protein Assay Dye Reagent Concentrate, it's active ingredient is Coomassie Brilliant blue G-250. When distilled water is added to the concentrated dye according to the ratio given on the Bio-Rad manual (1 part of concentrated dye 4 parts of dH2O), the colour changed from its darkish-red to a lighter brown colour. Then, to test how the water affects the dye, I continued adding more distilled water. As more water is added, the colour changed from light brown to green, then from green to aqua, then aqua to blue. When the colour stopped changing, no more water is added. The whole volume of the water-dye solution is now around 50mL, and it appears to be light blue. Before & during & after adding distilled water, the water-dye solution has remained at a pH of 1.5 (due to the presence of phosphoric acid in the dye). Is this colour change normal when water is added? This may or may not be related to the of the expiration by 1.5 years from its 1 year shelf life. Thanks.
I am in the middle of research in describing mode of action of antimicrobial extracted from plants ( still using raw extracts). there are 2 indicators i want to describe as mode of action, 1. leakage, 2 lysis. i have reviewed some papers reported that increasing number of absorbance at 260 nm and 280 nm will correlate with leakage while decreasing number of absorbance in 595 nm will correlate with lysis of bacterial cells. However, when i measured the absorbance, the 595 nm resulted absorbance below 0,1 even for bacterial suspension equal to 0,5 McFarland and 1,5 McFarland. the second one, when i tried analysis the absorbance spectrum from 200 nm to 800 nm, i did not observe peak at 595 nm and 600 nm. all absorbances were below 0. the peak was observed high in around 200 - 300 nm. have you experienced with this ? would you mind to share your suggestion. Thank you for helping me.
In the hemolysis assays after incubating the erythrocytes with my protein, centrifuged to remove the cells and measured the absorbance of the supernatant at 490 nm (according to the protocol) but, what is measured at that wavelength? I would appreciate your response as I want to know the basis of this step.
I am looking for a standard protocol for determining the Total Alkaloids in Tobacco Leaf by spectrophotometry. All papers on this topic don't have detailed information and some critical information in their followed procedure completely missing.
I wish I can find a database for plant phytochemicals and for plant Phyto-constituents.
For molecular Biology, so many manuals and step-by-step protocol for different purposes easily find, but for phytochemical analysis, such types of protocols do not exist.
I am trying to assess chlorophyll-a content from coral tissue using spectrophotometry (with SPECTROstar Nano, BMG). However, I always seem to obtain some negative values in my optical density raw data which then affects the chl-a calculations (Ritchie, 2008)
My extraction solvent is 95% etOH (laboratorios Roldan) and I use this as a blank (one blank well at the end of each line of wells filled with the samples)
I have tried different protocol settings in the spectrophotometer but still getting negative values:
Usual protocol settings:
BMG 96-well microplate
Path length corr.ection off
well scan: spiral average (4mm diameter)
settling time 0.5s
No. of flashes: 30
Shaking before plate reading: orbital 500rpm for 5s
well scan: orbital
settling time: 0.1
No. of flashes: 21
Thanks so much for your thoughts!!
I use the 1.5N HCl/EtoH method to extract the anthocyanidin from potato roots, then dilute 5 times with 0.025M potassium chloride, pH1.0 and 0.4M sodium acetate buffer,pH4.5 respectively. To measure the absorbance at 510nm and 700nm.
Set into the formula: Anthocyanin pigment concentration(mg/liter)=(A*MW*DF*1000)/(ɛ*1)
But now I have a problem is that the result of the A=(A510-A700)pH1.0-( A510-A700)pH4.5 is a negative value that I can’t have result from this formula.
So my question is Why A would be a negative value?
I was measuirng protein concentration using both UV spectrophotometry method and SEC-HPLC method at 214nm wavelength. But the protein concentration of using UV is higher than HPLC. What would be the possible reasons? The protein concentration range I got from UV is 0.06 - 0.07mg/ml, while from HPLC is about 0.04 - 0.05mg/ml.
I’ve been doing Sandwich Elisa with Greiner Bio-One High Binding Standard ELISA plates (clear polystyren, microlon600 surface treatment, Fisher scientific). It didn’t work well, but we’ve figured that antibodies didn’t have the affinity for the species of interest. Now we’ve changed to different antibodies, provider protocol is very similar to the old one. The main thing that differs in protocol , they suggest using Corning costar 9018 plates (Also clear polystyren, high bind surface treatment??, fisher scientific). They’re also high affinity plates and I cannot find significant enough difference between Corning 9018 plates and greiner bio-one plates. I plan to stick to the greiner plates so there’s less variability between experiments (apparently it’s an issue, according to some reviews). Does anyone has any info on Corning costar 9018 plates? Is there any reason to use them instead of greiner ones?
p.s. I’m analyzing IL-1 beta, 17 Kda, human.
Have any one performed edu assay by spectrophotometric means ?
I'm looking for a kit for that purpose.
I have Chinese kit for this purpose, I followed their protocol
but the result is not good enough
so, I'm looking for similar kits to apply some modifications on my protocol...
I'm having problems with the method based on cresol purple to measure pH in sea water. I made a 10 mM solution of pure cresol purple in Milli Q water, as did Easley & Byrne 2012 and Liu et al 2011. I adjusted the pH to 7.9, as specified in Dickson and did an absorbance spectra, using 50 ul of CP solution in 3 ml milli Q water. I got a nice peak at 434 nm but nothing at 578, so I obviously cannot have a ratio A578/ A 434 of 1.6 as asked in the SOP, What did I do wrong?
Thanks in advance, Elise
I want to measure protein and nucleic acid within conditioned medium. I used nanodrop with fresh medium as blank. Yet, I got a weird result, the blank absorbance is higher than the sample. However, I expected that the blank will have lower absorbance since it doesn't contain growth factors or other molecules released by cells. If I use PBS or ddH2O as blank, is it comparable? since, I used medium containing phenol red. Thank you
To study and analyze plant extract through GCMS and UV viz spectrophotometry, i want protocols that how can I prepare sample for both technique.
I am currently pursuing my research in Phycology. I'd like to know if there's any simple and cost-effective method to detect and analyze nutrients and vitamins in the algal matter. The experiment should be quite feasible in a basic research laboratory.
Isosbestic signals are usually associated with the spectrophotometry. What could be the possible explanation for such signals on cyclic voltammetry?
I am working with HUVECs and induction of oxidative stress. I have attempted to measure intracellular ROS using DCFDA analysis by spectrophotometry at the recommended excitation/ emission wavelength. To obtain more accurate results, I performed a cell viability test after and then normalized the values of both analyzes. However, the data were not consistent between independent experiments and the values were not accurate considering the positive (incubation with H2O2) and negative controls.
I have read that certain publications use a fluorescence microscope to measure the reaction, but I am not sure if it is the best way to measure intracellular ROS level. On the other hand, some researchers demonstrate the use of flow cytometry in the detection of reactive oxygen species, although I do not know how accurate it can be or if I must combine it with another complementary test to normalize the data.
I hope you can advise me an accurate detection method and if it is possible to recommend a specific protocol to apply to HUVECs.
How will 4% w/v SSA interfere with the NADP/NADPH ratio? Can this method of deproteinization be used as an alternative to 10 kDa cut-off spin columns in order to measure NADP/NADPH ratio?
In general, in a ligand-protein binding study, is there a limit to what percentage of a ligand can be used relative to the final reaction volume?
For instance, a ligand is dissolved in buffer A or water or any organic solvent and the reaction has to be set with a protein in a buffer B at some pH. Is there some established information regarding what the maximum percentage of that ligand can be employed such that the buffer properties of the final solution doesn't change?
Please tell me what to look into. Thank you.
Hi. I'm encapsulating a protein inside a liposome (lipid vesicle). I want to quantify encapsulated protein by UV since it absorbs and the lipids don't. But I need to dissolve both the liposomes and the protein to form a homogeneous traslucent phase in order to measure absorbance of the protein. Which solvents are useful to dissolve both the liposomes and the protein without precipitating the protein? I have tried mixtures of ethanol, methanol and chloroform and they all precipitate protein or form turbid/two phases. I have access to different solvents. Protein is hydrophilic, lipids are amphiphilic. Thank you.
I have recently done a research on spectrophotometric analysis of iron in selected vegetarian food items and done poster presentation International Youth Conference on Science, Technology and Innovation organised by NAST (Nepal Academy of Science and Technology), Ministry of Education and National Youth Council of Nepal on 21st October 2019. Now, I want my paper to get published. So, kindly help me out this.
Ankit BK, Bsc Chemistry
Prithivi Narayan Campus (TU, Nepal)
I am trying to extract RNA using trizol from very small parts of honeybees e.g. antennae and mouth-parts and was taking as much of the aqueous phase as possible in order to maximise yield. Following multiple problems with DNase treatments not working we decided that phenol carry over was to blame and are now trying to find a middle ground between decent quality (260/280 ~1.8-2 and 260/230 ~ 1.8-2) and decent yield. I have started using glycoblue as a co-precipitant to increase the yield and whilst the 260/280 remains high the 260/230 never gets above 1.3, is this the glycoblue?
Hi dear researchers
We used HRP (Biobasic) in phosphate buffer (pH: 6.5) and after adding TMB, the reaction turning to light blue without adding H2O2. After adding H2O2 to reaction media, the brown color was produced (not expected blue color).
There were no changes after decreasing TMB, HRP and H2O2 concentration.
We expect that after adding TMB to the enzyme without H2O2, the reaction doesn’t start and after adding H2O2, we expect that see blue color.
( I have checked and rechecked all calculations, buffer composition, pH etc. )
Based on references I've read, the dehydration of xylose to form furfural requires heating (most of them above 100°C) in the presence of a strong acid. However, I was wondering if it's possible to bypass the heating process.
I am confused that how to calculate total albumin or total globin? whether we have to do all the tests then sum them up to find total protein or we have to subtract any of serum proteins from the total proteins in serum using spectrophotometry kits
I am currently trying to estimate simvastatin concentration within microemulsion. The method I did before was diluting 12 mg simvastatin in 24 mL microemulsion (500 ppm) (tween 80, propylene glycol, oleic acid). To perform spectrophotometry, the solution was diluted to 50, 40, 30, 20, and 10 ppm with methanol. From other journals, it is mentioned that simvastatin wavelength would be around 238nm, and I've, kinda, proved this claim when trying to find simvastatin solubility within propylene glycol and oleic acid which is diluted with methanol, it showed that the peak was at 238nm for both. But, when I did it with microemulsion at 5 ppm diluted with methanol, even at 210 nm it already has an absorbance of 2.2 and peaked at 3.2 at 230 nm, and this is not suitable with Lambert's Law equation. Is there something that I missed here?
I am thinking of using a crockmeter to rub on a coated fabric that I have with a clean, white cotton fabric on top. I want to find a way of determining, the amount of coated substances that has smeared onto the cotton fabric, or a way to determine the color change on the fabric (that is not the standard color transfer scale). Anyone know of any method as such?
Hi everyone, can anyone please tell me if we zero the blank in spectrophotometer in DPPH assay? or we record the number of blank and continue reading the number for samples without doing zero for blank? Many thanks
It is said that light passing through a single slit forms a diffraction pattern. However, I couldn't find any research to say whether where this phenomenon starts? Before? Inside? Or after the single slit? At the inlet edges? Or at the outlet edges?
Please let me know if there is any reference for it.
I'm conducting a phytoremediation experiment on pesticides such as urea and 2,4 DCP from water, so I need suggestions on the best way to determine the concentration of these pesticides in the plant?
I'm trying to reduce He to HeO2 with Sodium Dithionite, protocol for measuting nitric oxide from the Hemoglobin assay (Murphy and Noak, 1994). But for some reason (my suspicion is a bad sodium dithionite) i can not get HeO2.
Should I use more sodium dithionite or change to another reducing agent?
we have "inherited" an older UV-Vis spectrometer - a Cary 100 Bio - which is still available now from Agilent.
As no one is experienced with this device i read available information i could find on how to properly use it.
But my "problem" is that the infos are not coherent.
My main question is how to measure properly in practice.
In the manual it is not described clearly - e.g.
for the scan program it is asked to add a blank/reference when using baseline correction, while for the zero nothing is told.
While in the simple reads program it says "Make sure that the sample compartment is clear (or you have a blank in position) and zero the instrument by pressing the Zero button"
In simple reads you have no options - but the readings differ when you add/dont add some sample in the read beam. Which tells me: simple reads might be using double beam. On the other hand the simple reads manual you can zero with a clear compartment - which would make the sample measurement with sample front and blank in back look strange.
On the webpage you find (https://www.agilent.com/en/support/why-do-we-perform-a-zero-baseline-in-scan-faq) that zeroing should be done "using the actual solution (without the sample) in both the Front Beam (Sample) and the Rear Beam (Reference)."
This practice also is used in an introductionary video by supreme science:
But in a video from Yale a blank is only added in the front beam holder