Science method

Spectrophotometry - Science method

The art or process of comparing photometrically the relative intensities of the light in different parts of the spectrum.
Questions related to Spectrophotometry
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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.
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Ortho-Phosphate, Antimony, Silica (silicon dioxide), and Tannins will react with molybdate but under different conditions. See the 1989 version of the AOAC (Association of Official Analytical Chemists).
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I have to find protein percent in a different type of fruit. Which protocol should be used if spectrophotometry is used in it.
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Thanks a lot. W.J. Colonna
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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?
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here's the link talking about using a spectrophotometer to determine the concentration of bacteria.
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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!
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Ethylene glycol can not be observed by UV analysis using HPLC. It must be derivatized to detect using UV. To measure it, you will need to resolve it away from all of the other compounds present in your sample so UV/VIS spectrophotometry is not select enough. Depending on what types of compounds are presnet in the mix, sample prep procedures will need to worked out, then methods using techniques such as HPLC with derivatization or GC may be used.
When researching basic questions, skip these web forums. Instead, try perform a simple keyword search on the web (via Google, Bing etc...) to find additional info on the topic and/or question. You can easily answer your own question(s) by looking up the structure of the compound, its UV absorbance, examples of papers describing analysis of the same compound in mixtures using different analytical techniques etc. Learning HOW to research a question is one of the most important skills you can learn.
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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.
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I think,
try complexometric titration with standard EDTA solution. First ash the sample and then titrate with standard EDTA solution.
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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.
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This is a typical calibration procedure. You will need multiple concentrations to develop a quantitative response relation that you will use to estimate concentrations in your samples
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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).
EXPT-1.
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
EXPT-2
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?
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Hello Rajarshi. First check again - Does quercetin dissolved in methanol?. Second can you try using Rutin in place of quercetin for the calibration curve? Just prepare concentration of 20-100microgram of rutin using serial dilution. That will give you a straight line graph with equation for the estimation of your flavonoid content.
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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?
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Thank you very much for your reply Malcolm Nobre
I read that some metabolically inactive (~dead) cells can still have intact membranes that escape staining… I will look into other assays as well.
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Sample = 0.5g .
After extraction process, 1ml of sample was mixed with anthrone and sulphuric acid.
Total 7ml.
Concentration of sample obtained from glucose standard curve equation =103 mg/l.
How to calculate total carbohydrate content in g/100g?
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Miss Sima Shirahmadi, can you share where you get that formula? And what is the unit measurement for the total carbohydrate using this formula?
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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.
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make sure that you use high sensitive spectrophotometer with two path lenght and that make a scan of your sample. beside you should yourself the maximum wavelenght of diclofenac don't use the one of litetraure.
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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?
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This was already done in the early or mid-1980s using a spectrophotometer which has the originality of using an optical fiber, marketed at the time by the Métrohm company.
Most commercial photometers or turbidimeters, designed for laboratory use, are - most often - poorly suited to field measurements. The use of optical fibers makes it possible to separate the measuring cell analyzer and to design open cells less likely to clog or deteriorate. The light from a 10 W halogen lamp passes through a gradient filter which (depending on the manufacturer) allows the wavelength to be fixed between 420 and 700 nm with a resolution of 10 nm. The light is then split into two optically modulated beams at two distinct frequencies using a rotating shutter. The reference light beam comes directly at the detector, the measurement beam takes the outward fiber, passes through the effluent sample, is reflected on a concave mirror and is guided by the return optical fiber to the photodetector. The system, therefore, operates in the double beam. The substantially monochromatic incident light allows the wavelength to be fixed without the need for an array of interference filters.
The outgoing and return optical fibers of the semicircular section are joined and joined together in a single guide which leads to the measuring cell. This consists of a stainless steel bracket supporting the concave mirror. We, therefore, have an open measurement cell. Its optical path is 20 mm. The digital display provides either an absorbance measurement or a transmittance measurement. The digital signal is neither damped nor integrated. The hardiness and ease of use of this photometer, although quite desirable for field measurements, are however accompanied by a few drawbacks, in particular the existence of an optical fiber that is too short, the absence of devices for integrating the signal, autozero or analogue output proportional to absorbance.
But, there was a new model (METROHM 662) marketed
since the beginning of 1985 and based on the same principle and which includes some improvements.
Regards
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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)
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This method has no benefit that I can see over the usual method of measuring the absorbance at a single wavelength and applying Beer's Law. It is also unrealistic in that absorbance spectra typical lack linear parts, except as approximations over short wavelength ranges. Nevertheless, below I examine the proposed method in detail.
Consider the absorption spectrum of a substance S in a dilute solution, such that Beer's Law pertains and the absorbance at any wavelength is directly proportional to the concentration. The constant of proportionality is called the extinction coefficient.
For the slope measurement, choose any 2 wavelengths (lambda 1 and lambda 2) at which the solution has a non-zero absorbance. The difference betwen the 2 wavelengths is Delta lambda. Measure the absorbances (A) of the solution at each wavelength: A1 and A2. If you draw a line between these two points on the spectrum, as for any two points in a plane, you can measure the slope of the line as (A2-A1)/(Delta lambda).
Now consider a solution of twice the concentration as before. Its absorbance values at the same two wavelengths will be 2A1 and 2A2, because for dilute solutions the absorbance is directly proportional to the concentration, according to Beer's Law. The slope of the line between the two points on the spectrum of this solution will be (2A2-2A1)/(Delta lambda) = 2(A2-A1)/(Delta lambda).
Thus, the slope of the line between the two points increases in direct proportion to the concentration. The result of the comparison of the spectra by this method is that you have determined the ratio between the concentrations of the 2 solutions, but not the actual concentrations. To get the actual concentrations, you either need to know the concentration of one of them to begin with, or to know the extinction coefficients at the two wavelengths.
Notice, by the way, that this calculation did not require that the two selected points on the spectrum lie along a linear portion of the spectrum. They could be anywhere on the spectrum. More to the point, the calculation achieved the same result as a measurement made at a single wavelength.
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I don't know how to turn third-derivate spectra in absorbance.
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Dear Jacueline! For correct scientific work, you should use the anlytical capabilities of the fourth or eighth derivative. The third derivative is yesterday and is not alwas accurate in operation and analytical work. Vladimir
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Hi, could you recommend a software compatible with spectrophotometry from various brands?
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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.
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Hello,
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.
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Hello,
when precise transmissive measurements are required, a detector and reference standard light source are used to measure transmittance of an object.
The spectral transmittance, τ(λ), of an object is expressed as the ratio of transmitted spectral flux, Φλt, emitted by a light source and an incident flux, Φλi, which can be measured with a detector.
For all translucent objects, some of the light is absorbed, some is reflected, and some is transmitted. The deviation between these three variables is defined by the characteristics of the object.
Based on the conservation of energy, it can be established that the total amount of light emitted by a light source directed towards an object is equal to the sum of absorption α, transmission τ, and reflection ρ of a particular object.
The detector can only measure the object’s transmission.
An object’s features can be measured using a spectrometer and a broadband reference standard. As not all wavelengths are transmitted through an object equally, it is essential to measure these properties, such as when measuring a material’s UV blocking properties. A spectrometer is capable of accurate measurement of transmittance for each wavelength. A different light source may be needed depending on the type of object to be measured.
Alternatively a colorimeter can be used to establish X, Y and Z values for transmittance based on the required test setup. This setup enables exact measurements for filters with high-density factors, when measurements must be performed at low luminance levels. The light source can again be chosen based on the type of object to be measured, but a white LED would be an ideal choice.
Y have some more info for you in added pdf files.
Best regards
Dr. A. Chandrinos
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I'm looking for a technique or adapter for a spectrophotometer to read the OD of liquid culture in Hungate tubes?
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If you do not have the appropriate machine, I think you can take a small amount of sample, from the hungate via syringe, dilute, and measure the OD using a normal Spectrophotometer.
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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.
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Can you suggest me some chromogenic agents for detecting Ca ions using spectrophotometry.
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Nagham Turkey Thank You for the information. I will look into that.
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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?
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Please look at the following below links which may help you in your analysis:
Thanks
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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?
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If there is a change in the absorbance spectrum when the metal binds to the small molecule, you can use that to measure the affinity.
You should be careful to keep the pH constant, since some metal solutions can affect the pH.
When keeping the small molecule concentration constant and titrating the metal, you should subtract the spectrum of the metal alone from the spectrum of the mixture at each metal concentration. Better still, if possible choose a wavelength to monitor at which the metal has no absorbance.
Make sure all the absorbances are in the linear range of the spectrophotometer.
The concentration of the small molecule being monitored should be well below the Kd of the complex in order to get a good fit of the data to a Langmuir isotherm, which assumes that the free and total concentrations of the binding partner are essentially equal.
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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 ?
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The statistical analysis is ahows the variability inherent in data in a quantitative form. The statiscs analysis ais very important for consistency, uncertainty, and authentic data or results for scientific research.
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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!
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RLT, which is a buffer used in the Qiagen RNeasy RNA isolation kits also contains a guanidine isothiocyanate-like component that may interfere significantly with RNA OD measurements. The RLT give large absorbance below 230 nm.FTI.
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HI
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?
thanks alot
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hi! may I know if you have learned the reason behind the negative result?
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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?
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If your species of interest are ionic and the color component is non-ionic (or can be made neutral by pH adaptation), smal solid-phase extraction columns can do the job of removing them.
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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
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Dear Fatih,
No problem, thanks for sharing. We had the same problem with this ELISA (mybiosource) very low values and also tried differnt dilutions. We tried another one (I have to look up the name and brand; working at home today) but this one worked with a standard diluent and a sample diluent and the sample diluent (without sample)already gave a huge signal (halfway the reference).
Best regards,
Herbert
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Hi,
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 :)
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Hi Kristina
Alfredo's tips are good. We also use dodecyl-maltoside for our complex IV assay but we use it at a much lower concentration (0.45mM) in the assay.
When you prepare reduced cytochrome c, do you get rid of surplus dithionite? If you carry over dithionite into the assay then you may not see any absorbance change because the cytochrome c will get reduced again by dithionite as soon as it has been oxidized by oxygen.
If you have too much IV activity in your assay the reaction may be over (out of oxygen, out of cytochrome c) by the time you start measuring which also would give the impression that the assay doesn't work. To test, run one cuvette at a time and start measurement immediately. You can also check whether the "steady state" absorption you observe is similar to a reaction with a buffer blank. If the absorption in the presence of enzyme is lower than with the blank then you missed the action.
Best of luck
Bernhard
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spectrophotometry, penicillin, bioactive molecules
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Your welcome dear.
Good luck
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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.
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Tryptophanase gene is regulated by catabolite repression and transcription attenuation. Its expression is induced by the presence of elevated levels of tryptophan in the media devoid of other catabolite-repressing carbon sources. So, prior growth in tryptophan broth may be helpful (if possible).
Kindly check out these articles
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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
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HPLC maybe! But this depends on the sample matrix, concentration... and is expensive!
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Hello
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.
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I used Mass spectrometry with reference amino acid (A9781 Amino acid standards for protein hydrolysates, Supleco) for quntification and identification amino acids within protein hydrolysates.
It take time 20-30 min per sample (60-90 min if 3 technical replication).
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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.
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The casein assay detects the peptides released from casein by the action of protease. The protein assay will also detect the peptidic inhibitors, potentially causing an over-measurement if the concentrations of inhibitors are high enough. One approach to deal with this problem would be to include a blank sample for each concentration of each protease inhibitor, omitting the casein. The protein measurement of the blank would then be subtracted from the measurement of the whole reaction.
Another approach would be to use a different protease assay. There is a version of the casein assay that uses fluorescein-labeled casein. The fluorescein labeling is quite dense, leading to self-quenching of fluorescence. Proteolysis releases fluorescein-labeled peptides, relieves the quenching, and results in a fluorescence increase. This can be measured using a fluorescence plate reader or spectrofluorometer. Since this assay measures fluorescence intensity rather than protein concentration, there is no interference from the peptidic protease inhibitors. Another advantage is that you don't have to separate the undigested casein from the released peptides.
If you are using a specific protease, you can also use a synthetic chromogenic or fluorogenic peptide substrate appropriate to that protease. The peptidic inhibitors would not interfere with this method either. The chromogenic substrates would allow you to use a spectrophotometer or absorbance plate reader.
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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!
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UV-Vis spectroscopy is never a good technique for quantitative phase determination of any material. Do a correlation study using Raman, FTIR and XRD analysis.
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  • Is There Any Feasible Method To Test The Efficiency Of Fluorescent Compounds Other Than UV Spectrometers ? Suggestions Would Be Appreciated !
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ThankYou Dear Salah Uddin Xuhua Wang
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We are looking for a buffer system for acetonitrile (pure, no water addition) with pHS around 16+ for comlexometric measurements with UV-Vis technique.
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pH is not only for aquous solution. It can be applied to any solvent. It is just a way to speak about the proton concentration in the medium. If you put very strong acid in acetonitrile, you will have a proton concentration, hence a pH. The pH will just be much higher than in water because the solvation energy of the proton is higher in the case of acetonitrile. Now, could you make a buffer in acetonitrile. I guess you could, but you dont make yourself an easy task. Usually you need a weak acid or base with its conjugated base or acid. So you would need to find this type of acid/base with a pKa that correspond to the pH you are aiming for. Harder to find than in water but not impossible.
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Hello!
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!
Nicolò
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Have you read this publications ?
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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.
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So your sample assay contains 150µg of protein in 5,1mL . According to the BioRad calibration curve, you should expect an OD at 595nm above 0.8... Did you check your reagent with fresh calibrated BSA and/or IgG solutions to get sure it is OK? If you don't run the latter you can't state if the problem is coming from your ovalbumin solution or from your Bradford reagent (or from both)? Further, you have to check your spec and read against a blank sample (0.1mL of buffer + 5mL of reagent and also reading at 595nm is enough. More generally if you don't run any control it is more than difficult to state on the results you get...
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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.
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Hello Ryan,
If you want to know whether your batch of Bio-Rad protein assay reagent is still good, just try to perform a standard curve with BSA according to the instructions of the manufacturer. It it works, don't worry about colour changes upon diluting with increasing volumes of water (although you may find it lots of fun, of course)
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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.
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You're welcome!
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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.
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Peter K Lauf Sir what about 540nm for hemolytic assay ? as it is widely available and astm method for hemolysis assessment is also based on it.
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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.
Thanks.
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Dear Ali Vafaei
The work of Manish Rai, K. N. Ramachandran and V. K. Gupta* contains good information. You can read (attached)
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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:
Endpoint
BMG 96-well microplate
wavelengths: 632,649,665,696
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
Variation:
well scan: orbital
settling time: 0.1
No. of flashes: 21
Thanks so much for your thoughts!!
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I'm not familiar with using wells for this type of application. However, by analogy with using conventional cells in a spectrophotometer, it sounds like mismatching and or contamination.
What happens if you fill the wells of a couple of plates with solvent and the wells of a couple more plates with the same chlorophyll extract?
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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?
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Dear Chiao-Yi Chang,
I have the same problem (negative A) when I used pH-different method for Peristrophe bivalvis (L.) Merr leave. You already solved the problem or not? How do you do? Could you give me the advice?
Thank you in advance!
Ngo Van Tai, from Vietnam.
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I am going to conduct research related to plasma activate water. I need to measure H2O2 level in treated water. I wonder that there is a method that sufficiently accurate to measure H2O2 with UV spectrophotometry???
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Hello, simple colorimetric assay of hydrogen peroxide based on iodide-catalyzed oxidation of 3,3,5,5-tetramethylbenzidine.
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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.
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Mr. Luan,
Neither UV-spectroscopy, nor chromatography appear suitable methods for such analysis. There should be used mass spectrometry, however within the framework of our innovative stochastic dynamic concept and new formulas for quantification of mass spectrometric experimental variables.
Please, cosnider the content of discussion [A] with the corresponding references show, therein.
[A]
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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.
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I've crossed compared different Elisa plates and found the grenier high binding plates to be the best and most consistent. I used the costar for screening purposes only.
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Dear all,
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...
Thank you
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What is analysis' sample? What is analyte? What are analyte concentration levels (range)?
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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
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Hi all, another question: how long is mCP is good for? We prepared some indicator solution a month ago for some tests in January. We have been keeping the dye in a fridge at 4 deg C. Are there any concerns with using that dye in the pH samples in January?
Thank you so much,
Nicolai
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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
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Thank you very much for all your answers. I think I will try to use basic medium as blank. However, I don't expect that there's nucleic acid within the conditioned medium, so I check it
Thank you, once again
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To study and analyze plant extract through GCMS and UV viz spectrophotometry, i want protocols that how can I prepare sample for both technique.
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The protocols will depend on what you want to measure
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Estimation of Protein Concentration by Spectrophotometry
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I would rather prefer buffers such as PBS, TBS, PBS-T, and TBS-T for protein estimation and have my proteins also diluted in the same buffers. And try to not make it a foamy dilution.
Thank you.
Varsha
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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.
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No.
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Isosbestic signals are usually associated with the spectrophotometry. What could be the possible explanation for such signals on cyclic voltammetry?
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Thank you Isam
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How to estimate anthocyanin content of vegetable sample from OD value in spectrophotometry method?
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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.
Thank you.
Lorena
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@Nai-kei, @debasish and @aman thank you so much for your advice. I'm going to use mitosox and cell-ros dyes for superoxide and total Ros measures, respectively. If I see any complication by fluorescence imaging analysis I will try by flow cytometric. Thank you again for your help. Really appreciate it.
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Tbars parameter assay
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Yes, you can use spectrophotometer in measurement of OD; BUT with further disadvantages such as:
1. Time-consuming as you need to use blank before measurement OD of each sample.
2. Mistakes during procedures.
3. Difficulties in adjustment of preferable wave length.
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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?
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Good Answer Achim Recktenwald
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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.
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There are several different problems here:
  1. Pipetting error is lowest when the volumes of all solutions are near identical. So if you mix an enzyme and a substrate solution, both should be near 50% of the final volume.
  2. Changes in composition are minimised either by making all solutions similar in composition (pH, ions...) or by minimising the volume of a deviating solution. For example, if your substrate is dissolved in an organic solvent like ethanol or DMSO, you want to limit the solvent content of the final mixture to a few percent. In this case the effect of solvent is minimised by having the same concentration in all assays. For example, if you use 1, 2,...,10 µL of substrate, you have to add 9, 8, ..., 0 µL of neat solvent. Of course, in such a case you would also do a compatibility study: What is the highest solvent concentration you can get away with, without significantly changing Vmax and Km.
  3. If your substrate is most stable at a pH different from the pH-optimum of the enzyme, you can minimise the effect of substrate addition by keeping the buffering capacity of the substrate solution as low as possible, and that of the reaction buffer sufficient so that changes of pH upon substrate addition are minimised.
  4. Aside: Some reactions change the pH, for example by producing protons. In such a case it is worthwhile to select a buffering agent with a pKa above the desired pH. Thus, the protons produced would initially increase the buffering capacity of the reaction mix. In case of proton consumption, you would, for the same reason, choose a buffer with pKa below the desired pH.
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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.
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David E. Bautista-Erazo Have you tried n-Octylglucoside? It's a non-ionic detergent and I tried to used it to construct protein-conjugated liposome before. Attached link is for the product from SigmaAldrich.
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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.
Regards,
Ankit BK, Bsc Chemistry
Prithivi Narayan Campus (TU, Nepal)
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1. Look for a journal you want to submit your manuscript to and determine if your study fits aim & scope of the journal.
2. Read the instruction provided by the journal carefully.
3. Follow the format required by the journal.
4. Submit into the journal.
For your field of interest, there are a couple of Elsevier food journals you may want to look into, such as Food Chemistry, Food Research International, LWT, etc.
Hope this helps.
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Absorbance value of 10 different samples were taken using spectrophotometric technique.
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Ankit
If you plan to use thos technique/method please read about some basics first. Otherwise you don't know anything about factors thay affect your result.
Remember that your matrix can strongly influence the measured value
In this cas a standard addition method sometimes is a must (instead of calibration curve).
Regards
GB
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I am following protocols using spectrophotometer.
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Even the question is too old but I will answer you in case someone needs it, I agree with Johan De meester because the obtained product is non-colored which is the uric acid, in this case, you have to measure the ABS in the UV range (298nm), if you want to see a colored control you need to see our method "the DED or Double Enzyme Detection" described in the link below, so, you can trace the uric acid in form of Quinoneimine colored in purple.
Good luck.
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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?
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YES. It does affect, both concentration and 260/230 ratios. Change to a glycogen without any dye and you´ll see how your ratios go up by a lot.
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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. )
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That's alright. You have mentioned virtually everything you did but what happened to the addition of SDS to promote the stabilization of the blue colour? It might be what is missing. Please see the attached document:
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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.
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With MPV catalysts you can get furfural at lower temperatures
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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
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Hello, Zohaib Saeed The most widely accepted assays so far for proteins are the Biuret (Gornall et al 1949), Lowry (Lowry et al 1951), Bradford (Bradford 1976, Tsaffrir Zor and Zvi Selinger 1996) Bromophenol Blue (Flores 1978), and Bromocresol Green (Lee Rodkly 1964)] methods. In this Biuret reaction is highly susceptible to interference by non-protein substances (Caraway and Kammeyer 1972, Elking and Kabat 1968, Parvin et al 1965, De La Huerga et al 1964). The bromocresol green method for determination of serum albumin is the most specific and sensitive of the dye binding techniques (Doumas et al 1972). The glyoxylic acid method measures tryptophan content which represents 8-10% albumin and 90-91% globulin. Since the bromocresol green method is specific and simple, it is the method of choice for albumin determination (Doumas et al 1971).
For all this methods and how to perform the analysis see attached info
Good luck!
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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?
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If you look at the formulas of tween-80 and oleic acid, you will notice that a double bond is present in their molecules. In this regard, both these substances have absorption maxima in the same place as simvastatin, which is what you see when you take their spectra separately. Thus, in a microemulsion there are three substances that absorb light in a near region. In this regard, due to the law of additivity of optical density, the spectrum in a microemulsion will be the sum of all three substances and have a very high optical density, which is what you observe. Against its background, changes related to the concentration of simvastatin will not be visible. It is necessary either to take the same microemulsion as a blank solution, or use a solvent (methanol), which does not absorb after 200 nm.
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Hi,
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?
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Dear M.Mussab,
The resistance of textile materials to abrasion as measured on a testing machine in the laboratory can be followed by couple of ASTM and ISO standard given below . Abrasion occurs during wearing, using, cleaning or washing process and this may distort the fabric, cause fibres or yarns to be pulled out or remove fibre ends from the surface. Abrasion ultimately results in the loss of performance characteristics, such as strength, but it also affects the appearance of the fabric. The main factors that reduce service life of the garment are heavily dependent on its end use. But especially certain parts of apparel, such as collar, cuffs and pockets, are subjected to serious wear in use.
Most abrasion tests depend on applying energy to the fabrics and measuring their response to it. The manner of transferring the energy from machine to the fabric is different for different machines, but the basic principles are the same. ASTM and ISO define several methods to quantify abrasion resistance of textile materials and introduce methods for the evaluation of abraded fabrics.
1. ASTM D 4966 - Standard Test Method for Abrasion Resistance of Textile Fabrics - Martindale Abrasion Tester
2. ASTM D 3884 - Test Method for Abrasion Resistance of Textile Fabrics - Rotary Platform DoubleHead (RPDH)
3.ISO- 19427-I,2,3,4- Determination of the abrasion resistance of fabrics by the Martindale method Part 1: Martindale abrasion testing apparatus - Martindale Abrasion Tester
A specimen is mounted in a holder and abraded uniformly in all directions in the plane and about every point of the surface of the specimen. The Uniform Abrasion Tester, consists of the abradant mounted at the lower end of a shaft, weights placed on the upper end of the shaft to produce constant pressure between abradant and specimen throughout the test, lever and cam for raising and lowering the abradant, shaft, and weights. Essentially, the surface of the abradant lies in a plane parallel to the surface supporting the specimen and presses upon the specimen. The abradant and specimen rotate in the same direction at very nearly but not quite the same angular velocity (250 rpm) on noncoaxial axes which are parallel to within 0.0025 mm (0.0001 in.). The small difference in speed is to permit each part of the specimen to come in contact with a different part of the abradant at each rotation. Each rotation is equivalent to one cycle (ASTM D 4158).
Evaluation methods of abrasion tests can be done by series of experiments conducted abrasion cycles for determination of weight loss. There are various measurement methods to measure abrasion properties of yarns, including yarn on yarn abrasion and yarn external abrasion, which comprises different test procedures.
Just refer this site for further study.
Hope it work out for you.
Ashish
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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 
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what is the reading of blank for DPPH method
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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.
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Finally, I finished making a device to check out where the diffraction starts. Here is a photo and I will explain the details.
Let's see how it is going on.
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Is there any online free course on planetary photometry (or photometry)? Pls, send me the link. Thanks!
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You can go through MIT courses on YouTube.
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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?
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The best way to identify the traces of pesticides such as urea and 2,4-DCP accumulated in plant from water is to analyze your samples by LC/GC-QTOFMS or HRMS depending upon the nature of an analyte molecule. It is most accurate as well as efficient method to detect even the small amounts of analytes. For this analysis, you need to prepare your samples from the plant parts which aids for phytoremediation by using different extraction methods like Ethyl acetate extraction. For your more understanding, I am herewith attaching some references which might help you for your experiments. Good Luck!
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Hello,
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?
Thanks
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beta mercaptoethanol is another easily available reducing agent.
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Could anyone suggest PMT with fast response
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The fastest kind of PMTs are microchannel plates e.g.
Among 'normal' PMTs the smallest PMTs are usually the fastest , e.g.
In many cases one can use 'fast' output SiPm, e.g. :
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Did spectrophotometry on 4 samples last night and they all have high Nucleic Acid concentration, but very low A260/230 ratio.
Any ideas on how I can RE-PURIFY the RNA and measure again so I don't lose the time spent growing cells for RNA extraction?
Thanks!
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Qiagen has described RNA purification protocol in an online manual. And it works!
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Hi,
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