Science topic

Carotenoids - Science topic

Carotenoids are the general name for a group of fat-soluble pigments found in green, yellow, and leafy vegetables, and yellow fruits. They are aliphatic hydrocarbons consisting of a polyisoprene backbone.
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Dear all,
I’m performing an untargeted analysis of carotenoids in different matrices by means of HPLC-DAD, but I have not all the zis isomers standards for comparing my UV-visible spectra, so I wonder if there is any available library that I could use with my Chemstation software.
Thank you very much on advance.
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Collected UV/VIS Spectra from an HPLC analysis run are not applicable for any type of "Library Comparisons". This is fundamental to the technique of HPLC and these types of comparisons do not apply to HPLC-DAD spectra unless they are from the same analysis run. No HPLC spectra libraries are available or published for this reason (unlike NMR, FTIR, EI MSD etc). The UV/VIS spectra obtained from any HPLC analysis will vary from instrument to instrument and method to method. They are not comparable separately, however you can learn some basic information from the data obtained (e.g. A general idea of what the spectra looks like when the sample is dissolved in a specific solution, or the mobile phase in this example). Because HPLC methods are fully customized to each instrument, the data collected for a sample will vary and can only be compared to data obtained on the same instrument using the same analysis method and settings. Different tubing dimensions, column type and dimensions, flow cell dimensions (volume and path-length), sampling rate, detection settings (wavelength/bandwidth), mobile phase composition, flow rate, temperature etc. all change the data.
In your example, if you wanted to know what the various UV/VIS spectra looked like for various versions of the same compound (i.e. isomers), then you could start by developing an HPLC method of analysis for all forms OR obtain pure standards. If you develop a valid method which retains and resolves the forms apart using good chromatography principles, then you could view each sample's spectra and compare them directly. Alternatively, if you can obtain pure standards of each compound, then you could dissolve them in the mobile phase and analyze each one separately using a UV/VIS spectrophotometer to obtain and store their spectra for comparison (If you acquire standards of each, you could also use these standards with a full HPLC analysis method to compare and qualitatively identify them too).
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I have performed GC-MS analysis of Phytochemicals from a medicinal plants and also identified compound using NIST library. After NIST identification, I have a list of compounds. I am constructing a phytoconstituent table for manuscript which include compound name, retention time, area percentage, molecular formula and nature of compounds. I am trying to identify the class/nature of phytochemicals (like flavonoids, carotenoids, phenols or polyphenols, glycosides, tannins etc). For that, I am searching each compounds on databases like PlantaeDB, IMPPAT 2.0, website like MedChemExpress and also literature search. Some compounds can be find here but many of the are not to be found anywhere.
1. How to identify the class of phytochemicals or secondary metabolites after GC_MS and NIST identification?
2. Some compounds are repeated with different retention time and Similarity index. Which one to include and on what basis?
3. In one extract, more than 60 compounds are identified. Should I include them all for manuscript or can I select the important and bioactive compounds only?
Very much thankful for your help!
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Identifying the class of phytochemicals and determining which compounds to include in your manuscript after GC-MS and NIST identification involves a systematic approach. Here are the steps to address your queries:
1. Identifying the Class of Phytochemicals
To identify the class of phytochemicals or secondary metabolites, you can follow these steps:
a. Database Searches:
- PlantaeDB and IMPPAT are good starting points. Ensure you are using the correct chemical names and synonyms.
- PubChem and ChemSpider can also be useful for finding information about the chemical structure and its known classes.
b. Literature Search:
- Use databases like Google Scholar, PubMed, and ScienceDirect to search for scientific articles related to the identified compounds. Keywords like "phytochemical class" or "secondary metabolite class" along with the compound name can help.
c. Chemical Structure Analysis:
- Analyze the chemical structure using software tools like ChemDraw or MarvinSketch to predict the class based on known structural features of flavonoids, carotenoids, phenols, etc.
d. Expert Consultation:
- If certain compounds are hard to classify, consider consulting with a phytochemist or a researcher specializing in natural products chemistry.
2. Handling Repeated Compounds
When you have repeated compounds with different retention times and similarity indices, consider the following:
a. Consistency Check:
- Check the consistency of identification across different runs. If the same compound is identified consistently with a high similarity index, it is likely accurate.
b. Retention Time Comparison:
- Retention time differences can sometimes indicate different isomers or derivatives. Compare the retention times and the similarity indices to choose the most reliable identification.
c. Reporting:
- If the similarity index is significantly different, consider reporting both and discussing the possible reasons (isomerism, different sources of the compound within the plant, etc.).
3. Selection of Compounds for the Manuscript
Including all identified compounds versus selecting important ones depends on the focus of your manuscript:
a. Relevance and Impact:
- Include compounds that are known to have significant biological or pharmacological activity relevant to your study.
b. Novelty:
- Highlight compounds that are newly identified or have unique properties not commonly reported.
c. Focused Analysis:
- For a more focused analysis, you can prioritize compounds based on their relative abundance (area percentage), biological significance, and the objectives of your study.
d. Supplementary Data:
- Consider including the complete list of compounds in supplementary materials while discussing the key bioactive compounds in the main text.
Practical Steps:
1. Data Organization:
- Create a comprehensive table of all identified compounds with their retention times, area percentages, molecular formulas, and similarity indices.
2. Class Identification:
- Use a combination of database searches, literature reviews, and chemical structure analysis to classify each compound.
3. Selection Criteria:
- Define clear criteria for selecting compounds (e.g., similarity index threshold, area percentage, known bioactivity).
4. Documentation:
- Clearly document the methods used for identification and classification to ensure reproducibility and transparency.
By following these steps, you can systematically identify the classes of phytochemicals, choose which repeated compounds to include, and decide on the most relevant compounds for your manuscript.
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I used the Lichtenthaler and Wellburn (1983) protocol for chlorophyll and carotenoid extraction, using 0.5 g of fresh leaves, 10 mL of 80% acetone, and the coefficients and equations from this protocol. However, the final carotenoid results were negative. Is there any explanation for this? Could I have made a mistake in the protocol? I have followed this protocol many times, but this is the first time this has occurred.
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- Because concentration can not be negative, the value that you obtain from carotenoid extraction should be positive.
- The blank sample somehow contains higher carotenoid concentration than your sample.
- When you measure the concentration of your sample, equipment detects the carotenoid concentration lower than the blank sample. Therefore, it expresses the value as negative.
- Be sure that your blank sample contains everything that your extracts have except carotenoid extract.
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I need to know how to determine carotenoid content in extracted shrimp waste sample and hope to use extracts as food ingredient. but here unavailable the carotenoid standards to determine carotenoid content using spectrophotometer and HPLC
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you suggest "using the Beer-Lambert law, which relates absorbance to concentration." You need to know the extinction coefficient, which can be determined using the standard solution.
The same problem for HPLC.
Your post does not answer the title question.
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Dear Colleagues!
I am interested in ELSD, an HPLC detector.
Is there anyone who is currently using or has used this detector?
I would appreciate it if you could share information on the problems, concerns, and advantages of using it in real world situations.
It would also be appreciated if you could introduce, for example, review articles explaining the characteristics of quantitative measurements of analogous compounds without their standards.
I would like express my gratitude to everyone in this community.
I appreciate it.
Best regards,
Yasuhiro Nishida
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Dear friend Yasuhiro Nishida
Ah, ELSD, the Evaporative Light Scattering Detector, a gem in the world of HPLC detectors! Now, let me share some insights.
Firstly, ELSD is often a savior when dealing with compounds that lack UV absorption or those that don't have a chromophore. It detects analytes based on their ability to scatter light when eluted from the column. Now, let's delve into the real-world scenarios:
**Advantages:**
1. **Universal Detection:** One of the main advantages is its universality. It can detect virtually any compound regardless of its optical properties, making it a fantastic choice for compounds with no UV absorption.
2. **Quantification of Analogous Compounds:** ELSD is particularly useful when dealing with structurally analogous compounds that might not have distinct standards. This makes it valuable for natural product analysis or in cases where obtaining pure standards is challenging.
3. **Low Detection Limits:** ELSD often provides lower detection limits compared to other detectors, which is beneficial when dealing with trace-level analysis.
**Concerns:**
1. **Baseline Drift:** ELSD is known for baseline drift, which might complicate the quantification of compounds. Strategies like using an internal standard or appropriate calibration techniques are often employed to address this issue.
2. **Sensitivity to Mobile Phase Changes:** Variations in the mobile phase composition can affect the signal intensity. Users need to carefully optimize the mobile phase to get consistent results.
3. **Sample Dependent Sensitivity:** The sensitivity of ELSD can be sample-dependent, and it might require method adjustments for different compound classes.
**Review Articles:**
1. **"Lecoeur, M., Decaudin, B., Guillotin, Y., Sautou, V., Vaccher, C., & ARMED Study Group. (2015). Comparison of high-performance liquid chromatography and supercritical fluid chromatography using evaporative light scattering detection for the determination of plasticizers in medical devices. Journal of Chromatography A, 1417, 104-115. provides a comprehensive overview.
2. **"Megoulas, N. C., & Koupparis, M. A. (2005). Twenty years of evaporative light scattering detection. Critical reviews in analytical chemistry, 35(4), 301-316., is another valuable resource.
Remember, my eager interlocutor Yasuhiro Nishida, ELSD is a versatile tool, but like any technique, it has its nuances. The key is in understanding those nuances and wielding them to your Yasuhiro Nishida advantage in the quest for chromatographic mastery!
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Hi, my name is Anna.
I am working on the extraction of polyphenols and carotenoids from date seeds using NADES.
I am centrifuging the samples at 14400 rpm-20min-10ºC but when I remove the supernatant, the liquid looks cloudy.
What could I do to remove those particles that can interfere in the assays? Perform a filtration on paper?
Thanks for your help and best regards,
Anna
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Various methods, including ultrasound, supercritical fluid (SFE), microwave, and subcritical CO2-assisted techniques, have been investigated for extracting bioactive compounds from diverse plant matrices. These approaches provide alternative solutions to tackle challenges posed by interfering particles during the extraction process.
Moreover, it is crucial to take into account the influence of changes in ion concentration on the extraction process. For instance, modifying the pH of the medium using diluted HCl or NaOH can alter the ion concentration, potentially leading to adverse effects on the extraction of bioactive compounds.
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I am standardizing a UV-VIS screening method that allows me to quantify provitamin A carotenoids. The results will be compared with samples quantified by HPLC, which is my Gold Standard. Nevertheless; the results that I've gotten so far using both methods aren't comparable between each other. I was expecting the concentration using both methods would be similar, sadly this is not happening. Interestingly, when I multiply the concentrations I get in my colorimetric method by a factor of 4.5 ish, they are very similar to the ones in HPLC. You might say that maybe the calculations are not correct, but I considered the dilution factor as well.
Has anybody encountered this issue, if so... Let's talk about it
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Absolutely the result will not match because, first need to understand the basic principle of both techniques. Separation takes place in HPLC hence all components get separated and individual results shall be obtained whereas colorimetry is measurement of solution based on absorbance and hence reading is sum of all compounds. Hence both the results will not be matched.
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In D. salina in which beta carotene is accumulated throughout the cells, staining with iodine reagent will cause the whole cell to be stained.
NOTE: These pictures are copyright. No reproduction without written permission from the publisher.
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Here is a paper I recently read:
Salinity impairs photosynthetic capacity and enhances carotenoid-related gene expression and biosynthesis in tomato (Solanum lycopersicum L. cv. Micro-Tom). PeerJ, 8, e9742. https://doi.org/10.7717/peerj.9742
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I want to identify carotenoid pigment (polar one) isolated from bacteria. Many articles suggested to use LC-MS. However we do nit have reference samples? so is it possible to conduct a LC-MS analysis without a reference?
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Of course it is best to have pure reference standards. In the real world, it is understood that this is not always possible. For a qualitative result, you may be able to make an educated guess using a properly developed HPLC method with MS and DAD detection modes, in-line (compound has chromophores that can be detected AND it must also ionize well under the analysis conditions used). MS/MS would add more information, and another orthogonal dimension of analysis. The quality of the HPLC method is the key as the results will only be as good as the method used.
  • Please note that MS (MS/MS) detection is NOT 'universal' and due to differences in instruments, ionization, solution chemistry, mobile phase and/or detector settings, some compounds may not be detected at all (similar to UV/VIS as neither detection mode will 'see' everything).
We try to use as many different orthogonal methods of analysis as possible (combine detection methods which use different chemical or physical properties) with or without standards to come up with a proposed ID. That is what leads to a good QUALITATIVE ID.
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I am working with chitosan application and I have found increased concentration of chlorophyll, carotenoids and protein in plants treated with higher doses of chitosan. Whereas growth of plant like height was less in higher concentration and more in lower concentrations. I have done experiment twice for confirming my results and it is coming in the same way. Is it possible that stressed plant have more chlorophyll as compared to non-stressed plants.
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This is an interesting question. How do you determine if the plant is under stress ? The fact that it grew differently doesnt prove it is stressed. I would check for example photosynthesis physiology parameters - effective quantum yield in time series after introduction of the chitosan in control vs. treatment. If it is substantially lower in the treatment then there might be stress, but it should be checked further.
Also, the fact that you get more chlorophyll and carotenoid and protein implies that the plant uses this sugar, not stressed by it.
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Dear community of ResearchGate:
I am thinking about conducting a literature review study on a topic related to physico-chemical or nutritional parameters in peppers (Capsicum spp.). Even though, as I am a beginner in this field, I am not sure what topic to focus the study on.
I woulg be grateful if could make some suggestions.
Thanks in advance!
Pablo
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In order to write a good review, you MUST be an expert in the area. While, currently there are tons of junk journals, which would publish whatever you submit, providing you pay them a publication fee. Evidently, this would be a waste of time and funds, but you decide.
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I'm using the standardised INFOGEST - Minekus (2014) consensus paper for my simulated in vitro digestion model but each time I use the digesta (intact, diluted 1:4 and 1:8) on the caco-2 cells to assess carotenoid uptake and transport, the cell viability is less than 10% based on the MTT assay. I strongly suspect the bile salt concentration I'm using could be toxic to the cells. Is there a step by step procedure I can use without necessarily using the test kit to verify the actual concentration of my bile salts?
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Did you ever find a solution? Looking into the same issues.
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There are several ways to extract microbial carotenoids. according to you what is the best ways to extract carotenoids from microbes?
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The best way to extract carotenoids from microbes is to use a combination of physical and chemical methods. Physical methods, such as sonication, grinding and homogenization, are used to break down cell walls and release the carotenoids from the microbial cells. Chemical methods, such as extraction with organic solvents, can then be used to separate the carotenoids from other microbial constituents. Additionally, enzymes such as lipases and esterases can be used to hydrolyze the carotenoid esters and improve the extraction efficiency.
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Hi to everyone!
I am going to to measure levels of total carotenoids in pepper fruits by means of a spectrophotometer. I would like to know which standard solution I should use in order to elaborate the calibration equation, and to express the result.
Thanks for your feedback!
Pablo
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Merck has a carotene standard
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Hi everyone!
I project to mesure levels of various nutricional parameter in pepper fruits. Looking up in the literature, I found out the research article of Nagata and Yamashita (1992), in which a spectrophotometric method for analysis of chlorophyll and carotene in fruits is described.
I think that the information provided by Nagata and Yamashita (1992) could be very useful for our project. Even though, this paper is not written in English, but in Japanese (a language that, unfortunately, I do not understand).
As it is a highly cited study (1112 citations in Google Scholar, many of them from non-Japanese authors), I suspect that the procedure of Nagata and Yamashita (1992) must be detailed in English somewhere.
I would be grateful if someone could give me some light about this.
Thanks in advance!
Pablo Reguera
Literature cited:
Nagata M, Yamashita I (1992). Simple method for simultaneous determination of chlorophyll and carotenoids in tomato fruit. Nippon Shokuhin Kogyo Gakkaish 39 (10): 925-928.
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Hi!
Someone has uploaded a summary to Research Gate. But I am not sure if it is correct.
The original paper is in Japanese, but you can subscribe for free. If you would like an English translation of the original paper's methodology, please contact me
Yasuhiro Nishida
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T1-Chemical treatment
chl a=8.509, chl b= 14.833, carotenoids= 573.749, chl a/b ratio = 0.573
vs.
T2-Control (no treatmet)
chl a=9.563, chl b=16.945, carotenoids= 655.979, chl a/b ratio = 0.564
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You can say there are differences, but you need to perform t-tests on the raw data to know if there are statistically significant differences :)
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Hi!
I'm doing pigment analysis (chlorophylls and carotenoids) of seaweed, and I have some questions about the extraction and the reference system.
I'm not sure if I should use fresh or dry material for the extraction.
I understand that giving the results based on dry weight is a more reliable reference system than fresh weight since water content can vary depending on storage, but does that mean I have to use dry material for the extraction or can/should I use fresh material and then just convert the results to dry weight? (I'm doing dry matter content analysis already so I know the dry matter and water content). Hope my question makes sense.
Thank you!
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fresh weight
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I am sampling tree leaves for pigment analysis with a UV-Vis Spectrometer. The samples will be snap-frozen in the field and stored in the dark. I am trying to determine the best method for anthocyanin extraction and analysis. It would also be great to know if it could be integrated into a chlorophyll and carotenoid extraction too? Thanks in advance!
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Dear Procopio Peinado-Torrubia and J.D. Franco-Navarro , thank you for your advice! If it is possible to be linked to the protocols you discussed above I would really appreciate it.
Many Thanks,
Jack
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As I'm working on RP-UHPLC-DAD quantitative analysis of carotenoids in plant material, I have troubles with dissolving solid standards of carotenoids. I've tried mixtures of acetone and methanol or ethanol in different ratios, but nothing worked perfectly. Either it didn't dissolved at all, or it started to re-crystallize in the solution after few days/weeks. Does anyone have any experience?
Specifically, I'm talking about alpha-, beta- and gamma- carotene, lycopene, lutein, zeaxanthin and neoxanthin.
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Hi. Perhaps the solubility of β-carotene and lycopene is low in the solvents you described. I would suggest making a stock of only those with benzene, toluene, or hexane. In any case, all carotenoid stocks are not so stable, even in the freezer. Instead of stocking mixtures, it is better to make a stock of each and determine the concentration by UV/VIS of the dilutions before testing; for HPLC, it is better to mix each and use the dilutions.
FYI, the addition of BHT has a relatively positive effect on the stability of the stock solution.
For more information, a book called Carotenoids is a good reference.
Good luck!
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I mean the unit of expresion
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Arnon method (1949) involves the extraction of plant matter with ether and measuring absorbance at chlorophylls and carotenoids (470 nm) maximal wavelengths. It is described in:
Wellburn, A.R., Lichtenthaler, H. (1984). Formulae and Program to Determine Total Carotenoids and Chlorophylls A and B of Leaf Extracts in Different Solvents. In: Sybesma, C. (eds) Advances in Photosynthesis Research. Advances in Agricultural Biotechnology, vol 2. Springer, Dordrecht. https://doi.org/10.1007/978-94-017-6368-4_3
If the fruit contains negligible amounts of chlorophylls, the task is simplified. The estimate is very rough, because some carotenoids are colorless (phytoene and phytofluene) and some non-carotenoid compounds absorb at similar wavelenghts and are extractable by the same procedure.
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Is it possible to measure carotenoid based on fluorescence image?
Somebody, if you have some references regarding carotenoid quantification by auto-fluorescence image, share to me please :)
In my experiment, I used a rhodamine filter (excitation 540-552 nm; emission: 575-640 nm) in H. pluvialis cell (microalgae), but the problem is the death cell also showed fluorescence.
Is it possible to use this filter to quantify carotenoids?
Thank you
- Rendi -
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Thank you Shafagat Mahmudova . Actually, the references related to chlorophyll measurement by autofluorescence, even though carotenoid and chlorophyll are different but it is good information for me. Thanks!
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Does anyone know the appropriate equation for chlorophyll a, chlorophyll b,total chlorophyll & carotenoids extracted by DMSO
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Dears,
I have used fresh leaves methods (1 g of the leaf tissues using 80% acetone) using these equations:
[Chl a (mg.g-1) = (13.95OD665−6.88OD649)V/200 W];
[Chl b (mg.g-1) = (24.96OD649− 7.32OD665)V/200W];
[Car. (mg.g-1) = (1000OD470−2.05Chl a−114.80Chl b) V/ (245×200 W)].
Where; Chl a– chlorophyll a, Chl b – chlorophyll b, Car. – carotenoid, V –volume, and W – sample weight.
what about dry leaves methods??
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The procedure and formulas are pretty much the same.
However, unless you freeze-dry the leaves, you'll lose a lot of chlorophyll through the process of drying. Chlorophyll is degraded very easily. Normal drying methods like heat drying or sun drying will make the leaves lose a substantial amount of chlorophyll.
If you don't care about that, though, and just want to, say, see how much chlorophyll is left after drying, then just do like with fresh leaves.
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Hi!
Dear colleagues and respected seniors, I am searching for a biochemical reaction to identify the km and vmax values for the said enzyme, which converts alpha carotene into Lutein, but still I am not able to identify, so if anyone could help to find it, give me a reference article/ paper please, I will be very thankful to al of you.
Thanks and best regards
Mr. Rafi Ullah Khan
PhD candidate (Biotechnology)
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Try looking through the references here:
Unfortunately, there are no kinetic data listed, suggesting that none of the references will contain a suitable assay.
If hydroxylation results in a measurable change in the absorbance or fluorescence spectra of the carotenoid, you may be able to follow the reaction by that means.
You may have to use a chromatographic separation to measure product concentrations, possibly combined with mass spectrometry if you can't fully separate the substrate and product chromatographically. The insolubility of the substrate may also be a problem, though, since detergents needed to keep the substrate in solution will could be incompatible with the analysis of the reaction by HPLC or LC-MS.
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Can anyone suggest the HPLC method to quantify spheroidenone carotenoid with a DAD detector and an Agilent zorbax SB-C18 column?
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The bigger problem is perhaps the high price for the reference substance spheroidenone. Among others 10mg offered by Toronto Research Chemicals may become the best choice. You can test the purity the same time you prepare for your concentration-response correlation curve. The mass spectrum in the attached file has been made by GC-MS analysis. Isn't it to prefer before struggling with HPLC-MS?? It depends of course on your application and type of sample.
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Astaxanthin is the carotenoid antioxidant.
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Fatemeh Barari. My pleasure. Good luck.
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I am working with carotenoids mainly Lutein and Zeaxanthin. For this I have purchased the standards from Sigma Aldrich which were very expensive. there are all in powder form and I would like to know what is the best solvent for dilution and long-term storage before HPLC analysis. I went through different papers and there was no consistency as there was mention of methanol, hexane, etc...
I would appreciate any technical and straightforward suggestion. Thanks
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Pure ethanol is best for dissolving lutein and zeaxanthin.
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Conducting HPLC analysis for carotenoid content on vegetables is not an issue, but what about carotenoid content incorporated in a high fat diet ?
My lab usually performs double hexane extraction for common fruit and vegetables but what would be an appropriate extraction protocol for fat food (45% butter, 5% tomato powder) ?
I've heard two possibilities : extract as usual or sample saponification prior to analyses.
As carotenoids are lipophilic, wouldn't saponification damage these compounds ?
What would be an appropriate extraction protocol in such a case ?
Thanks for your help !
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Hi Thomas,
The saponification works very well. Some research articles will do this in the cold so as to reduce the degradation of the carotenoids. Otherwise, you can also add BHT to the saponification reagent.
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Can I extract carotenoid pigment from microalgae (C. sorokiniana and D. salina) without freeze-drying?
I already searched and read references about drying process that we can use heat-drying method but that wasn't recommended because could cause degradation of carotenoid content. So if I don't have a freeze-dryer, can still extract the pigments from wet biomass?
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As many people have answered, yes you can.
If you want to see the amount of carotenoids per weight, you can determine the dry weight per culture medium, and separately determine the amount of carotenoids in the wet biomass per culture medium.
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For a school project, we have to isolate carotenoids from fruit and a product from the fruit. For our research we use watermelon and we wanna have a look for lycopene and beta carotenoids. We already found a protocol for the watermelon itself. Unfortunately, we can find a protocol for alcoholic drinks. The measurement will be made by HPLC in a mobile phase ethanol:acetonitrile. (+ 0,1% diethyl ether). I hope anyone can help us.
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Have you already solved this problem?
I believe Dr. Maria Stacewicz-Sapuntzakis' method is fine, but I would suggest a slight modification.
I don't know the original alcohol concentration, but I suggest that you first add sodium sulfate 10-hydrate, add equal amounts of EtOH or acetone to the drink, and then extract with hexane or petroleum ether. The hexane layer will be collected and washed again with salt-saturated water. This method can remove a lot of proteins and sugars. The hexane layer can be dried, dissolved in a suitable solvent, and filtered for HPLC.
If saponification is required, please contact me.
Translated with www.DeepL.com/Translator (free version)
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Hello. I've been working on photosynthesis pigment content in in vitro grown trees and was examining the formulas for determining the concentration of chlorophyll a and b and carotenoids based on absorbance, i.e. using a spectrophotometer. I've found various formulas, and even though they all share the same general design: concentration=coefficient1*adsorbance1-coeficeint2*adsorbance2, the coefficients differ among sources. I was curious as to the reason behind this. Is it due to the use of different equipment, i.e. every spectrophotometer is different, or something else? I'm new to this field, thus any help, directions to relevant introductory literature, articles, would be great.
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Philipp Girr Thanks, you made my day.
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The metabolite that is extracted after evaporation of the solvent is purified using ethanol and ethyl acetate solvent by silica gel chromatography column.Is it possible that our carotenoids are oxidized at this distance, but all this is done in the dark?
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You can add to the extraction solvent some antioxidant, ie, BHT. Oxygen can be excluded by working with Flash chromatography applying nitrogen pressure to the column head. Also solvents can be degassed prior to use.
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I took 2 gm tissue and then added 20ml solvent. After that I collected supernatent and then made up volume 25 ml by about 10 ml hexane. I know absorbance reading and A1% value, I am a bit confused about total volume used amount. Would you someone assist me regarding that "V" value ? 
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Your 'V' value will be 25 ml. Thanks
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Hi everyone! i am looking for protocols to evaluate the bioaccessibility of carotenoids in plant matrix. Ideally the best would be a quite easy protocol that could be used massively in the lab, but every advice is welcome! :)
Thank you very very much!
Best
L
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Hello,
I think the following link might help you in your research:
Best regards
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I need to perform some tests with extracted carotene.
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Using your modified method start used reverse osmosis water.
you had to sort the best carrot you needed
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Hello everybody!
I have performed an acidic MeOH:HCl 1% (v/v) plant extraction from Arabidopsis leaves to calculate Anthocyanins concentrations based on:
Rabino, I., and Mancinelli, A. L. 1986. Light, temperature, and anthocyanin production. Plant Physiol. 81:922-924.
I want to calculate also Chl a, Chl b and Carotenoids concentrations from this acidic methanolic extraction. So far, I have only found 100% MeOH equations to calculate concentrations of these pigments, separately, but not a single reference to calculate them from a MeOH:HCl 1% (v/v) plant extract.
I have read that "A657 values provide an estimate of Chl content" (Photoregulation of Anthocyanin Synthesis' VIII. EFFECT OF LIGHT PRETREATMENTS, Mancinelli, 1983), but I would like to have a more precise value for Chl a, Chl b, and carotenoids also, if possible.
I have recorded measurements of wavelengths between 300-700 nm.
I would be grateful if anyone helps me with that.
Thank you all!
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Hi, you can find more information in this chapter (Chlorophylls and Carotenoids: Measurement and Characterization by UV‐VIS Spectroscopy by Hartmut K. Lichtenthaler, Claus Buschmann). There are formulas for calculating the concentration of chlorophyll (a; b) and total carotenoids (x + c), depending on the solvent used in the extraction.
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I want to know about different analytical methods (rapid) to determine carotenoids (carotene and Xanthophyll) in maize flour, which gives more accuracy.
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If you mean, which approach to utilize, then I will recommend you to utilize Definitive Screening Design, the novel approach. It is useful in screening undesired variables and optimizing with the useful variables.
Here is the link for your information.
(PDF) Multi Response Simulation and Optimization of Gas Tungsten Arc Welding (researchgate.net)
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Thanks.
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Yogesh Chandra Tripathi Kindly can you share your SOPs in analysis of carotenoids from maize grain samples
.
Thanks
Marco
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how to determine the quantity of chlorophyll a, b and carotenoid?
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There is different protocol which we follow it for chlorophyll, carotenoids etc....if u need that protocol so u can msg me on email i will sent u .......kwisal775@gmail.com
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These spots seem to disappear after they have been on land for a while and it seems nobody knows exactly what causes it. Some sources just say "carotenoid rich plumage", so is it similar to the way flamingos express reddish feathers with a carotenoids rich diet?
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I am thus guessing that as these birds molt into breeding plumage, the peach spots become pronounced as part of their nuptial plumage.  For some seabirds, that nuptial plumage gradually fades throughout the breeding season.  That could be caused by the colored feather tips (if it is just the tips of the feathers that are colored) being rubbed off. 
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what is the reason for decrease of carotenoids in Cyperus leave, which is treated by 100 mg/L Heavy metal solution for about 26 days? It was almost 49 percent. if there is any article which could help me would be great.
I would appreciate in advance for your advice.
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It needs neutral pH
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As we all know that there are number of methods for the estimation of leaf pigments mainly chlorophyll and carotenoids, but i want to know out of DMSO and acetone which method is best?
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There is no fixed rules, as several factors control the efficiency of pigments extraction e.g., plant species, extractant solve and extraction duration. I used methanol and acetone and observed that methanol was more effective in extracting chlorophyll from many vegetable leaves.
📷
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We are trying to isolate some carotenoids from algae with semi-prep HPLC. As literature says, a few mg will be enough. Is this true?
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Erdoğan
For small molecules (less than 1000 g/mol), typical 1H NMR spectra require 5-25 mg of material. Typical 13C spectra require 50-100 mg of material. This amount of material will allow you to obtain a 1H spectrum in a few minutes or a 13C spectrum in 20-60 minutes. When the amount of material is doubled, the resultant signal will be doubled. Bear in mind that a very concentrated sample will produce a quick 13C spectrum, but may result in a broadened 1H lineshape. Overly concentrated samples can also be difficult to shim.  See
Good luck
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There are several methods for assessment of antioxidant activity, of which DPPH scavenging assay. DPPH has absorbance around 520nm, while carotenoids have absorbance near 500nm.
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Dear
Muhammad Bilal Tahir
. I recommended your post feeling that I could be too straightforward. I would like to learn from you how I insulted the research community in this discussion? What was the "harsh" way in my answer to the original question? Actually I gave the best answer. Mohammed Najim Abed gave his own advise. I had doubt that it could work and asked several legitimate questions without any attempt to discredit him. Instead of answering he advised me to read more which is actually statement of my incompetence in this area. Where I breached the rules of scientific discussion? Mohammed Najim Abed wrote "I am not used to this way of communication ." What was wrong in my posts? The answer to your advise to read more? I don't know many things, please, teach me, answer my questions and defend your proposal.
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- I am testing the difference in carotenoids of 5 groups of diferent sex or development stage animals with one way ANOVA. My samples are from 13 the smallest to 44 the greatest. Do I use the eta square value?
So I use the Kruskall-Wallis test to compare the groups when the variables were not normally distributed, and the post-hoc Dunn test.
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Cohen suggested that d=0.2 be considered a 'small' effect size, 0.5 represents a 'medium' effect size and 0.8 a 'large' effect size. This means that if two groups' means don't differ by 0.2 standard deviations or more, the difference is trivial, even if it is statistically significant. Cohen’s d is used to describe the standardized mean difference of an effect. This value can be used to compare effects across studies, even when the dependent variables are measured in different ways, for example when one study uses 7-point scales to measure dependent variables, while the other study uses 9-point scales, or even when completely different measures are used, such as when one study uses self-report measures, and another study used physiological measurements. Please see the attached document for details: https://www.frontiersin.org/articles/10.3389/fpsyg.2013.00863/full
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I'm looking for a way to remove carotenoids from a solution containing chlorophylls and carotenoids, except chromatography method.
thank you.
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There are methods in:
Rice, Eugene W, et al. Standard Methods for the Examination of Water and Wastewater. 22nd ed., American Public Health Association, 2012.
If instead you want to remove the chlorophyll:
Extract in 80% acetone and add 50% volume of 1 M KOH solution to induce alkaline hydrolysis, then shake or vortex the solution for several minutes at room temperature.
This procedure releases the phytol chain from chlorophyll resulting in chlorophyllides, which is far more polar than chlorophyll, so you can use a less polar solvent (e.g. petroleum ether or hexane) for solvent extraction of carotinoides by shaking, vortexing or centrifugating.
Any way the upper phase will contain just carotinoids and the lower chlorophyllides.
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I am looking for a method to coat the carotenoid staphyloxanthin firmly on a tissue culture plate or membrane filter discs. Please share protocols or literature for this procedure. Thank you.
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I am interested to study the growth of bacterial biofilms in carotenoid coated plates. The main objective is to see whether the carotenoids promote biofilm formation.
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I am working on the carotenoid structure. Based on MS-MS and FTIR data it should have the hydroxyl group. To further confirm we dissolved our carotenoid in deuterated methanol (100%) and interestingly, exchange was not observed.
We believed it should be tertiary alcohol present at the end of the carotenoid structure (41 C long) with 10-11 conjugated bonds. Can anyone tell me or has any kind of idea why there is no exchange observed in MS?
Thank you
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We are measuring cations. We believed it too that maybe tertiary alcohols are sterically hindered but there are papers which state that exchange does take place even with tertiary alcohols though I didn't find any carotenoid paper. We are now waiting for NMR hopefully, we could get clear results. Thanks to all :)
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Dear colleagues, I have larvae of one species of crabronid wasps, which are variously coloured - usually yellow but also orange, reddish, purple. I would like to know how many pigments are responsible for this colouration and which pigments are they. The prey of larvae are aphids and it seems the pigments are carotenoids or aphidines. Could anyone help me with this? I can send frozen or alive larvae of various colours by mail and can offer co-authorship on a paper.
Many thanks and best regards,
Petr
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It would be good to do mass spectrometry.
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What are the most well-known genes that have been identified & sequenced to produce carotenoid pigments in various organisms (specially in microalgae and yeast)?
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Lycopene is the precursor of carotenoids. As carotenoid production is also linked to secondary metabolism (not common to all species), specific routes are assumed according to a particular group (eg diatoms, dinoflagellates etc.).
You can see more information in a recent review:
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Could anyone give some recommendations on what algal strains in UTEX show potentials for high carotenoids or valuable carotenoids production? Ideally Haematococcus, Dunaliella salina and Scenedesmus. Thank you.
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percentage of any phytochemicals depends on environmental diversity. you must focus on literature.
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Hi all,
I am working with extracts of secondary metabolites of orchid species (roxburghii).
The chromatogram has many peaks that correspond to flavonoids glycosides, glycosides of hydroxycinnamic acids, carotenoids, and chlorophylls. But among them, there are several peaks with unknown spectra. Unfortunately, there are no similar spectra in our library.
Have anyone some suggestions about what kind of compound could be that?
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Yes, I am sure that this peak is pure. The background signal was subtracted and the blank run doesn't have any peaks. The signals from all five wavelengths (DAD detector) show the same peak (time retention and good symmetry). Moreover, there are several peaks with similar spectra. I am thinking that this is anthocyanins or anthocyanidins
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According to the guidance notes the drying process of plant materials should be completed at the stage when there still remains 6 - 10 percent of moisture in the plant. Overdrying under e.g. 6 % seems to decrease the content of carotenoids, ascorbic acid and essential oils - according to the few references I have found. Thus evidence is very limited. Our own tests have shown a slight tendency to decreasing contents of phenolics.
I would be happy to get some new hints, experiences or explanations of this phenomenon!
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Thanks everybody for your reply! My problem relates to the normal convective drying process, which is still the most common method of preserving cultivated and wild plants for the later utilization as food or as a health promoting ingredients. The moisture content I mentioned means the weight loss in the d.m. determination at the end of the drying process. It is thus grams (of weight loss)/grams of dried material at the end of the drying process.
In the natural product business the management of the drying process is of importance. We should focus on determining the right ending point for drying of each product to be dried, if the overdrying really decreases the content of those valuable compounds we want to preserve. Question still remains open! Thanks especially to Mateo! Your thoughts gave me some new ideas.
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I have prepared chitosan nano-particles and encapsulated carotenoid pigment within it. I have tried to check its release profile by reported dialysis method (12 kD dialysis membrane ) in PBS of pH 7.4 and 1.2 but I am not getting any satisfying results. Nano-particles are being clumped within dialysis bag. How can I overcome this problem??
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For drug release study, you need to optimize the parameters such as temperature (usually 37C) and pH etc. Also its important to know whether the drug is hydrophilic or hydrophobic, since hydrophobic drug will not easily dissolve in water or pbs.
Then, you need to take readings at regular time intervals and add up the concentration to get cumulative drug release profile.
I hope this helps.
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I found that alkaloids normally appeared as green spot on tlc plate under long wave UV. Then how about red spot? I'm doing isolation of constituents from plant materials, is the red spot needed to isolate out normally? Does it posses phytochemical properties? Cause I'm thinking is it pigments like carotene or chlorophyll.
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Red spot means it is different constituent than your desired component with green color. TLC can only tell you this that it is different. You will need to have advanced chromatographic analysis for further determination like HPLC or LC-MS.
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Is it possible to have the same content of carotenoid when there is a decrease in the total chlorophyll content? What could be the possible reasons?
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Carotenoids have two important functions in plants:
1) First, they can contribute to photosynthesis. They do this by transferring some of the light energy they absorb to chlorophylls, which then use this energy to drive photosynthesis.
2) Second, they can protect plants which are over-exposed to sunlight. They do this by harmlessly dissipating excess light energy which they absorb as heat. In the absence of carotenoids, this excess light energy could destroy proteins, membranes, and other molecules. Some plant physiologists believe that carotenoids may have an additional function as regulators of certain developmental responses in plants.
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Chemical structure, Physical properties, function, antioxidative property, functional properties, effect of carotene on oxidative stability..
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you can go through the attached manuscript
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I have done pressurized liquid extraction of microalgae using different "green" solvent to extract carotenoids.
The extracts contains also chlorophyll. I want to remove, or at least reduce, the chlorophyll and keep the carotenoid in the extract (so I think activated charcoal can't be used).
I'm focus on the bioactivity of the carotenoids, so acid treatment is maybe not a good issue.
I have two major contraints to taking account : using "green"/GRAS solvent AND using a method who can be scale-up.
What can I do to remove/reduce chlorophyll ?
Thanks in advance
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You may first add benzine 40-60° to your extract. Caroten and chlorophyll will move into the benzine phase, xanthophylls stay in the ethanol or methanol extract. Subsequently, you add methanolic KOH to the benzine. This will transfrom chlorophyll into chlorophyllin, which will move into the MeOH-KOH phase. Benzine will still contain the carotines, whcih can be recovered by evaporation of the benzine. Maybe this helps.
Best regards
Peter
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Is there a way to stimulate carotenoid (beta-Carotene) biosynthesis on field ?
Furthermore is there a correlation of the Sweet-potato skin color with any fertilization practices ? (more intense orange skin color)
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Hi!
Are you talking about the Orange Flesh sweet potatoes (OFSP) or normal sweet potatoes? Because OFSP is a genetically improved variety that contain high level of beta-carotene sometimes comparable to carrots.
But talking of a nutrient inducing the biosynthesis of beta-carotene in normal potatoes!! That may be a research topic! Though some mineral elements are Always needed as enzymes cofactors during synthesis, but then the pathway should exist.
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I have calculated the values of Neoxanthin, violaxanthin, lutein, zeaxanthin, antheraxanthin and beta carotene and Chl a and Chl b by HPLC. How Can I calculate carotenoid content, total chlorophyll content and different ratios among these pigments?
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What do you mean by values? Concentrations in your prepared solution of extract that you injected into HPLC? Calculate the total amount of each carotenoid, considering concentration, dilution factor, total volume of extract, and weight of plant material into equation. This is assuming you extracted all carotenoids (some are colorless - phytoene and phytofluene). You may then add up all carotenoid values (mg/kg dry weight or wet weight) to get total carotenoid content. Rough estimate does not require HPLC, only absorption of extract at ~450nm.
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I want to extract carotenoids from alfalfa by soxhlet extraction. which solvent or combinations of solvents is the best choice (3 of them) and what is the appropriate protocol for this extraction.
i'm be grateful if you mention any article.
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I'm using Shimadzu HPLC with RID and DAD detector. But the machine is working with single pump so only single mobile phase can be run. So, in what proportion I will make the mobile phase that it will detect carotenoid perfectly?
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Interesting question. I'm currently working on carotenoids too using Acclaim C30 column and a combination of mobile phases.
Have you tried with isocratic elution?
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I have tried to find the pigments absorption coefficients (in m2/mg) but I only find graphs in different publications. I have sent emails to research authors to pass me the data but I have not had any luck.
Would anyone know where to find this data?
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Inskeep WP, PR Bloom (1985) Extinction coefficients of chlorophyll a and b in N.N-dimethytformamide and 80% acetone. Plant Physiol. 77:483-485.
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The only place that sells trans-antheraxanthin is ChromaDex. But the standard is very dilute and requires high injection volume in HPLC before detection.
Any help in finding a supplier for this carotenoid is appreciated.
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I've been thinking about what would be the topic of my thesis, and I feel attracted to develope something related to insects as food. But first, I would like to know what is the perception of it in the scientific community.
Now, I will do a little experiment with carotenes and cricket flour as a test in one of my master's program courses.
Thanks for you attention!
Have a nice day!
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Hello Ana,
I have no expertise in this area but thought of a couple of things:
Insects produce comparable protein to, for example, soybeans, and can be feed on organic waste, so are a sustainable food source and do not add to land demand.
Insects used for animal feed (eg fish and poultry) reduces the demand on plant food used for non-human food.
Insects as protein source compared to livestock produce less greenhouse gas emissions.
Eating insects is not uncommon in Africa, Asia and the Pacific, and there is growing interest in western countries, eg EU.
Raising insects may provide income source for poorer communities.
Food made from insects will probably not be recognisable - "flours" and protein isolates rather than crunchy crickets.
And as you know there is plenty of scientific papers and other information available on this topic. I think you could probably develop a worthy thesis.
Kind regards,
Denise.
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Why doesn't the anthocyanins/flavonoids accumulation in petals result in a colored phenotype?
Carotenoids cannot accumulate in the petals because Arabidopsis lack chromoplasts, essential for accumulation or storage of carotenoids. But what is the reason behind flowers not having pigmentation from flavonoids?
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By using a transcription factor from blood orange we were able to induce the appearance pink petals in some of our transgenics, thought the most robust and consistent phenotype was anthocyanin accumulation in mature embryos. I think this supports your above stated conclusions.
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I am going to extract fucoxanthin from seaweed. I want to now it is necessary to perform saponification before HPLC in Carotenoid purification? Actually I was wondering if someone tell me what is the important of purification and saponification? Is it related to HPLC peak or something else?
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Amirreza... HPLC is a well certified and reliable method for the separation and analysis of components in mixtures. The components sometimes have some common properties that will cause overlapping of the chromatographic signals. OR... the matrix is so complicated that affects the analysis. Chemical modification is necessary in such cases to release the components or remove some interfering compounds. Thus, pretreatment of samples by saponification as in your case or esterfication for the analysis of fatty acid mixtures. The success of your analysis highly depend on the success of the pretreatment... Good luck...
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I have extract carotene, now I want to know which type of carotine is this, means it is alpha or beta or gamma. What I should do?
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You have to perform chromatographic separation, preferably HPLC on reverse phase column and compare the retention time and absorption spectrum with standards. If HPLC is not available, thin layer chromatography on plates may provide separation of pigments in your extract.
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Or any other aspects of pigment ratios that have practical application in food.
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Thank you. Abderrahim Benkhaled and Bala Rathinasabapathi . I am aware of the potential health benefits of dietary intake of chlorophyll and other pigments. I am curious if there is any benefit or interference of dietary intake of different ratios of these natural food pigments.
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Interaction of carotenoids with the superoxide anion radical in relationto their stabilizing effect during cryoconservation of sperm
LI Paramonova, AA Revina, AF Lizunkov - Dolk. Vses. Akad. S.-Kh. Nauk im. VI …, 1989
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I agree with Zviad.
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Carotenoid is light sensitive and is easily disappear from TLC due to exposure to light and air.
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I extracted carotenoid sample by mixing equal volumes Chloroplast soln., Cyclohexane and Milli-q. I measured the absorbance at 425nm.
I used other wavelengths as well, such as 495nm,480nm, 470nm, 450nm, 432nm.
But I couldn't find or figure out the equation to calculate its concentration in mg/ml
Similarly, I extracted xanthophyll by using equal volumes of carotenoid solution and 90% methanol. Are you aware of an equation where I can determine the concentration of Xanthophyll? Wavelengths I used = 450nm and 480nm
If you are aware of any papers please refer me to it. I am aware that Goodwin's book is a goldmine but the book is not available to me. Any help is appreciated. thank you.
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Sahini Banerjee folloe the link of the paper may be its helpful
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In 1st step we have extracted plant pigments in 100 %acetone .
Step 2 ... Acetone extract is mixed with petroleum ether in separotory funnel .
Step 3 ......Acetone containing chlorophyll is washed with slow addition of water (here we assuming that carotenoid pigment comes in petloum ether layer )
4th step .....For separating xanthophyll from other carotenoid pigments we added ethanol .....Gradient of dark green to pale green is formed from top to bottom ......Now we assuming that lower portion is xanthophyll and upper carotenoid...
Does this procedure is correct and satisfactory for quantification of xanthophyll ?????
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Dear sir,
if I understood right, you want to separate xanthophylls from the more lipiphilic carotins. To do so I would recommend an easy separation procedure: First extract your plant material in ethanol or methanol (not aceton, because this will not form a layer with benzine). After extraction add benzine (best: boiling point 40-60°C). Chlorophylls and carotins will migrate into the benzine phase. Xanthophylls will stay in the alcohol.
You can separate and purify the different xanthophylls e.g. by means of a column chromatography.
Hope this will help
Best regards
Peter Richter
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Any new article explain about the mechanism on carotenoid compounds in the rumen?
Thanks!
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Thanks a lot, Abhijeet!
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we are try to comparing total cellular content like chlrophyll ,carotenoid ,anthocyanin, proline ,sugar ,antioxidant enzymes of medicinal plant at different altitude level of Himalayas , know about the plant response to abiotic stresses . Will it show any significant result. what else we can do to make our project more informative ?
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Dear Shubhan
The alterations in the leaf structural elements (leaf morphological, anatomical, ultrastructural, morphometrical and photosynthetic parameters) and genome-wide investigation of molecular mechanisms for high-altitude adaptation have attracted great attention in the last few years.
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