Respected Sir,

In our lab, we use to follow the method of Harborne JB, 1973. Phytochemical methods. Chapman and Hall, London. using spectrometer as you asked for. The following is the method.

Take 100mg of fresh plant tissue and add to it 10ml of 80% acetone and grind it well in a mortar and pestle. Now centrifuge it at 3000rpm for 10 mins. Take the supernatant and discard the pellet. Make up the supernatant upto a known volume of 10 ml. Now read the OD values at 480 nm in UV-spectrophotometer.

Calculation:

Amount of carotenoids in 100mg plant tissue = 4 x OD value x Total volume of sample (i.e we have make up the supernatant volume as 10 ml) / Weight of fresh plant tissue (i.e. we have taken 100 mg plant tissue to grind ) = ....................mg/gm

Hi, We used HPLC for detection of carotenoids using the Fraser method. But there might be a way to standardize using a UV-Vis. I find atleast one paper with a quick google search and perhaps you can find more.

http://onlinelibrary.wiley.com/doi/10.1002/0471142913.faf0202s00/abstract

I think there are two things I will consider: accuracy and speed of quantitation.

Respected Sir,

In our lab, we use to follow the method of Harborne JB, 1973. Phytochemical methods. Chapman and Hall, London. using spectrometer as you asked for. The following is the method.

Take 100mg of fresh plant tissue and add to it 10ml of 80% acetone and grind it well in a mortar and pestle. Now centrifuge it at 3000rpm for 10 mins. Take the supernatant and discard the pellet. Make up the supernatant upto a known volume of 10 ml. Now read the OD values at 480 nm in UV-spectrophotometer.

Calculation:

Amount of carotenoids in 100mg plant tissue = 4 x OD value x Total volume of sample (i.e we have make up the supernatant volume as 10 ml) / Weight of fresh plant tissue (i.e. we have taken 100 mg plant tissue to grind ) = ....................mg/gm

I too follow the same method explained by Dr.Tresina

Respected Sir,

If my given methodology really helps you please upvote my answer to encourage me.

I use the method of Fish et al 2002. This method works well. Tomato fruits are finely chopped in a blender. Then, samples were homogenized using

a homogenizer at low speed with a 10-mm

shaft. Two grams of puree were weighed into the centrifuge

tube and extracted with HPLC-grade solvents of 10 mL of

hexane, 5 mL of ethanol, and 5 mL of acetone containing

0.05% butylated hydroxytoluene (Merck KGaA). Samples

were tightly sealed and placed on an orbital shaker

for 15 min at 320 rpm, and then 3 mL of deionized

distilled water was added and samples were shaken again

for 5-10 min (if phase separation did not occur within five minutes wait 5 more minutes to phase separation. . Afterwards, samples were put in a rack to allow

solvent phase separation. Read absorbance of the upper hexane layer at 503 nm for lycopene and 450 for beta carotene.

Fish WW, Perkins-Veazie P, Collins JK (2002) A quantitative assay for lycopene that utilizes reduced volumes of organic solvents. J Food Comp Anal 15: 309-317.

Ground one hundred mg fresh tissue from interveinal areas of leaves in 10 mL 80% acetone. Filtrate suspension and than read filtrate sample at 480 and 510 nm using the spectrophotometer for carotenoid estimation. The total carotenoid content was calculated using the following formula.

V

Leaf carotenoid content = 7.6 × (OD 480) – 1.49 × (OD 510) × –––––––––– mg g-1 FW

d ×FW×1000

Where, OD = Optical density of the extract at given wavelength

V = Final volume of chlorophyll extract in 80% acetone

FW = Fresh weight of leaves (g)

d = Length of the path of light (1 cm)

Thank you so much for all the suggestion.I am really appreciate it. I am looking forward to measure carotenoid on tomato fruit.

Same procedure I added above can be used for total caratenoid. If you read Absorbance of sample at 450 nm you will get total carotenoid

Formula: (Abs@450 nm x V x10000)/(A%1 1cm x amount of sample)

V= 10 ml because of hexane layer is used

A%1 1 cm=absorption coefficient =2500 for total carotenoid

Hello,

First of all, there is almost no spectral region of carotenoid absorption that does not overlap with chlorophyll (especially chl b) absorption. (480 nm in 100% acetone could roughly work - especially for lycopene which absorbs far more red than other carotenoids - here you can go beyond 500 nm). Therefore it is crucial to know whether you want to determine carotenoides in green parts of the plant (leaves, unripe fruits etc.) or the ripe fruits - this is the reason why you'll find so many formula for estimation of the carotenoid content (mainly lycopene is interesting in the latter case).

Secondly, I'm not sure which carotenoid you want to determine: Lycopene in the fruits, xanthophylls or carotenes in leaves?

They have considerably different absorption spectra. So it is hard to determine carotenoides exactly with just a single wavelength - better is to record the whole absorption spectrum (and substract the values measured at 750 nm - since there is no pigment absorption - just scattering)

References for the pigements in several solvents can be found in :

" Data for the identification of 47 key phytoplankton pigments"

Jeffrey, SW;Mantoura, RFC; Bjørnland, T

Phytoplankton pigments in oceanography: guidelines to modern methods. UNESCO, Paris

1997

For exact measurements it will be necessary to calibrate the spectrophotometric method against HPLC measurement.

very good description by Lars

Is there any spectrphotometric method for the determination of phytotene

Thank You Dr. Tresina 

But Why we are multiplying with 4 in the formulae.

With Regards

 I too follow the same method explained by Dr.Tresina

Yes you can. Carotenoids are highly unsaturated liposoluble hydrocarbons derived from isoprene, the distinguishing feature of which is a conjugated double bond system which constitutes the chromophore group, responsible for the energy absorption capacity in the visible spectrum. With Regards

A.M. Goula, M. Ververi, A. Adamopoulou, K. Kaderides, Green ultrasound-assisted extraction of carotenoids from pomegranate wastes using vegetable oils, Ultrasonics Sonochemistry (2016), doi:

http://dx.doi.org/10.1016/j.ultsonch.2016.07.022

should the volume of the solution be given in ml in the calculations? (i use 10ml like in dr teresin procedure)

and next question: should the phase be clearly visible after the centrifugation? What amount of supernatant should I take?

And don't we needs any standarts?

Yes.....

Please suggest a method for determining the concentration of purified zeaxanthin apart from HPLC. Also, Which solvent is compatible for the solubility of zeaxanthin.

I was using the beta carotene 95% pure to derive a calibration curve using the UV-vis spectrophotometric analysis. I had an initial stock solution of beta carotene (0.12 mg/mL). I made a couple of dilutions and ran at 450nm spectrophotometrically. After using the equation:

C (mg/mL) = (A*10)/A1%, where A1% = 2592 A.U

The final concentration calculated was 0.04 mg/mL. This shows ~68% loss from my initial stock solution. Did anyone have a similar situation. Or does anyone have a suggestion to avoid any errors.

Kindly go through the attached articles