A short investigation of the use of pumpkin seed extracts in peroxyoxalate chemiluminescence demonstrations

Pumpkin seeds, equation, pink chemiluminescence in test tube

Co-Authored by Iain A. Smellie*, Iain L. J. Patterson*, Andres Tretiakov**

*University of St Andrews, School of Chemistry, North Haugh, St Andrews KY16 9ST, United Kingdom

**St. Paul’s School, Lonsdale Road, London, SW13 9JT, United Kingdom

In recent years, the fluorescence properties of pumpkin seeds have been highlighted on social media. When illuminated with a UV lamp, pumpkin seed extract appears orange/red to the human eye (see figure 1), this is due to fluorescence associated with protochlorophyllide that is present in the seeds.1

Figure 1: Pumpkin seed extracts, illuminated with a 365 nm lamp (picture A) and 405 nm laser pointer (picture B).


Protochlorophyllide is the precursor to chlorophyll a (see figure 2), the latter is well known to emit red light when leaf extracts are exposed to UV light,2,3 so it is perhaps no surprise that protochlorophyllide has similar properties. Although similar, there are 2 key structural differences between chlorophylls and protochlorophyllide 1) chlorophylls possess a phytol side chain, in contrast, protochlorophyllide is not esterified 2) chlorophylls have a chlorin cyclic motif that complexes a magnesium ion, whereas protochlorophyllide has a fully conjugated porphyrin ring for binding to Mg2+.2,3   

Figure 2: Structures of chlorophyll a and protochlorophyllide


Chlorophyll extracts can also be used as a fluorescent dye in “glowstick” chemiluminescence experiments.4 The similarities between chlorophylls and protochlorophyllide raised the question, is it possible to use pumpkin seed extract as a fluorescent dye in chemiluminescence experiments? In this short article, some results are reported from attempts to use pumpkin seed extracts for chemiluminescence experiments. A summary of the reaction that produces light in glowsticks is shown in scheme 1 below.4,5 There are a range of aryl oxalate esters that can be used in peroxyoxalate chemiluminescence experiments,6 in this case bis(2,4,5-tribromophenyl) oxalate was selected since stocks were readily available from our laboratory store.


Scheme 1: Chemiluminescence from reaction of aryl oxalate esters with hydrogen peroxide in the presence of a fluorescent dye.


Extraction Method

Investigations focused on shelled fresh pumpkin seeds from a discarded Halloween pumpkin and some dried pumpkin seeds purchased in a grocery store (see figure 3).


Figure 3: Pumpkin seeds (L to R: fresh pumpkin seeds 1, shelled fresh pumpkin seeds 2, commercially available dried pumpkin seeds 3).


In each case, about 12-15 seeds were crushed using a mortar and pestle, ethyl acetate (10 mL) was added and the crushed seeds were allowed to soak for 10 minutes. The resulting extracts were pale green in colour and gave the expected orange emission when illuminated under UV light (see figure 4). The seed extracts were initially cloudy in appearance, so before further use, they required filtration (either through fluted filter paper or through a funnel with a cotton wool plug placed in the stem). The fresh seeds were found to be slightly easier to work with since the extract was less cloudy than the extract obtained from dried seeds.

Figure 4: Crushed pumpkin seeds in ethyl acetate illuminated in daylight (picture A) and 365 nm lamp (picture B).





Chlorophyll in Parsley

Video 1: Light emission from chlorophyll in the presence of glowstick mix (chlorophyll extracted from Parsley)


The chlorophylls in spinach extract have been previously shown to act as fluorescent dyes when added to an activated white light emitting glowstick,4 under these conditions red chemiluminescence is observed. We have found that red chemiluminescence is obtained when spinach, parsley or chive extract is added to peroxyoxalate based chemiluminescent systems that use hydrogen peroxide5 or sodium percarbonate7 as oxidants (an example using parsley extract is shown in figure 5). See video 1 of chlorophyll chemiluminescence above.

Figure 5: Chemiluminescence using an aryl oxalate ester/peroxide activating mixture and chlorophyll extract from parsley acting as fluorescing agent


In this study, the extract from fresh pumpkin seeds was added to aryl oxalate ester mixtures containing 30% hydrogen peroxide or sodium percarbonate (see procedures section), in both cases red/pink or red/orange emission was observed (see figure 6). See video 2 of protochlorophyllide chemiluminescence below.


Pumpkin Seed Extract

Video 2: Light emission from pumpkin seed extract in the presence of glowstick mix.


When the extract from dried pumpkin seeds was added to an aryl oxalate ester mix, the results were disappointing since weak chemiluminescence was observed when 30% hydrogen peroxide was used as the oxidant. Furthermore, all attempts to initiate chemiluminescence by addition of sodium percarbonate to the aryl oxalate ester/pumpkin mix failed.

Figure 6: Chemiluminescence using aryl oxalate ester/peroxide activating mixtures and chlorophyll extract from fresh pumpkin seeds acting as fluorescing agent. A = pumpkin extract in ethyl acetate under UV lamp B = pumpkin extract chemiluminescence using 30% hydrogen peroxide as oxidant C = pumpkin extract chemiluminescence using sodium percarbonate (hydrogen peroxide generated in-situ) as oxidant.


The disappointing results with the extract from dried pumpkin seeds in chemiluminescence tests led to an investigation of the composition of the extracts from fresh and dried seeds. UV-visible spectroscopy is frequently used to analyse the chlorophylls in leaf extracts,8,9 a similar approach was used for analysis of pumpkin seed extracts in this study. The spectra obtained showed that the fresh and dried extracts had different compositions (see figure 7). The extract from the fresh seeds showed a strong absorption (437 nm) in the blue region of the spectrum, in contrast, the extract from the dried seeds showed two strong absorptions (422 nm and 437 nm) in the blue region.

Figure 7: UV-visible spectra of ethyl acetate extracts from crushed pumpkin seeds. Plot A = Fresh seed extract, Plot B = Dried seed extract.


The shift of absorption to shorter wavelength in the sample from dried seeds is reminiscent of the effect seen in loss of Mg2+ from chlorophyll a to form pheophytin a at low pH (scheme 2).2,9 It is unclear at this stage what the composition differences between fresh and dried seeds are, however, investigations are ongoing.


Scheme 2: Conversion of chlorophyll a to pheophytin a under acidic conditions


Time required: 

Each procedure takes about 5-10 minutes to prepare and complete. Initial emission of fairly bright light usually lasts for 15-20 seconds, frequently longer, although dim light can sometimes still be observed after 60-90 seconds.


Pumpkin seeds are readily available, in this case from a discarded Halloween pumpkin, and can be stored for several months. 

Ethyl acetate

30% Hydrogen peroxide

Sodium salicylate

Sodium percarbonate

1 × Small mortar and pestle

1 × 25 mL Measuring cylinder

1 × 100 mL beaker or Conical flask (If using sodium percarbonate rather than hydrogen peroxide, it is best to use a conical flask that can be sealed with a rubber stopper). This will allow any carbon dioxide pressure build-up to be easily released).


Example chemiluminescence procedure using hydrogen peroxide

To a 100 mL conical flask, add the desired aryl oxalate ester (approximately 100 mg) and dissolve as much of this as possible in 40-50 mL of ethyl acetate. Transfer sodium salicylate (approximately 75 mg) to the reaction flask and mix the solution occasionally for a few minutes (some solid material may still be visible, this is not a problem). To initiate chemiluminescence, add 2-3 mL of pumpkin seed extract, followed by dropwise addition of 30% hydrogen peroxide until light is observed (3 or 4 drops of peroxide is usually sufficient).

Example chemiluminescence procedure using sodium percarbonate (adapted from ref 3)

To a 100 mL conical flask, add the desired aryl oxalate ester (approximately 100 mg) and dissolve as much of this as possible in 40-50 mL of ethyl acetate. Transfer sodium percarbonate (ca. 150 mg) to the reaction flask and mix the solution (the percarbonate is not particularly soluble in ethyl acetate). To initiate chemiluminescence, add 2-3 mL of pumpkin seed extract, followed by 1-2 mL of water to the mixture, and vigorously shake or mix the flask contents (Caution! vent CO2 pressure build-up regularly if you decide to stopper the flask and shake).

Waste Disposal

After a demonstration has been completed, the flask contents are poured into water (100 mL) and treated with sodium metabisulfite or sodium sulfite to destroy any residual hydrogen peroxide. The biphasic mixture is transferred to a separating funnel and the 2 layers separated. The ethyl acetate fraction should be disposed of as organic solvent waste and the aqueous fraction can be treated as low hazard aqueous waste.

Chemical Hazards

  • Ethyl acetate (CAS number: 141-78-6) is flammable and should be considered as harmful by inhalation, ingestion or skin absorption.
  • Hydrogen peroxide (CAS number: 7722-84-1) is a potent oxidant, that will be harmful in contact with skin, eyes or if ingested.
  • Sodium salicylate (CAS number: 54-21-7) does not pose a significant toxicity hazard but should be considered as a potential irritant.
  • Sodium percarbonate (CAS number: 15630-89-4) is a potent oxidant, that will be harmful in contact with skin, eyes or if ingested.
  • Bis(2,4,6-tribromophenyl) oxalate should be considered as a harmful irritant. Note that this compound is not commercially available, the material used in these experiments has been prepared by one of the authors. The reactivity and safety properties of the brominated analogue are expected to be very similar to commercially available bis(2,4,6-trichlorophenyl) oxalate (CAS number: 1165-91-9).10 The reaction may also work with other readily accessible bis(aryl) oxalates.6




1. Borthakur, P. R.; Barua, A. G. Fluorescence Studies of the Seeds of the Pumpkin (Cucurbita pepo L.). Natl. Acad. Sci. Lett. 2014, 37, 275.

2. Pucci, C.; Martinelli, C.; Degl'Innocenti, A.; Desii, A.; De Pasquale, D.; Ciofani, G. Light-Activated Biomedical Applications of Chlorophyll Derivatives, 2021, 21, Macromol. Biosci., 2100181.

3. Borah, K. D.; Bhuyan, J. Magnesium Porphyrins with Relevance to Chlorophylls, Dalton Trans., 2017, 46, 6497.

4. Kuntzleman, T. S; Rohrer, K.; Schultz, E. The Chemistry of Lightsticks: Demonstrations to Illustrate Chemical Processes, J. Chem. Educ., 2012, 89, 910.

5. Eghlimi, A.; Jubaer, H.; Surmiak, A.; Bach, U. Developing a Safe and Versatile Chemiluminescence Demonstration for Studying Reaction Kinetics, J. Chem. Educ., 2019, 96, 522.

6. Smellie, I. A. “A General Method to Prepare Halogen-Free Oxalate Diesters for Peroxyoxalate Chemiluminescence Demonstrations” www.chemedx.org/article/general-method-prepare-halogen-free-oxalate-dies... (Accessed 20th February 2022).

7. Smellie, I. A.; Aldred, J. K. D.; Bower, B.; Cochrane, A.; Macfarlane, L.; McCarron, H. B.; O’Hara, R.; Patterson, I. L. J.; Thomson, M. I.;  Walker, J. Alternative Hydrogen Peroxide Sources for Peroxyoxalate “Glowstick” Chemiluminescence Demonstrations, J. Chem. Educ., 2017, 94, 112.

8. Diehl-Jones, S. M. Chlorophyll Separation and Spectral Identification, J. Chem. Educ., 1984, 61, 454.

9. Dujardin, E.; Laszlo, P.; Sacks, D. The Chlorophylls: An experiment in bio-organic chemistry, J. Chem. Educ., 1975, 52, 742.

10. Orosz, G. The Role of Diaryl Oxalates in Peroxioxalate Chemiluminescence, Tetrahedron, 1989, 45, 3493.



General Safety

For Laboratory Work: Please refer to the ACS Guidelines for Chemical Laboratory Safety in Secondary Schools (2016).  

For Demonstrations: Please refer to the ACS Division of Chemical Education Safety Guidelines for Chemical Demonstrations.

Other Safety resources

RAMP: Recognize hazards; Assess the risks of hazards; Minimize the risks of hazards; Prepare for emergencies