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Abstract
Natural products are materials extracting from organic compounds that can contribute to many medical, scientific, and cultural advancements. This study investigates the extraction and examination of organic compounds found naturally, particularly the chromophores in grape leaves. The main objective revolved around isolating and recognizing specific compounds using methods like column chromatography and UV-Vis Spectrophotometry. The findings pointed to the presence of Chlorophyll A, Chlorophyll B, and Pheophytin A within the grape leaves. To identify these compounds, data was compared with existing literature values within reasonable anaylsis. Recommendations of future experiments highlight the constraints of the procedures and suggests alternatives such as flash chromatography and high-performance liquid chromatography to address error within the lab. By refining extraction methods and broadening comparative studies across various plant species, future experiments could be greatly enhanced. Extracting natural products plays a significant role in scientific exploration and is vital to continue research as modern medicine and science continues to expand.
Introduction
Natural products are a diverse group consisting of organic products produced by life which can range from wood and soil, to milk and plants. Natural products can be found in prokaryotes such as bacteria and archaea and eukaryotes such as fungi, plants, and animals. Natural product extraction is the isolation of various compounds such as enzymes, proteins, pigments, terpenes, and antibiotics. The use of extraction can be traced back to beginning civilizations who harnessed the healing and regenerative properties of plants, grains, and soils. Extraction is a key role in agriculture, food, cosmetics, and fragrances. Natural product extraction also plays a key role in medicinal advancements such as cancer research, drug discovery, and preventative medicine. In pharmaceuticals, 49% of the drugs are either natural products or derived from natural products. (Cragg, 2016). Morphine, opium from poppy, quinine from cochine bark, and aspirin from willow bark all are drugs synthesized from natural products as therapeutic agents. Antibiotics such as penicillin from fungus are another example of medicinal extraction. Natural extractions can be used in cancer research such as the compound, vincristine from the Madagascar Periwinkle w;hich can be used to treat leukemia. Taxol from the
Pacific Yew Tree has also been used in cancer therapy (Cragg, 2016). A useful organic product from plants are chromophores which give pigmentation and certain chemical properties to the organic compound. Chromophores play a crucial role in the plant's photosynthetic capabilities. During photosynthesis, light energy is converted into glucose, providing energy for metabolic processes, growth, and reproduction. Moreover, photosynthesis releases oxygen and contributes to the regulation of carbon dioxide levels. Chlorophylls, carotenoids, and anthocyanins are
examples of pigments found in various plants. UV-visible spectroscopy is a technique often used
to identify and examine the absorption of light by the sample of these chromophores.
Isolation of these natural products is a mutli-stage process consisting of extraction and purification. Extraction, purification, structural identification, and synthesis are all processes involving isolation of natural products, Extraction is the first step to separate the desired natural products from the raw materials. This is based on solubility and acid base properties. Purification
is based on chromatography and recrystallization. Identification of products can be found through many procedures such as Solvent extraction, thin layer chromatography, ultraviolet visible spectroscopy, infrared spectroscopy, nuclear magnetic resonance, mass spectrometry, atomic spectroscopy, and X-ray spectroscopy (Smith, 2020). Chromophores specifically are best analyzed through the use of column chromatography and UV-Vis spectroscopy. Chromophores are grouped into subgroups such as carotenes, pheophytins, chlorophylls, and xanthophylls. Derivatives of these subgroups involve α-carotene, β-carotene, phenophytin B and chlorophyll B (Smith, 2020). Science utilizes these methods to identify and analyze natural products and compounds. The compounds must be lysed, centrifuged, and analyzed with thin layer column chromatography. The absorption of pigments extracted from the plants is analyzed with UV-
spectroscopy. The analysis of these natural compounds derived from organic compounds plays a crucial role in development of medicine, pharmaceuticals, agriculture, and technology.
Methods and Results
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Week 1: Extraction of a Natural Product
Two grams of recently harvested green grape leaves were finely ground using a mortar and pestle provided by the laboratory instructor. Following the addition of 2 mL of acetone, the
resulting mixture was carefully transferred to a labeled centrifuge tube (Tube 1). The mortar underwent two additional rinses with acetone, ensuring a cumulative volume of at least 6 mL in Tube 1. After a thorough 2-minute vortexing, Tube 1 underwent centrifugation at 2000 rpm for 2 minutes, employing a counterweight. Subsequently, the tube was delicately handled to avoid disturbing the sediment, and the acetone fraction was poured into a separate labeled tube (Tube 2). Tube 2 was then supplemented with 3 mL each of
hexane and deionized water. Following a minute of vortexing
and centrifugation at 1000 rpm for 2 minutes, Tube 1 underwent
a
cleaning process to eliminate plant debris and was dried using
compressed air under the fume hood. The acetone/water layer
from Tube 2 was then carefully transferred back to Tube 1
utilizing a Pasteur pipette. Meanwhile, the hexane from Tube 2 was reserved, and 2 mL of hexane was introduced to Tube 1, followed by a minute of vortexing and centrifugation at 1000 rpm for 2 minutes. The acetone layer was discarded, and the amalgamated hexane solution from both tubes was prepared for subsequent steps. Figure 2: Sample tube after centrifugation. Sample
product assembled at the bottom of the tube.
Figure 1: Balanced centrifuge tubes ready for
centrifugation.
To establish a drying column, a Pasteur pipette was packed with cotton and sodium sulfate, affixed to a support, with a fresh centrifuge tube positioned below. The hexane layer was meticulously passed through the column. Following this, three TLC plates were readied, each delineated with a line positioned 1 cm above the bottom edge. Extract samples were applied to the plates using glass microtubes: four drops on the first
spot, eight drops on the second, and sixteen drops on the
third along the marked line. Three distinct jars containing
hexane with ratios of 90:10, 80:20, and 70:30 were
prepared. Each TLC plate was immersed in one of the jars
and retrieved when the solvent had traversed ¾ up the
plate. Subsequently, the samples were stored for evaporation, to be utilized in the upcoming week's procedures.
Week 2: Separation of Chromophores by Column Chromatography The procedure began with the combination of 4.0 grams of silica gel and 15 mL of hexane in a beaker. The contents for stirred eliminate air from the silica, and the mixture was set aside for later use. A column assembly involved gently compressing a small cotton piece at its base, securing the column to the workbench, and affixing a funnel on top. Approximately 2–4 mm of
sand was add to the sand and the column was leveled by tapping it gently with a plastic tool. 5 mL of hexane was slowly added using a Pasteur pipette. 15 mL of hexane was added with a collection beaker placed beneath.
Figure 3: TLC plates immersed in hexane filled jar to allow
the pigments to travel up the jar.
Silica gel was loaded into the column by pouring the stationary phase solution with a slow, steady flow, tapping the column to level the gel. Maintaining a
level solvent layer was crucial to prevent drying and potential
damage to the silica gel, which could compromise the separation
process. The hexane was run through the column until it nearly
reached the top of the silica gel. The column was then filled with
hexane, settled, and the process was repeated twice more. Sand
was reintroduced, and the hexane level was carefully drained to
a few millimeters above the sand.
The sample was dissolved in approximately ¾ mL of hexane and added to the column. Additional hexane was added while maintaining the sand's position to maintain the solvent flow. A solution of a 90:10 hexane to acetone mixture was introduced, collecting distinct bands
in separate test tubes. As the column progressed, solvent ratios were adjusted, and bands were collected, particularly focusing on the distinct yellow band. The ratios were altered to 80:20 and 70:30, respectively. The column was stopped after collecting the yellow bands using pure acetone.
During this process, TLC plates were prepared, marked,
and spotted with samples. They were immersed in a 70:30 hexane to acetone solution, and after solvent migration, the test tubes containing bands closest to a single pigment band were photographed, labeled, and stored for the upcoming week's experiment.
Figure 4: Separatory column filtering the sample though the
silica gel.
Figure 5: Separate bands filtered into test tubes after separatory column.
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Week 3: Compound Identification and Analysis Using UV-VIS Spectrophotometry The spectrophotometer was linked to the data acquisition system via USB. The initiation of absorption spectroscopy adhered to the specific manufacturer's guidelines, as directed by the laboratory instructor. Each test tube containing reserved color bands from the previous weeks' lab was mixed with hexane to form a liquid solution. The spectrophotometer was adjusted to measure within the 350–700 nm range in the assigned mode. 1.5 mL of DI water was added to a cuvette for the blank sample, and another cuvette was
filled with 1.5 mL of the sample mixed with hexane.
This process was repeated for each color band solution
saved from the previous week. The reference and
pigment samples were individually run through the
spectrophotometer to generate an absorbance graph. Following the separate run for each Figure 6: Pigment samples in cuvette ready for UV
analysis
Figure 8: Data set of Grape
Leaf Absorption Spectra
Figure 7: UV-VIS Spectrophotometry set up
sample, data was collected and saved for further analysis.
Discussion
The extraction of chromophores from grape leaves involved laboratory separation techniques, specifically TLC plating and UV-Vis Spectrophotometry. The column chromatography distinct samples with varied color bands or chromophores. The purest samples
giving the most accurate results, one through five, were stored for analysis in the subsequent week, determining the absorbance of the most purified chromophores through UV-Vis
Spectrophotometry. Selection for each sample was based on the extraction of a single color from a descending band in the column. Samples located between bands or displaying color shifts were omitted make sure only one compound was being examined at a time.
The chosen fractions underwent UV-Vis Spectrophotometry to retrieve the maximum wavelength and absorbance spectra. These measurements were then cross-referenced with established literature values to identify the compounds present in the grape leaves. The compounds successfully identified within these fractions were chlorophyll A (Experimental wavelength 413.5, 434.8, 543.4, 658.8), chlorophyll B (experimental wavelength 451, 590.6, 645.7), and pheophytin A (experimental wavelength 412.4, 530, 670). Limitations in the lab equipment accuracy hindered obtaining a perfect reading of the chromatophores therefore the closest results were chosen within reason keeping in mind the outliers. For example, fraction 5 exhibited a wide range.
After examining the original spectra, abnormalities in the curves of the graphs were noted. This could have been attributed to equipment error or improper calibration with the dilution sample. The ideal range was 400-480, anything outside this range was considered an outlier. To enhance accuracy and comparability between samples and external sources, the data
and graphs were normalized, setting the maxima of the y-axis at 1, resulting in clearer slopes and clearer peak observation.
The experimental results successfully identified various compounds, including chlorophyll A (fraction 4), chlorophyll B (fractions 2 and 3), and pheophytin A (fraction 1). However, some common compounds such as beta carotene, pheophytin B, and flavonoids were
not observed in the preserved extractions, indicating a limitation in the experiment. The
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absence of these compounds could be attributed to either non-testing or extraction, highlighting
an experiential limitation that could have been alleviated by testing all collected samples. Identified errors during column chromatography included insufficient silica gel quantity, erratic control of hexane flow, and potential inaccuracies in the collection of distinct fractions. The sand-to-silica gel ratio was recognized as a potential source of error impacting column height and flow rate.
To address the identified errors in column chromatography, flash chromatography was suggested as a potential alternative. This technique, utilizing air pressure for rapid separation, could ensure even layer settlement and provide superior control over band width and shape compared to gravity-fed chromatography. Another alternative, high-performance liquid chromatography, was mentioned but dismissed due to its high cost and inefficiency in the lab setting.
Analysis of TLC plates revealed deviations in RF values outside the expected range of 0.01-0.3 in gravity column chromatography. The experimental results ranged from a low of 0.25 to a high of 0.7 on the highest analyte. Probable sources of error in the plating process, such as spotted size discrepancies, concentration issues, or omitting a sample, were identified. To enhance plating accuracy, samples were dabbed on the plates eight to 12 times, ensuring limited spotting distance would not be an issue, and samples could be easily observed. This process was conducted in both regular and UV-light spectra, enabling the visualization of UV-
reactive compounds that might have been otherwise missed.
Conclusion
The extraction of compounds from natural products serves as a valuable method for analyzing their chemical and physical properties, which advances scientific, environmental, and
medicinal research. Chromophores are crucial in the specified plant’s photosynthetic capabilities.
Photosynthesis allows for conversion of light energy into glucose, providing energy for metabolic processes, growth, and reproduction. Photosynthesis also releases oxygen and regulates carbon dioxide levels. Chromophores such as α-carotene, β-carotene, phenophytin B and chlorophyll B reflect colors different shades of yellow, green, blue, and gray. The chromophores identified through Compound Identification and Analysis Using UV-Vis Spectrophotometry were a Pheophytin A for fraction 1, Chlorophyll B for fraction 2 and 3, Chlorophyll A for fraction 4, and a mix of Chlorophyll A and Pheophytin A for fraction 5. The methods used to extract the pigment natural products from the grape leaf were centrifugation, thin-line chromatography, column chromatography , and UV-Vis Spectrophotometry. The experiment revealed the presence of Pheophytin A, Chlorophyll, and Chlorophyll B in the grape leaf. To optimize the data collection and increase the accuracy, multiple trial runs of the experiment could have been performed. Comparison of separate chromatophore data could have been compared to give more conclusive results. Other recommendations for future experiments are comparing multiple plant sample chromophores of different species. Refinement of extraction technique is necessary to produce the most precise results such as the TLC chromophore traveling up the plate and proper use of centrifugation. Natural products are vital compounds used in various areas of scientific and societal advancement. Extraction of these organic compound products can be achieved through extraction, purification, structural identification, and synthesis.
References
Barkovich, M. (2013, October 2). High Performance Liquid Chromatography
. Chemistry LibreTexts.https://chem.libretexts.org/Bookshelves/Analytical_Chemistry/
Supplemental_Modules_(Analytical_Chemistry)/Instrumentation_and_Analysis/
Chromatography/High_Performance_Liquid_Chromatography
Cragg GM, Newman DJ (2016). Natural Products as Sources of New Drugs from 1981 to 2014. J
Nat Prod. 79(3):629-61. doi: 10.1021/acs.jnatprod.5b01055. Epub 2016 Feb 7. PMID: 26852623.
Regenstein, J. M., & Regenstein, C. E. (1984). Column Chromatography - an Overview | ScienceDirect Topics
. Www.sciencedirect.com. https://www.sciencedirect.com/topics/agricultural-and-
biological-sciences/column-chromatography
Scheer. (1991a). Chlorophyll a - awiSc. https://epic.awi.de/id/eprint/28828/1/Jef1997j.pdf
Scheer. (1991b). Chlorophyll b - awi. https://epic.awi.de/id/eprint/28829/1/Jef1997k.pdf
Scheer. (1991c). Pheophytin a - awi. https://epic.awi.de/id/eprint/28856/1/Jef1997al.pdf
Zhang, QW., Lin, LG. & Ye, WC (2020). Techniques for extraction and isolation of natural products: a comprehensive review. Chin Med
13
. https://cmjournal.biomedcentral.com/articles/10.1186/s13020-018-0177-x
Appendix
RF Values:
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Test
Tube
0
Test
Tube
1
Test
Tube
2
Test
Tube
3
Test
Tube
4
Test
Tube
5
Test
Tube
6
Test
Tube
7
Test
Tube
8
Test
Tube
9
#1 Analyte Top
0.70
N/A
0.70
0.34
0.36
0.35
0.36
0.36
0.27
0.28
#2 Analyte
0.34
N/A
N/A
N/A
N/A
N/A
0.30
0.30
0.26
0.26
#3 Analyte
0.30
N/A
N/A
N/A
N/A
N/A
N/A
0.29
N/A
N/A
#4 Analyte
0.28
N/A
N/A
N/A
N/A
N/A
N/A
0.28
N/A
N/A
#5 Analyte
0.27
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
#6 Analyte
0.26
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
Test
Tube
10
Test
Tube
11
Test
Tube
12
#1 Analyte (Highest spot)
0.26
N/A
N/A
#2 Analyte
0.25
N/A
N/A
#3 Analyte
N/A
N/A
N/A
#4 Analyte
N/A
N/A
N/A
#5 Analyte
N/A
N/A
N/A
Week 3 Data: Spectrum (i.e. Fraction 1, spectrum 1) Compound
Peak
Wavelengths
of sample
Peak Wavelengths
from Literature
Literature Source
Fraction 3
Chlorophyll a
429.8
430
The Light-Dependent Reactions of Photosynthesis. OpenStax CNX.
Oct 9, 2013 http://cnx.org/contents/f829b3bd-472d-4885-
a0a4-6fea3252e2b2@11.
Fraction 4
Chlorophyll a
430.6
430
The Light-Dependent Reactions of Photosynthesis. OpenStax CNX.
Oct 9, 2013 http://cnx.org/contents/f829b3bd-472d-4885-
a0a4-6fea3252e2b2@11.
Fraction 5
Phenophytin
B
410.8
410
The identification of chlorophyll and its derivatives in the pigment mixtures: HPLC-
chromatography, visible and mass spectroscopy studies - Scientific Figure on ResearchGate. Available from: https://www.researchgate.net/figure/The-
absorption-spectra-of-chlorophyll-A-pheophy-
tin-B-and-chlorophyllide-C_fig1_258696540 [accessed 9 Nov, 2022]
Fraction 6
Beta Carotene
452.4
453
Hagos, M., Redi-Abshiro, M., Chandravanshi, B. S., & Yaya, E. E. (2022). Development of Analytical Methods for Determination of
β
-Carotene in Pumpkin (
Cucurbita maxima
) Flesh, Peel, and Seed Powder Samples.
International journal of analytical chemistry
,
2022
, 9363692. https://doi.org/10.1155/2022/9363692
Fraction 7
Phenophytin
B
411.8
410
The identification of chlorophyll and its derivatives in the pigment mixtures: HPLC-
chromatography, visible and mass spectroscopy studies - Scientific Figure on ResearchGate. Available from: https://www.researchgate.net/figure/The-
absorption-spectra-of-chlorophyll-A-pheophy-
tin-B-and-chlorophyllide-C_fig1_258696540 [accessed 9 Nov, 2022]
Spect
rum (i.e. Fract
ion 1,
spect
Comp
ound
Peak
Wavel
engths
Peak
Wavel
engths
Literature Source
rum 1)
of
sample
from
Literat
ure
Fracti
on 1
Pheop
hytin A
412.4 (530,6
70)
410 (534.9,
665.5)
https://www.researchgate.net/figure/The-absorption-spectra-of-
chlorophyll-A-pheophy-tin-B-and-chlorophyllide-C_fig1_258696540
Fracti
on 2
Chlor
ophyll
B
451.3 (645.7)
456.9 (596.7,
645.5)
https://epic.awi.de/id/eprint/28829/1/Jef1997k.pdf
Fracti
on 3
Chlor
ophyll
B
452.7 (644.4)
453 (596.7,
645.5)
https://epic.awi.de/id/eprint/28829/1/Jef1997k.pdf
Fracti
on 4
Chlor
ophyll
A
434.8 (534.2,
662.1)
430 (534.2,
662.1)
https://www.researchgate.net/publication/
258696540_The_identification_of_chlorophyll_and_its_derivatives_in_t
he_pigment_mixtures_HPLC-
chromatography_visible_and_mass_spectroscopy_studies
Fracti
on 5
mix of
Chlor
ophyll
A and Pheop
hytin A
418.3 (512.3,
545.6, 670.2)
418.3 (512.3,
545.6, 670.2)
https://pubs.acs.org/doi/10.1021/acs.jnatprod.2c00720#:~:text=The
%20absorption%20spectra%20of%20flavonoids,tails%20to%20400–
450%20nm
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- A student weighed out 0.150 g of protein powder and dissolved it in 100 mL of water (Solution 1). The student then diluted this solution by transferring 1 mL into a 25 mL flask and diluting with water (Solution 2). Finally, 1 mL of that solution was transferred to a test tube and combined with 4 mL Bradford reagent. The absorbance of the solution in the test tube was 0.187. Assuming that the best fit linear line of the standard curve was y = 0.04144 x + 0.01521 (μ g mL), calculate the percent protein by mass in the original protein powder.arrow_forwardHere is the protocol for a UV-Vis spectrophotometer to detect water and chlorine-carbon. 1.Dissolve the water and chlorine-carbon compounds in a solvent, such as water. 2.Prepare a standard solution of known concentration that is similar to the sample being measured. 3.Calibrate the spectrophotometer using the standard solution. 4.Measure the absorbance of the sample using the spectrophotometer. 5.Calculate the concentration of the compounds in the sample using the calibration curve obtained from the standard solution. How is the spectrophotometer calibrated with standard solutions? When is the blank solution placed in the spectrophotmeter?arrow_forwardHow is light and energy used in gas chromatography- mass spectrometry (GC-MS)?arrow_forward
- Quantitative Analysis by External Standard Method Calcium in a juice sample is determined by atomic absorption spectrophotometry. A stock solution of calcium is prepared by dissolving 1.834 g of CaCl, 2H20 in water and diluting to 1000.0 mL. A substock calcium solution was prepared by transferring 10.00 mL of the stock to a 100.00 mL volumetric flask and diluting to the mark. Three standard solutions of calcium were prepared by transferring 2.500 mL. 5.00 mL and 10.00 mL of substock to 50.00 mL volumetric flasks and diluting to the mark. The sample is prepared by transferring 1.000 mL of fruit juice from the original bottle to a 25.00 mL flask and diluting to the mark. Strontium chloride is added to all solutions before dilution to avoid phosphate interference. Instrumental response of the standards is measured against the blank, and the following readings are recorded: 9.10, 18.6 and 37.0. The sampie reading is 28.1. Using the multipoint method, find the concentration of calcium in…arrow_forwardThe spectroscopic data in the table is generated with five solutions of known concentration. Concentration (M) 0.0133 m= 0.0266 0.0532 0.106 0.213 Absorbance 0.1271 What is the intercept of the linear regression line? 0.08531 0.5388 1.069 Use a spreadsheet program, such as Microsoft Excel, to graph the data points and determine the equation of the best-fit line. 1.954 What is the slope of the linear regression line formed by these points? M-1arrow_forward0.1120 g of a solid sample containing copper was dissolved by acid and transferred to a 250.00 mL volumetric flask and volume was made up with distilled water. The solution was then diluted by a factor of 25. The final solution was analyzed by atomic absorption spectrophotometry and the concentration was found be 2.982 ppm, What is the weight percentage of copper in the original solid sample? Keep four significant figures in your final answer.arrow_forward
- A sample of drinking water is analyzed colorimetrically for ammonia nitrogen concentration. If the sample colour when viewed through a depth of 5.5 cm corresponds in intensity to a 1 mg/L standard sample when viewed through a depth of 40 cm, what is the concentration of ammonia nitrogen in the sample?arrow_forwardThe absorbance of an unknown dye solution is measured to be 1.06 by a spectrometer. The calibration plot of the spectrometer is provided below. What is the molar concentration of the unknown sample (in mole L)? Absorbance vs. Molarity 18 y3 67024x-0.0343 16 14 12 1 0.8 0.6 0.4 0.2 01 0.2 0.000005 00001 0000015 Concentration of solutions, mol/L 0.00002 0.000025 000003 0 163e-5 O 2.35e-6 0 163 0 235 Absorbancearrow_forwardA sample solution containing quinine was analysed by fluorescence spectroscopy. 5 mL of the sample solution was diluted with 0.1 M HCl to a final volume of 100 mL. The fluorescence of the solution was measured and gave a value of 54. A reference solution containing 0.086 mg/mL of quinine gave a fluorescence reading of 38. A blank solution gave a fluorescence reading of 21. Calculate the quinine content of the sample solution in mg/mL.arrow_forward
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