3.2.1 Nuclear Magnetic Resonance Spectrometer (NMR)
NMR provides spectrum that has the information on the number of magnetically distinct atom present in the compound studied. In this project, 1H-
& 13C-NMR, HMBC and HMQC spectra were obtained by using trimethylsilane (TMS) as internal standard and reference. The frequency used for 1H-NMR analysis was 400 MHz; while 13C-NMR was performed at 100 MHz. The samples were prepared by dissolving a small amount of sample in
38 sufficient acetone-d6 in order to fill up the NMR tube to a height of 4-5 cm and the cap was affixed to the tube and wrapped with parafilm to avoid solvent evaporation
3.2.2 Infrared Spectrophotometer (IR)
Through the IR analysis, IR spectrum was obtained. IR spectrum was used to indicate the presence of certain functional group in the compound analysed. In this project, Perkin Elmer 2000-FTIR spectrophotometer was used for sample analysis and the range used was 4000 cm-1 – 400 cm-1. The sample was grinded with KBr powder in the ratio of 1:10. Then, the mixture of the sample with KBr powder was compressed under a high pressure to form KBr sample pellet.
3.2.3 Ultraviolet-Visible Spectrophotometer (UV-Vis)
The UV-Vis spectrum was used to determine the qualitative information and the position of the hydroxyl group in xanthone block. From the transition energy of the highly conjugated organic compound, colour produced by the compound can be identified. In this project, Perkin-Elmer Lambda (25/35/45) UV-Vis spectrophotometer was used and the samples were prepared by using absolute ethanol to dissolve the sample. Then, the sample was measured by using quartz cuvette in the range of 190 nm to 400 nm.
39 3.2.4 Mass Spectrometer (MS)
Mass spectrometry was used to provide the information on the molecular mass, molecular formula and the structure of the molecule. This was done by analysing the molecular ion and fragmentation pattern of the compound. In this project, the sample was analysed by using Agilent 5975 MSD (nominal mass) mass spectrometers.
3.2.5 Melting Point Instrument
Melting point instrument was used to provide melting point of the tested compounds. By obtaining the melting point, the characteristic and the purity of the tested compound can be determined by comparing the range difference with the pure compound. In this project, Barnstead Electrothermal 9100 melting point instrument was used. Heamatocrit capillaries were used to contain the solid form sample for testing.
40 3.3 Methodology
3.3.1 Synthesis of 1, 3, 6, 8-Tetrahydroxyxanthone
14.1 g (75 mmol) of 2, 4, 6-trihydroxybenzoic acid monohydrate was added with 75 ml of Eaton’s reagent and warmed in water bath at 80 oC for 10 minutes whilst stirring. The mixture was cooled at room temperature and was stirred for 20 minutes. The mixture was poured into 500 mL beaker initially filled with ice and was left to soak in ice bath for 30 minutes. The precipitate was then filtered via Buchner filtration and the precipitate was placed in oven at 50 oC overnight, while, the filtrate was extracted with ethyl acetate. After drying overnight in the oven, the dried precipitate was then extracted with acetone. Both the extracts were dried under reduced pressure separately.
41
2,4,6-trihydroxybenzoic acid and Eaton's reagent
1. The mixture was warmed in water bath at 80oC for 10 minutes while stirring.
2. The mixture was cooled at room temperature and stirred for 20 minutes.
21 3
45 6 7 8 9
1 1
0 2 3
45 6 7 8 9 11
500 mL beaker filled with ais 3. The mixture was poored into the beakers filled with ais and left to soak for 30 minutes.
hotplate stirrer
4. The precipitate formed was filtered with Buchner filtration and dried in the oven overight at 50oC.
5. Dried precipitate was extract with acetone.
seperating funnel
6. The filtrate was extraceted with ethyl acetate.
7. Both extracts were dried under reduce pressure seperately.
Figure 3.1: Flow chart on the Synthesis of 1,3,6,8-tetrahydroxyxanthone
Figure 3.1: Flow chart on the synthesis of 1, 3, 6, 8-tetrahydroxyxanthone Separating
funnel 5. The filtrate was extracted with ethyl acetate.
6. After overnight drying in the oven, the dried precipitate was extracted with acetone.
3. The mixture was poured into the beakers filled with ice and left to soak for 30 minutes.
7. Both extracts were dried under reduce pressure separately.
1. The mixture was warmed in water bath at 80 oC for 10 minutes while stirring.
2. The mixture was cooled at room temperature and stirred for 20 minutes.
4. The precipitate formed was filtered with Buchner filtration and dried in the oven overnight at 50 oC.
42 COOH
OH OH
HO
+ COOH
HO OH
OH
Eaton's reagent, 80 oC, reflux 10 minutes
O
OH
OH HO
HO
O
Figure 3.2: Synthesis pathway of 1, 3, 6, 8-tetrahydroxyxanthone
3.3.2 Alkenylation of Xanthonic Block in Organic Solvent
A mixture of 1.04 g (4 mmol) of 1, 3, 6, 8-tetrahydroxyxanthone and 1.656 g (12 mmol) of K2CO3 was stirred for 5 minutes in 100 mL acetone under room temperature. 2.384 g (16 mmol) of 5-bromo-1-pentene was added into the solution. The mixture was refluxed for 6 hours at 65 oC. The solid was filtered and the filtrate was dried under reduced pressure.
43 Round flat bottom flask filled with the mixture
of 1,3,6,8-tetrahydroxyxanthone and K2CO3 in 100 mL aetone.
21 3
456 7
8 9
1 1
0 2 3
456 7
8 11 9
hotplate strirrer 1. The mixture was stirred for 5 minutes in room temperature.
2. 5-bromo-1-pentene was added into the mixture and the mixture was reflux for 6 hours at 65oC.
3. The solid was filtrated and the filtrate was dried under reduce pressure.
Figure 3.3: Flowchart on the synthesis of o-alkenylated xanthone 2. 5-bromo-1-pentene was added into the mixture and the mixture was refluxed for 6 hours at 65 oC
3. The solid was filtered and the filtrate was dried under reduced pressure
1. The mixture was stirred for 5 minutes at room
Flat bottom flask filled with the mixture of 1, 3, 6, 8-tetrahydroxyxanthone and
K2CO3 in 100mL acetone
44 O
OH
OH HO
HO
O
K2CO3 in 100 mL acetone, 5-bromo-1-pentene, reflux for 6 hours at 65 oC
O
OH
OR2 HO
R1O
O
(1) R1 = R2 = pent-4-en-1-yl (2) R1= H, R2 = pent-4-en-1-yl
Figure 3.4: Synthetic route for synthesising o-alkenylated 1, 3, 6, 8- tetrahydroxyxanthone