Rachel Klein
Instrumental Chemistry
Real World Project
Introduction:
Vitamin C, or ascorbic acid, helps slow down or prevent cellular damage. It is also needed to
maintain healthy body tissues and immune systems. Finally, it helps the body absorb iron from plant
foods. The disease scurvy is caused by a vitamin C deficiency. Fruits and vegetables are the best source
for vitamin C; it is recommended to get between 75-90 mg/day. Vitamin C can also be found in many
breakfast drinks. In this experiment, we will address the question of which breakfast drink has the most
amount of vitamin C in it. This will prove which drink is the best source of Vitamin C.
Objective:
The purpose of this experiment is to test apple juice, orange juice, cranberry juice, and grape
juice to determine levels of vitamin C. These samples will be compared with 5 different standard
concentrations of vitamin C.
Method 1: HPLC
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Make sure there is enough degassed methanol and water.
Turn on computer and check pump lines A and B for air bubbles
Turn on detector (SPD-10 AV)
On desktop, select HPLC, select Shimadzu icon, select the Methods icon and choose TSW.met
Set parameters by clicking the yellow box icon, click Parameters and set as following:
a. Flow rate A: .500 mL/min
b. Flow rate B: .500 mL/min
c. Flow rate C: 1.000 mL/min
d. Max pressure:6000 psi
e. Min pressure: 1 psi
f. Wavelength: 256 nm
Click download and OK
Select Single Run icon and make sure method is still TSW.met, choose start
When computer indicates “Waiting for trigger”, insert 1µl of sample and switch knob to inject,
and leave syringe in after injection
Select analysis icon and click report and area % icon and record information
To shut down, select method icon and choose LOWFLOW.met and set parameters
a. Flow rate A: .010 mL/min
b. flow rate B: .010 mL/min
c. Flow rate C: 1.000 mL/min
11) Click download and turn detector off, leave computer and pumps on
Method 2: IR-Raman
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Fill the blue dewar with liquid nitrogen and wait 20 minutes then top off
make sure the FTIR is on
Turn on the laser power supply switch and then turn the key to the on position
Make sure the lever between the Raman and FTIR is up, the lever between the Raman and
microscope is down, and the internal lever in the Raman should be up, meaning bypass closed
Double click on the Varian Resolutions Pro icon to open software
From the current scan menu select Raman scan
Optics parameters should be:
a. IR source: off
b. Beam: right
c. Detector: Raman Ge
d. Beamsplitter: quartz near IR
e. Accessory: FT-Raman
f. ATR Crystal: none
g. Optical filter: holographic notch
h. Aperture: open
Select the laser tab and click turn on diode
Press the shutter switch on the front of the Raman (to open) and turn the power to its highest, 3
Under the laser tab, adjust laser control current until the Raman display reads between 600-700
mW
Click setup. If filled with noise, click autoscale (the four arrows)
Adjust same with X, Y, and Z, knobs until the centerburst is at its maximum
Click scan
To shut down: collect menu >Raman scan>Laser tab and click turn diode off. Press the shutter
button (closed) remove your sample, turn off the laser power supply and turn hey to off
position, and exit Varian Resolutions Pro. Leave everything else on
Calculations:
Example Calculation of fruit juice concentration:
Apple:
𝑦+197897
x=
y= 46579x-197897
x=
344054+197897
46579
46579
= 12 𝑝𝑝𝑚
Percent Recovery Calculation (spiked w/ 500 mg)
Mg in Apple:
500
=
𝑥𝑚𝑔
x mg=26.4 mg
6528139 344054
6528139+197897
Conc. Of Spiked Apple: x=
Mg in Spiked Apple:
46579
ppm = mg/ L
Percent Recovery:
=114 ppm
mg= (114 ppm)(0.250L) = 28.5 mg
𝑠𝑝𝑖𝑘𝑒𝑑−𝑎𝑐𝑡𝑢𝑎𝑙
28.5−26.4
𝑚𝑔 𝑠𝑝𝑖𝑘𝑒𝑑
500 𝑚𝑔
Percent Recovery (spiked w/ 50 mg)
Mg in Apple:
50
=
𝑥𝑚𝑔
10281535 344054
Conc. Of Spiked Apple:
=1.67 mg
10281535+197897
46579
=225 ppm
Mg in Spiked Apple: mg = (225 ppm)(0.250L)=56.25 mg
Percent Recovery:
56.25−1.67
50 𝑚𝑔
=109.16%
=0.42%
Data:
***All spectra can be found in lab notebook
Table 1. Standards for Calibration Curve
Ret. Time
Area Area %
10 ppm
1.408
307100 99.572
20 ppm
1.458
745400 81.378
30 ppm
1.483
1132929 87.917
40 ppm
1.508
1606466 92.306
50 ppm
1.533
2205540 99.994
Height
30690
69914
106560
153740
217231
Height %
99.662
90.042
93.522
92.214
99.950
Calibration Curve
2500000
y = 46579x - 197897
R² = 0.9931
Area
2000000
1500000
1000000
500000
0
0
10
20
30
40
50
60
Concentration (ppm)
Table 2. Concentrations of fruit juices
Concentration
Orange
49 ppm
Grape
23 ppm
Apple
12 ppm
Cranberry
26 ppm
Table 3. Summary table of information of fruit juices and fruit juices spiked with ascorbic acid
Ret. Time
Area
Area &%
Height Height %
Apple
1.567
344054
75.934
47568
91.205
Orange
1.683
2077012
98.501
148930
97.449
Grape
1.625
853583
67.686
107219
74.607
Cranberry
1.633
1021223
76.418
142170
81.828
Spiked Apple
1.600
6528139
99.821
824785
99.860
Spiked Orange
1.600
4365632
97.055
536584
99.110
Spiked Grape
1.883
27333634
91.225
999298
84.689
Spiked Cranberry
1.675
11326920
99.762
998171
99.545
Conclusion:
Ascorbic acid was successfully identified in all standards and samples using the FT-Raman.
Ascorbic acid was successfully quantified using the HPLC. We made an error by spiking the juices with
500 mg because this was an extreme amount and our percent recovery was extremely low. We then respiked orange juice and apple juice with 50 mg and re-calculated percent recovery and it was in the
range we had expected. Also, the concentrations of the juices were as we had expected. Overall, orange
juice had the highest concentration of ascorbic acid.