Lab 1: HPLC
Introduction:
Liquid chromatography is the most widely used of all separation techniques. The HPLC
is the high performance liquid chromatography used for separation of compound mixtures. This
instrument uses column separation technique where the mobile and stationary phases are
separated in an analyte under extreme pressure.
Methods:
*The LC was not working.
Day 1:
1.
2.
3.
4.
5.
6.
7.
Check the degassed methanol and distilled water levels.
Check lines coming from methanol and water to ensure there are no air bubbles.
Turn on the detector and oven
Start up program
File, Open Method, Taylor test, Test method one.
Hit download and allow pressure on pumps to raise to 275-300 pressure.
Make stock solution using 100 mg of caffeine in 100 mL of distilled water in a 100 mL
volumetric flask.
8. Make 5 different dilutions (50, 40, 30, 20 and 10 ppm).
9. Click acquire, single run, name the sample ID, sample name and the data file and hit
OK.
10. Use a syringe to take up some sample
11. Inject sample into hole in the center of the knob on the oven that is set to load, until
sample begins to drip into waste beaker.
 Leave syringe in the hole
12. Quickly turn the knob to inject.
13. Allow the scan to run for 10 minutes.
 Run all 5 dilutions
14. Boil 200 mL of distilled water
15. Add tea bag (regular tea) for about a minute.
16. Allow sample to cool to room temperature
17. Add 1 mL of the caffeinated water to a 100 mL volumetric flask and fill with distilled water
Day 2:
1. Weigh out 0.1011g of caffeine
2.
3.
4.
5.
6.
7.
Add to 100 mL volumetric flask and fill with distilled water
Make same percentage standards using the reserved stock
Use previous computer and injection techniques
Dissolve 1 aspirin tablet in 200 mL of hot water
Allow mixture to cool to room temperature
Add 1 mL of dissolved aspirin to a 100 mL volumetric flask and fill with distilled water
Day 3:
1. Grind up one aspirin tablet and add to a 100 mL volumetric flask
2. Add 1 mL of 95% ethanol and swirl
 Repeat addition of ethanol 3 times
3. Run sample
4. Add 1 aspirin tablet to a 10 mL volumetric flask and fill with 95% ethanol
5. Run sample
6. Add 1mL of 95% ethanol in 100 mL volumetric flask and fill with water
7. Dispose of ethanol in water hood
Calculations:
Stock Solution:
1 𝑚𝑔
0.001 𝑔
1𝑔
100 𝑚𝑔
=
=
=
1𝑔
1,000,000 𝑔 100 𝑚𝐿
1000𝑚𝐿(
) 1000 𝑔
1 𝑚𝐿
Dilutions:
M1V1=M2V2
1000(x)=(50)(50)
=
1000(x)=2500
=2.5mL
1000(x)=(40)(50)
=
1000(x)=2000
=2.0 mL
1000(x)=(30)(50)
=
1000(x)=1500
=1.5 mL
1000(x)=(20)(50)
=
1000(x)=1000
=1.0 mL
1000(x)=(10)(50)
=
1000(x)=500
=0.5 mL
Tea:
461435 = 35348(𝑥) + 68771
392664 = 35348(𝑥)
11.109 = 𝑥
11.109 𝑝𝑝𝑚
Aspirin dissolved in hot water:
313434 = 34064(𝑥) + 133715
179719 = 34064(𝑥)
5.276 = 𝑥
5.276 𝑝𝑝𝑚
Aspirin dissolved in ethanol and diluted with water:
253464 = 34064(𝑥) + 133715
119749 = 34064(𝑥)
3.515 = 𝑥
3.515 𝑝𝑝𝑚
Aspirin dissolved in ethanol and diluted in ethanol:
262046 = 34064(𝑥) + 133715
128331 = 34064(𝑥)
3.767 = 𝑥
3.767 𝑝𝑝𝑚
Results:
Concentration vs. Area
2.010 0 6
Area (nm)
1.510 0 6
1000000
y= 35348x+68771
R2= 0.9518
500000
0
0
20
40
60
Concentration (ppm)
Figure 1. Concentration curve of Concentration vs. Area for five different concentrations
(10,20,30,40 and 50 ppm) on day one. The concentration at 40 was a significant outlier.
Concentration vs. Area
2.010 0 6
Area (nm)
1.510 0 6
1000000
500000
y = 34064x + 133715
R2=0.9834
0
0
10
20
30
40
50
60
Concentration (ppm)
Figure 2. Concentration curve of Concentration vs. area of the second days standard solutions
(10,20,30,40 and 50 ppm).
Conclusion:
There were some issues with this lab. First the LC did not work and we only worked on
the HPLC. Secondly on the second day the oven detected an error, LK.S, telling us that the
leak sensor was broken. Also our first linear regression line had a seemingly low R2 value.
Despite these issues we were able to analyze the 5 different dilutions on both days and design
a graph with a linear regression line. Figure 1 illustrates the concentration curve for the first
day’s standards. The R2 value could be so low due to improper pipeting or insufficient mixing of
the components. We were also able to analyze caffeinated tea (there was no decaffeinated
available at the time) on this day and the analysis report can be found on note book page 16 of
the lab notebook. The second day new stock solutions were made and analyzed, giving a not
perfect but much better R2 value. The first aspirin run (pg. 24) in water led us to believe that the
caffeine concentration in the aspirin was about 15 ppm because the peak was in between our
standard of 10 and 20 ppm. This solution appeared cloudy and didn’t seem to completely
dissolve. The second aspirin test dissolved in ethanol and then diluted with distilled water gave
a very small caffeine peak and seemed to be too small of a concentration for the aspirin tablet.
This was also believed to not be completely dissolved. It was then decided to dissolve the
aspirin tablet in all ethanol to ensure that the tablet was completely dissolved. This gave us a
solid peak with a much larger peak (believed to be ethanol) right in front of it (pg.28). To ensure
that the large peak in front of the caffeine was ethanol we ran a strictly ethanol sample which
gave us an extremely large peak in the same location (pg.29) In conclusion, the aspirin tablet
was best dissolved 95% ethanol and will have an ethanol peak at a retention time of 1.943.