HPLC, LC/MS, IC
Introduction
Ion Chromatography (IC) separates aqueous samples and analyzes them based on their ion
levels. Liquid Chromatography-Mass Spectroscopy (LC/MS) and High Performance Liquid
Chromatography (HPLC) work great together as a team. The LC/MS can determine the makeup of a
sample while the HPLC tells us how much of it there is.
Purpose
To learn how to use the HPLC, LC/MS, and IC to their full potential in analyzing aqueous
solutions.
Procedure
Day one: HPLC, LC/MS
-Create calibration curve with caffeine standards on both instruments
Day two:
-Run tea samples on both instruments (3 brands, two caffeinated, one decaf.)
Day one: IC
-Create standard anion concentrations
-Create calibration curve with anion mixture and run standards
Day two:
-Make new calibration curve with standards then run water samples
-Adjust standards accordingly so water samples wall on curve.
Data
HPLC
Caffeine: 0.22g
(See lab notebook for spectra’s of 50, 100, 200, and 400ppm solutions)
400ppm was too concentrated…flat peak
Day two: made new solutions (50, 100, 200, and 300ppm) and ran
(See lab notebook for those spectra)
Area
Concentration V. Area
9000000
8000000
7000000
6000000
5000000
4000000
3000000
2000000
1000000
0
y = 29350x - 845900
R² = 0.9509
0
50
100
150
200
250
300
350
Concentration (ppm)
Unknown
10% English Teatime Caffeinated
10% English Teatime Decaffeinated
10% Guaranteed Value Brand Tagless Teabags
Area
569661
59293
496207
LC/MS
Using day two solutions ran along with 10% tea solutions
10% tea solutions made by taking 10mL of tea and placing it in 100mL volumetric flask and diluting to
the line
(See lab notebook for spectra)
Intensity (cps.)
Concentration V. Intensity
1.00E+07
9.00E+06
8.00E+06
7.00E+06
6.00E+06
5.00E+06
4.00E+06
3.00E+06
2.00E+06
1.00E+06
0.00E+00
y = 20136x + 3E+06
R² = 0.8366
0
50
100
150
200
Concentration (ppm)
250
300
350
Unknown
10% English Teatime Caffeinated
10% English Teatime Decaffeinated
10% Guaranteed Value Brand Tagless Teabags
Intensity
3500000
2400000
3700000
Unknown
10% English Teatime Caffeinated
10% English Teatime
Decaffeinated
10% Guaranteed Value Brand
Tagless Teabags
HPLC Calculated Concentration
48.23ppm
LC/MS Calculated Concentration
24.83ppm
30.84ppm
-29.80ppm
45.73ppm
34.76ppm
Unknown
English Teatime Caffeinated
English Teatime Decaffeinated
Guaranteed Value Brand Tagless
Teabags
HPLC Original Concentration
482.3ppm
308.4ppm
LC/MS Original Concentration
248.3ppm
-298ppm
457.3ppm
347.6ppm
IC
Problems were encountered while trying to run standard anion solution. Conductivity stated at zero
except when it went negative for a bit of time at the start of the run.
No data collected for IC
Calculations
HPLC and LC/MS
1 microgram per L= 1ppm, so 400milligrams per L is 400ppm
0.2𝑔 200𝑚𝑔
=
0.5𝐿
0.5𝐿
Dissolve 0.2g of caffeine in a 500mL volumetric flask and dilute to line
200ppm:400𝑝𝑝𝑚 × 𝑋𝑚𝐿 = 200𝑝𝑝𝑚 × 50𝑚𝐿X=25mL of 400ppm in a 50mL volumetric flask and
dilute to line
100ppm:400𝑝𝑝𝑚 × 𝑋𝑚𝐿 = 100𝑝𝑝𝑚 × 50𝑚𝐿X=12.5mL of 400ppm
50ppm: 400𝑝𝑝𝑚 × 𝑋𝑚𝐿 = 50𝑝𝑝𝑚 × 50𝑚𝐿X=6.25mL of 400ppm
Day two:
From 1000ppm stock solution repeat similar steps to make 300, 200, 100, and 50ppm solutions
Ex:
1000𝑝𝑝𝑚 × 𝑋𝑚𝐿 = 300𝑝𝑝𝑚 × 50𝑚𝐿15mL of 1000ppm stock in a 50mL Volumetric and
dilute to line
200ppm: 10mL
100ppm: 5mL
50ppm: 2.5mL
HPLC: Y=293500X-845900
LC/MS: Y=20136X+3+E06
Ex: HPLC English Teatime Caffeinated
𝑋𝑝𝑝𝑚 × 10𝑚𝐿 = 48.23𝑝𝑝𝑚 × 100𝑚𝐿
𝑋𝑝𝑝𝑚 =
48.23𝑝𝑝𝑚 × 100𝑚𝐿
10𝑚𝐿
𝑋𝑝𝑝𝑚 = 482.3𝑝𝑝𝑚
IC
No calculations
Conclusion
The liquid chromatography lab was a mixed bag of results. The HPLC was a complete success; it
is designed to quantify the data. The approximate concentration of the tea samples were calculated and
everything seems to work out (decaffeinated has a lower concentration of caffeine than in caffeinated
samples). The LC/MS worked and gave us a decent chromatogram which is meant to qualify the data.
Using that data to quantify is not what this instrument is meant to do. So it is understandable that the
calculated concentrations from it are fairly off (nearly 500ppm vs. approximately 250ppm). The IC did
not work at all. The seven anion standard was attempted to be ran but we were given a conductivity of
zero. Learning the general use of these instruments was successful except for the IC.