HPLC/LC-MS Lab Report Introduction: Liquid Chromatography similar to gas chromatography is the separation of compounds. There are several types of liquid chromatography but the main ones are High Pressure Liquid Chromatography (HPLC) and liquid chromatography mass spec (LC-!). The HPLC separates compounds that are dissolved in a solution and they are separated b y in"ecting a small amount of the sample into a column. The components pass through the column at different rates due to the partitioning of the samples. The HPLC is generally used for identifying# purifying# and quantifying components of a mi$ture. !ome applications of HPLC include drug analysis# and protein levels in compounds. The LC-! is mainly used for the detection and identification of chemicals in a mi$ture. !ome applications of LC-! include pharmaco%inetic analysis and drug development. Purpose: The purpose of this e$periment &as to determine the caffeine levels in various standard solutions of coffee# tea# and pure caffeine. The samples concentrated samples &ere run through both the HPLC and LC-!. Methods: 'ay *e &or%ed &or%ed on the HPLC for this lab period. +irst &e made various concentrated standards of pure pu re caffeine using ,,mL volumetric flas%s. *e calculated the number of mols that &ould be needed need ed for our standards. *e made made # # and solution standards of caffeine each , mols &ith ./g# , mols &ith .,g# and , mols &ith .//g respectively and then made un%no&n concentrations of regular and decaf coffee. *e first ran all our standards each by in"ecting a small amount into the column and letting it run for , minutes and obtained a chromatogram for each standard. Then &e used the same method for the un%no&ns.
'ay This day &e &or%ed on the LC-!. *e first chec%ed the degassed methanol and &ater levels. *e ended up having to ma%e more degassed methanol for the instrument but other than that everything &as correct. *e turned on the compressed nitrogen tan% in the gas room as &ell as on the bench top. Loaded all the samples at once and started the soft&are. 0nce the instrument &as ready &e let do its &or%. *e used the same samples from the HPLC and this &ould give a mass spec of the samples. 1ach sample ran for / minutes and gave us a chromatogram and mass spec &ith the desired pea% s for caffeine. *e *e also made a calibration curve for these results to find the molar concentration o f the un%no&n caffeine and tea samples. sa mples. Results: 2ll of the chromatograms and mass spec data can be found in the lab noteboo%. The retention time# 3 area# and height &ere measured for each chromatogram on the HPLC and the LC-! only had retention time and height measured for results. 2 calibration curve &as done for each instrument to test ho& &ell the instrument &or%s and performs. *e used the calibration curve equation to find the molar concentration of caffeine in the tea and coffee c offee samples.
HPLC results
Concentration(mol) 9egular Coffee 'ecaf Coffee
Retention time (min) .4/ .6/ .6/ .86 .4,7
% rea ,.56 /.5 58.77 .477 8.84
Hei!ht /#,57 ,#66 5#// ,#86 8#4,,
Calibration equation y : .,,,,$ ; .,5 : y : .,,,,(,#86) ; .,5 : .8 regular y : .,,,,(8#4,,) ; .,5 : ./7 decaf LC-! results Concentration(mols)
Retention time (min) .75
Hei!ht (cps) 6
6.2348 x 10
6
.7
2.6125 x 10
.7/
4.2459 x 10
'ecaf Tea 9egular Coffee
6
6
.78 .77
5.0776 x 10
6
8.8857 x 10
Calibration 1quationy : .,/8$ ; .54568 :y: .,/8(
6
5.0776 x 10 ¿
; .54568 : .,
decaf y : .,/8(
6
8.8857 x 10 ¿
; .54568 : .,8 regular
Conclusion: The e$periment &as successful and &e &ere able to figure out the molar concentrations of the un%no&ns from both the HPLC and LC-!.
as it should have been and the standard should have been the smallest. !omething &ent &rong some&here during the e$periment because the results should have been similar to that of the HPLC results# e$cept for the tea since there &as nothing to compare it to. To fi$ this error# more trials could have been done to ensure that it &ould yield the correct results and minimi>e errors and get a better concentration of caffeine.
