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Manish Kumar
Chemical Engineering
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5.545
Solvents
Analytes
Objectives
(i)
(ii)
(iii)
(iv)
(v)
learn the basics of liquid chromatography and to learn how to operate the WATERS
600E HPLC.
Optimize the separation of two compounds (sugars) using isocratic elution methods.
Obtain following data from EMPOWER (HPLC software): retention time (Tr), peak
widths (w), and peak areas for each peak.
(iv)Calculate following parameters on the basis of data obtained from HPLC:
Resolution between peaks (Rs), and Plate number or efficiency (N) for each peak.
Plot the calibration curve for mixture of both compounds and quantify the
concentration of unknown compound
Procedure
1. Switch on the HPLC in following sequence: Pump unit > degasser > autosampler > RI
Detector > computer.
2. Prepare 2L of 0.005MH2SO4 as mobile phase, and samples of analytes and keep in
ultrasonic bath for degassing purpose.
3. Select a mobile phase stream out of four lets say A and put in the mobile phase.
4. Set the flow rate 4 ml/min through controlling software (EMPOWER) and pass the
mobile phase through bypass valve for 5min for removing the air bubbles from the
stream.
5. Set the flow rate according to the separation method and maintain the same flow for 20
min to condition the column.
6. Start purging mobile phase in detector for 15 min.
7. Set auto zero in detector.
8. Keep samples in the autosampler in proper sequence.
9. Develop the method in EMPOWER, which includes several parameters like flow rate,
column temperature, autosampler temperature, sample volume, run time etc.
10. Run the method
11. Take data and chromatograph as PDF file.
12. Repeat procedure for finding the retention times, plotting calibration curves, and
quantifying the concentration of unknown sample.
Observations:
1. Prepare two tables - first for data obtained from the HPLC and second for calculated
parameters (see in Objectives).
2. Attach the chromatographs obtain from EMPOWER.