HPLC

The HPLC or High Performance Liquid Chromatography instrument. Since this link was created the Monmouth College Chemistry Department has purchased a new HPLC, so an updated procedure and pictures will be added to this page during my senior year.

Description

The following video is a very helpful short introduction to how the HPLC works.

HPLC for Beginnners Video

The solvent, or mobile phase, is held in a reservoir. A pump regulates a specified flow rate of the mobile phase, we used 1 mL/min. The autosampler injects the samples into the continuous stream of mobile phase, which carries the samples to the column. The column has a packing material that separates each component in a mixture. The detector separates the compound bands as they eluate from the column. The HPLC collects the separated band containing the purified compound for further study.

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  Figure 1. Photo of the High Performance Liquid Chromatographer

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  Figure 2. Photo of the pump inside the HPLC

Procedure for the New HPLC

1) Using the computer software, turn on the pump until you have reached a stable pressure.

2) Fill HPLC vials with samples and standards.

3) Place the vials in the autosampler, taking note of which sample is in which slot (or label each vial before placing in the slot).

4) To be finished in fall of 2020.

Procedure for the Old HPLC

The following steps were used to run the HPLC. A method was previously created to run with our caffeine samples that allows for the analysis of absorbance at 273 nm, which is the lambda max for caffeine.

1) Fill HPLC vials with samples and standards.

2) Place the vials in the autosampler, taking note of which sample is in which slot.

3) Close the autosampler.

4) Click "file" --> "new sample set" --> "sample set"

5) Set flow rate and solvent to the desired settings --> click "go."

6) Click inside the sample set box and name each sample, set a method, and change the time, making sure the name of the sample matches the vial number of where the vial is located. (make sure to run the first sample twice, to ensure an accurate reading)

7) Select a stop method for the last sample to ensure the machine does not run continuously.

8) Click "run sample set"

Exporting Data from the Old HPLC

The following is a description of how to properly export the data from the HPLC.

1)Click "Sample Sets"

2) Click "File" --> "New Method" --> "Export Method".

3) Click "File" in the top left of the pop-up tab.

4) Open desired export method.

5) Click the "Raw Data" tab.

6) Click the ASCII box under file and choose the folder you wish to export the data to.

7) Name the file.

8) Choose "Save" from the "File" button in the top left of the pop-up tab.

9) Click the "Find Data" and then choose the "Sample Sets" tab.

10) Right click on the sample set you wish to export.

11) Choose export.

Data from the Old HPLC

Below is some example data we have analyzed from the HPLC. These chromatograms show where each standard absorbed at 273 nm, so the coffee samples can be tested against these standards for caffeine levels.

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  Figure 1. Chromatgram of 62ppm Caffeine Standard

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  Figure 2. Chromatgram of 125ppm Caffeine Standard

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  Figure 3. Chromatgram of 250ppm Caffeine Standard