HPLC Standard Operating Procedure

High-performance liquid chromatography (HPLC) is a technique that allows for separation and quantification of solutions containing a mixture of organic compounds. Solutions are pushed through a column (with varying functional groups) at high pressure. Different organic compounds (with varying functional groups) will have different retention times in the column, based on the column type as well as other programmable conditions (flowrate, temperature, eluent type, etc.). This separation allows for identification and quantification of analytes by using either the refractive index detector (RID) or photodiode array (PDA) detectors.

Instrument startup

Power switch lower left side of instrument (usually already on)

Power button on instrument panel

Input “00000” into instrument panel

Sample setup

The HPLC uses an autosampler

Four racks (1-4) each have numbered slots (1-54) for samples

Mobile-phase setup

The top of the instrument houses different solvents that can be changed as needed based on the observed compounds (up to four different solvents, A-D, can be used for one technique)

Column setup

The column can be changed by opening the housing on the right side of the instrument

Columns can be changed by simply switching the input (i.e. from sample to column) and output (i.e. from column to detector) tubing to the desired column

*ensure solvent compatibility with column before use*

Instrument operation

PC password is “hplc” Open “labsolutions”

Analysis screen

Method setup File, new method

In method editor, all variables can be manually set (time, flow rate, temperature, mobile phase, volume, etc.). Exact settings need to be optimized based on the specific system observed. See literature for starting points.

Running samples

In the “quick batch” tab

Method file can be loaded, and sample position selected and queued. Make sure you note the volume of solution injected

Samples should be named. When “Start” is selected, the run will begin unless the instrument is still waiting for the

oven to reach the proper temperature. Make sure you have enough eluent!

Shut down

Make sure PDA is off

Power button on instrument panel

Notes

It’s advised to equilibrate the system by running the eluent through (with the PDA off, pump on, oven on), without a sample, for 15 minutes before using. The system should also be cleaned by flowing mobile phase after analysis. ADDITIONALLY, it may be good practice to make your first sample the supporting electrolyte only.

Make sure PDA lamp is off when not in use

*Do not make any software changes on the computer*

Contacts

Salesman: Rick Koeritz

rnkoeritz@shimadzu.com

Technician: Zachary Lilla

ztlilla@shimadzu.com