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
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Power switch lower left side of instrument (usually already on)
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Power button on instrument panel
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Input “00000” into instrument panel
Sample setup
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The HPLC uses an autosampler
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Four racks (1-4) each have numbered slots (1-54) for samples
Mobile-phase setup
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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
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The column can be changed by opening the housing on the right side of the instrument
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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”
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Analysis screen
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Method setup
File, new method
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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
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In the “quick batch” tab
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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