HPLC Analysis Procedure on Shimazdu System
Alex Engler
This document serves as an instruction set for the Shimazdu HPLC system found in room 047 of the
Fenske Chemical Engineering Building. It will guide new research undergraduates through the process
of analyzing an already prepared sample using the system. This involves
The “Sequence” is the
setting the system up, preparing a sequence, and collecting the resulting
information the computer sends
data for analysis. Also included are a list of components for the system’s
the machine, telling it how
setup and an exceptions section for troubleshooting. The final data will
many samples are being run
contain information regarding the composition of the samples tested, as is
common with all chromatography analysis methods. Understanding the
raw data is not covered in the scope of this document, only the procedure for obtaining it.
Contents
1. Equipment List…………………………………………………………………………………..Page 2
2. Preparing the Machine……………………………………………………………………….Page 3
a. On the System…………………………………………………………………………..Page 3
b. On the Computer………………………………………………………………………Page 3
3. Running the Sequence………………………………………………………………………..Page 5
a. On the System…………………………………………………………………………..Page 5
b. On the Computer………………………………………………………………………Page 5
4. Analysis……………………………………………………………………………………………..Page 7
5. Exceptions………………………………………………………………………………………....Page 8
Equipment List
-
Computer: The computer set up in the lab is a fairly standard PC for campus labs.
o The software icon on the desktop is EZ Start
Shimazdu System: This is the primary hub on the top of the system (See Figure 1.)
Pump: The pump is on the bottom (Figure 1) and labeled as LC-10AD. This pushes the mobile
phase through the collumn
Detector: The detector is shown in Figure 1 to the right of the pump. It is labeled as SPD –
10AVP. The detector contains the lamp used in analysis.
Sample Tray: The Sample Tray (Figure 5) holds the vials containing the samples to be
analyzed.
Sample Cabinet: The Sample Cabinet houses the sample tray during analysis. It is located
between the Shimazdu System and the Pump. (Figure 1)
Metal Absorbers: The metal absorbers are the two tips connected to the pumps by tubing.
Their primary function is to get the mobile phase flowing through the column.
Column: The column is the red tube connecting the pump to the sensor (Figure 1.) There is a
short and long column. See a Post Doc or Grad Student about switching columns.
Mobile Phase: The Mobile Phase is housed in a glass container, usually on top of the system to
allow for easier downward flow. See the “exceptions” section for information on making and
replacing Mobile Phase.
Figure 1: System Setup
Images
Preparing the Machine
The machine’s pump and lamp need to “warm up” for some time before
the sequence can be run. It is recommended that this step be performed
before preparing the samples so that time can be most efficiently used.
This step is performed in two parts, physical interaction with the
hardware, and an on-screen interaction with the software.
Note: If system is not already on, power the system [top
component], pump[LC-10AD], and detector[SPD-10AVP] using
physical buttons labeled “power” on the device.
On System
Figure 2: Mobile Phase
1.) Ensure that the mobile phase bottle (Figure 2) is filled up over
200ml and labeled with the required mobile phase specific to
your experiment. If this is not the case, please see the
“Exceptions” section at the end of this document.
2.) Press the pump button, seen on the left hand side of the
pump component (Figure 3)
3.) Confirm that pump is running. Pressure reading on LC-10AD
screen should read above 500 psi.
Note: If the pump’s pressure is incorrect, please see the
“Exceptions” Section at the end of this document for instructions
on manually purging the system.
Figure 3: Pump
Component
On Computer
1.) Run EZ start from desktop (not the offline version.)
2.) When “Instrument Wizard” window pops up, hit “OK.”
3.) Turn on lamp
a. On "Instrument Setup" window (Figure 4,) change "lamp" from "off" to "D2."
i. Click "apply" to confirm setting, and then click "download method" to send
setting to machine.
ii. If done correctly, "Lamp preheating" will display on SPD-10AVP.
Figure 4: Primary Window
4.) “Purge” the system.
a. Click Control – Direct Control - Purge
b. Click Control – Preview Run
i. The display will show readings from the sensor. Wait about 45 minutes for
the line to become horizontal, and then continue to “Running the Sequence.”
Running the Sequence
Like the previous step, “Running the Sequence” is divided into two parts; placing your samples into the
sample cabinet, and telling the computer to run the proper sequence. This is a very quick step, but
afterwards there is a lot of waiting before analysis can be run. If you will be running multiple
sequences, it is recommended that you prepare the second sequence after this step.
On System
1.) Place sample tubes numerically into the sample tray.
2.) Place the sample tray into the sample cabinet by lining up the three prongs in the cabinet with
the holes on the underside of the tray. See Figure 5 for reference
Figure 5: Sample Tray
On Computer
1.) Fill out the “Sequence” window (Figure 4) with all of the information for the samples. If there is
no option under the column “Method” that matches the experiment, see an adviser/grad
student.
a. If this is the final sequence for the day, set the “Run Type” setting for the last vial to
“Shutdown.” This will shut the pump and lamp off upon completion of the sequence to
save power and mobile phase.
Tip: If the samples being used have long names; fill out the information for the first vial, then
right click and choose “fill down.” This will copy the information onto the following vials, then
minor changes can be made to each entry.
2.) Under File – Sequence – Save As, save sequence with a unique name.
3.) Under Method – Advanced, change the “Path Export” folder (Figure 6.)
Caution: Remember this folder; it is where the data will appear in the “Analysis” section.
Figure 6: Advanced Method Options Window
4.) Exit the window. A message will pop up asking to create the folder. Click yes.
5.) Click Control – Sequence Run. This will open the window shown in Figure 7.
a. Ensure that “All” is selected under “Run Range.”
b. If it is desired, the “Begin Run” tab in the bottom left corner can be used to have the run
start after a specified time.
Figure 7: Run Sequence Window
6.) Click “Submit” to start the sequence. If the run is set to start immediately, the needle in the
sample cabinet can be seen moving to draw out sample.
Caution: If the method has been changed, a window will pop up that asks to save the method.
Click yes on this window. If this message is not addressed, the run will not start, but mobile
phase will be used.
7.) Wait for the sequence to run, this time varies from experiment to experiment to experiment,
but is usually several hours.
Analysis
Retrieving the data for analysis is relatively simple. Once the sequence is finished, the data is all
located in individual files.
1.) Minimize the EZ Start Window. Open the EZ Start (Offline) icon on the desktop.
2.) Click File – Open – Data and navigate to the folder where the method is saved. Choose one of
the data points.
3.) The reading will appear on the screen. Click Report – View – Area% to get the data in terms of
raw numbers (Figure 8.)
Figure 8: Data Window
4.) Copy the numbers needed, and then repeat steps 1-4 for each sample.
Exceptions
This final section covers two of the most common issues that can be run in to while performing HPLC
analysis. If a difficulty is experienced that is not covered in this section or the rest of this procedure, an
adviser/post doc/grad student should be consulted.
Inadequate Pump Pressure
If the pump fails to reach a high enough pressure to run a sequence, the pump may need to be
manually purged. To do this, twist the pump dial (Figure 9) counterclockwise while the pump is
running. Then extract some of the mobile phase using the syringe attached to the tubing. This “pulls”
some of the mobile phase to eliminate air bubbles.
Figure 9: Manual Purging Tools
Inadequate/Incorrect Mobile Phase
When switching out the mobile phase bottle to either refill the current phase or change phases, simply
take out the metal knobs (Recall Figure 2) and wash with DI water and a wipe, then place the knobs
into the new bottle and seal with a lid or parafilm. If there are no bottles with the needed mobile
phase available, see a post doc/grad student for the formula needed to make a new batch.