SOP For Agilent HPLC (LC Chem Station)
Please schedule training with an experienced lab member before using the Agilent
HPLC. This procedure is written for general operating steps. This SOP therefore is not
self-explanatory. It does not explain how to create a method but you may consult senior
or read operation manual by yourself.
Before Start

Check the “List of Authorized Users” for Agilent HPLC; If you name is not
on this list, please contact assigned responsible user to set up a training;

Sign up the “Log Book” with “User name”, “Start time”, “No. of samples”,
“End time” and “Note”;

Make sure the HPLC is properly connected and initialized. For more details, refer
to the hardware manuals that came with the system.

Make sure a suitable column is installed.

Make sure all modules are switched on.

Make sure the solvent bottles are full and waste bottle is empty!

Prepare appropriate solvent depending on the type of column!
Set Up a Sequence
1. Go to Instrument 1(online); it will display the method and run control that allows
you to monitor and control the instrument and run sequences;
2. Click on the “pump”, and select set up pump from the menu;
3. In the control group, gradually increase the flow rate to desire value according to
Appendix;
4. Click “OK” to close the dialog box;
5. Click on the “column”, set up the “Temperature” according to Appendix.
6. Click “SequenceLoad Sequence”;
7. Click to “chem32”, then “1” and select appropriate file location;
8. Go to sequence and open one of the old sequences;
9. Back to the original system Diagram, Click on “Sequence Save Sequence As”;
10. Name your sequence;
11. Click on P1 Agilent plate, go to “Sequence Table”;
12. Enter the sample location on the plate; then name it; choose a method; set up the
type of sample (i.e. calibration, control or sample); enter the number of injections
for this location; enter injection volume;
13. Click “Append line” to add new line to the table, and complete the line as shown
in step 12;
14. Click “OK” to close the sequence table after completing your sequence;
15. Click “Sequence”  “Sequence Parameters”; set data path to your personal folder
(please create a folder under C:\chem32\1\data\ if you don’t have one yet)
16. Save sequence again by clicking “Save Sequence” icon;
17. You may also use sequence wizard to create a sequence.
Run Samples
1. Place the 96 well-plate with samples in the plate holder of auto-sampler;
2. Ensure that your samples are in the correct positions in the auto sampler tray
according to their entries in the sequence table;
3. Click “Start” to start the sequence run.
Data Analysis and Batch Report
1. Open the “Instrument 1 offline”;
2. Click on “Batch  Load batch”;
3. Select a data fileclick OK; set up batch window will show up;
4.
Select all, and then click ok;
5. After done with your analysis, go back to “Batch Output Batch Report”;
6. The batch report is located in the data folder and shown as an excel file names
“report1”;
After Finish
1. Adjust flow rate to 0.2 ml/min if nobody want to use Agilent HPLC afterwards;
2. Make sure the solvent bottles are full and waste bottle is empty!
Appendix
1. Typical conditions for a HPX-87H column
Column temperature: 65 ºC; mobile phase: 5 mM sulfuric acid; flow rate: 0.6
ml/min; injection volume: 20 uL; sugar concentration: 0-25 g/L; running time: 15
min/sample for sugar measurement or 70 min/sample for organic aicd, HMF and
furural measurement.
2. Typical conditions for a HPX-87P column
Column temperature: 80 ºC; mobile phase: DDI water from Milipore water
system; flow rate: 0.6 ml/min; injection volume: 20 uL; sugar concentration: 0-25
g/L; running time: 30 min/sample for sugar measurement.
3. Follow the recipe below to make 5 mM Sulfuric Acid mobile phase for HPX-87H
Column; Mix appropriate amount of 72% w/w H2SO4 with DDI water from
Milipore water system; no degas needed;
Target Volume (L)
1L
2L
2.5 L
4L
5L
72% w/w H2SO4 (uL)
417
833.6
1042
1667
2084
List of Authorized Users for Agilent HPLC
If you name is not on this list, please contact responsible user to set up a training.
ID User name (please type)
Signature
Date authorized Note
1
Mirvat Ebrik
3-23-2010
responsible user
2
Jian Shi
3-23-2010
responsible user
3
Taiying Zhang
3-23-2010
4
Qing Qing
3-23-2010
5
Rajeev Kumar
3-23-2010
6
Deepti Tanjore
3-23-2010
7
Jaclyn DeMartini
3-23-2010
8
Heather McKenzie
3-23-2010
9
Xiadi Gao
3-23-2010
10 Hongjia Li
3-23-2010
11 Yi Jin
8-15-2010
12 Xiaoqi Sun
3-15-2010
13
14
15
16
17
18
19
20
Responsible users:
Jian Shi, 951-781-5747, jian@cert.ucr.edu
Mirvat Ebrik, 951-781-5655, mirvat@cert.ucr.edu
Log Book for Agilent HPLC
User name
Start time
Example
10 am, 03-24-10
No. of
samples
96
End time
Note
10 am, 03-25-10
Normal