Sauer:HPLC protocol: Difference between revisions

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(New page: HPLC usage: Buffers: A: 0.06% TFA in dH2O. B: 0.054% TFA in 80% Acetonitrile When preparing, filter with Chemically Resistant ZapCap. 1) Turn on pumps from bottom to top. (there are 4 pa...)
 
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HPLC usage:
{{Sauer lab sidebar}}
== HPLC usage: ==


Buffers:
A: 0.06% TFA in dH2O.
B: 0.054% TFA in 80% Acetonitrile
When preparing, filter with Chemically Resistant ZapCap.


1) Turn on pumps from bottom to top. (there are 4 parts that need to be turned on)
=== Buffers: ===
Uncap waste container
2) Turn on computer, open Class VP, open Instrument 1.
3) To turn on computer control, click on icon that looks like a stamped letter. (upper left-hand corner.)


Column should be stored in 100% Buffer B.
*A: 0.06% TFA in dH2O.
*B: 0.054% TFA in 80% Acetonitrile
*When preparing, filter with Chemically Resistant ZapCap.
----


4) Flush into Buffer A before run.
Under Instrument Set-up, set %B to 0, and T.Flow to 1mL/min.


To run, click Download
# Turn on pumps from bottom to top. (there are 4 parts that need to be turned on)
##Uncap waste container
# Turn on computer, open Class VP, open Instrument 1.
# To turn on computer control, click on icon that looks like a stamped letter. (upper left-hand corner.)


5) To run a method, open method (start with basic.met, obtain copy and create your own folder for this.)
'''Column should be stored in 100% Buffer B.'''
6) To start the method, click on the Blue arrow.
 
''Flush into Buffer A before run.''
* Under Instrument Set-up, set %B to 0, and T.Flow to 1mL/min.
 
'''To run, click Download'''
 
# To run a method, open method (start with basic.met, obtain copy and create your own folder for this.)
# To start the method, click on the Blue arrow.
Select a name for your file.  Make sure this will be in your own directory.
Select a name for your file.  Make sure this will be in your own directory.


Loading Sample:
'''Loading Sample:'''
7) Wash the syringe a couple times with Buffer A, shooting the waste into the waste container.
 
8) With the knob turned all the way to the right (clockwise) insert the needle.
# Wash the syringe a couple times with Buffer A, shooting the waste into the waste container.
9) Turn the knob counterclockwise all the way, and then inject your sample.
# With the knob turned all the way to the right (clockwise) insert the needle.
10) Turn the knob back in the clockwise direction.
# Turn the knob counterclockwise all the way, and then inject your sample.
# Turn the knob back in the clockwise direction.
'''Triggering the Run: '''
 
# Press Start on the control box.
# To trigger the run to start, form a circuit between the two exposed wires.


Triggering the Run:
'''Shutdown:'''
11) Press Start on the control box.
12) To trigger the run to start, form a circuit between the two exposed wires.


Shutdown:
# Go to 100% Buffer B for 10 min.
13) Go to 100% Buffer B for 10 min.
# Change flowrate to 0.
14) Change flowrate to 0.
# Close down Class VP
15) Close down Class VP
# Shut-down pumps from top to bottom.  
16) Shut-down pumps from top to bottom.  
# Clean the Syringe with Buffer A. Leave syringe in HPLC.
17) Clean the Syringe with Buffer A. Leave syringe in HPLC.
(otherwise the next person could end up with your sample as a contaminant)
(otherwise the next person could end up with your sample as a contaminant)

Latest revision as of 09:18, 23 June 2009

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HPLC usage:

Buffers:

  • A: 0.06% TFA in dH2O.
  • B: 0.054% TFA in 80% Acetonitrile
  • When preparing, filter with Chemically Resistant ZapCap.


  1. Turn on pumps from bottom to top. (there are 4 parts that need to be turned on)
    1. Uncap waste container
  2. Turn on computer, open Class VP, open Instrument 1.
  3. To turn on computer control, click on icon that looks like a stamped letter. (upper left-hand corner.)

Column should be stored in 100% Buffer B.

Flush into Buffer A before run.

  • Under Instrument Set-up, set %B to 0, and T.Flow to 1mL/min.

To run, click Download

  1. To run a method, open method (start with basic.met, obtain copy and create your own folder for this.)
  2. To start the method, click on the Blue arrow.

Select a name for your file. Make sure this will be in your own directory.

Loading Sample:

  1. Wash the syringe a couple times with Buffer A, shooting the waste into the waste container.
  2. With the knob turned all the way to the right (clockwise) insert the needle.
  3. Turn the knob counterclockwise all the way, and then inject your sample.
  4. Turn the knob back in the clockwise direction.

Triggering the Run:

  1. Press Start on the control box.
  2. To trigger the run to start, form a circuit between the two exposed wires.

Shutdown:

  1. Go to 100% Buffer B for 10 min.
  2. Change flowrate to 0.
  3. Close down Class VP
  4. Shut-down pumps from top to bottom.
  5. Clean the Syringe with Buffer A. Leave syringe in HPLC.

(otherwise the next person could end up with your sample as a contaminant)

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