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5.5 HPLC (PDF version)

 

1. SAMPLE PREPARATION (approx 1mg per mL):

- Weigh mg of homogenous sample mixture
- Dissolve sample in column solvent mixture of the method
- Take up in 1 ml syringe
- Filter through special filter into HPLC vial

SETUP/CONNECT

2. CONNECT COLUMN

- Tighten the screw to the right as it is shown in the picture

3. SET UP SOLVENTS

- Change solvents if necessary (from MeOH which is connected when not using the machine to i-PrOH and n-Hexane when in use)
- If using non-HPLC grade solvent filter through filter paper using special buchner funnel
- Always filter water if using as a solvent

- New solvents connected (i-PrOH and n-Hexane when in use)

4. TURN ON MACHINES

- Order! Left to right (and then wait 10 min after turning all on):

1. Degasser (Turn ON button behind right bottom)


 

2. Pump (Turn ON button on the left side)

 

 

3. Autosampler (Turn ON button behind left)

4. Detector (Turn ON button in the front)

- Wait 10 minutes

5. SYRINGE TO REMOVE AIR

- Introduce syringe
- Open the valve DRAW-OFF (half turn)

- Syringe out air/solvent (approximately 2 mL)
- Close valve

- Remove syringe and dispose of the solvent in the RESIDUS HPLC

- Repeat the process for the bottom valve

6. TURN ON COMPUTER

- Do not start program until detector shows it is on!

- Turn on the computer of the HPLC

- Load software (Empower)

- Enter username and password (1.system 2.manager)

- Press Run samples

- Load samples/name

- Select the first option

- Load instrument method

If you wish to create a new method (ramp up for 1 min and 5 min ramp down, considered to be waiting time after the ramp up):

- Press Edit near Instrument Method

 

- Select the icon PCM/15xx

 

- Write the wanted Isocratic Flow A/B (the High Pressure Limit should be set to 900)

 

- Save the newly created method with the same model name as specified in the image

7. Ensure lever is set to purgar

- And don't forget to insert the tube in the RESIDUS HPLC jar

8. PURGE PUMP

- 2 ml/min each pump

- Click tap icon and set with the instructions that follow

FLOW A 2.0
FLOW B 0.0
RAMP 1.0
Wait 5 min

- And then:

FLOW A 0.0
FLOW B 2.0
RAMP 1.0
Wait 5 min

- Then click-stop (red button on bar)

9. Change the lever from "purgar" to "no purgar"

10. PURGE & PREP COLUMN

- Click tap icon and set max pressure to 900 (because if the machine goes over this value, it will automatically stop)
- For the 80:20 hex IPA set ramp for 20 min

Note: When leaving to ramp, make sure that the flow A/B keeps going up from 0.2X to 0.8 and from 0.0X to 0.2, respectively.

- Then leave it for about 20 min until the pressure is stable

SETUP SAMPLE

11. LOAD METHOD

- Check if method set & instrument method are the same

- Click Monitor
- Click Setup
- Check absorbance it is at approx 0 for 2 min
- Press Abort (red button below actions menu)

12. TYPE NAME

IC=column type

- Use this format: IC_BB123_254_HEX_iPrOH_8_2

13. SET RUN TIME

- For approximately 30 min

14. PUT SAMPLE

- Go to: 2707 autosampler window and right click on "move tray forward"

- Put sample in place: A1, A2, etc.
- Edit

NOTE: MAKE SURE THERE IS SOLVENT TO CLEAN THE NEEDLE IN THE AUTOSAMPLER!

- Click plates icon (below help menu)

- Ensure is set to: ANSI-48vial2mlHolder

15. INJECT

- Make sure all the parameters are correct (Inject samples; A1 position; 30 min Run Time)

- Click inject
- PUT SAMPLE - NAME - RUN TIME (repeteadly in a cycle)
- SIMPLE SAMPLE SAMPLES (for multiple samples to run continuously)

16. EXPERIMENT RUNS

 

- To repeat for other samples change name and go back to PUT SAMPLE and repeat the sequence

 

17. END RUN

- Click abort or allow RUN time to end
- DO NOT CLICK tap button crossed out icon
- Change solvents quantities:
0.8 to 0.0
0.2 to 0.0
- Set ramp for 5 min

 

18. PROCCESS SPECTRA

- Process method wizard
- Mark samll peak with cursor
- Then baseline with cursor
- Then large peak
- Save result
- Click NEXT 3 times - answer NO each time

- Method name = a result pestina
- Update
- Right button
- Preview publisher
- Injection summary report

 

19. CLOSE DOWN

- End use completely
- Change solvents to MeOH
- Both pumps to 0.3/0.3
- 15 min Purgar
- Close software
- Close machines
1. Detector
2. Autosampler
3. Pump
4. Degasser
- Close computer
- Remove column

 

20. Temporarily (for re-use the next day)

- Close software
- Close machines
1. Detector
2. Autosampler
3. Pump
4. Degasser
- Close computer

 


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