An Example of Multi Dimensional Chromatography: Desalt and Ion Exchange

In this video we learn how to do multi dimensional chromatography using a desalting column and some anion exchange columns on an AKTA Avant. To start we are building on an a resin selection project we did earlier see below. This method will allow us to test many different conditions using multiple buffers at the same time! For example the sample can be in high salt with low pH conditions and the method will still work. This is an 'advanced' technique, so if you're new try some of my other videos first then come back. Setup video to follow.
Resin selection video: • Selection of Ion Excha...
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Main method:
0.00 Base: CV, Vc=0.962 {ml}, (HiTrap Q FF, 1 ml)#Column
0.00 Phase: Method Settings
0.00 Base: SameAsMain
0.00 Alarm pre column pressure: Enabled, (0.50)#Pre column pressure limit {MPa}, 0.00 {MPa}
0.00 Wavelength: 280 {nm}, Off {nm}, Off {nm}
0.00 Noise reduction UV: 5.0 {sec}
0.00 Injection valve: Manual load
0.00 Outlet valve: Out-Waste
0.00 pH valve: In-line, In-line
0.00 Alarm air sensors: Enabled, Enabled, Disabled
0.00 Inlet A: (A1)#Inlet A
0.00 Inlet B: (B1)#Inlet B
0.00 Pump wash: (A1)#Wash A, (B1)#Wash B, Off, (Off)#Wash Sample Buffer Inlet
0.00 End_Block
0.00 Phase: System Flush
0.00 Base: Volume, Any
0.00 Block: Desalting flow rate
0.00 Base: SameAsMain
0.00 System flow: (4.000)#Desalting Flow rate {ml/min}, Pre column pressure
0.00 End_Block
0.00 Injection valve: Inject
10.00 End_Block
0.00 Phase: Desalting Column Equilibration
0.00 Base: CV, Vc=5.027 {ml}, HiTrap Desalting
0.00 Injection valve: Manual load
0.00 Gradient: 0.0 {%B}, 0.00 {base}
0.00 Column position: (Position 5)#Desalting Column Position, Down flow
(3.00)#Column Equilibration length End_Block
0.00 Phase: Desating Load Sample
0.00 Base: Volume, Any
0.00 System flow: 0.000 {ml/min}, Off
0.00 Injection valve: Direct inject
0.00 Auto zero UV
0.00 Sample inlet: (S1)#Sample inlet
0.00 Sample flow: (4.000)#Desalting Loading flowrate {ml/min}, Off
0.00 Injection mark
0.00 Hold until: Air sensor Sample inlet valve, Equals, Air, (2.00)#Desalting Sample Load Volume
{base}
0.01 Block: Reset system
0.00 Base: SameAsMain
0.00 Sample flow: 0.000 {ml/min}, Off
0.00 Injection valve: Manual load
0.00 Block: Desalting flow rate
0.00 Base: SameAsMain
0.00 System flow: (4.000)#Desalting Flow rate {ml/min}, Pre column pressure
0.00 End_Block
0.00 End_Block
0.01 End_Block
0.00 Phase: Desalting Elution
0.00 Base: CV, Vc=5.027 {ml}, HiTrap Desalting
0.00 Watch: UV 1, Greater than, (100.0)#Start desalting collection threshold {mAU}, Start desalting
collection
0.00 Base: SameAsMain
0.00 Outlet valve: Out 1
Comment: Sample loop load
0.00 Block: Delay A
0.00 Base: Time, Any
0.05 End_Block
0.00 Watch: UV 1, Less than, (100.0)#Stop desalting collection threshold {mAU}, Stop desalting
collection
0.00 Base: SameAsMain
0.00 Outlet valve: Out-Waste
Comment: Sample loop closed
0.00 End_Block
0.00 End_Block
(3.00)#Length of Desalting run End_Block
0.00 Phase: IEX Equilibration
0.00 Base: CV, Vc=1.000 {ml}, Any
0.00 Watch off: UV 1
0.00 Block: IEX flow rate
0.00 Base: SameAsMain
0.00 System flow: (1.000)#IEX flow rate {ml/min}, Off
0.00 End_Block
0.00 Block: Delay B
0.00 Base: Time, Any
0.10 End_Block
0.00 Column position: Position 1, Down flow
8.00 End_Block
0.00 Phase: IEX Sample Loadting
0.00 Base: Volume, Any
0.00 Injection valve: Inject
0.00 Peak fractionation parameters: Level, 0.150 {min}, (75.000)#Start IEX Frac Threshold {mAU},
100.000 {mAU/min}, (75.000)#End IEX FracThreshold {mAU}, 75.000 {mAU/min}
0.00 Peak fractionation: Volume, 96 deep well plate, (2.00)#IEX Frac volume {ml}, Next tube
5.00 End_Block
0.00 Phase: IEX Column Wash
0.00 Base: SameAsMain
0.00 Injection valve: Manual load
(5.00)#Length of IEX column wash End_Block
0.00 Phase: IEX Graident Elution
0.00 Base: SameAsMain
0.00 Auto zero UV
0.00 Gradient: (100.0)#Target IEX Elution Concentration {%B}, (10.00) #Length of IEX Elution {base}
15.00 End_Block
0.00 Phase: Reequilibration
0.00 Base: SameAsMain
0.00 Stop peak fractionation
0.00 Gradient: 0.0 {%B}, 0.00 {base}
(10.00)#Length of reequilibration End_Block
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[@giantbird]

THANK YOU MY FRIEND .

[@UnicornStuff]

I'm glad you liked this video! I worked really hard on it and to find that someone found it useful is great!

[@tylerfield6447]

These videos are super helpful. I don't think there are any other resources out there like this for AKTA.
I was wondering, if you have a loop valve with 5 loops, can you run scouting using five different inlets that each would store different proteins in each of the five loops. Then each loop could be desalted one by one? The first step would be IEX and the second step would be desalting.

[@UnicornStuff]

Yeah you could, you'd either need two valves plumbed like the outlet valve or one like the column valve. There's no reason you couldn't have a second column valve you'd just have you use your imagination when it came to understanding the flow path.