Autosampler Vented Column

Using Autosampler and Vented Column on LTQ1

Meng-Qiu Dong

Source: John V and Akira

8/19/05

vented column parts and accessories

Upchurch (1-800-426-0191)

MicroTee for 1/32 inch OD tubing, part# P-885 ($90 each, need 2)

MicroTight True ZDV (zero dead volume) Union for 1/32 inch OD tubing, P-771 ($29 each)

Valco Instruments Co. (1-800-367-8424)

Sleeve, 5/pk, part# C-NL.35L-5 ($20/pk, need 1 pk)

Sleeve with 1 mm steel screen/frit, part# C-NLS1.35 ($25 each, need 3)

360 mm OD fused silica capillary column

ABgene, Advanced Biotechnologies Ltd. (+44(0) 1372-723-456)

distributed in the US by Marsh Bio Products (1-800-445-2812/www.marchbio.com)

0.2 ml skirted 96-well PCR plates, 25/box, cat# AB-0800

adhesive sealing sheets, 100/box, cat# AB-1115

for Edwin’s homemade fritted column:

Upchurch (1-800-426-0191) MicroTight True ZDV Union P-720 ($29 each)

“Cut it smooth and straight!” says Akira.

Hold the fused silica capillary firmly between the thumb and index finger, make a firm point cut with the soft/smooth edge of a ceramic cutter, then pull straight apart (no bending at the cut). Check under the microscope. A slanted or jagged silica edge increases the dead volume inside a Tee, but an overhang of the coating is no concern.

A point cut means sliding the cutter perpendicularly against the capillary over a very tiny distance.)

1. Pack a pulled 360 mm OD, 100 mm ID capillary column with 7-10 cm reverse phase resin, equilibrate;
2. Clean the capillary ends, sleeves, ferrules, and MicroTee bodies with compressed air to ensure an open passage.
3. Assemble the desalting column with a 360 mm OD, 100 mm ID capillary, a MicroTee for 1/32 inch tubing, and a sleeve with 1 mm steel screen. Pack 5 cm reverse phase and equilibrate;
4. Assemble the HPLC line as illustrated in the figure. Notice there are two valves:
1. The valve in the mass spec toggles between “to detector” and “to waste” and is controlled by a blue button to its right. In the “to waste” mode, flow from 5 to 6 is open, while 3 to 2 is blocked. Conversely, flow from 3 to 2 is open and 5 to 6 is blocked in the “to detector” mode.
2. the valve in the autosampler toggles between “fill” and “inject” and can be controlled from Tune Plus. The fill mode means filling the sample loop (i.e. from port 2 to 1 to 4 to 3) and in the mean time the buffer can only flow from 5 to 6. In the inject mode, the buffer flows from 5 to 4 to 1 to 6, injecting the content of the sample loop into the desalting column (if the valve in the mass spec is set to detector).
5. if the autosampler is taken out of setup, activate it by doing the following:
1. close Xcalibur
2. open Instrument Configuration from the destktop and add Surveyor AS to the list of configured devices
3. re-open Xcalibur

Note: leave the autosampler in the setup even if it is not needed. It can be inactivated in the “run sequence” window (one that pops up everytime you start a run/sequence). Click the “change instruments” button and take the autosampler out of use with another click. In this way, you don’t have to disconnect and later reconnect the autosampler);

6. Test the assembly, purge the line, and fix any leakage or flow direction errors:
1. Set the mass spec valve to waste.
2. Open Tune Plus/Inlet direct control window.
3. Select the autosampler tab/Set Injector Position, choose fill and apply.
4. Pick up the Surveyor MS pump tab, set 100% buffer A, flow rate 100 ml /min, click start and watch the pressure go up (but no higher than 100 bar) and liquid come out of the splitter and the tip of the pulled column (this may take a while as the empty line gradually fills up).
5. To purge the 20 ml sample loop, Set Injector Position to “inject” from the autosampler tab and click “Apply”.
6. Wait1 min, then set the injector position back to fill, apply.
7. Pick up the Surveyor MS pump tab and drop the flow rate to 10 ml/min.
8. Set the mass spec valve to detector, watch the pressure rise and in quick response adjust the flow rate so the pressure is never above 200 bar. Test the flow rate from the method file for sample loading (usually 5 ml/min) and make sure that the pressure is stably below 200 bar and the line doesn’t blow.
9. Set the mass spec valve back to waste and stop the flow.
7. Set up method and sequence files
1. Each sample goes through two steps–first loading then a gradient. So theree is a loading method and a gradient method. If the two methods are merged into one, the instrument waits after loading a varying amount of time before starts the gradient, which causes large variations in retention time.
2. Loading method (D:\CDriveData\johnv\methods\AS_loading.meth)
   Divert Time (min): 0.00, 0.10, 19.50 and Valve State: to waste, to source, and to waste, respectively.
   Contact Time (min): 0.00, 0.05, 5.05 and Valve State: open, closed, and open, respectively.
   MS detector aquires full scans only.
3. Gradient method (D:\MQ\Methods\120min_SinglePhase_AS.meth)
   Divert Time (min): 0.00, 0.05, 0.15 and Valve State: to waste, to source, and to waste, respectively.
   Contact Time (min): 0.00, 0.05 and Valve State: open, closed, respectively.
   MS detector aquires 1 full scans followed by 3 MS/MS scans.
8. Load 96-well plates
1. Load samples into a 96-w PCR plate and seal with an adhesive sheet.
2. Place the PCR plate into the sample tray, with the well A1 on the left and away from you. The tray has 3 positions A (front) through C (back) for 96-w plates. Make sure the plate sits squarely over one of them.
3. Push the tray all the way back.
4. Make sure locations of samples match exactly their indicated locations in the sequence file.
9. Run samples . Yay!
10. P.S.: Illustration