Simple In-House Ultra-High Performance Capillary Column Manufacturing with the FlashPack Approach

Simple In-house Ultra-high Performance Capillary Column Manufacturing with the FlashPack Approach. The goal of this procedure is to introduce an optimized protocol for fast and easy ultra high-performance capillary column packing for LC-MS proteomics analysis. Modern proteomics is based on mass spectrometry coupled with ultra-high performance capillary liquid chromatography.

Peptide separation is mostly done in capillary columns with an internal diameter of 75 micrometers. On par with commercial products, it is possible to prepare capillary columns in-house. Customly prepared columns demonstrate separation qualities similar to commercial analogies, while saving a lot of money and allowing sorbent and column size customization.

Here we demonstrate an optimized packing procedure for in-house capillary column manufacturing, which we call FlashPack. The idea behind the optimization is to pack from very high sorbent slurry concentration, while still using the same common 100 bar pressure bomb packing setup. This requires continuous breaking up sorbent clusters, which form around the column entrance at high sorbent concentration and prevent new sorbent material from entering the capillary.

Technically, we achieve that by using a magnet bar as a hammer, constantly heating and moving around the capillary inside the sorbent wire. Properly applied FlashPack increases packing rate up to 10 times. That makes it suitable for packing up ultra high performance sorbents of softer micro metricbeat size and for manufacturing very long columns up to a meter length in reasonable times.

Column preparation procedure consists of five steps. Three preparatory steps, which include packing station assembly and capillary and sorbent slurry preparation. Then the capillary is first packed in the pressure bomb at 60 to 100 bar pressure.

It is followed by connection of the packed capillary to the HPLC system, packing up the sorbent at high pressure and column cutting to the size. The FlashPack procedure requires changes to be made in the steps three and four of the common protocol. First of all, we prepare a packing station.

It consists of a nitrogen gas tank with at least 50 bars outlet pressure. The tank is connected to the vent valve of the pressure bomb, which is placed in the magnetic stirrer. The outlet of the vent valve is inserted into a water bottle through narrow ID PEEK capillary, which allows fine control on depressurization.

Next, we prepare a capillary. Packing can be done either into a fritted capillary with an integrated glass frit formed from Kasil and formamide. Another option is to pack into a pulled capillary prepared by sutter laser puller.

The capillary must be made 10 to 15 centimeters longer than the intended column length. The following video is prepared on the example of a pulled emitter capillary packing. Protect a pulled emitter end with a cut gel-loading pipette tip.

For details, see the text version of the manuscript. Next stage is sorbent slurry preparation. First, put around 50 milligrams of dry sorbent into 1.5 milliliter centrifuge tube.

That will be a stock sorbent vial. This video was prepared with the use of Reprosil Pur C18 AQ 1.9 micro metric sorbent from Dr.Maisch. Add one milliliter of methanol into the stock sorbent vial.

Vortex for 10 seconds. And sonicate in a sonication bath for another 10 seconds. Let the sorbent soak thoroughly for 20 to 30 minutes, then vortex and sonicate it once more.

Prepare a working sorbent vial. It must be a conical bottom vial which fits into the bomb. It can be either another 1.5 milliliter centrifuge tube or any other vial depending on the particular pressure bomb design.

We use a skirted conical bottom screw cap tube cut to the height of the pressure bomb. Re-suspend the sorbent in the stock sorbent vial and transfer 500 microliters into the working sorbent vial along with a magnet bar two by three millimeters size. Top to the working vial to one milliliter with methanol.

Let the working vial stand on the table for 10 minutes for the sorbent to settle by gravity. The result of this step must be a conical bottom vial with the bottom layer of loose sorbent around four millimeters high. If the layer is too low, and some more sorbent from the stock vial.

Thus prepared working vial is intended for multiple columns'preparation over months time. If the working sorbent vial stays without stirring for more than two hours, it must be vortexed, sonicated and settled by gravity again before packing. When everything is prepared, proceed to stage four, packing in the pressure bomb.

Always wear protective glasses when working with fused silica capillaries and the pressure bomb. At the same time, protective gloves are not recommended. They severely reduce the sense of touch required for proper handling of small diameter capillaries and lead to mistakes.

Place the sorbent vial into the pressure bomb and fix all the knots tightly. Start the rotation at 60 to 100 rounds per minute, Insert the fritted or pulled emitter capillary into the bomb. Push it to the very bottom of the vial, then lift it up two to three millimeters and fix the knot.

Apply minimum required force to fix the capillary. Check that the capillary is properly fixed. It must be impossible to move the capillary by pulling it out by hand.

Very slowly, open the pressure bomb valve while keeping the open end of the capillary pointed away from your face. Watch the initial steps of the packing process. Immediately upon pressurization, the sorbent fills the capillary and it becomes non-transparent for the whole length.

As soon as the sorbent starts to pack inside the distal end, the back-pressure increases, the flow slows down and the even sorbent slurry inside the capillary reforms into several sorbent packets, separated by sorbent free gaps. Already packed sorbent is visible as a densely colored continuously growing region. Keep the sorbent filled regions to be at least 70%of the capillary length with small sorbent free gaps for the whole duration of the packing process.

There are several common issues to watch during the packing process. They require on-the-flight setup adjustment to keep efficient sorbent delivery into the capillary. The most frequent issue is when new sorbent stops entering the capillary while the sorbent already inside keeps moving.

In most cases, the capillary entrance gets blocked by self-aggregating sorbent clusters. Apply these steps one by one until the sorbent flow is restored, then skip the rest of the issue-related steps. Increase the rotation speed to 500 rounds per minute, and immediately reduce it back to 60 RPM.

Most of the time it restores the sorbent flow. Check the rotational speed to be at least 60 RPM for the rest of the packing process. If it does not help, briefly vent the packing bomb and immediately pressurize it back.

If it does not help, or the blocking happens again, reposition the capillary inside the sorbent layer. The absence of the sorbent can be due to the capillary open-end being either too high above the magnet bar, so the column end does not touch it. Or the capillary is positioned too low and sticks into the vial bottom.

First, vent the bomb completely, then loosen the nut, push the capillary to the bottom, and then pull it two millimeters back. Fix the nut. Open the valve to pressurize the bomb and continue packing.

More details on the packing issues are described in the text version of the method. Keep on packing the column till target column length plus five to seven centimeters is achieved. Stop the rotation and very slowly depressurize the bomb.

Open the bomb valve a little and wait for the bubble burst inside the water bottle to subside. Then open the valve a little wider and again wait for the bubble burst to slow down. Thus in incremental steps, release the pressure until no gas comes out of the valve.

Do not open the valve all the way at once. It will lead to bubbling inside the capillary and the sorbent going back into the vial. If that happens, pressurize the bomb back and wait for the column to get packed again.

When the gas stops coming out of the vent valve, take the packed capillary out of the pressure bomb. Do not let the column dry out. If not connected to the HPLC system immediately for further packing, put the packed capillary into storage by submerging it whole into 10%ethanol solution.

Disconnected HPLC columns are stored in the same manner. If no more packing is planned for today, take out the sorbent vial from the bomb and close it tightly. Keep it for further the column packing.

Stage five, HPLC packing up. Connect the packed capillary to the HPLC system via HPLC connection. Start the flow of high organic solvent.

Adjust the flow according to the packed length targeting around 300 bars. For 40 centimeter packed capillary use the flow rate of 200-300 normal liters per minute. Watch for loose sorbent inside the capillary being packed, adding to the total packed length.

Without stopping the flow, dip the column body two times into the sonication bath. It is important to immerse only part of the column body. Do not immerse column ends and capillary connections.

When the sorbent bed stops shrinking, dip the column body into the sonication bath two times more without stopping the flow. Run the column for additional 10 minutes at 300 bars. Stop the flow, wait for the pressure to drop to below three bars and disconnect the column.

Visually inspect the column for the lack of gaps and discolorations. If any are found, sonication under the flow can be repeated. For critical experiments, consider making a new column.

Cut the column to the desired length. Properly done cutting is a prerequisite for column efficiency. Make a notch in polyamide coating with the scribe.

Partially crack the capillary, and pull two pieces apart. Polish the column front-end on a ceramic wafer or with lapping film. Reconnect the column to the LC system using ultra high pressure connection.

Start the working flow rate at 2%B. Working flow rate is adjusted according to column parameters. For instance, 100 micrometer ID columns are run at 500 nanoliters per minute.

Wait for the pressure to equilibrate and check the column back-pressure. A properly prepared column gives back-pressure proportional to its length and sorbent characteristics. For example, 30 centimeter capillary column packed with two micrometers sorbent has back-pressure of 450 to 500 bars at 2%solvent B.If the produced column gives a back-pressure within the expected range, it is ready to use.

If the back-pressure is well outside the expected range, something is wrong either with the column or with the LC system. Packing with FlashPack produces a capillary packed for 50 centimeters with 1.9 micrometers sorbent in less than one hour at 100 bar packing pressure. As an example, we produce 30 centimeter 100 micrometer ID ultra high-pressure column packed into a pulled emitter capillary with 1.9 micrometer ReprosilPur C18 AQ sorbent.

Packing was done at 60 bars into 50 centimeter long pulled emitter capillary. The capillary was packed to 40 centimeters in 40 minutes with some more sorbent inside the capillary not packed. The packed capillary was connected to the HPLC system and run at 300 nanoliters per minute with solvent B, which in our case was 80%acetonitrile, 0.1%formic acid.

The final packed length was 43 centimeters. The column was cut 30 centimeters. At 500 nanolitres per minute working flow with 99%solvent B the column gave the back-pressure of 275 bars, which is close to the expected value.

To test the column, we separated 50 fmol of a tryptic digest of cytochrome C protein in a 15 minute gradient from 2-50%B. Chromatographic peaks were highly symmetrical with minimum tailing. Average FWHM was around three seconds.

The described protocol is able to produce UHPLC columns in a common 100 bar packing set up in reasonable time. Short HPLC or UPLC columns can be packed in minutes time, while long 30-50 centimeter columns are packed in less than one hour. The method is efficient for packing up sorbents with RP and any other chemistry into any column IDs.

Produced columns are reproducibly as effective as any other commercial column of similar size and sorbent parameters.