FAQ

Please find an overview of the most frequently asked questions below. If you have any other questions, get in touch!

Are there any special requirements for washing/re-equilibrating the µPAC™ columns?

No, standard washing with a high percentage of organic solvent should be sufficient. Due to the extended length of the µPAC™ columns, however, ensure that enough time is included in the HPLC method to re-equilibrate the column before subsequent injections.

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Instructions for use of the 200 cm µPACᵀᴹ column, 50 cm µPACᵀᴹ column

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What degree of sample carry-over is usually observed?

Can I order the µPAC™ column with nanoViper fittings?

No, attached to both ends of the µPAC™ columns are fused silica capillaries protected by PEEK tubing (blue). Each end is fitted with stainless steel unions. Both sides of the inlet union connects 1⁄32″ tubing; whereas the outlet union connects 1⁄32″ tubing to 360 µm O.D. fused silica. Provided with every µPAC™ column is a µPACᵀᴹ 1⁄16″ Connecting PEEKsil™ capillary with 1⁄16″ (labelled: to valve) and 1⁄32″ (labelled: to µPAC™) finger tight fittings that facilitates connection of the µPAC™ column to standard HPLC switching valves that utilise nanoViper fittings. To guarantee column performance, the PEEK tubing and fittings must never be removed.

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Instructions for use of the 200 cm µPACᵀᴹ column50 cm µPACᵀᴹ column

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Which HPLC systems can I use with the µPAC™ columns?

In what respect is the design of the µPAC™ column different compared to my conventional column?

Conventional HPLC columns are fabricated by stacking (packed beds) or depositing (monoliths) material into a capillary. The µPAC™ (micro pillar array column) technology from PharmaFluidics is unique because the design is based on precise micro-machining of the chromatographic separation beds into silicon wafers. This approach creates three crucial and unique characteristics for our µPAC™ columns: (i) perfectly-ordered free-standing pillars; (ii) high-permeability that results in a low backpressure; and (iii) a chromatographic bed with a solid backbone.

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The µPACᵀᴹ brochure

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Can I use the µPAC™ column in both directions?

How should I correctly ground a µPAC™ column?

The most effective means of grounding the µPAC™ column is to connect the outlet union of the µPAC™ column to a grounding pin on your instrument (usually located on the backside of the HPLC or mass spectrometer). This is achieved with the Ground it! blue coiled grounding cable (swallow tail and crocodile clip options; P/N: 201901TLK).

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How do I know if the µPAC™ column is not correctly grounded?

Can I reduce the sample loading time with a 200 cm µPAC™ column?


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Yes, you can load the sample onto the µPAC™ column at a higher flow rate. For example, when you perform your gradient separation at 300 nL/min, you can load the sample at 750 nL/min. As a consequence of the low backpressure of our columns, this is feasible as you will not reach the upper pressure limit (350 bar or 5,000 psi).

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Instructions for use of the 200 cm µPACᵀᴹ column50 cm µPACᵀᴹ column

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Is there a difference between the inlet and outlet unions on my µPAC™ column?

Yes, the inlet contains a through-bore 1⁄32″ stainless steel union (VICI ZU.5T); whilst the outlet is a 50 µm bore stainless steel union (VICI C360RU.5FS2). The outlet union can be identified by a small groove. This outlet union is used to ground the column before applying high voltage.

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µPACᵀᴹ connectivity

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Can I apply high voltage directly to the metal union after the µPAC™ column?

Can I use the µPAC™ column in both directions?

Yes, our µPAC™ columns are fabricated by etching from a crystalline silicon wafer following lithographic definition of a pillar array. This means the µPAC™ columns consist of one piece of solid material. The columns are completely symmetrical and do not contain any frits. So, you can easily backflush the column without the risk of moving or breaking particles from the separation bed. To reverse the column, you just need to swap the inlet and outlet unions.

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How easily can I clog the µPAC™ columns?

Do I need to ground the µPAC™ column if the ESI source of my mass spectrometer is already grounded?

Yes. Our µPAC™ columns are semi-conductive (the columns contain a microchip), thus when high voltage is applied to the ESI emitter you must ground the µPAC™ column. This is achieved by connecting the outlet union of the µPAC™ column to a grounded point on your HPLC or mass spectrometer with the blue coiled Ground it! grounding cable.

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µPACᵀᴹ connectivity

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How should I correctly ground a µPAC™ column?

How easily can I clog the µPAC™ columns?

Our µPAC™ columns are fabricated by etching from a crystalline silicon wafer following lithographic definition of a pillar array. This means the µPAC™ columns consist of one piece of solid material. The columns are completely symmetrical and do not contain any frits. So, you can easily backflush the column without the risk of moving or breaking particles from the separation bed. Consequently, our µPACᵀᴹ columns are less vulnerable to clogging. We have analysed a range of challenging samples that failed with conventional packed-bed columns. No significant change in column backpressure or separation performance was apparent. More information is provided in our technical note on column robustness.

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Technical note

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Do I need to filter a HeLa lysate before injection?

Does temperature influence analyte separation?

Of course, we always place our µPAC™ columns inside the column compartment of the HPLC system. Therefore, the temperature is precisely controlled. Similar to other columns, temperature changes will affect analyte retention time. With the µPAC™ column, however, it is not necessary to heat the column to 50°C or 60°C to operate the column at moderate HPLC pump pressures.

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At what temperatures can I use the µPAC™ columns?

Can I reuse a µPAC™ column and how should I do so?

Yes, our µPACᵀᴹ columns are fabricated by etching from a crystalline silicon wafer following lithographic definition of a pillar array. This means the µPAC™ columns consist of one piece of solid material and there is no risk from moving or breaking beads. The columns are completely symmetrical, do not contain any frits and every chip is identical. Therefore, our customers have experienced that the µPACᵀᴹ columns are very robust and highly-reproducible when compared to traditional columns. We have run an experiment where a µPACᵀᴹ column on one of our nanoLC instruments was continuously used for 6 months. We performed serial injections of a tryptic digest of cytochrome C, followed by a tryptic digest of HeLa cells, followed by a blank. After a total of 3,500 injections, no effect on the retention time or pressure profile was apparent. More information is provided in our technical note on column robustness.

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Technical note

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Can I use the µPAC™ column in both directions?

We have a Thermo Scientific™ Ultimate 3000 nanoRSLC connected to a Q Exactive Plus or Lumos™. Are the connections on your µPAC™ columns compatible with our liquid junction?

Yes, you can use a liquid junction. You will, however, need to connect the outlet union of the µPACᵀᴹ column to the liquid junction with a 360 µm O.D./20 µm I.D. fused silica capillary. Here, you can find information on the part numbers required for the union. This configuration is commonly-adopted at our expert centres and the additional connection between the column and the mass spectrometer neither results in any issues nor leads to any negative effects on the chromatography.

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µPACᵀᴹ connectivity

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Can I apply high voltage directly to the metal union after the µPAC™ column?

Can I apply high voltage directly to the metal union after the µPAC™ column?

No, you cannot apply high voltage to the outlet union of the µPAC™ column. Our column is comprised of a microchip and as such is semi-conductive. Therefore, when coupled to a mass spectrometer where the high voltage is applied to an emitter (e.g Thermo Scientific™ or Waters instruments) the outlet union must be used to ground the µPAC™ column. Grounding is achieved by connecting the outlet union of the µPAC™ column to a grounding pin on your mass spectrometer or HPLC with the blue coiled Ground it! grounding cable (swallow tail and crocodile clip options; P/N: 201901TLK).

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We have a Thermo Scientific™ Ultimate 3000 nanoRSLC connected to a Q Exactive Plus or Lumos™. Are the connections on your µPAC™ columns compatible with our liquid junction?

Is it possible to reduce the length of the capillaries that connect the µPAC™ column to obtain better separation results?

No. To guarantee column performance, the PEEK tubing and fittings must never be removed. The internal volume of these capillaries, however, is only a few nanolitres. Therefore, the effect on the separation efficiency is negligible.

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Should I use direct injection, or do you recommend using a trap column to protect the µPAC™ column from clogging?

You can work in direct injection mode or with a trap column. PharmaFluidics now offers their own µPAC™ trap column that is fully compatible with our analytical µPAC™ columns. We do have customers that use classical trap columns in conjunction with our µPAC™ columns. Nevertheless, such configurations can influence the performance of our analytical columns because, in general, conventional trap columns have slightly different retention capabilities compared to the µPACᵀᴹ columns.

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Is it possible to reduce the length of the capillaries that connect the µPAC™ column to obtain better separation results?

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