How to Produce a Premium Nano-Capillary LCMS Column
Many scientists find it best to purchase premium nano-capillary columns from commercial vendors, while some researchers (especially those in academics) wish to make their own nano-capillary LCMS columns for economic reasons. Although PremierLCMS tries to provide premium columns at economical prices, we also provide several column packing kits which include everything needed to produce high quality columns. These kits include detailed instructions that teach users how to produce top quality nano-capillary columns that provide high efficiency, robustness and reproducibility for most LCMS applications, and those instructions are provided below for anyone interested in packing their own columns.
Choosing the Best Packing Material
The first step to producing high quality LCMS columns is to start with a premium LC column packing material. PremierLCMS offers three generations of Magic LC packing materials to cover most LCMS applications. They are made from spherical type B silica which provides high purity, low metal content, high surface area and broad pH stability. These materials were developed specifically for LCMS applications to insure operation over a wide pH range with no column bleed. They are also well end capped to eliminate residual silanols so that MS compatible mobile phases (low volatile buffer concentrations and no ion pairing agents) can be used without impacting sensitivity or resolution. Examples of LCMS issues from non premium materials are shown below:
The following LCMS base peak traces show the separation of 100 fmol of the TP4 column test mix on four commercial 3µ 0.1 x 150 mm RP columns, using a gradient of ACN in 0.1% Formic Acid/Water.
Column A packing contains free silanols, which can adsorb basic peptides (peaks 1 & 3).
Column B shows the impact of excess hydrophobicity which may result in the loss of larger nonpolar peptides (peak 4).
Column C contains an irregular shaped packing, which can result in bed shifts that cause voids, resulting in peak tailing.
Column D shows the ideal separation of the TP4 column test mix on a well packed column with a material specifically developed for proteomics LCMS.
Choosing the best LC packing particle size depends on your LC instrumentation (HPLC or UHPLC), column packing equipment (pressure bomb, high pressure pump or ultra high pressure pump) and application requirements (simple or complex samples). We recommend using 4-5u particles when using a pressure bomb column packer, 2.5-3.5u particles when using a high pressure packing pump and 1.5-2.5u particles when using an ultra high pressure packing pump. The size of the molecules of interest determine the optimum packing material pore size, with 60-100A pores best for small molecules (100-5,000 daltons), 200-300A pores for medium molecules (500-20,000 daltons), 400-500A pores for large molecules (5,000-100,000 daltons) and 800-1000A pores for very large molecules (> 100,000 daltons).
Preparing Column Packing Material Slurry
Well packed nano-capillary columns require air free, well solvated particles in a solvent mix that provides a dense, well balanced slurry to prevent aggregation, clogging and settling during the packing process. Since the physical and chemical properties of column packing materials varies widely, there is no optimum slurry solvent for all column packing materials. We have found that mixtures of our Column Packing Solvents A and B provides optimum balanced density slurries for most packing materials (packing material should stay suspended without settling or clumping for at least 8 hours).
Preparing the Capillary Tube
Nano-capillary columns can be made using stainless steel tubes, Peeksil tubes or fused silica tubes, but premium nano-capillary columns are generally made using fused silica tubing (available from a variety of commercial vendors). Although a packed tip nano-capillary column offers the lowest outlet extra column volume possible, these columns are very fragile and tip breakage or fouling results in the loss of the entire column (although we do recommend a packed tip for nano columns with an internal diameter less than 75 microns. For nano-capillary columns with an internal diameter greater than or equal to 75 microns, we recommend our dual screened nano union columns with a separate nanospray emitter (Fossil Ion Technologies or equivalent) as they are more robust, easier to handle and when properly coupled to a spraytip, provide minimal band spreading compared to a packed tip column (and if the spraytip is broken or fouled, it can be replaced without impacting the column).
Packing the Capillary Tube
Although it is relatively easy to fill a capillary tube with LC column packing material, getting a well packed, reproducible, robust and stable column bed takes some expertise. This is especially true as the LC column packing particle size gets smaller. For 4-5 micron particles, bomb loading (1-2 KPSI) of capillaries is often sufficient to obtain a decent column, while particles from 2.5-3.5 micron require high pressure packing (8-10 KPSI) and 1.5-2.5 micron particles require ultra high pressure packing (15-18 KPSI).
Nano-capillary column packing time also increases with decreasing particle size and decreasing column ID. Bomb loading of 4-5 micron particles generally only requires 30-60 minutes to fill and depressurize the capillary, while 2.5-3.5 micron particles require 8-12 hours to fill and depressurize the capillary and 1.5-2.5 micron particles require 16-24 hours to fill and depressurize the capillary. Once a LC column is packed under pressure, the pressure must be released through the column outlet to prevent backflow and disruption of the column bed.
Once the column is fully packed and depressurized, the inlet should be terminated with a nano union containing a one micron screen and the outlet attached to a nano-electrospray emitter via a second screened nano union. The column can then be tested by LC/MS to verify performance.