EASY columns

The specific chromatography medium used in each column is specified in the product description table. Currently all materials are sourced from Dr. Maisch GmbH, Germany

C18-A1 and C18-A2 are Proxeon codes for stationary phases.

C18-A1:          5µm, 120 Å, ReproSil-Pur C18-AQ (Dr. Maisch, GmbH, Germany)
C18-A2:          3µm, 120 Å, ReproSil-Pur C18-AQ (Dr. Maisch, GmbH, Germany)

It is highly recommended that a standard sample is run on a regular basis to assess overall system and column performance. We recommend a tryptic digest of bovine serum albumin, available from Bruker Daltonic (Item no: 217498).

First remove the column from the flow path to check if the increased back pressure is generated by the column. If the backpressure remains high the problem lies elsewhere in the system flow path.

Note: a column may still perform effectively with only a small increase in back pressure. Assess column performance using a standard sample.

If performance is unacceptable and/or back pressure too high, begin by flushing the column with a high percentage of organic mobile phase in order to remove adsorbed material that may have partially blocked the column. Alternatively, run a series of blank gradients.

Increasing column temperature (up to 60⁰C) may also help to increase the effectiveness of flushing the column.

As a last resort, try cutting away a small piece of the column at the HPLC end (top of the column) as this may remove any blocked material. However, this will affect column performance.

If these measures did not help, replace the column with a new one.

Note: a column may still perform effectively with only a small decrease in back pressure. Run your chromatographic standard to assess the chromatographic performance of the column.

Note: Running your standard sample may reveal a delay in normal peak elution times (due to reduced flow rate through the column) and help to confirm the presence of a leak.

• Thoroughly inspect your system for leaks - this is the most likely cause of low back pressure.
  
• If no leaks are visible, remove the column from the flowpath and inspect the column visually to ensure that it is still fully packed with packing material. A stereo loop or low magnification microscope may be helpful in this process. If the column has lost some or all of its contents material, the column must be replaced as the frit that holds back the column material is probably damaged and there is a risk of material leaking into the detector.
• If the packing material is intact, reconnect the column to the system and continue the search for the leak. Try increasing flow rate to see if the leak can be spotted more easily.

The packed length of column material may shrink slightly when subjected to alternating solvent compositions (organic/aqueous) at flow rates that create high back pressure. In order to ensure a reproducible retention time between columns we recommend pre-conditioning an analytical column with alternating solvent compositions at flow rates that create high backpressure before trimming the column end to the precise length required. To facilitate this process, EASY-Column analytical columns are packed and quality controlled with an additional 10 mm of packing material.

With a one-column set-up it is simpler and easier to minimize peak broadening caused, for example, by dead-volumes in connections. The eluent is constantly infused into the MS and hence non-retained peptides will also be analyzed.

A two-column set-up is typically based on a shorter pre-column (trap-column) with a larger inner diameter with the advantage of increased loading capacity (depend on actual dimensions of the pre-column) and faster loading. A further advantage is that "contaminants" from the samples or salt plugs are not injected into the MS, but redirected to waste which may reduce the need for cleaning the MS source.