EASY-Column, nano LC column, capillary column, nano LC, LC MS, LC separation, LC analysis

Frequently Asked Questions

Q: What packing materials are used in EASY-Column products?

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

Q: What do C18-A1 and C18-A2 mean in the EASY-Column product names?

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)

Q: Why should I run a standard sample for my EASY-Column and which standard should I use?

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).

Q: What backpressure should I expect from my EASY-Column?
Please refer to the chart below for approximate back pressures:
 

 Prod. No.

 Product name

 Approximate back
 pressure

 Column volume 
 (without packing
 material)

 SC001

 EASY-Column, 2cm, 
 ID100µm, 5µm, C18-A1

 50  bar (+/- 10 bar,
 at 3 µL/min, 22⁰C)

 0.393 µL

 SC200

 EASY-Column, 10cm,
 ID75µm, 3µm, C18-A2

 85 bar (+/- 20 bar,
 at 300 nL/min, 22⁰C)

 0.442 µL

Q: What should I do to reduce too high back pressure on my EASY-Column?

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.

Q: Why is the back pressure on my EASY-Column too low?

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.
Q: How do I obtain the best reproducibility between columns?

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.

Q: What are the advantages and disadvantages of one-column or two-column set-ups?

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.

Downloads

For further information please login to download the file below. ->
EASY-Column instructions for use (173.5 kB)
EASY-Column C-18 MSDS sheet (272.8 kB)
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