Sunday, January 27, 2013

Optimizing your nanoLC gradient and number of MS/MS events to your column length, part 1: How to calculate your gradient delay

First of all, this is a big topic and one that is definitely beyond the scope of what I could possibly write here today.  I'm going to break it into several parts throughout the week.
With that out of the way, I needed to get some ideas out there.  I recently visited one lab that was using 50cm nanoLC columns and trying to get efficient peptide separations and IDs using 15 to 30 minute gradients for whole cell digests.  The following week I visited a lab that was using 10 cm columns and 180 minute gradients to study single protein digests.  While both approaches can work, it just served to outline the extremely different approaches that our being used in the field.  I'm, by no means, a great chromatography expert, but I will walk you through what I consider when establishing a nanoflow set up.
1)  The very first thing I consider is the total dead volume of my LC system.  I start by taking a ruler and measuring the total length of my output line from my mixer to my LC column.  I take the internal diameter (ID) of these lines and the length I measured and calculate my empty dead volume.  There are good calculators at, but I usually use the MSBioworks App on my Ipad or phone.  I then make the same calculation for my column. While the column is filled with stationary phase, I ignore this effect because it's permeable and because it is just simpler that way.  When I add the two together, I have my total system dead volume.

2)  I divide the total system dead volume by my base mobile phase flow rate to get what I call my gradient delay, the amount of time it takes for what I have in my mixer to actually elute from the tip of my emitter.
On some systems this is pretty small.  If you are using nanoflow tubing with a 20 um ID and a 10 cm picofrit column (column + emitter) and 20 cm of line to connect your LC to your nanosource, you are looking at a total system dead volume of ~1 uL.  At 200nL/min, you will see a 5 minute gradient delay.  If you are using a system with 75 um ID lines where your LC isn't exactly beside your instrument (55cm inlet lines are pretty common) and a 50 cm column, you are looking at a dead volume of ~4.6 uL.  Your gradient delay at 200 nL/min is going to be 23 minutes.  In other words, if your gradient runs to 30% B in 30 minutes, 30% organic will not actually be eluting from the tip of your emitter until 53 minutes into your run.  If your gradient is 45 minutes in length, your peptides will be eluting during the following run.

On to part 2!
On to part 3!


  1. Thanks Ben for opening this crucial topic. Looking forward to read the next part. By any chance can you suggest any andriod apps for LC?

  2. Unfortunately, I don't know of one. I've become very Apple centric recently. If you do happen across one, please let me know!

  3. Hi Ben, I've been trying to re-do your math.

    For "a 20 um ID and a 10 cm picofrit column (column + emitter) and 20 cm of line," should the volume be calculated as 3.14*(20e-6/2)^2 * 30e-2? That gives me 0.1 ul.

    How did I do it wrong?