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First published July 22nd, 2001

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How Much Protein Should I Load?

This is a difficult question to answer because not all mass spectrometers are created equal and even UV detectors vary in sensitivity.  Below we have listed a range that should get you into the correct ball park.  For example if you have one of the newest generation of electrospray mass spectrometer (circa 2001-4) you may be able to work at the lowest levels listed here and most likely you will be able to do better.  The high end of the range is listed to be at the boarder of overloading the column.  You will be able to tell if you overload the column because the peaks will begin to broaden and tail in LC/MS.  If you are just starting out we would suggest starting  in the mid range and then working down as you perfect your technique.

    Detection (peptide digest in pmol)

Column (ID in mm)

Flow Rate (ul/min)

0.150 1 NA

0.005 to 0.200

0.300 4 5 to 10 0.100 to 10 
2.1 200 50 - 2,000 10 to 2,000
4.6 1000 500 - 10,000 50 - 10,000

UV detection

Flow cell size is important.  The standard flow cell (6 - 12 mm path length, 6-12 ul volume) will not perform well at flow rates below 100 ul/min because peaks will become diluted in the large UV cell.  Remember UV detectors are concentration dependent detectors and sensitivity and separation will be lost in the large mixing volume.  Since peaks will become diluted in this volume it will also hurt your MS sensitivity.  At 200 ul/min researchers will often split the flow before the UV flow cell and send half (or less) to the mass spectrometer and the other portion will be directed to UV detection and possibly fraction collection.  Remember the mass spectrometer is more or less a concentration dependent detector so a solution infused at 10 ul/min or 200 ul/min at the same concentration will most likely give you the same MS result.  Again, more sample introduced into the mass spectrometer is not always better and is often the same or worse.

      In addition splitting the flow to the MS can be advantageous for the following reasons:  

  1. Some mass spectrometers nebulize more efficiently at lower flow rates.
  2. A pre-UV split to the MS will mean better MS sensitivity since peaks will not be diluted in the UV cell prior to MS analysis.
  3. The mass spectrometer will see less sample and will require less cleaning maintenance.
  4. It allows for fraction collection so if you find something you want to investigate further just go back to the fraction you collected.   

Capillary UV flow cells used for low flow rate are often constructed from small diameter fused silica and will not contribute to peak dilution.  Check with the manufacture and always ask, "What's the flow cell volume?"  There is something called the 10% rule for UV flow cells, this means the flow cell should not be larger than 10% of the flow rate.  For example the flow cell used with a 300 um ID column flowing at 4 ul/min should not exceed 400 nl in volume, this is the upper limit!  Use the 10% rule only as guide, actually the optimal cell size will be the one that gives the best sensitivity and linearity of response. Our preference for the 300 um column is a Z or U shaped fused silica cell with a calculated flow cell volume of 30 nl.  Check out our capillary UV detection page in our capillary HPLC tutorial.




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