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Reduce Baseline Noise For Peptide Gradient LC-UV Analysis

The following is a guest LabNote written by Dr. Stephanie Schuster of Advanced Materials Technology

Reduce Baseline Noise for Peptide Gradient LC-UV Analysis


LC pump mixing noise (high frequency waves in the baseline) can be a problem when you’re analyzing low concentration peptide samples using trifluoroacetic acid (TFA)-modified mobile phases with low wavelength UV detection and low signal attenuation (low mAUFS).  A good way to reduce pump mixing noise is to use a larger volume mixer on your instrument. In the example chromatograms below, the results obtained with a 20 µL mixer (top trace) are compared to the results obtained with a 180 µL mixer (middle trace). The baseline for the 180 mL mixer run is smoother, but the initial resolution between peaks 6 and 7 was lost. The gradient conditions were then changed by reducing the hold time at the beginning of the gradient to compensate for the increase in dwell volume due to the 180 mL mixer. When the initial hold time was reduced from 0.3 minutes to 0.1 minute using the 180 µL mixer (bottom trace), the resolution between peaks 6 and 7 was restored.

When you choose a larger volume mixer, you increase the total gradient dwell volume* of the system.  Changes to dwell volume may either improve or adversely change the original separation.  For this example, using a larger mixer with a minor change to the initial gradient hold time allows you to keep the original separation and improve your baseline noise.  However, there are instances where a large volume mixer is not recommended. Two examples are:  (1) when using MS detection and (2) when using fast gradients with short columns (20 or 30 mm) with fast re-equilibration and cycle times. Depending on the UHPLC/HPLC instrument manufacturer, there will be different mixer recommendations for different detectors and different mobile phases. A larger volume mixer (e.g., > 180 µL) is recommended when using photodiode array detection (PDA) and solvents containing TFA. A smaller volume mixer (e.g., < 20 µL) is recommended for LC-MS separations. Depending on the analysis conditions and the goals of the method, an appropriate volume mixer should be chosen.

*Gradient dwell volume (delay volume) is the volume of the system from the point where solvents are mixed to the column inlet. The dwell volume should be considered and the gradient modified when adjusting hardware as it may cause selectivity changes.

   Peak Identities:

  1. Gly-Tyr
  2. Val-Tyr-Val
  3. Angiotensin 1/2 (1-7) amide
  4. Met-enk
  5. Angiotensin 1/2 (1-8) amide
  6. Angiotensin II
  7. Leu-enk
  8. Ribonuclease A
  9. Angiotensin (1-12) (mouse)
  10. Porcine Insulin
  11. Angiotensin (1-12) (human)


Test Conditions:  Same for all tests except for gradient conditions

Column:  3.0 x 50 mm, 2 µm HALO 2 Peptide ES-C18

Part Number:  91123-402

Mobile Phase A:  water/0.1% TFA

Mobile Phase B:  80/20 ACN/water/0.1% TFA

Gradient Programs:

  • Top trace, 20 µL mixer) Hold at 12.5% B until 0.3 min. then ramp to 63% B from 0.3 to 1.0 min.
  • Middle trace, 180 µL mixer) Same gradient as for top trace
  • Bottom trace, 180 µL mixer) Same gradient as top and middle trace, but hold only 0.1 min. at 12.5% B

Flow Rate: 2.2 mL/min

Temperature:  60 °C

Detection:  PDA at 215 nm

Flow Cell:  Volume (1 µL), Path length:  10 mm

Data Rate:  200 Hz

Response Time:  0.025 sec.

LC System:  Shimadzu Nexera X2 UHPLC

Note:  Peaks 3, 5, and 9 are shorter in bottom trace because sample was re-prepared between middle and bottom chromatographic runs.




  1. The Physicochemical Causes of Baseline Disturbances in HPLC, Part I – TFA-Containing Eluents, K. Choikhet, B. Glatz and G. Rozing, Agilent Technologies Deutschland GmbH, Waldbronn, Germany. LC•GC Europe February 2003, pp 2-9.
  2. MAC-MOD HPLC LabNote, “HALO® Peptide ES-C18 for Ultrafast and High Resolution Separation of Peptides and Small Proteins”, 7/29/2010.



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