Considerations When SEC Separated Components Tail and/or Resolution Decreases

 

a. Connecting an ACQUITY UPLC Protein BEH SEC Column to an ACQUITY UPLC System

It is critical that the ACQUITY UPLC Protein BEH SEC Column be correctly installed onto the UPLC System. Figure 5 details the effects of proper vs. improper column connections on chromatographic performance. It is strongly recommended that new column inlet and outlet connections be made whenever a new ACQUITY UPLC Protein BEH SEC Column is installed onto the UPLC System.

Figure 5. Effects of column-to-LC-system connections on chromatographic performance.

For more information on proper column connections, reference these videos:

How to Install a UPLC Column onto an ACQUITY UPLC System with an Active Column Preheater

How to Connect an ACQUITY UPLC Column to an ACQUITY UPLC System

The effect of a purposely-created, 600-µL gap between the ACQUITY UPLC System solvent line and the BEH SEC column inlet is shown in Figure 6. This gap leads to a subtle increase in peak tailing, which can result in variability in the integrated area of the low-level LMW1 peak eluting as a trailing peak after the large mAb monomer peak. The HMW and LMW2 peaks are unaffected as they are baseline resolved from the monomer peak.

Figure 6. Effect of improper column-to-system connection on a mAb separation.

 

b. LC System Dispersion Effects on a Waters ACQUITY UPLC Protein BEH SEC Separation

In SEC, analytes elute within a single column volume during the isocratic separation. It is therefore critical to appreciate that the total LC system volume, including the injector, tubing, and detector volumes, affect the obtained separation. In general, and as shown in Figures 7 and 8, the lower the total LC system dispersion volume relative to the column volume, the narrower the peaks. The more dramatic effect of LC system dispersion volume on the ability to resolve challenging and closely eluting peaks (e.g., mAb monomer from LMW mAb clip 1) is shown in Figure 8. Most often, and for ACQUITY UPLC Protein BEH SEC separations, it is recommended that the total UPLC system dispersion volume be less than 15 µL.

Figure 7. Effect UPLC system dispersion and SEC column length have on mAb component resolution.

Figure 8. Effect of LC system dispersion on an ACQUITY UPLC Protein BEH SEC 200Å, 1.7 µm Column separation of a human polyclonal IgG antibody.

Note: Unlike the mAb sample used in Figures 1, 4, 6, and 7, the sample shown here did not contain either the LMW clip 1 (i.e., 100,000 Da) or LMW clip 2 (i.e., 50,000 Da).

 

c. Determining the LC System Dispersion Volume

  1. Replace the column with a Zero-Volume Union (p/n: 700002636). The LC tubing should be 0.005" I.D.
  2. Purge all LC solvent, wash, and purge lines with water, then 50/50 water/acetonitrile.
  3. Set the detector to 273 nm and collect data at >40 points per second.
  4. Flow rate: 0.5 mL/min
  5. Run time: 1 min
  6. Sample: 0.16 mg/mL caffeine in 50/50 water/acetonitrile
  7. Injection volume: 0.5 µL
  8. Inject three mobile-phase blanks followed by five caffeine sample injections
  9. To calculate the LC system volume:
    1. Measure the caffeine peak width (in minutes) at 4.4% peak height.
    2. Multiply the peak width by the flow rate to determine the peak volume width in mL. 
    3. Multiply the peak volume width in mL by 1000 to determine the peak volume width in µL.

Note:  The average ACQUITY UPLC H-Class System volume dispersion, when measured using 4-Sigma Method, should be =10.0 µL when used with CH-A, =12 µL for CM-A, and <22.0 µL for 30CH-A column heater. If your value is greater, determine the source(s) of the deleterious extra peak dispersion volume and correct.

 

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