Liquid Chromatography-Mass Spectrometer (LC-MS) Flow Splitting and Sample Injection

Capillary Liquid chromatography/mass spectrometry (LC/MS) has been extensively used in life science for the analysis of peptides and proteins, especially in proteomics area.  Because it shows higher resolution and sensitivity over conventional LC.  Capillary LC utilizes columns with smaller inner diameters (< 0.5 mm) than conventional LC and flow rates from some few µL /min down to the nL/min or even pL/min range.   Smaller ID columns produce higher and narrower chromatographic peaks for a fixed amount of injected material as the same amount of analyte elutes at a higher concentration as well as a better separation efficiency.  The peak height is inversely proportional to the cross-sectional area of the column.  Meanwhile the lower solvent flow rate provides higher ionization and sampling efficiency allowing a higher percentage of analytes in the solution phase to be transmitted to the MS detector.  Combination of these effects provide better detection limit for mass spectrometry.  This allows the use of much less sample input which is critical for those analysis with limited sample volume available.   

The use of capillary column requires careful planning due to the lower loading capacity and the nanoliter injection volume requirements.  Small column ID results in high hydraulic resistance and low flow rate to drive the sample loading on the column and achieve best separation. Advance LC/MS systems utilize split-less nano-flow pumping systems to provide a flow rate as low as a few nL/min.  However, this adds extra cost and is still not enough for some ultrasensitive solution which requires the use of a few micron ID column and Pico-scale flow rate.   Further flow reduction with an external pump and a flow splitter as well as restriction capillary becomes an alternative option.  Below describes an application by tuning such setup to achieve Pico-scale flow rate in a 2 µm ID column.  

Figure 1. Schematic Diagram of Experimental Setup

Components are used in this application:

• Syringe pump (TJP-3A/W0109-1B or LSP02-1B)
• 6-port electric rotary valve (ASP-ERV-O1.2-06)
• Connection capillary with ID 150 µm and length of 10 cm
• Micro-flow splitter/regulator
• Restriction capillary ID 10 µm
• Injection capillary ID 2 µm

The Reynolds number is generally defined as:  Re= Q*DH/ν*A=Q* DH *ρ/(μ *A)

• DH is the hydraulic diameter of the capillary tubing
  
• Q is the volumetric flow rate (m^3/s)
• A is the capillary’s cross sectional area (m^2)
• μ is the dynamic viscosity of the fluid (Pa.s)
• ν is the kinematic viscosity 
• ρ is the density of the fluid (kg/cm^3) 

Table 1 Parameter of Fluidics Properties 

The Reynolds number is much less than 1 in this case and laminar flow is in the capillary.  Flow rate Q in the capillary tubing is proportional to the applied pressure drop across it.  The hydrodynamic resistance of the circular cross-sectional capillary is determined by the total length L, radius and the dynamic viscosity of the delivered fluid: Rh=8*μ*L/pi*R^4 

The ratio of the hydraulic resistance can be tuned by the length and selection of the radius of the capillary tubing.  The length and ID are listed in Table 2.  The sample concentration is about 25 ng/ μL and the injection time is about 2.94 mins. The total sample injected from syringe pump is about 2.65 μL, thus the sample loaded is about 66.25 ng. Sample injection splitting ratios are calculated in Table 2.  The flow rates in these three cases are about 449.78 pL/min, 719.42 pL/min and 479.74 pL/min.  The sample injected through 2 μm capillary are about 33.10 pg, 52.96 pg and 35.31 pg.

Table 2 Capillary Tubing Dimensions 

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