Syringe Pump Application Note AN6
Theory
The Van Deemter equation is the key to under-standing the fundamentals of chromatography.According to the Van Deemter equation:H = A + B/µ + Cµwhere: H is the column’s plate height, and µ is themobile phase linear flow rate. A, B, and C are constants.A smaller plate height H indicates higher separationresolution.
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The constant A is the eddy diffusion term. Eddy diffu-sion results from multiple flow paths in the column andis independent of mobile phase flow rate. Due to thepacking particles, analyte molecules can follow multiplepathways of differing path lengths. These multiple path-ways of differing length spread the analyte moleculesapart and cause peak broadening. The A term dependson the compactness of the stationary phase. Voids inthe stationary phase contribute to peak broadening dueto channeling. In contrast, smaller packing particlesoffer smaller differences in path length, thus reducingpeak broadening.B is the longitudinal diffusion coefficient. It dependson the diffusion coefficient of the analyte molecules inthe mobile phase. Faster mobile phase flow rates reduceresident time, and shorter resident time of the analytemolecules in the column reduces the effects of longitu-dinal diffusion. This reduction contributes to betterseparation efficiencies since the analyte molecules haveless opportunity to spread out through diffusion, thusexplaining the 1/v factor.C is the analyte mass transfer coefficient. It dependson the time needed for the analyte molecules to equili-brate between the mobile and stationary phases. If thisequilibration is too slow, then some of the analyte mole-cules, which did not have enough time to bond to thestationary phase, will flow down the column with themobile phase; whereas, the other molecules, which didnot have enough time to detach from the stationaryphase, are left behind. Higher mobile phase flow rateswill contribute to the spreading out of the analyte mole-cules, thus explaining the v factor. Smaller stationaryphase particles are expected to have shorter equilibra-tion time.As stated above, smaller stationary phase particlesor beads should contribute to smaller A and C values.The A term will contribute less to H and allow for higherresolution. As the C term becomes less significant to thevalue of H, increasing mobile phase flow rates will notsacrifice separation performance as much. This wouldallow for faster separations with the same resolution.Even though smaller column beads may not directlyaffect the B value, the higher flow rates reduce its contri-bution to H. Smaller column beads would allow forfaster separations with higher resolution.
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Smaller beads will pack with smaller interstitialspaces, thus reducing the A value, but offer higher resis-tance to solvent flow. In turn, this requires highersolvent pressures. With higher optimal separation flowrates, column back pressure increases as an inversecube of the stationary phase particle size; i.e., P
α
1/d
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Typically, pressures of 1,000 psi are required for HPLCwith 5µm column beads, whereas 20,000 psi or above isrequired for Ultra-HPLC (UHPLC) with 1.7µm beads.
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Figure 2: Gradient Solvent Delivery System
Pump refilland outlet valvesPumpmodulesPump andgradient controllerSolvent in-linefiltersPump isolationcheck valvesStaticmixerSampleinjection valve
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