watersUPLC初学者手册（适合初学者）
Table of Contents
Introduction................................................................................................................................................................................................................................................................................7
Bands, Peaks and Band Spreading..............................................................................................................................................................................................................9
How a Chromatographic Band is Formed..............................................................................................................................................................................9
How a Chromatographic Band Becomes a Peak.......................................................................................................................................................10
What is a Chromatogram?...................................................................................................................................................................................................................11
Band Spreading..................................................................................................................................................................................................................................................12
Addressing Extra-column [Instrument] Band Spreading................................................................................................................................14
The Influence of Tubing Length and Diameter...........................................................................................................................................................16
The Impact of Detector Settings on Extra-column Band Spreading...............................................................................................17
The Promise of Small Particles....................................................................................................................................................................................................................19
The Relationship Between Resolution and Intra-column Band Spreading.............................................................................19
The Relationship Between Resolution, Efficiency and Particle Size...............................................................................................20
Understanding van Deemter Curves........................................................................................................................................................................................22
The Impact of Extra-column [Instrument] Band Spreading on van Deemter Plots.......................................................30
Maintaining Separation Integrity without Compromise.................................................................................................................................32
Understanding Column Resolving Power [L/dp]........................................................................................................................................................34
Measuring Gradient Separation Performance [Peak Capacity].............................................................................................................37
The Consequence of Improved Performance..............................................................................................................................................................................39
Addressing System Pressure.............................................................................................................................................................................................................39
Elevated Temperature.................................................................................................................................................................................................................................42
Improving Productivity with UPLC Technology.......................................................................................................................................................................44
Holistic System Design.............................................................................................................................................................................................................................44
Maximizing Separation Power.........................................................................................................................................................................................................45
A Single System for HPLC and UPLC Separations................................................................................................................................................47
Conclusion .............................................................................................................................................................................................................................................................................49
References for Further Reading...................................................................................................................................................................................................................50
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List of Figures
Figure 1 ACQUITY UPLC System........................................................................................................................................................................................................7
Figure 2 Versatility of UPLC Technology – achieving resolution, sensitivity and speed..........................................8
Figure 3 Representation of an HPLC system........................................................................................................................................................................9
Figure 4 Understanding how a chromatographic column works – analyte bands.........................................................10
Figure 5 Concentration profile of analyte molecules within the analyte band.................................................................10
Figure 6 Digital creation of a peak by a detector....................................................................................................................................................11
Figure 7 Sources of band spreading [instrument and column]................................................................................................................13
Figure 8 Band spreading as a statistical function...................................................................................................................................................13
Figure 9 Impact of system [instrument + column] band spreading on peak shape......................................................14
Figure 10 Equation for determining plate count...........................................................................................................................................................15
Figure 11 Impact of instrument band spreading on column performance..................................................................................16
Figure 12 Analyte band spreading as a function of tubing ID and length..................................................................................17
Figure 13 Impact of detector settings on perceived performance..........................................................................................................18
Figure 14 Fundamental resolution equation.......................................................................................................................................................................19
Figure 15 Impact of individual chemical and mechanical factors on resolution.................................................................20
Figure 16 Relationship between resolution and efficiency..............................................................................................................................20
Figure 17 Relationship between efficiency and particle size for
constant column length...................................................................................................................................................................................................21
Figure 18 Relationship between optimal flow rate and particle size
for constant column length.........................................................................................................................................................................................21
Figure 19 Relationship between efficiency, peak width and peak height.....................................................................................21
Figure 20 Comparing performance gains of a 1.7 μm particle to a 5 μm particle to prove
theoretical predictions......................................................................................................................................................................................................22
Figure 21 Simplified equation to determine HETP.....................................................................................................................................................23
Figure 22 Diffusion-related processes occurring within a chromatographic column.....................................................23
Figure 23 van Deemter equation........................................................................................................................................................................................................24
Figure 24 Individually plotted terms of the van Deemter equation.....................................................................................................24
Figure 25 Summation of individual terms of the van Deemter equation
to form a van Deemter curve....................................................................................................................................................................................26
Figure 26 van Deemter plot comparing 10 μm and 5 μm particles.....................................................................................................26
Figure 27 Influence of particle size on the A term....................................................................................................................................................27
Figure 28 Influence of linear velocity on longitudinal diffusion.............................................................................................................28
Figure 29 Mass transfer [diffusion] into and out of a chromatographic pore............................................................................28
[ List of Figures ]
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6
Figure 30 Impact of linear velocity on mass transfer
and analyte bands [same particle size].......................................................................................................................................................29
Figure 31 Mass transfer differences related to particle size.........................................................................................................................29
Figure 32 van Deemter plot comparing particle size [5.0, 3.5 and 1.7 μm]...........................................................................30
Figure 33 Result of large instrument band spreading on
van Deemter plots [2.5 μm vs. 1.7 μm]......................................................................................................................................................31
Figure 34 Result of small instrument band spreading
on van Deemter plots [2.5 μm vs. 1.7 μm]............................................................................................................................................32
Figure 35 Correlation between van Deemter plots [HETP vs. u] and plate count vs. flow rate........................33
Figure 36 The compromise between resolution and speed in HPLC....................................................................................................33
Figure 37 Dependence of particle size on optimal flow rate.........................................................................................................................34
Figure 38 Calculating the column length-to-particle size [L/dp] ratio..............................................................................................35
Figure 39 Comparing the L/dp ratio as a function of separation index...........................................................................................35
Figure 40 Achieving faster separations while maintaining resolution [L/dp]...........................................................................36
Figure 41 Equation for determining peak capacity...................................................................................................................................................37
Figure 42 Applying the peak capacity equation to a fast separation...............................................................................................37
Figure 43 Peak capacity comparison of HPLC vs. UPLC Technology..................................................................................................38
Figure 44 The relationship between optimal pressure and
particle size for a constant column length.............................................................................................................................................39
Figure 45 Comparing column pressure differences between a 1.7 μm particle
and a 5 μm particle at constant column length...............................................................................................................................40
Figure 46 The relationship between optimal pressure and particle size
for differing column length........................................................................................................................................................................................41
Figure 47 Comparing column pressure differences between a 1.7 μm particle
and a 5 μm particle at constant L/dp..............................................................................................................................................................41
Figure 48 The effect of column temperature on efficiency.............................................................................................................................42
Figure 49 The dependence of column performance on temperature and pressure..........................................................43
Figure 50 Attributes of holistic system design................................................................................................................................................................44
Figure 51 Improvements of efficiency, resolution, peak shape and
peak height related to holistic system design....................................................................................................................................45
Figure 52 Combining elevated temperature with UPLC Technology to maximize plate count..........................46
Figure 53 Combining elevated temperature with UPLC Technology to maximize peak capacity..................47
Figure 54 A single system for HPLC and UPLC separations..........................................................................................................................48
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