Running UPLC

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16 Air Gap Effects Preparing to Run UPLC; Injection and Detection Paramters pre – air gap is more critical than post – air gap automatic Pre air gap Post air gap Peak area Peak height % area difference Automatic (1) Automatic (1) 38889 27156 0,0 4 0 38488 26796 98.97 4 4 38179 26592 98.17 0 4 30866 21404 79.37 0 0 30748 21413 79.07 Air Gap Settings Automatic settings — Pre and post air gap — 4 µL each — 1 µL for PLUNO Manual settings — Lower values: 0,4 o Sample diffusion providing peak area and peak height reduction — Higher values: o Can effect the chromatogram • Higher peak height RSD • Loss of resolution 4,4 4,0 Air Gap Dr. Shula Levin, Medtechnica Large air gap volume can effect the chromatogram. Detection Mechanisms System Configuration Detector Designs Importance of Sampling Rate Detector Requirements
17 Preparing to Run UPLC; Injection and Detection Paramters Designing for UPLC ACQUITY UPLC columns produce small volume peaks To avoid band spreading and maintain concentration the flow cell volume must be corresponding low If you use conventional flow cells the path length must be reduced which results in a loss of sensitivity UPLC detection requires cells that are designed for minimal dispersion, low cell volumes and optimum light throughput Importance of Sampling Rate Must ensure enough points are collected across a peak to adequately define the peak shape: 20-50 points across the peak. Peak detection algorithms require a minimum number of points across a peak to distinguish it from baseline noise and correctly determine peak lift off and touch down A peak which does not have enough data points will be difficult to integrate and therefore have irreproducible peak areas and heights. Dr. Shula Levin, Medtechnica ACQUITY UPLC TUV and PDA – Features Light guided flow cell design Two flow cell options Low noise electronics — High brightness lamp Support for data rates up to 80 Hz — Independent data rate and filter constants Console Control Effect of Sampling Rate on Chromatography – TUV AU 0.080 0.070 0.060 0.050 0.040 0.030 0.020 0.010 0.000 1 pt/s 5 pts/s 40 pts/s 0.50 0.52 0.54 0.56 0.58 0.60 0.62 Minutes 0.64 0.66 0.68 0.70 0.72 0.74
Page 1 and 2: 1 םירטליפה םג ןכו , HP
Page 3 and 4: 3 Bottle Caps Preparing to Run UPLC
Page 5 and 6: 5 Preparing to Run UPLC; Injection
Page 7 and 8: 7 THREE injection methods available
Page 9 and 10: 9 Partial Loop Injection Weak Wash
Page 11 and 12: 11 Summary of Partial Loop using Pr
Page 15: 15 Strong Contaminations Preparing
Page 21 and 22: 21 Teflon ® AF light guiding flow

Running UPLC

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