Fast and Comprehensive 2-Dimensional Liquid Chromatography of Block Copolymers through Temperature Gradient Driven Trapping

Transcription

1 Fast and Comprehensive 2-Dimensional Liquid Chromatography of Block Copolymers through Temperature Gradient Driven Trapping International Symposium on GPC/SEC and related techniques Bastiaan Staal & Joshua Fuchs

2 Example of a conventional 2D-LC Solvents 1 st D GPEC x SEC Solvents 2 nd D Characteristics: 1 D: linear gradient over 20 h, 0.03 ml/min flow Upto 300 µl (10 %) of sample injected Transfer Interface Effluent is collected in storage loops with a volume of around 100 µl each Pumps Injector Pumps 2 D: isocratic, 1.5 ml/min flow, 15 min run time Problems: Long analysis time of several hours Dilutions of analytes and broad injection through loops 1 D column 100 µl Storage Loop Optional Detector Waste 2 D column Detector Significant solvent carry-over from 1 D to 2 D Adjustment of flow rates and storage volume, 2 D run time and valve switching Disadvantages: Not suitable for routine analysis due to analysis time Need of highly sensitive detectors because of dilution Refractive index detectors need steady solvent composition Hindered transfer of proper working 1 D methods because of excessive dependency of all parameters Is not fully comprehensive (recovery <50%) Low 2 D-resolution 2

3 Differences of our 2D-LC Solvents Challenges: Cost factor: faster, easy applicable, robust H 2 O TH F Pump High resolution in 2 D at high flow rates Free detector selection Pump Injector Column Bank 90 1 C D-column 0 C Cooling Bath Tra p C18 Trap Column Waste Column Bank 90 C 2 D-column Detector Comprehensive and match of 1D GPEC and SEC with marginal 2D plots Independency of 1 D and 2 D so that 1D methods can be transferred Wide application for different analytes Solutions: UPLC in 2 D high flow rates High pressure resistant columns Trap column no solvent carry-over High temperature to low temperature gradient trap column and easy setup Water as 1 D co-solvent flexible 3

4 Detailled setup for analysis of a PS-b-PB copolymer Fast ~90 min (down to 60 min with only 2 SEC columns) Fully comprehensive (recovery >80 %) Reproducible (done >100 runs in 2 month) Easy method development or transfer (SAN separation in 3 runs) 1 D: H 2 O/THF (75/25, during Injection; 85/15, 1 min, 90/10, 90 min), 0.6 ml/min 10x1 µl (10 %) Injection GPEC at 90 C C18 column Temperature drop causes turbidity and precipitation After 84 s: valve switch and reinjection into 2 D (6 s) 2 D: isocratic THF 0.6 ml/min High temperature SEC with 200, 450 and 900 Å pore size column Detection through UV (254 nm) and RI Valve switching, Inject signal processing and 2D/SEC distinction done by Arduino Minicomputer 4

5 GPEC /min PS-b-PB 2D-chromatogram SEC /min Norm. Intensity 5

6 PS-b-PB: 1 D-Size Exclusion Chromatography Detector Signal/a.u. 0,12 0,1 0,08 UV RI Kalibration bc a Molecular Mass/Da 0,06 d ,04 e 100 0,02 f ,5 1 1,5 2 2,5 3 1 Time SEC /min 6

7 PS-b-PB: SEC as sum of 2D-cuts a bc d UV e f 0 0,5 1 1,5 2 2,5 3 Time SEC /min 0 0,5 1 1,5 2 2,5 3 Time SEC/min 7

8 PS-b-PB: SEC as sum of 2D-cuts 0 0,5 1 1,5 2 2,5 3 8

9 PS-b-PB: SEC sum of 2D-fractions 0 0,5 1 1,5 2 2,5 3 9

10 ELSD/a.u. PS-b-PB: 1 D-Gradient Polymer Elution Chromatography compared to 2D 0,45 0,40 0,35 0,30 0,25 0,20 0,15 0,10 0,05 0, GPEC Time/min THF/% Exact same method on another HPLC system ELSD GPEC chromatogram could be recovered Raw 2D chromatogram 10

11 PS-b-PB: Recovery = f(injection Time, ) Recovery/ 0,9 0,8 0,7 0,6 0,5 90 1, Theoretical - Measured 0,4 0,3 0,2 0, Injection Time/s 11

12 PS-b-PB: Reproducibility 27. Jan 15. März Done over 100 2D-runs Columns only suffered from pressure peaks (> 1000 bar) Trap column also works as pre-column. As trapping performance went done, singly switching to a new one restored performance. 12

13 PS-b-PB: decoupled flow rates GPEC /min SEC /min 0.4 ml/min 1 D-flow ml/min 1 D-flow SEC /min 79.3 % recovery 77.2 % recovery 2 D-flow was 0.6 ml/min in both experiments

14 Polystyrene-stat-Polyacrylonitrile copolymer (SAN): 2D-LC is easy applicable GPEC 1 st 2D run H 2 O/THF (95/5 to 0/100 in 90 min) SEC GPEC SEC 3 rd 2D run H 2 O/THF (20/80 to 10/90 in 90 min) SEC 70 % recovery without any further optimization 14

15 PS-b-PB: artificial oligomers 20 Injects 80 Injects

16 PS-b-PB: UV vs RI dection GPEC /min SEC /min SEC /min

17 Conclusions: As long as the trap columns and the HPLC column have the same stationary phase, 2D-LC will work. The 2D will work with a DRI detector, but requires a more intelligent way of setting a baseline. With a single trap column, never 100% recovery can be obtained. A full 2D measurement can be done within 90 min. Reducing to strong the number of transfer injections, will lead to artificial separations.