LECTURE 2. Advanced Separation Science Techniques Present and Future Separation Tools

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1 LECTURE 2 Advanced Separation Science Techniques Present and Future Separation Tools Jack Henion, Ph.D. Emeritus Professor, Analytical Toxicology Cornell University Ithaca, NY Lecture 2, Page 1

2 Contents HPLC HILIC Porous Graphitic Carbon UPLC Nano HPLC Capillary Electrophoresis (CE) Differential Mobility Spectrometry (DMS) Lecture 2, Page 2

3 Schematic of HPLC System Lecture 2, Page 3

4 The Resulting Chromatogram t R t 0 C A B w b minutes Question: Is this an isocratic or gradient separation? What is the clue? Lecture 2, Page 4

5 The Chromatographic Process Injected Sample Mixture Most Retained Moderately Retained Least Retained Lecture 2, Page 5

6 Retention and Differential Migration in LC Mobile Phase C m A m Equilibrium distribution of compounds C and A between stationary and mobile phases C s A s S Lecture 2, Page 6

7 Compound Polarity Differences Require Different Stationary Phases Like Dissolves Like Choose a stationary phase that best suits your sample mixture composition Lecture 2, Page 7

8 Normal Phase Comparison of Normal Phase vs. Reversed-Phase Reversed-Phase Lecture 2, Page 8

9 Reversed-phase The Five HPLC Modes ion pair size exclusion normal phase ion exchange Lecture 2, Page 9

10 HPLC Pumping Systems Gradient Former HPLC Pump(s) Injector (Autosampler) HPLC Pump Characteristics: * Constructed of chemically inert materials. * Pulse-free and reproducible flow rate. * Flow rates from 0.05 ml/min to 2 ml/min. * Compatible with gradient elution. * Pressures up to 6000 psi. (400 bar). Most common types of pumps: * Reciprocating pistons. Key Issues: * Low pressure vs. high pressure mixing (helium sparging or no degassing). * Pulse dampner required which contributes delay to volume to gradient mixing. * Limitations regarding micro HPLC with gradients. Lecture 2, Page 10

11 HPLC Column Dimensions Lecture 2, Page 11

12 HPLC Column HPLC Pump(s) Injector Detector Column sizes: 4.6 mm i.d.: Flow = 1.0 ml/min 2.1 mm i.d.: Flow = 0.2 ml/min 1.0 mm i.d.: Flow = ml/min 0.3 mm i.d.: Flow = ml/min mm i.d.: Flow = ml/min (200 nl/min) Is the condition of the bed packing important? Why Lecture 2, Page 12

13 Relationship Between Column Diameter and Particle Size Lecture 2, Page 13

14 HPLC Resolution Resolution is a function of three different factors: 1. Capacity factor, k 2. Column plate number, N. 3. Separation factor, alpha, or band spacing. Changes in these three parameters are shown in figure. Effect of different separation conditions on LC resolution (from J. W. Dolan and L.R. Snyder, Troubleshooting LC/Systems, Human Press, Clifton, NH 1989, p.97 Lecture 2, Page 14

15 Gradient HPLC System Two pumps and shorter run times Lecture 2, Page 15

16 Key Parameters in HPLC Columns Column length Long: (10-25 cm) gives more plates, more backpressure, longer runs Short: (1-3 cm) gives fewer plates, lower backpressure, and short run times Column inside diameter (i.d.) Large: (4.6 mm and higher) employs higher mobile phase flow, reduced LC/MS sensitivity Small: (75 microns to 2.1 mm) employs lower mobile phase flow, higher sensitivity Lecture 2, Page 16

17 Chromatographic Peak Shape In Gradient Mode HPLC Contributed by: Tzipi Ben-Tzvi and Prof. Eli Grushka Institute of Chemistry The Hebrew University Jerusalem, Israel Lecture 2, Page 17

18 Gradient U A -velocity of mobile phase U 1 =U front U 2= U rear Lecture 2, Page 18

19 How Do We Characterize Peak Shape? 1.Tailing Factor = B/A Lecture 2, Page 19

20 How Do We Characterize Peak Shape? 2. Skew M 3 3/2 2 ( M ) Lecture 2, Page 20

21 Objectives 1. Comparing Peak shape in gradient and isocratic separations 2. The dependence of peak shape on the time of gradient starts 3. The dependence of peak shape on the steepness of the gradient Lecture 2, Page 21

22 Amount Amount Examples of peaks Gradient starts after 570 transfers Skew= Transfer Gradient starts after 30 transfers Skew= Transfer Lecture 2, Page 22

23 Amount Amount Amount 0.12 Grad start at Grad start at K=3 Skew2=0.706 K=1 Skew2= K=0.5 Skew2= K=3 Skew2= K=1 Skew2= K=0.5 Skew2= Transfer Grad start at Transfer K=3 Skew2=0.259 K=1 Skew2=-1.16 K=0.5 Skew2= Transfer Lecture 2, Page 23

24 Amount Results step gradient 1.4 Gradient start after 900 transfers, Skew2= Transfer The chromatogram is in the limited range Lecture 2, Page 24

25 Conclusions 1. The peak shape gradient elution can be different than in isocratic separations. 2. The peak shape is a function of the gradient starting point. 3. Step gradient influences the peak shape more than continuous gradient. 4. When the slope of the gradient is higher the absolute values of the skew are greater. 5. A more accurate picture about the peak shape is obtained with skew measured in the limited range. Lecture 2, Page 25

High Performance Liquid Chromatography

High Performance Liquid Chromatography What is HPLC? It is a separation technique that involves: Injection of small volume of liquid sample Into a tube packed with a tiny particles (stationary phase).