Sorbents. [ A] [ A] aq. Strong affinity = large K D. Digital chromatography: all-or. or-nothing LC mechanism to extremes K =

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1 Theory of SPE 1

2 SPE Nonequilibrium,, exhaustive removal of chemical constituents from a flowing liquid sample via retention on a contained solid sorbent and subsequent recovery of selected constituents by elution from the sorbent M.J.M. Wells, Essential guides to method development in solid-phase extraction, in I.D. Wilson, E.R. Adlard,, M. Cooke, and C.F. Poole, eds., Encyclopedia of Separation Science,, Vol. 10, Academic Press, London, 2000, pp

3 Sorbents Strong affinity = large K D K = D [ A] solid phase/ solid supported liquid [ A] aq Digital chromatography: all-or or-nothing LC mechanism to extremes Infinity K D during retention Zero K D during elution 3

4 Plate Theory and RP-SPE Series of equilibrations occur (batches), K d is very large Typical SPE contains only 20 plates or less (10,000 plates for HPLC) Not for separation, but for simple isolation of analytes in an on/off mode Sample Sorbed analyte Equilibrium steps in the SPE column Ref.: E.M. Thurman, and M.S. Mills, Solid-Phase Extraction Principles and Practice, John Wiley & Sons, Inc. (1998) Sample effluent 4

5 Four Typical Steps of SPE Conditioning Loading Adsoption Washing Elution 5

6 Conditioning Make the sorbent compatible with sample solution for close contact in small channels The sorbent should not be dry at any stage Silica substrate Silica substrate C-18 activated with n-hexanen 6

7 Loading/Adsorption Gentle vacuum, or pump At reasonable rate, depend on column dimension, particle size Small particles, more efficient, permit faster flow rate The sorbent should not be allow to go dry at any point Air in the column prevent efficient interfacial contact between liquid and solid phase 7

8 Washing Remove interferences coadsorbed from the SPE column The wash solution must not be too strong to partially eluted the analyte of interest For RP-SPE, when the eluting solvent is immiscible with water the sorbent must be dried so that the eluting solvent can interact with all area of the sorbent Gentle vacuum for a few minutes Compressed air or N 2 Centrifugation 8

9 Elution Eluting solvent should be strong enough to completely removed adsorbed analytes from the sorbent as small a volume as possible (small k) Compatible with the analytical method (i.e., Low BP GC) Free from impurity Low cost and non toxic 9

10 Choice of Eluting Solvent Reversed Phase SPE Nonpolar solvent Methanol Acetonitrile Ethyl acetate Acetone Methylene chloride Normal Phase SPE A function of the eluotropic strength 10

11 Solvent Eluotropic Strength and Polarity Solvent Acitic acid, glacial Water Methanol 2-Propanol Pyridine Acetonitrile Ethyl acetate Acetone Methylene chloride Chloroform Toluene Cyclohexane n-hexane Eluotropic Strength, E >0.73 > Polarity, P

12 Eluting Solvent 12

13 Parameters Affecting N Flow rate Particle size and plate number Column length Temperature 13

14 Sample Breakthrough The analytes are no longer retained by the sorbent. The capacity of the sorbent has been exceeded or overloaded The maximum sample volume that can be passed through the SPE column Retention volume (V R ) and the number of theoretical plates (N) Frontal Chromatography 14

15 Breakthrough SPE UV Detector Sample itself is the mobile phase V m Solute Absorbance V R A o V b V b = Volume at A = 1%A 0 Ref.: M.C. Hennion and V. Pichon, Environ, Sci. Tech., 28, 576A-583A (1994) 15

16 Estimation of Breakthrough Volume V R = V o (1+k w ) k w = Capacity factor of solute eluted by water obtained by linear extrapolation from methanol-water solutions k w can be estimated from a linear relationship between the average log k w and log P oct P oct Log k w = (±0.051)( log P oct (±0.060)( oct = Octanol-water partition coefficient (The affinity of the solute for the stationary phase) Ref.: T. Braumann, J. Chromatogr., 373,, (1986) 16

17 Compound Log k w k w Compound Log k w k w Anilines Halogenated benzene Aniline Fluoro Nitro Chloro Chloro Dichloro 3.5 3,200 Phenols Trichloro ,000 Phenol Alkyl benzene Methyl Benzene Chloro Methyl Dichloro 3.0 1,000 Dimethyl 3.2 1,600 Polar benzenes Ethyl 3.4 2,500 Acetophenone n-propyl ,000 Benzyl alcohol n-butyl ,000 Benzaldehyde Fused-ring aromatics Benzonitrile Naphthalene 3.3 2,000 Nitrobenzene Anthracene ,000 Nitrobenzene Pyrene ,000 2-Nitro Nitro 4-Methyl T. Braumann, J. Chromatogr. 373 (1986)

18 Estimation of Breakthrough Volume C = C/C o V b V R 4σ v V R = V o (1+k) N for typical SPE ~ 20 V V R v b V V o V N = 16 w w = 4σ σ = = = V R R ( 1 + ) v 2 R 2 N - 2σ k w v t = t/t R Ref.: C.F. Poole, A.D. Gunatilleka, and R. Sethuraman, J. Chromatogr. A, 885, 17 (2000) 18

19 Example Calculation of Breakthrough Volume Using k w Chrysene, log P oct = 5.80 Log k w = 0.988(5.80)+0.02 = K w = 562,860 Extracted with 100 mg C-18 C Bonded silica Average density 0.6 g/ml Average porosity 0.65 N = 20 V o = 0.12 ml/100 mg V R = 0.12(1+562,860) = 67,543 ml ~67.5 L σ v = 0.12(1+562,860)/ 20 = 15,103 ml ~15.1 L V b = (15.1) ~ 37.3 L 19

20 Calculated breakthrough volume of some PAHs Breakthrough volume(l) PAHs Log P oct Log k w C-18, 500g N=10 N=15 Naphthalene Acenaphthene Acenaphthylene Fluorene Phenanthrene Anthracene Fluoranthene Pyrene Benzo(a)anthracene Chrysene Benzo(b)fluoranthene Benzo(k)fluoranthene Benzo(a)pyrene Perylene Dibenz(a,h)anthracene Benzo(g,h,i)perylene Coronene

21 Plate Number on Breakthrough Percent Breakthrough N = 50 N = 10 Volume (ml) Ref.: E.S.P. Bouvier, Solid-phase extraction: A chromatographic perspective, Waters Column, V(1), 1-5 (1994) 21

22 k' on Elution Volume Percent Recovery k' = 0 k' = 0.5 k' = Elution Volume (ml) Ref.: E.S.P. Bouvier, Solid-phase extraction: A chromatographic perspective, Waters Column, V(1), 1-5 (1994) 22

23 Sorbent Mass vs. Breakthrough Weak Sorbent-analyte Interaction mechanism Sorbent mass Strong Sorbent-analyte Interaction mechanism Breakthrough volume Ref.: M.J.M. Wells, Principles of extraction and the extraction of semivolatile organics from liquids, in S. Mitra ed., Sample Preparation Techniques in Analytical Chemistry, Wiley-Interscience, John Wiley & Sons, Inc., Hoboken, NJ (2003) 23

24 Sample ph Sample volume Sorbent mass Sample concentration Eluting Strength Solvent volume Recovery Recovery = sorption efficiency x desorption efficiency 24

25 Sample Volume nitroaniline 4-nitrophenol phenol aniline Volume (ml) Ref.: J. Patsias and E. Papadopoulou-Mourkidou, J. Chromatogr. A, 904, 171 (2000) 25

26 Sample ph Recovery MOP MPEP MBP MPRP MEP MMP ph Ref.: T. Suzuki, K. Yaguchi, S. Suzuki, and T. Suga, Environ. Sci. Technol., 35, 3757 (2001) 26

27 % Recovery Sorbent Mass DMP DEP DNBP BBP BEHP DNOP Phthalates Lower Column Upper Column Ref.: M.J.M. Wells, Principles of extraction and the extraction of semivolatile organics from liquids, in S. Mitra ed., Sample Preparation Techniques in Analytical Chemistry, Wiley-Interscience, John Wiley & Sons, Inc., Hoboken, NJ (2003) 27

28 % Recovery Eluting Solvent MMP MEP MPRP MBP MPEP MOP Phthalic acid mono ester CH 2 Cl 2 Diethyl Ether Benzene Ref.: T. Suzuki, K. Yaguchi, S. Suzuki, and T. Suga, Environ. Sci. Technol., 35, 3757 (2001) 28

29 % Recovery Eluting volume DMP DEP DNBP BBP BEHP DNOP Phthalates 5 ml 10 ml Ref.: M.J.M. Wells, Principles of extraction and the extraction of semivolatile organics from liquids, in S. Mitra ed., Sample Preparation Techniques in Analytical Chemistry, Wiley-Interscience, John Wiley & Sons, Inc., Hoboken, NJ (2003) 29

30 SPE Method Development What is solute structure? The clue to effective SPE Identify the goal Obtain physical constants Choose the mode (mechanism) of SPE Elute the SPE cartridge Perform the sorption experiment and determine breakthrough 30

31 Troubleshooting Incomplete elution Increase eluting solvent, change solvent, ph adjustment, back elution Breakthrough of analyte Increase the amount of sorbent, change the sorbent, reduce flow rate Interfering substances Wash with solvent selectively elute the interferences, clean up the eluent with another sorbent SPE bleed, wash the sorbent, change the solvent 31

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