Theory of SPE

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 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. 4636-4643.

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 Sorbents
Strong affinity = large KD

[A ]solid phase/ solid supported liquid

KD =
[A ]aq
Digital chromatography: all-or-nothing
LC mechanism to extremes
Infinity KD during retention
Zero KD during elution

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 Plate Theory and RP-SPE
Sample
Series of equilibrations
occur (batches), Kd is very Sorbed
large analyte
Typical SPE contains only
20 plates or less (10,000 Equilibrium
plates for HPLC) steps in the
Not for separation, but for SPE column
simple isolation of analytes
in an on/off mode

Sample
Ref.: E.M. Thurman, and M.S. Mills, Solid-Phase Extraction effluent
Principles and Practice, John Wiley & Sons, Inc. (1998)
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 Four Typical Steps of SPE
Loading
Conditioning Adsoption Washing Elution

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 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-hexane

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 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

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 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 N2
Centrifugation

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 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

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 Choice of Eluting Solvent
Reversed Phase SPE Normal Phase SPE
Nonpolar solvent A function of the

Methanol eluotropic strength

Acetonitrile

Ethyl acetate

Acetone

Methylene chloride

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Solvent Eluotropic Strength and Polarity
Solvent Eluotropic Strength, E Polarity, P

Acitic acid, glacial >0.73 6.2

Water >0.73 10.2
Methanol 0.73 6.6
2-Propanol 0.63 4.3
Pyridine 0.55 5.3
Acetonitrile 0.50 6.2
Ethyl acetate 0.45 4.3
Acetone 0.43 5.4
Methylene chloride 0.32 3.4
Chloroform 0.31 4.4
Toluene 0.20 2.4
Cyclohexane 0.03 0.0
n-Hexane 0.00 0.06

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Eluting Solvent

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 Parameters Affecting N

Flow rate
Particle size and plate number
Column length
Temperature

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 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 (VR) and the number of
theoretical plates (N)
Frontal Chromatography

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 Breakthrough
SPE UV Detector

Sample itself is the mobile phase

Vm
Ao
Absorbance

Solute

VR

Vb

Vb = Volume at A = 1%A0

Ref.: M.C. Hennion and V. Pichon, Environ, Sci. Tech., 28, 576A-583A (1994)
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 Estimation of Breakthrough Volume

VR = Vo (1+kw)

kw = Capacity factor of solute eluted by water obtained by

linear extrapolation from methanol-water solutions
kw can be estimated from a linear relationship between the
average log kw and log Poct

Log kw = 0.988 (0.051) log Poct + 0.02 (0.060)

Poct = Octanol-water partition coefficient

(The affinity of the solute for the stationary phase)

Ref.: T. Braumann, J. Chromatogr., 373, 191-225 (1986)

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 Compound Log kw kw Compound Log kw kw

Anilines Halogenated benzene

Aniline 1.1 13 Fluoro 2.3 200
4-Nitro 1.4 25 Chloro 2.8 630
4-Chloro 1.8 63 Dichloro 3.5 3,200
Phenols Trichloro 4.4 25,000
Phenol 1.3 20 Alkyl benzene
Methyl 1.8 63 Benzene 2.1 125
Chloro 2.3 200 Methyl 2.7 500
Dichloro 3.0 1,000 Dimethyl 3.2 1,600
Polar benzenes Ethyl 3.4 2,500
Acetophenone 1.8 63 n-Propyl 4.0 10,000
Benzyl alcohol 1.3 20 n-Butyl 4.6 40,000
Benzaldehyde 1.7 50 Fused-ring aromatics
Benzonitrile 1.8 63 Naphthalene 3.3 2,000
Nitrobenzene Anthracene 4.6 40,000
Nitrobenzene 1.9 80 Pyrene 5.0 100,000
2-Nitro 1.9 80
4-Nitro 1.7 50 T. Braumann, J. Chromatogr. 373 (1986) 191
4-Methyl 2.4 250 17
 Estimation of Breakthrough Volume

N for typical SPE ~ 20

C = C/Co

VR = Vo (1 + k w )
VR 2
VR
N = 16 2
w
VR = Vo (1+k) w = 4v
Vb
4 v v = VR N
Vb = VR - 2 v

t = t/tR

Ref.: C.F. Poole, A.D. Gunatilleka, and R. Sethuraman, J. Chromatogr. A, 885, 17 (2000)
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Example Calculation of Breakthrough Volume
Using kw
Chrysene, log Poct = 5.80
Log kw = 0.988(5.80)+0.02 = 5.7504
Kw = 562,860
Extracted with 100 mg C-18 Bonded silica
Average density 0.6 g/mL
Average porosity 0.65
N = 20
Vo = 0.12 mL/100 mg
VR = 0.12(1+562,860) = 67,543 mL ~67.5 L
v = 0.12(1+562,860)/ 20 = 15,103 mL ~15.1 L
Vb = 67.5-2(15.1) ~ 37.3 L

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Calculated breakthrough volume of some PAHs
Breakthrough volume(L)

PAHs Log Poct Log kw

C-18, 500g
N=10 N=15
Naphthalene 3.36 3.340 0.48 0.63
Acenaphthene 4.03 4.002 2.2 2.9
Acenaphthylene 4.06 4.031 2.4 3.1
Fluorene 4.18 4.150 3.1 4.1
Phenanthrene 4.46 4.426 5.9 7.7
Anthracene 4.45 4.417 5.8 7.6
Fluoranthene 5.22 5.177 33 44
Pyrene 5.03 4.990 22 28
Benzo(a)anthracene 5.70 5.652 99 130
Chrysene 5.80 5.745 123 161
Benzo(b)fluoranthene 6.42 6.363 509 669
Benzo(k)fluoranthene 6.50 6.442 610 803
Benzo(a)pyrene 6.20 6.146 308 406
Perylene 6.39 6.328 470 618
Dibenz(a,h)anthracene 6.52 6.462 639 840
Benzo(g,h,i)perylene 7.10 7.035 2389 3144
Coronene 7.64 7.568 8162 10739 20
Plate Number on Breakthrough
N = 50
Percent Breakthrough

N = 10

Volume (mL)

Ref.: E.S.P. Bouvier, Solid-phase extraction: A chromatographic

perspective, Waters Column, V(1), 1-5 (1994)
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 k' on Elution Volume
100 k' = 0
Percent Recovery
k' = 0.5 k' = 1
95

90

85

1 2 3 4
Elution Volume (mL)

Ref.: E.S.P. Bouvier, Solid-phase extraction: A chromatographic

perspective, Waters Column, V(1), 1-5 (1994)
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Sorbent Mass vs. Breakthrough
Weak
Sorbent-analyte
Interaction mechanism

Strong
Sorbent-analyte
Interaction mechanism
Sorbent mass

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)
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 Recovery
Recovery = sorption efficiency x desorption efficiency

Sample pH
Sample volume
Sorbent mass
Sample concentration
Eluting Strength
Solvent volume

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 Sample Volume
120
100 4-nitroaniline
80 4-nitrophenol
60 phenol
aniline
40
20
0
0 50 100 150 200

Volume (mL)

Ref.: J. Patsias and E. Papadopoulou-Mourkidou, J. Chromatogr. A, 904, 171 (2000)

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 Sample pH
110
100 MOP
90
80 MPEP
Recovery

70 MBP
60
50 MPRP
40
30 MEP
20
10 MMP
0
0 2 4 6 8
pH

Ref.: T. Suzuki, K. Yaguchi, S. Suzuki, and T. Suga, Environ. Sci. Technol., 35, 3757 (2001)

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 Sorbent Mass Lower Column
100
90 Upper Column
80
70
% Recovery

60
50
40
30
20
10
0
DMP DEP DNBP BBP BEHP DNOP

Phthalates
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
 Eluting Solvent
100
90
80
CH2Cl2
70
% Recovery

Diethyl Ether
60
50 Benzene
40
30
20
10
0
MMP MEP MPRP MBP MPEP MOP

Phthalic acid mono ester

Ref.: T. Suzuki, K. Yaguchi, S. Suzuki, and T. Suga, Environ. Sci. Technol., 35, 3757 (2001)
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 100 Eluting volume
90
80
70
% Recovery

60
5 mL
50
40 10 mL
30
20
10
0
DMP DEP DNBP BBP BEHP DNOP

Phthalates
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)
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 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
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 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

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