"Using Mechanisms of
Solid Phase Extraction to
Improve Your
Bioanalytical Results"
United Chemical Technologies, Inc.
2731 Bartram Road, Bristol, Pennsylvania 19007
800-541-0559
www.unitedchem.com
1
mtelepchak@unitedchem.com
215-781-3850
2
The force known as
solid phase extraction
is unusually strong.
3
Types of Base Materials for
SPE Packings












Silica – NA and K Silicates
Fluorosil® Mg Silicates
Alumina
Carbon
Polystyrene
Polystyrene – Divinyl benzene
Polystyrene – N-Vinylpyrrolidone
Cellulose
Hydroxyapatite
Fullerenes
Cyclodextrin
Agarose
4
MSMS EIC 20 ng/mL Salbutamol – 1 mL Urine 78:20:2 Elution Solvent
(Dichloromethane:Isopropanol:Ammonium Hydroxide)
RT: 0.00 - 4.00
NL: 7.80E6
m/z= 221.60-222.60 F:
+ c ESI w Full ms2
240.10@32.00 [
65.00-500.00] MS 81
cerex
100
80
60
1.90
40
20
0
100
NL: 7.80E6
m/z= 221.60-222.60 F:
+ c ESI w Full ms2
240.10@32.00 [
65.00-500.00] MS 81
certify
1.97
80
60
40
20
0
100
NL: 7.80E6
m/z= 221.60-222.60 F:
+ c ESI w Full ms2
240.10@32.00 [
65.00-500.00] MS 81
clean scrn
1.95
80
60
CLEAN SCREEN®
40
20
1.20
0
100
NL: 7.80E6
m/z= 221.60-222.60 F:
+ c ESI w Full ms2
240.10@32.00 [
65.00-500.00] MS 81
oasis
80
60
40
20
1.94
0
0.0
0.5
1.0
1.5
2.0
Time (min)
2.5
3.0
3.5
4.0
5
Benzoylecgonine Recovery Comparisons from Clean Screen and Cerex Solid Phase Columns Sample is 1
mL equine urine, Positive Control is commercially obtained CONDOA multiconstituent control
10 ng/mL Equine Urine 1 mL BEG-TMS CleanScreen
PC: CONDOA BEG-TMS CleanScreen
Abundance
Abundance
Ion 240.00 (239.70 to 240.70): 3790.D
Ion 240.00 (239.70 to 240.70): 3788.D
6000000
6000000
4000000
4000000
2000000
2000000
Time-->
0
5.00
6.00
7.00
8.00
9.00
10.00
11.00
12.00
Abundance
Ion 240.00 (239.70 to 240.70): 3793.D
6000000
6000000
4000000
4000000
2000000
2000000
Time-->
0
5.00
6.00
7.00
8.00
9.00
10.00
5.00
11.00
6.00
7.00
8.00
9.00
10.00
11.00
12.00
PC: CONDOA BEG-TMS Cerex
10 ng/mL Equine Urine 1 mL BEG-TMS Cerex
Abundance
0
Time-->
12.00
Time-->
Ion 240.00 (239.70 to 240.70): 3795.D
0
5.00
6.00
7.00
8.00
9.00
10.00
11.00
12.00
6
SPE Steps

Condition sorbents

Apply sample

Wash interferences

Dry sorbent

Elute analyte
7
Types of Sorbent-Analyte
Interactions





Polar
Non-polar
Ion-exchange
Covalent
Copolymeric
8
The Real Mechanism of
Solid Phase Extraction
9
Polar Extractions








Also called hydrophilic or normal phase
Unequal distribution of electrons
Involves hydrogen bonding, pi-pi and dipole/
dipole interactions
Sorbents - silica, diol, diethylamino,
cyanopropyl
Applications - lipids, oil additives,
carbohydrates, phenols, oil soluble vitamins
Analytes - amines, hydroxyls, carbonyls,
aromatic rings, heteroatoms (O, S, N, P)
Matrix - non-polar, organic
Elution solvents - medium to high polarity
10
Non-Polar Extractions








Also called hydrophobic or reverse phase
Interactions between sorbent C-H bonds and
analyte C-H bonds
Involves van der Waals / dispersion forces
Sorbents - C2, C3,C4, iC4, tC4, C5, C6, C7, C8,
C10, C12, C18, C20, C30 phenyl and cyclohexyl
Applications - drugs of abuse, TDM, pesticides
Analytes - protonated / neutral state, aromatics
& alkyl chains
Matrix - biologicals, water, aqueous buffers
Elution solvents - typically non-polar to
moderately polar
11
Sample pH vs. Recovery
CH3
(CH3) 2CHCH2
OH
C C
O
H
Ibuprofen
Copolymeric DAU Column
A/N Extraction
Hydrophobic Retention
12
Sample pH vs. Recovery of
Ibuprofen
3 4
3 4
5
5
1
2
2
1
Sample added at pH 6
1
2
3
4
5
Ibuprofen Sample
Meprobamate
Glutethimide
Phenobarbital
Phenytoin
added at pH 5
13
Chain Length Effect
(Recovery & Extract Cleanliness)
4
3.0e4
3
4
3.0e4
5
3
2
2.0e4
2.0e4
1
1.0e4
1.0e4
0
0
0
2
4
6
8
96%
94%
94%
96%
98%
1
0
C18
endogenous
peaks:
area = 71,628
5
2
2
4
6
8
C2
1
2
3
4
5
Butabarbital
Amobarbital
Pentobarbital
Secobarbital
Glutethimide
64%
87%
88%
89%
78%
endogenous
peaks:
area = 11,257
14
Chain Length Effect
(Recovery & Extract Cleanliness)
4
3.0e4
3
4
3.0e4
5
3
2
2.0e4
2.0e4
1
1.0e4
1.0e4
0
0
0
2
4
6
8
96%
94%
94%
96%
98%
1
0
C18
endogenous
peaks:
area = 71,628
5
2
2
4
6
8
C2
1
2
3
4
5
Butabarbital
Amobarbital
Pentobarbital
Secobarbital
Glutethimide
64%
87%
88%
89%
78%
endogenous
peaks:
area = 11,257
15
Chain Length Effect
(Recovery & Extract Cleanliness)
4
4
3.0e4
5
2.0e4
3.0e4
IST
D
3
2
2.0e4
3
IST
5 D
1
2
1.0e4
1.0e4
1
0
0
0
2
4
6
8
10
0
Ct4
endogenous
peaks:
area = 1,336
28%
73%
84%
98%
70%
2
4
6
8
10
Cn4
1
2
3
4
5
Butabarbital
Amobarbital
Pentobarbital
Secobarbital
Glutethimide
93%
97%
98%
98%
96%
endogenous
peaks:
area = 18,271
16
Yoda teaches Luke
the ways of the tube.
17
Ion Exchange Mechanisms







Ionic interactions occur between charged sorbent &
analyte of opposite charge
pH is manipulated to ionize analytes functional group
Ionic bonds are strong & retain analyte
Hydrophobic interferences washed away with organic
solvents
Polar interferences removed with aqueous or weak
aqueous / organic washes
Elute solvents containing stronger counterions or by
changing pH
For ionic/hydrophobic analytes, elute by simultaneously
disrupting both interactions
18
Cation Exchange Extractions

Cation exchange sorbents negatively charged

Basic analytes manipulated to carry positive charge

Opposites attract forming strong bonds

Sorbents
– Benzenesulfonic acid (strong)
– Propylsulfonic acid (strong)
– Carboxylic acid (weak)


Applications include basic drugs, catecholamines,
pharmaceuticals, herbicides
Analytes
– Amines
– Pyrimidines (cations)


Matrix - aqueous
Basic elution solvents to neutralize analyte
19
Anion Exchange Extractions






Anion exchange sorbents positively charged
Acidic analytes manipulated to carry negative charge
Opposites attract forming strong bonds
Sorbents
–
–
–
–
1, 2 amine
Aminopropyl (weak)
Quaternary amine (strong)
Diethylamino (weak)
Applications include phosphates, acidic drugs, organic
acids, fatty acids, vitamins
Analytes
– Phosphates
– Carboxylic acids
– Sulfonic acids (cations)


Matrix - aqueous
Acidic elution solvents to neutralize analyte
20
Copolymeric Extractions

Hydrophobic & ionic retention mechanisms

Reverse phase sorbent with cation OR anion exchange

Acidic, basic & neutral analyte applications

Matrix - aqueous

Selective washes

Elution solvents mixture of organics with acid or base

Superior sample clean up
21
C8 vs. Copolymeric
Extraction
0
4
8
12
16
20
0
4
8
12
16
Time (minutes)
Time (minutes)
C8
Column
Copolymeric
Column
20
22
23
pKa, pH & Ionization
% of Compound in Ionic State
Functionality
Acid
Base
Ionization State
Anion (-)
Cation (+)
pH units away from pKa
2<
1< at pKa 1> 2>
1
99
9
91
50
50
91
9
99
1
24
Specialty Anion
Exchange Columns
Ion exchange columns possess charged
functional groups which allow analytes
to bind upon sample application. Prior
to column use, these groups require
counter ions at these charged sites.
The standard counter ion for cation
exchangers is the hydronium ion and for
anion exchangers is the chloride ion .
From time to time during sample
application, a charged analyte is not
strong enough to displace the counter
ion & therefore does not bind to the
column. In cases such as these, a
weaker counter ion is required. Two
such columns with weaker counter ions
(Quaternary amine with acetate counter
ion) & (Quaternary amine with
hydroxide counter ion) are commercially
available. In terms of strength, the
acetate ion is stronger than the
hydroxide ion.
CAQAX
Silica Backbone
Quaternary Amine anion exchanger
Acetate counter ion
(Standard anion exchanger carries Cl- )
CHQAX
Silica Backbone
Quaternary Amine anion exchanger
Acetate counter ion
(Standard anion exchanger carries Cl- )
25
Relative Counter ion Selectivity
Cations
Larger numbers reflect greater
ability of the ion to displace other
ionic materials from the
bonded surfaces.
Strong Cation Exchanger
SO-3
Si
Benzenesulfonic Acid (BCX)
Strong Anion Exchanger
+
- Si - (CH2)3 N (CH3)3
Quaternary Amine (QAX)
Ba2+
2+
Ag
Pb2+
Hg2+
Cu+
Sr2+
Ca2+
Ni2+
Cd2+
Cu2+
CO2+
Zn2+
Cs2+
Rb+
K+
Fe2+
Mg2+
Mn2+
NH4+
Na+
H+
Li+
8.7
7.6
7.5
7.2
5.3
4.9
3.9
3.0
2.9
2.9
2.8
2.7
2.7
2.6
2.5
2.5
2.5
2.3
1.9
1.5
1.0
0.8
Anions
Benzene Sulfonate
Citrate
IPhenateHSO4CIO3NO3BrCN HSO BrO
NO2
CI HCO3IO33Formate Acetate Propionate FOH -
500
220
175
110
85
74
65
50
28
27
27
24
22
6.0
5.5
4.6
3.2
2.6
1.6
1.0
Standard cation exchange counter ion
26
I.
ROBINUL
(GLYCOPYRROLATE)
FROM EQUINE
URINE BY LCMSMS
(CLEAN UP® CUCCX-2)
500 mg / 14 mL
CONDITION SPE COLUMN
1.
Wash with 2 x 2.5 ml MeOH
2.
Wash with 2 x 2.5 ml phosphate buffer (0.1m, pH 7.0)
II. SAMPLE PREPARATION
1.
Buffer 5 ml of urine to pH 7.0 by adding 3 mL of
0.1M phosphate buffer (pH 7.0)
2.
Add (12.5 ng) of mepenzolate (internal standard)
3.
Add 5 ml of water to the sample
4.
Vortex or shake thoroughly
5.
Centrifuge for 5 min at 800g
6.
Apply supernatant to SPE column
III. WASH COLUMN
1.
Wash column with 5 ml of MeOH
2.
Wash column with 5 ml of H2O
IV.
V.
VI.
DRY COLUMN
1.
5 min. with vacuum at 25 mm Hg
ELUTE OF GLYCOPYRROLATE
1.
Elute with 4 ml of methanol –
0.5M ammonium acetate buffer pH 3.00
BLOWN DOWN
1.
Blown down eluent at 60°C under nitrogen and
reconstitute with 0.1 mL of MeOH
27
LCMSMS of Glycopyrrolate
28
Meprobamate
O
NH2
C
O
CH3
O
CH2
C
CH2
O C NH2
CH3
Polar Drug
Copolymeric DAU Column
A/N Extraction
Hydrophobic Retention
29
A/N Drug Recovery vs.
Changes in Elution Solvents
Hexane/Ethyl Acetate
1
Methylene Chloride
2
1
1
2
2
Ibuprofen
Meprobamate
30
Amphetamine Structures
CH2
CH NH
CH 3
Amphetamine
pKa = 9.9
2
Methamphetamine
pKa = 9.9
CH 2
CH NH 2 CH3
CH 3
H H H
C C N
OH CH3
CH3
Ephedrine
pKa = 9.6
31
Recovery vs. Different Elution
Solvents
1
2
3
4
5
6
7
8
9
10
11
12
13
14
d-Amphetamine
d-Methamphetamine
PPA
Pseudoephedrine
Meperidine
Lidocaine
PCP
Methadone
Propoxyphene
Cocaine
Codeine
Diazepam
Nordiazepam
Chlordiazepoxide
4
2
3
1
Elution:
MeCl2 / IPA / NH4OH
(78/20/2)
32
Recovery vs. Different Elution
Solvents
1
2
3
4
5
6
7
8
9
10
11
12
13
14
d-Amphetamine
d-Methamphetamine
PPA
Pseudoephedrine
Meperidine
Lidocaine
PCP
Methadone
Propoxyphene
Cocaine
Codeine
Diazepam
Nordiazepam
Chlordiazepoxide
2
3
4
1
Elution:
EA / NH4OH
(98/2)
33
Solubility
Best wash solvents are those in which
the compound of interest is insoluble.
Example:Vancomycin
Insoluble in Methanol
Wash: 100% methanol
Soluble in H2O
Elution:80:20 methanol/H2O
34
Technical Document P-105
Purification of Small Molecule Libraries
Desalting Samples Using Pharmasil™ Reverse Phase SPE
Principle: The generation of small molecule libraries for screening against
biological targets has emerged as an area of intense interest in the
pharmaceutical industry. SPE has been demonstrated to expedite work up and
purification of organic molecules synthesized in solution, and in the
automated construction of small molecule libraries. Samples that have been
synthesized in aqueous salt, buffer solutions, or low polarity organic solvents
containing salts may require the removal of those salts prior to analysis.
Pharmasil TM Reverse Phase SPE can be used to desalt these libraries.
Application: This application details the use of Pharmasil™ CEC18, a
highly loaded reverse phase sorbent, for desalting synthetic mixtures. In
combinatorial chemistry and organic synthesis salts are sometimes present in
the reaction mixtures. Once the reaction is complete, it is usually necessary to
separate the products of the reaction from the salts. If the salt is not removed
it can interfere with further testing as well as ruin expensive analytical
equipment. This can be done using a highly loaded reverse phase SPE
column to selectively remove the salt from the reaction mixture.
35
Chemistry of Pharmasil™ CEC18 Sorbent
Advantages of Pharmasil™
Based Sorbents
CH3
H3C
Si
CH3
O
O
Si
(CH2)17
O
H3C
Si
CH3
• Complete removal of salts
• Clean background
• High recoveries
• High levels of purification of anaytes
• Applicable to a broad range of
compounds
• Simple easy to develop methods
CH3
CH3
36
Purification Profile
This profile is based on the use of a Pharmasil™ CEC18 500 mg column (columns are available with varying
volumes). This column is capable of removal of salts. The method can be scaled up as necessary by using columns
of higher bed mass of sorbent and increasing the solvent volumes proportionately
The following profile is meant to be a guideline for these types of purifications. Each drug class has its own
specific requirements based on solubility, stability, and pKa and may require slight adjustments in
methodology. Therefore think of the following profile as a beginning rather than a final method.
Sample Pre-treatment
Samples may or may not require pretreatment before addition. The primary concern using desalting columns is to
adjust the pH of the compound of interest so that it is totally molecular. This may require the addition of an acid or
base. Desalting can be done out of low polarity organic solvents such as hexane or methylene chloride as long as the
compound of interest is protonated.
Column Conditioning
Condition the column 1 ml of Methanol followed by 1 ml of water.
Column Equilibration
Condition the column with buffer: If sample is a base, you want the pH to be >9
If sample is an acid, you want the pH to be<2.5
Apply the sample to the column under gravity. The salts will flow through the column and the sample will stick to
the column. The volume of the sample is not important and will probably be dictated by the equipment you use. The
critical factor is concentration and capacity of the sorbent. If the concentration of the compound of exceeds the
capacity of the sorbent you will not get the highest recovery. If you think this is a problem use a larger bed mass.
Product Purification
Wash the column with 1ml of DI water or hexane.
Product Elution
Elute compound of interest with 1ml of methanol, ethyl acetate, or the organic solvent of your choice.
37
Technical Document P-102
Purification of Small Molecule Libraries
TIN (Sn) Removal by Pharmasil™ Ion Exchange SPE
Principle: The generation of small molecule libraries for screening against biological
targets has emerged as an area of intense interest in the pharmaceutical industry. Ion
exchange chromatography has been demonstrated to expedite work up and purification
of organic molecules synthesized in solution, and in the automated construction of
small molecule libraries. The advantage of ion exchange chromatography over more
traditional small molecule purification modes such as flash chromatography or HPLC
is that one can reliably predict the elution characteristics of a broad range of molecules
solely by the presence or absence of an ionizable site on the molecule.
Application: This application details the use of Pharmasil™ TAX, a highly loaded
weak cation exchange sorbent, for the removal of tin catalysts from organic synthesis
mixtures. In combinatorial chemistry and organic synthesis tin compounds are
common catalysts. Once the reaction is complete, it is usually necessary to separate the
products of the reaction from the catalysts. If the catalyst is not removed it can
interfere with further testing as well as ruin expensive analytical equipment. This can
be done using a highly loaded weak cation exchanger to selectively remove the tin
catalyst from the reaction mixture.
38
Chemistry of Pharmasil™ TAX Sorbent
COOH
CH2
Si
H2
C
H2
C
H2
C
N
CH2CH2
N(CH2COOH)2
Advantages of Pharmasil™ Based Sorbents
• Complete removal of tin catalyst
• Clean background
• High recoveries
• High levels of purification of anaytes
• Applicable to a broad range of compounds
• Simple easy to develop methods
39
Purification Profile
This profile is based on the use of a Pharmasil™ TAX 500 mg column (columns are available with varying
volumes). This column is capable of removal of up to50mg of tin. The method can be scaled up as necessary by
using columns of higher bed mass of sorbent and increasing the solvent volumes proportionately
The following profile is meant to be a guideline for these types of purifications. Each drug class has its own
specific requirements based on solubility, stability, and pKa and may require slight adjustments in
methodology. Therefore think of the following profile as a beginning rather than a final method.
Sample Pre-treatment
Samples may or may not require pretreatment before addition. The primary concern using ion exchangers is to
adjust the pH of the compound of interest so that it is totally ionized. This may require the addition of an acid or
buffer. Ion exchange can be done out of organic solvents such as methanol or ethyl acetate as long as the compound
of interest is ionized... Tin catalysts are strong cations and are charged across the complete pH range.
Column Conditioning
Condition the column 1 ml of Methanol followed by 1 ml of water.
Column Equilibration
Condition the column with buffer: If sample is a base, you want the pH at 7-8.
If sample is an acid, you want the pH at 3-4.
Sample Application
Apply the sample to the column under gravity. The tin will stick to the column. The volume of the sample is not
important and will probably be dictated by the equipment you use. The critical factor is concentration and capacity
of the sorbent. If the concentration of the tin of exceeds the capacity of the sorbent you will not get the highest
removal of tin. If you think this is a problem use a larger bed mass.
Product Purification
Wash the column with 1ml of buffer used in column equilibration.
Product Elution
Elute compound of interest with 1ml of methanol.
40
Technical Document P-103
Purification of Small Molecule Libraries
Palladium (Pd) Removal by Pharmasil™ Ion Exchange SPE
Principle: The generation of small molecule libraries for screening against biological
targets has emerged as an area of intense interest in the pharmaceutical industry. Ion
exchange chromatography has been demonstrated to expedite work up and purification
of organic molecules synthesized in solution, and in the automated construction of
small molecule libraries. The advantage of ion exchange chromatography over more
traditional small molecule purification modes such as flash chromatography or HPLC
is that one can reliably predict the elution characteristics of a broad range of molecules
solely by the presence or absence of an ionizable site on the molecule.
Application: This application details the use of Pharmasil™ TAX, a highly loaded
weak cation exchange sorbent, for the removal of palladium catalysts from organic
synthesis mixtures. In combinatorial chemistry and organic synthesis palladium
compounds are common catalysts. Once the reaction is complete, it is usually
necessary to separate the products of the reaction from the catalysts. If the catalyst is
not removed it can interfere with further testing as well as ruin expensive analytical
equipment. This can be done using a highly loaded weak cation exchanger to
selectively remove the tin catalyst from the reaction mixture.
41
Chemistry of Pharmasil™ TAX Sorbent
COOH
CH2
Si
H2
C
H2
C
H2
C
N
CH2CH2
N(CH2COOH)2
Advantages of Pharmasil™ Based Sorbents
• Complete removal of palladium catalyst
• Clean background
• High recoveries
• High levels of purification of anaytes
• Applicable to a broad range of compounds
• Simple easy to develop methods
42
Purification Profile
This profile is based on the use of a Pharmasil™ TAX 500 mg column (columns are available with varying
volumes). This column is capable of removal of up to50mg of palladium. The method can be scaled up as necessary
by using columns of higher bed mass of sorbent and increasing the solvent volumes proportionately
The following profile is meant to be a guideline for these types of purifications. Each drug class has its own
specific requirements based on solubility, stability, and pKa and may require slight adjustments in
methodology. Therefore think of the following profile as a beginning rather than a final method.
Sample Pre-treatment
Samples may or may not require pretreatment before addition. The primary concern using ion exchangers is to
adjust the pH of the compound of interest so that it is totally ionized. This may require the addition of an acid or
buffer. Ion exchange can be done out of organic solvents such as methanol or ethyl acetate as long as the compound
of interest is ionized... Palladium catalysts are strong cations and are charged across the complete pH range. Adjust
the sample to pH 9 with buffer or ammonium hydroxide.
Column Conditioning
Condition the column 1 ml of Methanol followed by 1 ml of water.
Column Equilibration
Condition the column with buffer of pH 9.
Sample Application
Apply the sample to the column under gravity. The palladium will stick to the column. The volume of the sample is
not important and will probably be dictated by the equipment you use. The critical factor is concentration and
capacity of the sorbent. If the concentration of the palladium exceeds the capacity of the sorbent you will not get the
highest removal of palladium. If you think this is a problem use a larger bed mass.
Product Purification
Wash the column with 1ml of buffer used in column equilibration.
Product Elution
Elute compound of interest with 1ml of methanol.
43
Technical Document P-104
Purification of Small Molecule Libraries
TFAA Removal by Pharmasil™ Ion Exchange SPE
Principle: The generation of small molecule libraries for screening against biological
targets has emerged as an area of intense interest in the pharmaceutical industry. Ion
exchange chromatography has been demonstrated to expedite work up and purification
of organic molecules synthesized in solution, and in the automated construction of
small molecule libraries. The advantage of ion exchange chromatography over more
traditional small molecule purification modes such as flash chromatography or HPLC
is that one can reliably predict the elution characteristics of a broad range of molecules
solely by the presence or absence of an ionizable site on the molecule.
Application: This application details the use of Pharmasil™ CHQAX, a highly loaded
quaternary amine exchange sorbent, for the removal of acid catalysts from organic
synthesis mixtures. In combinatorial chemistry and organic synthesis TFAA is a
common catalyst. Once the reaction is complete, it is usually necessary to separate the
products of the reaction from the catalyst. If the catalyst is not removed it can interfere
with further testing as well as ruin expensive analytical equipment. This can be done
using a highly loaded quaternary amine exchanger to selectively remove the acid
catalyst from the reaction mixture.
44
Chemistry of Pharmasil™ CHQAX Sorbent
Si
H2
C
H2
C
H2
C
+
N
(CH3)3OH
Advantages of Pharmasil™ Based Sorbents
• Complete removal of acid catalyst
• Clean background
• High recoveries
• High levels of purification of anaytes
• Applicable to a broad range of compounds
• Simple easy to develop methods
45
Purification Profile
This profile is based on the use of a Pharmasil™ CHQAX 500 mg column (columns are available with varying
volumes). This column is capable of removal of up to 50mg of TFAA. The method can be scaled up as necessary by
using columns of higher bed mass of sorbent and increasing the solvent volumes proportionately.
The following profile is meant to be a guideline for these types of purifications. Each drug class has its own specific
requirements based on solubility, stability, and pKa and may require slight adjustments in methodology. Therefore
think of the following profile as a beginning rather than a final method.
Sample Pre-treatment
Samples may or may not require pretreatment before addition. The primary concern using ion exchangers is to
adjust the pH of the compound of interest so that it is totally ionized. This may require the addition of a pH 7 buffer.
Ion exchange can be done out of organic solvents such as methanol or ethyl acetate as long as the compound of
interest is ionized... acid catalysts are strong anions and are charged across the complete pH range.
Column Conditioning
Condition the column with 1 ml of methanol followed by 1 ml of DI water.
Column Equilibration
Condition the column with pH 7 buffer.
Application
Apply the sample to the column under gravity. The TFAA will stick to the column. The volume of the sample is not
important and will probably be dictated by the equipment you use. The critical factor is concentration and capacity
of the sorbent. If the concentration of the TFAA exceeds the capacity of the sorbent you will not get the highest
removal of TFAA. If you think this is a problem use a larger bed mass.
Product Purification
Wash the column with 1ml of buffer used in column equilibration.
Product Elution
Elute compound of interest with 1ml of methanol.
46
The Determination of
Trace Metal Concentrations in
Acid Mine Drainage
By
Michael W. Beneteau
Advisor: Dr. Carol M. Babyak
47
TAX Column % Recovery of Pb
at Various Concentrations
120
100
80
60
40
20
0
0.250 ppm
0.050 ppm
1.00 ppm
2.00 ppm
48
Metal Recoveries on Various Phases
Cu (ll)
Zn (ll)
As (V)
Sn (Vl)
Se (lV)
Hg (ll)
Cr (lll)
PSA
5.50
4.47
0.00
9.11
23.82
57.07
3.39
BCX-HL
18.01
19.21
0.00
29.43
0.62
58.97
9.81
CCX
0.67
0.03
0.00
16.99
0.00
10.66
0.55
TAX
3.73
3.58
0.00
33.69
0.00
23.97
0.55
THX
10.02
0.78
0.67
29.10
11.42
58.97
0.46
NAX
5.92
2.61
0.00
2.20
21.03
46.80
0.85
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50
51
52
53
54
55
56
57
58