Instructionsforuse
BAKERBOND spe™ Column Instructions
Please read the Material Safety Data Sheet before use.
BAKERBOND spe™ columns are disposable polypropylene
columns prepacked with high capacity sorbents contained
between two 20 µm polyethylene frits. The sorbent choices
are reverse phase, normal phase or ion exchange silica gel
based bonded phases of 40 µm particle size, and 60Å or 275Å
pore size. Columns are also available packed with normal
phase adsorbents or size exclusion gel.
Columns are available in 1 mL, 3 mL or 6 mL sizes with
sorbent weights of 100 mg, 200 mg, 500 mg or 1,000 mg.
BAKERBOND spe column configurations allow extraction of
compounds from sample solution volumes ranging from a
few hundred microliters up to 1 liter.
BAKERBOND spe columns can be processed by vacuum,
positive pressure or centrifugation. The columns can be
conveniently processed by vacuum using the BAKER spe-12G
or BAKER spe-24G Column Processing System.
Solid Phase Extraction Steps
Solid phase extraction is a sample preparation
technique based on the separation mechanisms of liquid
chromatography (LC). In LC, the solubility and functional
group interactions of sample. sorbent and solvent are
optimized to effect a separation. In solid phase extraction
these interactions are optimized to effect retention or elution.
Solid phase extraction is performed in the following steps:
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Bakerbond Spe Column Processing
1. Sample Preparation: Prepare sample for extraction as
specified in the procedure. If the sample matrix is complex
or strong, matrix modification may be necessary to
facilitate analyte extraction.
2. Column Conditioning: Condition the column with the
volumes of solvents specified. Since the volumes of the
conditioning solvents are not critical, they can be applied
easily with a squeeze bottle or automatic pipette.
DO NOT ALLOW COLUMN TO DRY BEFORE THE SAMPLE IS
APPLIED.
The rate of solvent flow through the extraction columns may
vary individually.*
3. Sample Addition: Accurately transfer the sample to the
extraction column. If required, an Internal Standard may
be added with the sample. Draw the sample through the
column. Flow rates of 5 mL/min or less are best. For sample
volumes greater than the extraction column reservoir
capacity, use an adaptor (Product No. 7122-00) to attach a
15 mL reservoir (Product No.7119-01) or a 75 mL reservoir
(Product No.7120-03) to the top of the extraction column.
Gross contaminants and particulate matter can be
eliminated from the sample by positioning a 1, 3, or 6
ml filtration column containing 20 µm frits (Product
No.7121-01, 03 or 06) with an adaptor ((Product
No.7122-00) above the extraction column.
4. Column Wash: Wash with the specified volume of
solvent. Air dry the columns, if required, under a vacuum.
The 1 mL column can be dried in 1–5 minutes, the 3 mL
and 6 mL columns require 3–10 minutes. Times may vary
with sample type. Turn vacuum off after drying.
Successive solvents should be miscible or the columns
should be dried between solvents. For aqueous samples,
residual water can be removed from the extraction
columns after sample extraction by adding 25 to 100 µL of
a nonpolar water- miscible solvent prior to analyte elution.
The volume should be well below the void volume of the
column.
5. Sample Elution: Pipette the specified amount of eluting
solvent into the extraction column. Aspirate or push the
elution solvent through very slowly. Maximum recovery
may be achieved by using two or more successive aliquots
of eluting solvent. The minimum elution volume for
most sorbents is two (2) void volumes (1 void volume =
1.0–1.2 µL/mg sorbent). The following table gives the
minimum eluting volumes typically required for the
various column sizes:
Eluting Extraction Column
Sorbent Weight
Solvent Volume
1 mL
100 mg
2 x 100 µL
3 ml LD (low displacement)
200 mg
2 x 200 µL
3 mL
500 mg
2 x 500 µL
6 mL
500 mg
2 x 500 µL
6 ml HC
1000mg
2 x 1000 µL
The collected eluates are either analyzed directly or evaporated and reconstituted in a suitable solvent.
Instructions For Specific Sorbent Types
Note: "Column volumes" referred to below are as follows:
For 1 mL, 100mg
For 3 mL, 200 mg
For 3 mL, 500 mg
For 6 mL, 500 mg
For 6 mL, 1000 mg
1 mL
2–3 mL
2–3 mL
5–6 mL
5–6 mL
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Normal Phase
Moderately polar to polar analytes are extracted from
nonpolar solutions onto polar sorbents.
Adsorbents:
Silica Gel (SiOH)
Kieselguhr (SiOn)
Florisil® (Mg2Si03)
and Alumina (Al203)
Bonded Phase Sorbents:
Cyano (CN)
Diol (COHCOH)
Amino (NH2)
Conditioning: Condition column with 1–2 column volumes
of the nonpolar solvent in which the sample is dissolved.
Sample Application:
1. If the analyte is to be retained by the sorbent, the sample
solution should be a nonpolar solvent in which the analyte
has limited solubility.
2. If the analyte is to be passed through the column and
polar interferences remain, the sample solution should be
a nonpolar solvent in which the nonpolar analyte is very
soluble.
Column Washing: Wash with a nonpolar solvent as required.
Analyte Elution: Elute the retained analyte with two (2) or
more void volumes of a polar solvent.
Reversed Phase
Nonpolar to moderately polar analytes are extracted from
polar solutions onto nonpolar sorbents.
Sorbents:
Octadecyl (C18)
Octyl (C8)
Cyclohexyl (C6H11)
Phenyl (C6H5)
Ethyl (C2)
Cyano (CN)
WP Butyl (C4)
Conditioning:
1. Condition column with 1–2 column volumes of methanol.
Other water miscible solvents such as ethanol, isopropanol,
or acetonitrile may be used.
2. Condition column with 1–2 column volumes of polar
solvent similar to the sample solution (water, buffer, etc.).
Keep sorbent bed wet during conditioning.
Sample Application:
1. If the analyte is to be retained by the sorbent, the sample
solution should be a polar solvent in which the analyte has
limited solubility.
2. If the analyte is to be passed through the column and
nonpolar interferences remain, the sample solution should
be a polar solution in which the polar analytes are very
soluble.
Column Washing: Wash with polar solvent as required.
Analyte Elution: Elute the retained analyte with two (2)
or more void volumes of a nonpolar to moderately polar
solvent. Basic analytes strongly retained by unbonded
silanols can be eluted with solvents modified with an acid or
an amine, such as diethylamine or triethylamine.
Ion Exchange
Ionized analytes are extracted from solution onto a sorbent
of opposite charge.
Anion Exchange Sorbents:
Amino (NH2)
1°.2°-Amino (NH2/NH)
WP PEl (NH)
Quaternary Amine (N+)
Cation Exchange Sorbents:
Carboxylic Acid (COOH)
Aromatic Sulfonic Acid (C6H5SO3H)
Propyl Sulfonic Acid ((CH2)3SO3H)
WP Carboxylic Acid (COOH).
Conditioning:
1. Optional: Wash sorbent with 1–2 column volumes of
methanol.
2. Condition column with 1–2 column volumes water or
0.01–0.1 M buffer at same pH as sample solution. Keep
sorbent bed wet during conditioning.
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Sample Application:
Sorbent: Sephadex® G-25
1 .Acidic compounds are extracted by an anion exchange
column from a sample solution 1–2 pH units above the pK. of
the analyte.
Column Preparation Procedure:
2. Basic compounds are extracted by a cation exchange
column from a sample solution 1–2 pH units below the pK. of
the analyte.
Column Washing: Wash with HPLC grade water or buffer .
Analyte Elution: Analytes are eluted from the column using
an eluting solution containing a high concentration of
competing counterions or by adjusting the pH of the eluting
solution to change the charge on the analytes or extraction
column. Some ionizable analytes may have limited water
solubility in the molecular form and cannot be eluted with
an aqueous elution solvent. The aqueous elution solvent
modified with a water-miscible organic solvent will often
elute these analytes.
1. Elute acidic compounds with a solution 1–2 pH units below
the pK. of the analyte. Modify the solution with a watermiscible organic solvent, if necessary.
2. Elute basic compounds with a solution 1–2 pH units above
the pK. of the analyte. Modify the solution with a watermiscible organic solvent, if necessary.
3. Elute analytes with a high ionic strength buffer (>0.5M).
Size Exclusion
Compounds differing significantly in MW are extracted by
a dextran gel which swells in water or buffered aqueous
solutions. The smaller molecules (<10,000 MW) enter the
pores of the hydrated carbohydrate polymer and are
significantly retarded as they percolate through the gel. The
higher MW compounds are excluded from the gel and elute
through the gel in the void volume. The dextran gel is often
used to desalt protein solutions or for buffer exchange.
1. Remove cap from column and add 2–4 mL of selected
buffer.
2. Re-cap column and immediately shake vigorously until
packing is completely hydrated.
3. If required, insert enclosed extra frit above the gel. Use a
clean inert flat surface to insert frit.
4. Condition with three (3) column volumes of selected
buffer.
DO NOT ALLOW SORBENT TO DRY AT THIS STAGE!
5. Introduce sample. Optimum extraction depends on the
volume of sample solution applied to the column.
The volume should be <200 µL to achieve an effective
extraction.
Elution: Elute with a buffered aqueous solution.
A NOTE ON SOLVENTS
The purity of any extraction is highly dependent on the
quality of solvents used. We recommend J.T.Baker HPLC
or ULTRA RESI-ANALYZED® solvents be used for solid phase
extraction procedures.
For More Detailed Information
J.T.Baker's manual, Solid Phase Extraction for Sample
Preparation. is available for more information on solid phase
extraction techniques and method development. Also, our
collection of BAKERBOND spe Application Notes has specific
methods for over 100 different applications.
Please visit www.avantormaterials.com
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Phillipsburg, NJ 9001: 2008 & 14001: 2004
Paris, KY 9001: 2008
Mexico City, Mexico 9001: 2008
Deventer, the Netherlands 9001: 2008 & 14001: 2004 & 13485: 2003
Selangor, Malaysia 9001: 2008
About Avantor™ Performance Materials
Avantor Performance Materials manufactures and markets high-performance chemistries and materials
around the world under several respected brand names, including the J.T.Baker®, Macron Fine Chemicals™,
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Lit Number: U-PP-BakerbondSPECoulumnInstructions-10V1-E-8006
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