AppliCations No.

6
Size-Exclusion Chromatography
for purification of biomolecules
Size-exclusion chromatography (SEC) is a popular method to separate biomolecules
based on their size. Primarily, it is applied to the separation of biopolymers such as
proteins and nucleic acids, i.e. water-soluble polymers. This system is also called gel
filtration, typically with beads of dextran or agarose serving as gel matrix. Smaller
molecules pass significantly slower through the column than larger molecules. Not to
be mixed up with gel electrophoresis, there are big differences in terms of the
separation principle. SEC does not require electric current and the sieving effect will
not separate small molecules first.

Keywords It is indeed correct that smaller molecules pass more slowly through the matrix than larger molecules. This is due to the
longer path the smaller molecules must travel. The longer path arises from the pores of the beads. The smaller molecules can
Gel filtration matrix enter the pores and go inside the beads. Entering the pores creates a longer path for the smaller molecules. There is a
­relationship between the delay this causes and the molecular size. The larger molecules can not enter the pores and­
cross-linked
therefore have a shorter path. The larger molecules therefore travel faster through the column than smaller molecules.
dextran beads

Desalting Advantages of SEC/Gel Filtration

Some important features of the technique have to be mentioned. First, that the separation of the large molecules from the
Buffer exchange small ones is very effective and is performed under mild conditions does mean that biomolecules retain their biological
­activity. The volume of the eluate may be kept small and therefore the technique is suited for concentrated samples.
Nucleic acid/
protein purification The Matrix
AppliChem‘s size-exclusion chromatography columns are packed with AppliXchange-G25 M, a beaded composite material
Dye terminator removal
composed partially of polymerized dextran. It exhibits high selectivity, high resolution and chemical stability. Molecules
purified with AppliXchange-G25 M are separated according to size. The chemical interaction of gel matrix and molecules to
be separated is negligible. Little or no absorption or binding takes place. Buffer and pH effects on resolution are kept minimal.
Therefore, buffer conditions, pH value and temperature during the filtration process may be adapted according to the needs
of the molecules but not the column material. Essential co-factors or ions necessary for the function or stability of proteins
may be included in the eluent as well, since they largely don‘t interfere with the separation. The size exclusion cut-off for
AppliXchange-G25 M is set at 10 kD for proteins and 10 bp for nucleic acids. Purified biomolecules are not significantly
­diluted when processed using AppliXchange-G25 M.
Experience shows that columns may be used repeatedly, up to four times. It is essential to include an additional washing step
of the columns with elution buffer after separation to remove residual molecules.
The number “25” correlates to the “water regain” of the matrix. A grade “25” means that the dry matrix will take up 2.5
times its weight of water (1 g AppliXchange-G25 M absorbs 2.5 g water). This is a water regain of 2.5. Likewise, a grade of
“50” gives a water regain of 5 grams (1 g AppliXchange-50 plus 5.0 g water.) The ability to absorb water is dependent on the
extent of cross-linking. Tighter cross-linking limits the ability of the beads to swell Instructions for Hydration (Bulk)
and as a consequence the water regain is lower. Less cross-linking gives a more AppliXchange-G25 M is provided as a dry powder. Before use, it must be hydrated.
flexible bead and allows additional swelling. The extent of cross-linking has been Do not stir the hydrating AppliXchange excessively during hydration, as this will
optimized for a particular water regain. The water regain/extent of cross-linking damage the beads. Do not use magnetic stirrers.
affects the separation properties of the gels. Larger water regain/less cross-linking AppliXchange-G25 M medium will swell to a volume of approximately 5 ml for
results in larger pores, which in turn retains larger molecules. Less water regain/ every one gram of dry AppliXchange-G25 M. As a general rule, use 6.5 to 10 ml
more cross-linking results in smaller pores, which in turn retains only the smallest hydration buffer or water for every gram AppliXchange-G25 M that is to be
molecules. The separation efficiency is ultimately dependent on the AppliXchange ­hydrated.
grade, flow rate, column diameter, column length, buffer eluent and size ­difference Choose the bed volume required for your application, determine the number
of the sample components. of grams of AppliXchange-G25 M required and then determine the volume of
The superfine, fine, medium and coarse designations describe the average size of ­hydration buffer or water required.
a fraction of bead diameters. Superfine refers to 20 – 50 µm diameters, fine 20 – 80 Hydrate AppliXchange-G25 M at room temperature for 3 hours or at 90°C for
µm, medium 50 – 150 µm, coarse 150 – 200 µm, where 80 % of the fraction is 1 hour. Hydrated AppliXchange-G25 M may be sterilized at neutral pH by
within the given diameter range. The average diameter distribution affects the ­autoclaving for 30 minutes at 120°C. For best results, the hydrated slurry should
flow rate through the medium. Typically, plates use superfine, spin columns use be degassed before use.
superfine or fine, smaller gravity columns use medium and larger columns use For re-use, the hydrated gel can be washed with 2 column-volumes of 0.2 M NaOH,
medium or coarse. rinsed with 2 column-volumes water, and re-equilibrated with 2 – 3 column
­volumes of buffer.
DextraSEC NA10 and DextraSEC PRO10 For storage, antimicrobial agents should be added to the suspension to prevent
“NA” stands for nucleic acids, “PRO” for protein. The number “10” correlates to contamination (0.001 % phenyl mercuric salts, 0.005 % thimerosal, 0.05 %
the volume of the sample to be processed, i.e. columns are for the purification of ­chlorobutanol, 0.002 % chlorhexine, 0.02 % sodium azide, or 20 % ethanol are
1.0 ml nucleic acid / protein sample. Typically, oligonucleotides are purified from acceptable). Hydrated AppliXchange-G25 may be stored at room temperature
salts, ammonia, and other small molecules. For example, after a labeling reaction (preservative included!) or at +2 to +4°C. Do not freeze! Allow the gel to­
with biotin or a dye, the excess biotin or dye is removed by using this column. The equilibrate to room temperature before use.
oligonucleotide size should be 10 bp or more to obtain satisfactory recovery.
­Comparably, proteins may be desalted or purified from dye. The protein size
should be 10 kD or more to obtain satisfactory recovery.
Instructions for Column Handling
The DextraSEC products are available both in column or plate (96- or 384-well
plates) formats.
1. Column Preparation
Remove the red cap from the top and then the bottom cap of
Stability of the Matrix
the DextraSEC NA10 Column. Allow excess column fluid
The issue is one of unwanted microbiological growth and not a physical problem
to drain (via gravity) into a suitable waste reservoir.
associated with the AppliXchange. The hydrated matrix may be stored at room
temperature indefinitely when hydrated in the presence of a preservative, such as
sodium azide, thimerosal, or other commercially available biocides. We use
Proclin300.
2. Column Equilibration
If the AppliXchange matrix is hydrated with water, or worse, in PBS without a
Choose a buffer for your specific application and use this
preservative, significant and visible bacterial or fungal growth will eventually be
same buffer for both equilibration and elution steps. To
observed. When this will occur depends on the conditions in which the­
equilibrate the column, allow the equilibration buffer to
AppliXchange was hydrated, the cleanliness of the containers used, the purity of
enter the gel bed completely and continue elution until
the water or buffers, etc. Since these are all uncontrolled conditions, we
­approximately 15 ml of buffer has been eluted.
recommend to store rehydrated AppliXchange-G25 M (without preservative) at
2 to 8°C for no longer than one week.
We have hydrated AppliXchange-G25 M using very clean glassware, highly ­purified
and sterile water, and then stored at 6°C without observing any bio-growth for over
3. Sample Application
6 weeks. We have also stored the same for up to 2 to 3 days (weekend) at room
Transfer up to 1 ml of your sample to the DextraSEC NA10
temperature without problems. Please note that phosphates act like fertilizers. If
Column. Allow the sample to enter the gel bed completely.
desired, PBS is a fine hydration and/or elution buffer. But the columns should not
If the sample volume is less than 1 ml, add enough equili-
be stored at room temperature in PBS without preservatives!
bration buffer so that the combined volume equals 1 ml
before ­applying it to the column.
Overload
“Load” can refer to either the sample volume or concentration. Too much of
either will overload the column and this incorrect use of the product can be
4. Elution
­corrected by simply reducing the load. Do NOT add more volume than recommen-
Place a tube for sample collection under the DextraSEC
ded and do NOT add an “unreasonably” high concentration of impurities and/or
NA10 Column. Transfer 1.5 ml of elution buffer to the
the molecules to be purified. This will lead to a less than satisfactory purification.
­column and elute the cleaned sample.
If there is an exact, definable application, then we may provide a maximum
allowed concentration and sample volume on request. Without this, you may
have to determine your own parameters for your particular application.
Protein Purification Oligonucleotide Purification

Conductivity
Absorption

Absorption

Conductivity
0,0 1,0 2,0 3,0 4,0 5,0 6,0 7,0 0,0 1,0 2,0 3,0 4,0 5,0 6,0 7,0
Elution volume [ml] Elution volume [ml]

Protein Desalting from 0.8 M NaCl Oligo Desalting from 0.8 M NaCl
Protein (280 nm): black line NaCl (µS): pink line Oligo (260 nm): black line NaCl (µS): pink line
1 mg IgG anti-Rabbit in 1 ml 0.8 M sodium chloride. Elution with pure water. 1 mg oligonucleotide (18-mer) in 1 ml 0.8 M sodium chloride.
Absorption

Absorption

0,0 1,0 2,0 3,0 4,0 5,0 6,0 7,0 0,0 1,0 2,0 3,0 4,0 5,0 6,0 7,0
Elution volume [ml] Elution volume [ml]

Removal of FITC from IgG Removal of Rhodamine from Oligonucleotide Labelling

IgG (280 nm): black line FITC (490 nm): pink line Oligo (260 nm): black line TAMRA (550 nm): pink line
1 mg IgG anti-Rabbit and 0.1 µmol FITC in 1 ml DMSO/NaHCO3. Removal of excess FITC 1 mg oligonucleotide (18-mer) and 0.5 µmol TAMRA in 1 ml DMSO/NaHCO3.
after coupling reaction. Elution with PBS. Removal of excess TAMRA after coupling reaction.

14
14

13
13
1,00
12
12
Absorption
Absorption

11
11
pH
pH

10
10

9
9

8 8

0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5 4,0 0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5 4,0
Elution volume [ml] Elution volume [ml]

Removal of ammonia (33 % NH3) from Dextran Blue Removal of ammonia (33 % NH3) from Nucleic acid
Dextran Blue (620 nm): black line NH3 (pH): pink line Oligo (260 nm): black line NH3 (pH): pink line
0.5 mg Dextran Blue (Mr 2,000,000) in 1 ml ammonia (33 %). Elution with pure water. 1 mg oligonucleotide (18-mer) in 1 ml ammonia (33 %)
after cleavage from solid support and protection group removal.
 A95,E

Typical Analytical Data of AppliXchange-G25 M

CAS Number 9041-35-4

HS No. 39139000990
Appearance white solid
Fractionation range (globular proteins) – Mr 1000 – 5000
Fractionation range (dextrans) – Mr 100 – 5000
Bead structure Cross-linked dextran composite
Bead size (Dry Particle Size) 50 – 150 µm
Bead size (Wet) 85 – 260 µm
Bed Volume in Water 4 – 6 ml/g
Maximum operating pressure Obeys Darcy’s Law
Chemical stability All commonly used buffers, including:
0.2 M NaOH; 0.2 M HCl; 1 M Acetic acid;
8 M Urea; 6 M Guanidine HCl; 1 % SDS,
24 % Ethanol; 30 % Propanol;
30 % Acetonitrile.
pH stability 2.0 to 13.0,
Autoclavable at 121°C, pH 7 for 30 minutes
Storage ambient (Do not freeze!)

High Performance Results: • Over 90 % recovery

• Easy-to-use
• High chemical stability
• High resolution

Description Product No. Quantity Sample Volume

DextraSEC NA10 A8870,0050 50 Columns 0.5 – 1 ml
DextraSEC PRO10 A8822,0050 50 Columns 0.5 – 1 ml
DextraSEC 96W A8595,0296 2 Plates max. 15 µl/well*
DextraSEC 96W A8595,2596 25 Plates max. 15 μl/well*
DextraSEC NA2 A8590,0050 50 Columns 0.15 – 0.25 ml
DextraSEC PRO2 A8710,0050 50 Columns 0.15 – 0.25 ml

coming soon: DextraSEC 384W 384-Well Gel Filtration Plates

for sample volumes of max. 10 µl

*Main application: dye terminator removal in automated DNA sequencing procedures

09/2009
4t Matthes + Traut · Darmstadt

Germany AppliChem GmbH | Ottoweg 4 | D-64291 Darmstadt | Phone +49 6151 93 57-0 | Fax +49 6151 93 57-11 | eMail [email protected] | Internet www.applichem.com
Asia AppliChem Asia Pte Ltd | TradeHub 21 8 Boon Lay Way #03-10 | Singapore 609964 Singapore | Phone +65 65154616 | Fax +65 65150220 | eMail [email protected]
USA AppliChem, LLC | 3998 FAU Blvd., Suite 210 | Boca Raton, FL 33431 USA | Phone +1 561 750 6120 | Fax +1 561 750 6140 | eMail [email protected] | Internet www.applichem.us