J. Biochem. Biophys.

Methods 64 (2005) 216 – 225

www.elsevier.com/locate/jbbm

Purification of a synthetic oligonucleotide by anion

exchange chromatography: Method optimisation
and scale-up
Jamil Shanagar *
GE Healthcare, Amersham Biosciences AB, SE-751 84 Uppsala, Sweden
Received 2 June 2005; received in revised form 11 August 2005; accepted 13 August 2005

Abstract

A single-step chromatographic method for purification of a synthetic 20-mer oligonucleotide is

described. Method optimisation was conducted at laboratory scale where 30 mg crude sample was purified
per run with a yield of 17 mg pure oligonucleotide. The protocol was scaled-up in steps to achieve 5-, 58-
and a final 230-fold scale-up. At the final scale, 7.0 g of crude material was purified with a yield of 4.1 g
product. The purity of the oligonucleotide was in all scales higher than 97%. The cycle time was 110 min,
which corresponds to a purification capacity of about 90 g crude oligonucleotide material per 24 h.
D 2005 Elsevier B.V. All rights reserved.

Keywords: SOURCEk 15Q; RESOURCEk Q; Polymer matrices; Anion exchange chromatography; HPLC;
Oligonucleotide; Scale-up

1. Introduction

Synthetic oligonucleotides (SON) are short single-stranded DNA sequences consisting of a

number of nucleotide units linked together by phosphodiester bridges. They are primarily
destined for applications in antisense drug therapy but also have wide applications in molecular
biology and other life sciences [1–5].
The growing antisense market challenges oligonucleotide manufacturers to develop cost-
effective and scalable processes.
SONs are produced on a solid support consisting of cross-linked polystyrene beads by
phosphoramidite chemistry [6]. The idea of synthesizing oligonucleotides on solid support was

* Tel.: +46 18 6120708; fax: +46 18 6121844.

E-mail address: jamil.shanagar@ge.com.

0165-022X/$ - see front matter D 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.jbbm.2005.08.004
 J. Shanagar / J. Biochem. Biophys. Methods 64 (2005) 216–225 217

first introduced by Letsinger [7], in an analogous fashion with MerrifeldsT work on peptides [8].
With the use of ÄKTAk oligopilot DNA/RNA Synthesizer, SONs can be produced with high
coupling efficiency [9]. However, the product obtained contains many impurities and short
failure sequences. Since highly pure SONs are a prerequisite for therapeutic and other
applications, purification is required.
Most purification procedures are based on either reversed phase chromatography (RPC) [10–
13] or anion exchange chromatography (AEC) [14,15].
AEC is an attractive technique for the purification of synthetic oligonucleotides because the
separation is performed in aqueous conditions without the use of high cost eluents, at low to
moderate operating pressures. Furthermore, during the purification process, it is of vital
importance to avoid aggregation of self-complementary or GC-rich oligonucleotides [10]. It is,
therefore, preferred to perform the purification in mild alkaline conditions. Attempt to optimize
separation of oligonucleotides and DNA using computer simulations modeling methods has
been reported [16]. The modeling based optimization method offers a more effective manner to
predict the elution behaviour and also offers significant time and cost saving over trial-and-error
approaches [16].
SOURCEk 15Q (GE Healthcare) is a polymer-based anion exchanger consisting of 15 Am
monosized beads. This anion exchanger has several advantages, such as a wide operating pH-
range, low back-pressure and the good recovery making the medium ideal for use in process-
scale purification of oligonucleotides [15]. Furthermore, the high pH stability of SOURCE 15Q
allows purification of oligonucleotides under denaturing conditions at pH 12. It is thus possible
to capture and purify oligonucleotides directly from the crude ammonia solution obtained after
cleavage of the oligonucleotide from the synthesis support.
The objective of this work is to develop a single-step chromatographic protocol for
purification of oligonucleotides with high purity and recovery using SOURCE 15Q medium. It
is also aimed to show the successful scalability of the developed method from a small lab-scale
to a preparative scale for purification of large amount of oligonucleotides.

2. Materials and methods

2.1. Oligonucleotide sample

The oligonucleotide with a sequence of 5V-ATA CCG ATT AAG CGA AGT TT-3V used in
this study was synthesized with an ÄKTA oligopilot DNA/RNA Synthesizer (GE Healthcare)
using its trityl-off mode [6,9]. Purity of the full-length oligonucleotide product (FLP) in the
crude reaction mixture was 68% after cleavage from the synthesis support. This sample was
applied directly onto the anion exchanger columns without any pretreatment.

2.2. Chromatography purification systems

All runs were done with columns packed with SOURCE 15Q, using ÄKTAexplorerk 10,
ÄKTAexplorer 100 or ÄKTApilotk systems (GE Healthcare). The ÄKTAexplorer systems were
comprised of a model P-900 HPLC pump, a model UV-900 monitor, a model pH/C-900 pH/
conductivity monitor and UNICORNk software system control. The column effluent from
ÄKTAexplorer systems was fractionated using a Frac-950 Fraction Collector (GE Healthcare)
although the effluent from ÄKTApilot system was collected manually. The volume of each
fraction collected in all the preparative purification experiments was 0.5 column volume (CV).
218 J. Shanagar / J. Biochem. Biophys. Methods 64 (2005) 216–225

The chromatographic conditions are described in Section 3.1 and in the legend for each
respective Figure.

2.3. Materials and columns

The SOURCE 15Q stationary phases used for the purification of oligonucleotide were
obtained from GE Healthcare in prepacked analytical columns (RESOURCE Q and SOURCE Q
PE 4.6/100) and in bulk packs. For scale-up studies, the SOURCE 15Q bulk medium was
packed in Tricornk (i.d. 10 mm), FineLINEk Pilot 35 (i.d. 35 mm), and FineLINE 70 (i.d. 70
mm) columns from GE Healthcare. Details on packing of the columns are described in the next
section). For desalting of the purified material, a HiPrepk 26/10 Desalting Column (GE
Healthcare) was used. All other chemicals used in these studies were of analytical grade and
were purchased from Merck (Darmstadt, Germany).

2.4. Packing and evaluation of columns

2.4.1. Tricorn column

The column was packed with SOURCE 15Q media using a constant flow rate of 10 ml/min.
The column was connected to a packing tube [17] and subsequently connected to the
ÄKTAexplorer 10 system. An appropriate amount of medium required for packing a 10 cm bed
height was slurried in 50% ethanol at a ratio of 1 ml sedimented medium/ml solvent (50%
ethanol) and loaded into the packing tube. Ethanol (solution of 50%) was then pumped through
the system at a flow rate of 10 ml/min until the packing was completed.

2.4.2. FineLINE columns

The FineLINE Pilot 35 and FineLINE 70 columns were packed with the SOURCE 15Q media
to bed heights of 10 cm using the axial compression technique [18]. The medium was slurried with
50% ethanol at a ratio of 1 ml sedimented medium/ml solvent. Typically, 100 and 400 ml
sedimented medium were slurried for the FineLINE Pilot 35 and FineLINE 70 columns,
respectively. The slurry was poured into the column and then the adaptor was mounted and lowered
to the top of the slurry. The column was then packed by pumping a solution of 20% ethanol through
the hydraulic chamber at a constant pressure of 10 bar. Thus, the adaptor unit started moving down
as the medium packed. When the adaptor unit stopped moving downwards and stabilized at its
lowest position, the adaptor unit was locked with the locking bar and locking screws.
The efficiency of the packed columns was evaluated with NaCl as a test probe. The columns
were equilibrated with a solution of 0.4 M NaCl at a flow rate of 75 cm/h. A solution of 1% of
bed volume containing 0.8 M NaCl was introduced into the column with a loop injector. The
conductivity curve was recorded and the plate count was determined. Plate counts of 20 000–22
000 plates/m and asymmetries of 1.0–1.3 were obtained for the columns.

2.5. Quantification of oligonucleotide

The oligonucleotide was detected by monitoring UV absorbance at 260 nm. The fractions
were diluted properly to obtain absorbance into the linear response range. The mass estimates of
the oligonucleotide was calculated from spectrophotometric absorbance measurements at 260
nm. The measured optical density (OD)/ml was converted into mg/ml values by using the factor
of 1 OD = 0.033 mg oligonucleotide.
 J. Shanagar / J. Biochem. Biophys. Methods 64 (2005) 216–225 219

2.6. Analytical characterisation

Analyses of the collected fractions (from the purification experiments) and the crude
oligonucleotide material were performed by anion exchange HPLC on a non-porous medium
(DNAPack PA-100 analytical column [4/250 mm]) from Dionex Corporation, Sunnyvale, CA,
USA) and/or by capillary electrophoresis [19].
The anion exchange HPLC analysis was conducted on Hewlett-Packard (Palo Alto, CA,
USA) Model 1100 liquid chromatography fitted with the DNAPac column, thermostatted to 25
8C and operated at a flow rate of 480 cm/h. The column was equilibrated with 5 column volumes
(CVs) of the equilibration buffer of 90% A and 10% B (A = 0.001 M Tris containing 0.01 M
NaClO4; pH 9.3 and B = 0.001 M Tris containing 0.3 M NaClO4; pH 9.3). Twenty-five
microliters of each fraction was injected into the column and eluted with a 7.6 CVs linear
gradient from 10% to 70% B. The column effluent was monitored at 260 nm.
All capillary electrophoresis experiments were performed using a 20 cm (effective length 18
cm)  50 Am i.d. capillary. The capillary was treated with PlusOnek Bind-Silane (GE
Healthcare) and filled with polyacrylamide cyclodextrin gel. Buffer was consisted of 0.1 M Tris-
0.25 M boric acid, pH 8.5, containing 7 M urea. Injection was made at 1 kV for 5 s. Analysis
was performed at 1.5 kV for 30 min.

3. Results

3.1. Method optimisation

Method scouting was first carried out using a small sample load (10 Ag crude oligonucleotide)
on a 1 ml RESOURCEk Q column (GE Healthcare), which has a bed height of 30 mm. The
column was equilibrated with solution A (0.01 M NaOH, pH 12). Elution of the sample was
performed with gradient formed with solutions A and B (0.01 M NaOH, pH 12 containing 2 M
NaCl). Different start conditions and elution profiles, which made up of steps and linear
gradients were tested to optimize the purification. Both the start conditions and slope of the
gradient were varied until an acceptable resolution was achieved. The optimal condition was
found to be a 40 CVs linear gradient from 15% to 35% B. This corresponded to 0.30 to 0.70 M
NaCl in a 0.01 M NaOH solution, pH 12 (see Fig. 1a–b). To further optimise the method, the bed
height was increased to 100 mm using a SOURCE Q PE 4.6/100 column. Except for the change
in bed height, all chromatographic conditions were kept the same as in the 30 mm bed height
column. Fig. 1C shows the resulting chromatogram where resolution has been further improved.
Fractions from each experiment were collected and the fractions of interest were analysed on the
DNAPac PA-100 analytical column to determine the purity. Fig. 1d shows a purity of 99%
achieved with SOURCE Q PE 4.6/100 column.

3.2. Frontal analysis

To determine the binding capacity for the oligonucleotide, a frontal analysis experiment was
carried out on the RESOURCE Q column. The column was equilibrated with 5 CVs of 0.01 M
NaOH solution. The crude oligonucleotide sample was diluted to a concentration of 1.5 mg/ml
with the same equilibration solution and was applied continuously into the column at a flow rate
of 360 cm/h until the column was saturated. Thus, the effluent UV absorbance at 300 nm was
similar to that of the applied sample. Fig. 2 shows the resulting breakthrough curve. From this
220 J. Shanagar / J. Biochem. Biophys. Methods 64 (2005) 216–225

a b
mAU mAU
RESOURCE Q 1 ml RESOURCE Q 1 ml
150 0-50% B in 30 cv 150 15-35% B in 40 cv

100 100

50 50

0 0

0.0 5.0 10.0 15.0 20.0 25.0 30.0 min 0.0 10.0 20.0 30.0 40.0 50.0 min

c d
mAU SOURCE Q PE 4.6/100
15-35% B in 40 cv

mAU
150 DNAPac PA-100
(4/250) analytical
column
15.0
100

10.0

50
5.0

0.0
0

0.0 10.0 20.0 30.0 40.0 50.0 60.0 70.0 min 8.0 9.0 10.0 11.0 12.0 13.0 14.0 min

Fig. 1. a–c. Effect of gradient shape and slope on the resolution of oligonucleotide. Sample: Crude oligonucleotide 20-
mer (10 Ag). Flow rate: 360 cm/h. Solution A: 0.01 M NaOH, pH 12. Solution B: 0.01 M NaOH + 2 M NaCl, pH 12. a)
Column: RESOURCE Q 1ml. Gradient,: 0–50% B in 30 column volumes. b) Column: RESOURCE Q 1ml. Gradient:
15–35% B in 40 column volumes. c) Column: SOURCE Q PE 4.6/100. Gradient: 15–35% B in 40 column volumes.
Fig. 1d. Analysis of a fraction collected from the main peak in Fig. 1c on the DNAPac PA-100 (4/250) analytical column.
Column: DNAPac PA-100 (4/250). Flow rate: 480 cm/h. Volume injected: 25 Al. Buffer A: 0.001M Tris + 0.01M NaClO4.
Buffer B: 0.001M Tris + 0.3 M NaClO4.Gradient: 10–70% B, 7.6 CVs.

curve, the capacity of the SOURCE Q media was calculated to be 30 mg oligonucleotide /ml
medium.

3.3. Preparative purification

The SOURCE Q PE 4.6/100 column was used in a small lab-scale preparative purification of
the oligonucleotide. Since the result from frontal analysis measurement showed that capacity of
the medium was 30 mg oligonucleotide/ml, it was decided to load the medium with the
 J. Shanagar / J. Biochem. Biophys. Methods 64 (2005) 216–225 221

mAU

800

600

400

200

0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 min

Fig. 2. Breakthrough curve for the crude 20-mer oligonucleotide. Column: RESOURCE Q 1 ml (6.4[ i.d].  30 mm).
Flow rate: 360 cm/h. Sample: 20-mer oligonucleotide (1.5 mg/ml in 0.01M NaOH, pH 12). Detection: UV-300 nm.

oligonucleotide at 60% of the dynamic binding capacity. In this case, 30 mg crude

oligonucleotide, corresponding to 18 mg oligonucleotide/ml medium was applied at a flow rate
of 360 cm/h. To secure the maximum binding of the oligonucleotide to the matrix at this high load
the sample application was done on the column, which was equilibrated with solution A (0.01 M
NaOH, pH 12 in the absence of NaCl). The unbound material was washed from the column with 5
CVs of the equilibration buffer (0.01 M NaOH, pH 12) and followed by a 40 CVs linear gradient
from 15% to 35% B (0.01 M NaOH, pH 12 containing 2 M NaCl), corresponding to 0.3 to 0.7 M
NaCl in 0.01 M NaOH. The yield was 17 mg 20-mer oligonucleotide with a purity of 97% and a
recovery of 83% (see Fig. 3a and Table 1). The same load was used during scale-up.

3.4. Scale-up

A 5-, 58- and 230-fold scale-up was performed by increasing the column diameter while
keeping other parameters such as linear flow rate, sample load/ml medium and column bed
height constant.
A Tricorn column (10 mm inner diameter), a FineLINE Pilot 35 column (35 mm inner
diameter) and a FineLINE 70 column (70 mm inner diameter) were packed with 7.85, 96 and
385 ml of SOURCE 15Q media, respectively and used for the scale-up experiments.
Fig. 3 shows the scale-up results of the purification of the 20-mer oligonucleotide from a
sample load of 18 mg/ml medium. When the protocol was scaled-up 5-fold, 141 mg crude
material was processed giving a yield of 70 mg. The 58-fold scale-up was performed using the
same method but on ÄKTAexplorer 100 system. From 1 825 mg crude sample a product yield of
980 mg was obtained. At the final scale using ÄKTApilot system, 7.0 g of crude material was
purified in one run resulting in a yield of 4.1 g product. This corresponds to a 230-fold scale-up
from the small laboratory scale. The final yield and the purities are shown in Table 1. These
results correspond well to the result achieved with the small lab-scale column (1.66 ml
prepacked SOURCE Q PE 4.6/100) and clearly demonstrate the scalability of the method. The
cycle time was 110 min, which corresponds to a purification capacity of about 90 g crude
oligonucleotide material per 24 h using a FineLINE 70 column.
The purity of the oligonucleotide purified by analytical anion exchange chromatography and
capillary electrophoresis was greater than 97% at all scales used. Fig. 4 shows an example of
analysis by capillary electrophoresis of the crude oligonucleotide and the pooled fractions from
the separation of the 20-mer oligonucleotide, shown in Fig. 3c.
 a b

222
mAU
2500

2000
mAU

1500 2500

2000

J. Shanagar / J. Biochem. Biophys. Methods 64 (2005) 216–225

1000
1500

1000
500 I II
500 I II

0 0
0.0 10.0 20.0 30.0 40.0 50.0 60.0 70.0 80.0 min 0.0 10.0 20.0 30.0 40.0 50.0 60.0 70.0 80.0 min
c d
mAU
mAU
3000

2500 4000

2000 3000

1500
2000
1000
1000
500
I II I II
0 0
0.0 10.0 20.0 30.0 40.0 50.0 60.0 70.0 80.0 min 0.0 10.0 20.0 30.0 40.0 50.0 60.0 70.0 min

Fig. 3. Scale-up of the optimised purification method for 20-mer oligonucleotide using 1.66, 7.85, 96 and 385 ml columns packed with SOURCE 15Q. Sample: Crude
oligonucleotide 20-mer. Flow rate: 360 cm/h. Solution A: 0.01 M NaOH, pH 12. Solution B: 0.01 M NaOH, pH 12 + 2 M NaCl. Gradient: 15–35% B in 40 column volumes.
a) Column: SOURCE Q PE 4.6/100 (1.66 ml). Sample load: 30 mg. b) Column: Tricorn (10/100 mm) packed with SOURCE 15Q (7.85 ml). Sample load: 141 mg. c) Column:
FineLINE Pilot 35 (35/100 mm) packed with SOURCE 15Q (96 ml). Sample load: 1825 mg. d) Column: FineLINE 70 (70/100 mm) packed with SOURCE 15Q (385 ml). Sample
load: 7000 mg.
 J. Shanagar / J. Biochem. Biophys. Methods 64 (2005) 216–225 223

Table 1
Comparison of yield and purity at different scales
Sample Yield Recovery Purity
load (mg) (mg) (%) (%)
Small lab-scale (SOURCE Q PE 4.6 / 100 mm) 30 17 83 97
5-fold scale-up (SOURCE 15Q [10 / 100 mm]) 141 70 73 97
58-fold scale-up FineLINE Pilot 35 (SOURCE 15Q [35 / 100 mm]) 1825 980 80 97
230-fold scale-up FineLINE 70 (SOURCE 15Q [70 / 100 mm]) 7000 4067 85 97

3.5. Desalting of the purified oligonucleotide

After purification of the 20-mer oligonucleotide by AEC, the purified material contained a
high concentration of salt. The salt was removed from the purified 20-mer by a desalting
method using Milli-Qk ultrapure water (Milliporek Corporation, Billerica, USA) as eluent. A

a b
mAU mAU

2.5 2.5

Crude material Pool I

2.0 2.0

1.5 1.5

1.0 1.0

0.5 0.5

0 0
0 5 10 15 min 0 5 10 15 min

c
mAU
2.5

Pool II
2.0

1.5

1.0

0.5

0
0 10 20 min

Fig. 4. Analysis by capillary electrophoresis from the separation of the 20-mer oligonucleotide shown in Fig. 3c.
Capillary: 50 Am  18 cm filled with polyacrylamide cyclodextrin gel. Buffer: 0.1 M Tris, 0.25 M borate/7 M urea.
Running conditions: 1.5 kV/30 min. Sample application: 1 kV/5 s. a) Crude material. b) The eluted failure sequences
(pool I in Fig. 3c). c) The purified 20-mer oligonucleotide (pool II in Fig. 3c).
224 J. Shanagar / J. Biochem. Biophys. Methods 64 (2005) 216–225

mAU

2500

2000

1500

1000

500

0
0.0 10.0 20.0 30.0 40.0 50.0 60.0 70.0 ml

Fig. 5. The elution profile of oligonucleotide desalting. Column: HiPrep 26/10 Desalting. Flow rate: 60 cm/h. Eluent:
Water. Sample: 15 ml purified 20-mer oligonucleotide. Detection: UV at 260 nm (—) and conductivity measurement (–).

HiPrep 26/10 desalting column was employed for desalting 15 ml of the purified 20-mer
oligonucleotide per run. The oligonucleotide was detected by monitoring UV absorbance at
260 nm and the salt by conductivity measurements. The recovery from the desalting was 98%
(see Fig. 5).

4. Discussion

The development of a single-step chromatographic purification method of oligonucleotide

requires a large number of experiments under varied conditions. Therefore, method scouting was
carried out on 1 and 3 ml prepacked SOURCEk Q columns using analytical load (10 Ag crude
oligonucleotide). Different gradient elutions were tested until the desired result in terms of
resolution and purity was achieved. The optimal condition was found to be a 40 CVs linear
gradient from 0.30 to 0.70 M NaCl in a 0.01 M NaOH solution, pH 12 on a column with a bed
height of 100 mm (see Fig. 1).
These conditions, except the load, were then used in a small lab-scale preparative and
scaled-up purification experiments. In the preparative purification experiments a load of 60%
of the dynamic binding capacity, which was corresponding to 18 mg oligonucleotide/ml
SOURCE Q medium was used. In a single run 30 mg crude oligonucleotide was applied and
resulted in a yield of 17 mg product with a purity of 97% and a recovery of 83% (see Fig. 3a
and Table 1).
This protocol was scaled-up in steps to achieve 5-, 58- and a final 230-fold scale-up
by increasing the diameter of the column but keeping the bed height constant (see
Section 3.4). This allows the sample to have the same residence time in the packing and the
same chance to interact with packing materials at all scales using the same linear velocity. The
final yield and the purities are shown in Table 1. It can be seen that at the 230-fold scale-up
experiment 7.0 g of crude material was purified in one single run resulting in a yield of 4.1 g
product with a purity grater than 97%. These results are very high compared to the small lab-
scale column (1.66 ml prepacked SOURCE Q PE 4.6/100) and clearly demonstrate the
scalability of the method.
Desalting of a small volume (15 ml) of the purified oligonucleotide using HiPrep 26/10
desalting column showed a high recovery (98%) and good efficiency (see Fig. 5). However,
desalting of larger quantities can be performed on a larger column packed with the same
desalting media since it is easily scalable.
 J. Shanagar / J. Biochem. Biophys. Methods 64 (2005) 216–225 225

5. Simple description of the method and its application

A single-step lab-scale chromatographic purification of an oligonucleotide was developed,

using SOURCE 15Q matrix, and was then scaled-up. The high pH stability of this matrix
allowed purification of oligonucleotide, directly from the crude ammonia solution under
denaturing conditions at pH 12. Using the 230-fold scaled-up method seven grams crude
oligonucleotide was purified per cycle with a recovery of 85% and a purity higher than 97%. The
developed method can be further scaled-up for production of larger quantities.
In this method one cycle takes less than 2 h, which corresponds to a purification capacity of
about 90 g crude oligonucleotide per day using a FineLINE 70 column.

Acknowledgments

The author is grateful to Israel Solomon for performing some chromatographic optimisation
experiments. Dr Akos Vegvari, BMC, University of Uppsala, is sincerely appreciated for the
HPCE analyses.

References

[1] Matsukura M, Shinozuka K, Zon G, Mitsuya H, Reitz M, Cohen JS, et al. Proc Natl Acad Sci U S A
1987;84:7706 – 10.
[2] Matsukura M, Zon G, Shinozuka K, Robert-Guroff M, Shimada T, Stein CA, et al. Proc Natl Acad Sci U S A
1989;86:4244 – 8.
[3] Shaw JP, Kent K, Bird J, Fishback J, Froehler B. Nucleic Acids Res 1991;19:747 – 50.
[4] Miller PS. Biotechnology (N Y) 1991;9:358 – 61.
[5] Zamecnik PC, Stephenson ML. Proc Natl Acad Sci U S A 1978;75:280 – 8.
[6] Beaucage SL, Caruther MH. Tetrahedron Lett 1981;22:1859 – 62.
[7] Letsinger RL, Mahadevan V. J Am Chem Soc 1965;87:3526 – 7.
[8] Merrifeld RB. J Am Chem Soc 1963;85:2149 – 54.
[9] GE Healthcare. ÄKTA oligopilot DNA/RNA Synthesizer. Data file 18-1144-66 AC (2003).
[10] Arghavani MB, Romano LJ. Anal Biochem 1995;231:201 – 9.
[11] Hoffman E, Liao JC. Anal Chem 1977;49:2134 – 231.
[12] Gilar M, Bouvier ESP. J Chromatogr A 2000;890:167 – 77.
[13] Gilar M. Anal Biochem 2001;298:196 – 206.
[14] Drager RR, Regnier FE. Anal Biochem 1985;145:47 – 56.
[15] Deskmukh R, Cole DL, Sanghvi YS. Methods Enzymol 1999;313:203 – 26.
[16] Simantini D, Satish CM, James TH. J Liq Chromatogr Relat Technol 2000;23:1809 – 19.
[17] GE Healthcare. Tricorn instructions 56-3154-71 AB, 2002.
[18] GE Healthcare. FineLINE columns user manual 18-1122-88 AD, 2003.
[19] Vegvari A, Hjertén S. J Chromatogr A 2002;960:221 – 7.