Electrophoresis in organogels

Article abstract of DOI:10.1021/ac049606m

A new matrix for electrophoresis, a low molecular weight organogel, is described. Dansylated amino acids and peptides were separated by planar and capillary electrophoresis in acetonitrile gels of trans-(1S,2S)-1,2-bis- (dodecylamido)cyclohexane. The superior separation ability of the organogel over its corresponding buffer solution in capillary electrophoresis is illustrated. Organogels provide all the advantages associated with planar matrixes with 100% efficient recovery and transfer of the analytes to a mass spectrometer. We demonstrate that the planar gel can be liquefied and injected as is into an ESI-MS to identify the separands.

Full text of DOI:10.1021/ac049606m

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Anal. Chem. 2004, 76, 5399-5404
Electrophoresis in Organogels
Shaul Mizrahi, Jenny Gun, Z. Gidon Kipervaser, and Ovadia Lev*
The Casali Institute of Applied Chemistry, The Chemistry Institute, The Hebrew University of Jerusalem,
Jerusalem, 91904, Israel
common. The aggregation occurs through noncovalent interaction,
A new matrix for electrophoresis, a low molecular weight
organogel, is described. Dansylated amino acids and
peptides were separated by planar and capillary electro-
phoresis in acetonitrile gels of tr a n s-(1 S,2 S)-1 ,2 -bis-
(dodecylamido)cyclohexane. The superior separation abil-
ity of the organogel over its corresponding buffer solution
in capillary electrophoresis is illustrated. Organogels
provide all the advantages associated with planar matrixes
with 1 0 0 % efficient recovery and transfer of the analytes
to a mass spectrometer. We demonstrate that the planar
gel can be liquefied and injected as is into an ESI-MS to
identify the separands.
usually though not exclusively hydrogen bonding. Often one
gelator combines several types of interactions such as π-stacking,
van der Waals, and hydrophobic interactions with hydrogen
bonding. The resulting structures are twisted fibers which
intertwine to trap solvent molecules, thus causing gelation.
While much of the focus has been on discovering new gelators
and understanding the process of gelation, very little success has
been recorded in finding applications for these materials. Several
preliminary discoveries about organogels have been made which
will help lead to applications. The ability to incorporate electrolytes
into gels has been shown.6,21 Organogels have been used as
templates for inorganic, sol-gel-derived chiral materials.10,22 They
have been found to have light-scattering properties which may
make them suitable for electrooptical displays.23 Gelators that can
selectively gel one of a mixture of solvents have been found.24,25
This type of gelation may be useful in dealing with oil spills.
Finally, electrolytic cells incorporating gelators have been fabri-
cated.26 Despite this progress, none of the above advances has
led to, to our knowledge, a functional material. In this paper we
describe for the first time the use of an organogel as an
electrophoresis matrix. The inherent thermoreversibility and
thixotropy of organogels can bring about modes of MS coupling
that are unattainable with any other type of solid network.
Electrophoresis, in both its planar and capillary forms, is a
widely used separation technique. One of the challenges in
electrophoresis is identification of the separated compounds. The
advent of capillary electrophoresis allowed for direct interface with
electrospray mass spectrometry (ESI-MS). However, planar elec-
trophoresis, which remains the heart of protein analysis, is not
suitable for direct MS detection. The compounds must be located
by staining, cut out of the slab, extracted from the matrix, usually
For the past decade or so, a new class of gels formed by small
organic molecules has attracted considerable attention.1 These
organogels are formed by self-aggregation of small concentrations
(typically <3%) of the gelator molecule in a solvent. The gels,
which are formed in a variety of organic solvents or water, are
thermoreversible. Upon heating they form liquid solutions. They
are also thixotropic, becoming liquefied upon mechanical agitation.
The exact requirements for gelation are still not known, and it is
not possible to predict whether any particular molecule will form
gels. However, most of the known gelators contain one of a
number of functional groups, the most common among them
being sugars,2-5 amino acids,6-9 amides,10-12 ureas,13-15 and
cholesterols.16-18 Alkanes can serve as gelators as well.19,20 Despite
the wide variety of gelators, they all have certain features in
(1) For recent reviews see: (a) Gronwald, O.; Snip, E.; Shinkai, S. Curr. Opin.
Colloid Interface Sci. 2 0 0 2 , 7, 148-156. (b) Adballah, D. J.; Weiss, R. G.
Adv. Mater. 2 0 0 0 , 12, 1237-1247.
(2) Jung, J. H.; Shinkai, S.; Shimizu, T. Chem.sEur. J. 2 0 0 2 , 8, 2684-2690.
(3) Kobayashi, H.; Koumoto, K.; Jung, J. H.; Shinkai, S. J. Chem. Soc., Perkin
Trans. 2 2 0 0 2 , 1930-1936.
(4) Battacharya, S.; Acharya, S. N. G. Chem. Mater. 1 9 9 9 , 11, 3504-3511.
(5) Yoza, K.; Amanokura, N.; Ono, Y.; Akao, T.; Shinmori, H.; Tackeuchi, M.;
Shinkai, S.; Reinhoudt, D. N. Chem.sEur. J. 1 9 9 9 , 5, 2722-2729.
(6) Hanabusa, K.; Hiratsuka, K.; Kimura, M.; Shirai, H. Chem. Mater. 1 9 9 9 ,
11, 649-655.
(16) Jung, H. J.; Kobayashi, H.; Masuda, M.; Shimizu, T.; Shinkai, S. J. Am. Chem.
Soc. 2 0 0 1 , 123, 8785-8789.
(17) Lin, Y.; Kachar, B.; Weiss, R. G. J. Am. Chem. Soc. 1 9 8 9 , 111, 5542-5551.
(18) Lin, Y.; Weiss, R. G. Macromolecules 1 9 8 7 , 20, 414-417.
(19) Abdallah, D. J.; Weiss, R. G. Langmuir 2 0 0 0 , 16, 352-355.
(20) Abdallah, D. J.; Lu, L.; Weiss, R. G. Chem. Mater. 1 9 9 9 , 11, 2907-2911.
(21) Placin, F.; Desvergne, J. P.; Lassegues, J. C. Chem. Mater. 2 0 0 1 , 13, 117-
121.
(7) Hanabusa, K.; Okui, K.; Karaki, K.; Kimura, M.; Shirai, H. J. Colloid Interface
Sci. 1 9 9 7 , 195, 86-93.
(8) Ragunathan, K. G.; Bhattacharya, S. Chem. Phys. Lipids 1 9 9 5 , 77, 13-23.
(9) Imae, T.; Takahashi, Y.; Muramatsu, H. J. Am. Chem. Soc. 1 9 92, 114, 3414-
3419.
(10) Bied, C.; Moreau, J. J. E.; Vellutini, L.; Wong Chi Man, M. J. Sol-Gel Sci.
Technol. 2 0 0 3 , 26, 583-586.
(22) Jung, J. H.; Ono, Y.; Hanabusa, K.; Shinkai, S. J. Am. Chem. Soc. 2 0 0 0 ,
122, 5008-5009.
(11) Yasuda, Y.; Iishi, E.; Inada, H.; Shirota, Y. Chem. Lett. 1 9 9 6 , 575-576.
(12) Hanabusa, K.; Yamada, M.; Kimura, M.; Shirai, H. Angew. Chem., Int. Ed.
1 9 9 6 , 35, 1949-1951.
(13) Wang, G.; Hamilton, A. D. Chem. Commun. 2 0 0 3 , 310-311.
(14) Estroff, L. A.; Hamilton, A. D. Angew. Chem., Int. Ed. 2 0 0 0 , 39, 3447-
3450.
(23) Mizoshita, N.; Suzuki, Y.; Kishimoto, K.; Hanabusa, K.; Kato, T. J. Mater.
Chem. 2 0 0 2 , 12, 2197-2201.
(24) Trivedi, D. R.; Ballabh, A.; Dastidar, P. Chem. Mater. 2 0 0 3 , 15, 3971-
3973.
(25) Bhattacharya, S.; Krishnan-Ghosh, Y. Chem. Commun. 2 0 0 1 , 185-186.
(26) Kubo, W.; Murakoshi, K.; Kitamure, T.; Yoshida, S.; Haruki, M.; Hanabusa,
K.; Shirai, H.; Wada, Y.; Yanagida, S. J. Phys. Chem. B 2 0 0 1 , 105, 12809-
12815.
(15) van Esch, J.; Schoonbeek, F.; de Loos, M.; Kooijman, H.; Spek, A. L.; Kellog,
R. M.; Feringa, B. L. Chem.sEur. J. 1 9 9 9 , 5, 937-950.
10.1021/ac049606m CCC: $27.50 © 2004 American Chemical Society
Published on Web 08/12/2004
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polyacrylamide, cleaned of the stain, and only then injected into
the MS for identification. The reversible nature of organogels
makes them suitable for direct injection into a MS. In the simplest
form of electrospray-MS coupling, which is demonstrated here
for small molecules, the gel can be liquefied locally by mechanical
agitation with the needle of a syringe and injected into the MS.
This simple technique allows the entire matrix to be analyzed by
mass spectrometry.
Another advantage of organogels is that they can be made in
organic solvents in addition to water. Current electrophoresis gels
are aqueous polymeric gels. The ability to create organic gels
opens the door to the analysis of hydrophobic compounds which
do not function in aqueous gels, as well as an entire group of
new solvents which can be used as the separation matrix.
Additionally, capillary electrophoresis with polymeric gels requires
long and complicated procedures for in-capillary polymerization.27
The thermoreversibilty of organogels allows them to fill the
capillaries as hot solutions and then cool to form gels, in a simple,
quick, one-step procedure.
In this work, we introduce for the first time the basic concepts
of organogel electrophoresis and demonstrate planar electro-
phoretic separations of dansylated amino acids in an organogel
in several different solvent systems. Amino acids are commonly
used model compounds which are derivatized to allow for easier
detection by UV. The gel was agitated locally and injected
sequentially into an ESI-MS without separating the organic gelator
from the analytes. All compounds separated were identified by
their molecular ion peaks. A group of amino acids was also
separated in the organogel by capillary electrophoresis and
compared to the separation in the corresponding buffer. The effect
of changing different parameters such voltage, ionic strength of
the buffer, and gelator concentration was examined.
Figure 1. Dependence of the mobilities of dansylated amino acids
[serine (1), phenylalanine (2), tryptophan (3), proline (4), and histidine
(5)] on solvent concentration. Dotted line and right axis represent the
viscosity of acetonitrile-methanol mixtures.
55 °C. Dansylation was performed by mixing 0.1 mL of 7.2 mM
aqueous amino acid with 1 mL of 10 mM pH 9 carbonate buffer
and 0.9 mL of 1 mM dansyl chloride in acetone and heating for
10 min at 70 °C.
Instrumentation. Planar electrophoresis experiments were
performed in a 12-cm long, 2.6-cm wide Teflon cell. Gel length
was 6 cm. Power was supplied by a Consort (Turnhout, Belgium)
E862 power supply operating between -200 and -300 V. Gels
were formed by adding 6 mg of 1 to 2 mL of buffer, heating until
dissolution, and casting into the cell. The buffer was 1 M acetic
acid and 25 mM ammonium acetate in acetonitrile or mixtures of
acetonitrile with methanol or PEG. Visual detection was performed
with a UV lamp (Vilber Lourmat 215M, Torcy, France). Experi-
ments were performed in triplicate. Samples for injection into the
MS (LCQ, Finnigan, San Jose, CA) were taken by pushing a plastic
grating (4-mm holes with 1-mm spacing) into the gel and
withdrawing the samples with a 10-µL syringe, after gentle stirring
for 1 s of the gel in each compartment by the needle of the syringe.
The MS was scanned in the m/ z range of 200-450 in both positive
and negative ionization mode. Capillary electrophoresis was
performed in a Dionex CE instrument with a 42-cm long, 100-µm
i.d. capillary (Biotaq, MD). Injection was done electrophoretically.
UV detection was carried out at 260 nm. Gel-filled capillaries were
made by injecting hot organogel solution (60 °C) into the capillary
and allowing it to cool. Relative viscosity measurements were made
by flowing the solvents through an HPLC system (Finnigan) and
measuring the pressure drop through the packed column. Real
values were determined by correspondence to reported values of
the pure solvents.
EXPERIMENTAL SECTION
Materials. (1S,2S)-1,2-Diaminocyclohexane, lauroyl chloride,
dansyl chloride, PEG 200, triethylamine, amino acids, and peptides
were purchased from Sigma-Aldrich (St. Louis, MO). Acetic acid
was from Frutarom (Haifa, Israel). Ammonium acetate and THF
were from Mallinckrodt (Phillipsburg, NJ). Acetonitrile, methanol,
and chloroform were from J. T. Baker (Deventer, Holland).
BDACH, trans-(1S,2S)-1,2-bis(dodecylamido)cyclohexane (1 ) was
synthesized according to the procedure of Jung et al.2 Briefly,
lauroyl chloride (0.768 g, 3.5 mmol) was added to a solution of
(1S,2S)-1,2-diaminocyclohexane (0.2 g, 1.75 mmol) and triethy-
lamine (1.77 g, 17.5 mmol) in 50 mL of tetrahydrofuran in a
nitrogen atmosphere. The mixture was refluxed for 3 h and
allowed to cool. The solvent was removed by rotoevaporation. The
remaining solid was dissolved in chloroform and washed in a
separatory funnel with water. The solvent was removed, and the
product dried in a vacuum desiccator overnight. The product was
recrystallized from methanol. Yield: 78%. TLC (9:1 chloroform/
RESULTS AND DISCUSSION
Cell Electrophoresis. Figure 1 shows the mobilities of five
dansylated amino acids in planar gels of different acetonitrile-
methanol mixtures. The compounds all moved in the direction of
the anode, indicating their negative charge in the buffers. In all
cases the five compounds were resolved by visual inspection of
the UV-illuminated spots. The vertical uncertainty bars were
calculated from the radius of the fluorescent spots. The nonlinear
dependence of the mobilities on solvent composition requires
explanation. The electrophoretic mobility (µ) is the proportionality
constant between the electrophoretic velocity (v) and the electric
field strength (E) and is given by the Stokes equation
1
ethyl acetate): Rf ) 0.4. H NMR (300 MHz, CDCl3): δ ) 5.90
(d, 2H), 3.65 (q, 2H), 2.10 (t, 4H), 1.81-1.49 (m, 11H), 1.25 (s,
35H), 0.88 (t, 6H). IR (KBr): ν ) 3280, 1638, 1549. Elemental
analysis calculated (%) for C30H58N2O4 (478.79): C, 75.26; H, 12.21;
N, 5.85. Found: C, 74.48; H, 12.12; N, 6.46. Sol-to-gel transition:
v
E
q
(27) Huang, A.; Chen, Y.; Zhu, T.; Fang, X.; Sun, Y. J. Chromatogr., A 1 9 9 6 ,
755, 138-141.
µ )
)
(1)
6πηr
5400 Analytical Chemistry, Vol. 76, No. 18, September 15, 2004

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Table 1. Selectivity in Acetonitrile-Methanol Mixtures
selectivity between amino acid pairs
dansylated
amino acid
MeCN
10% MeOH
25% MeOH
50% MeOH
histidine
2.23
2.30
1.35
1.15
2.02
1.97
1.31
1.24
1.92
1.74
1.36
1.23
2.39
1.51
1.42
1.28
proline
tryptophan
phenylalanine
serine
Figure 2. Dependence of mobilities of dansylated amino acids
[serine (1), phenylalanine (2), tryptophan (3), proline (4), and histidine
(5)] on addition of PEG 200 to acetonitrile.
where q is the charge of the analyte, r is its Stokes radius, and η
is the viscosity of the buffer. As the solvent accounts for 99.7% of
the gel, the local viscosity encountered by the analytes is assumed
to be the viscosity of the solvent. The viscosity of acetonitrile-
methanol mixtures is also given in Figure 1. The minimum in the
viscosity corresponds to the maximum in the mobility. According
to eq 1, the relation between the mobility and the inverse of the
viscosity should be linear. Indeed line fits of the 1/ µ versus η
gave linear relationships though the correlation coefficients of the
fits were rather low ranging between 0.79 and 0.93 for the five
spots. This deviation can partly be attributed to the change in the
Stokes radius as a function of solvent properties. It also reflects
the uncertainties in measurement of the location of the spots in
the 6-cm planar gel.
The selectivity between consecutive ion pairs in the different
solvents is given in Table 1. These results show the versatility
and adaptability of organogel systems to electrophoresis. Different
solvents can be used in order to control the separation of a group
of analytes or a specific analyte pair. Proline and tryptophan have
the best resolution in pure acetonitrile, while phenylalanine and
serine separate better in the 50% mixture. Analytes that are difficult
to resolve can potentially be resolved with a change in solvent.
Poly(ethylene glycol) (PEG) was shown to improve the
strength of organogels, without interfering with their conductivity.6
Since by addition of PEG it is possible to tune the viscosity over
a much larger range (compared to a mixture of solvents) we have
investigated the possibility of altering the selectivity of the gel by
addition of different levels of PEG. The mobilities of the dansylated
amino acids were measured with differing concentrations of PEG
200 in a 0.3% gelator acetonitrile organogel. The gelator concentra-
tion was kept constant in all these tests. The observed mobilities
in the planar electrophoresis configuration are shown in Figure 2
and selectivities between adjacent amino acid pairs listed in Table
2. Indeed, increasing the level of the modifier decreased the
observed mobilities by up to 70%, probably due to the increased
viscosity. The selectivities here too show a different dependence
on the modifier for each pair of analytes. The histidine-proline
separation improves with added PEG while the proline-tryp-
tophan selectivity is worse. Specific separations can be optimized
with addition of the right type and amount of oligomer modifier.
The voltage dependence in the cell was not checked due to
the small voltage range (up to 300 V) in which the planar gel was
stable. The dependence was checked however with capillary
electrophoresis and will be discussed in that section. The mobili-
ties of the analytes are strongly dependent on the ionic strength
of the buffer, decreasing with increasing ionic strength. Over the
Table 2. Selectivity in Acetonitrile-PEG Mixtures
selectivity between amino acid pairs
dansylated
amino acid
MeCN 10% PEG 20% PEG 30% PEG 40%PEG
histidine
2.23
2.30
1.35
1.15
2.58
2.12
1.31
1.13
2.39
1.78
1.34
1.13
2.76
1.57
1.38
1.11
3.00
1.52
1.47
1.13
proline
tryptophan
phenylalanine
serine
range of 0.1-1 M, the mobilities of the dansylated acids in
acetonitrile buffer decreased linearly with the square root of the
ionic strength. The Onsager equation expresses the mobility (µ)
as a function of ionic strength (I). For univalent electrolytes it is
given as28
8.204 × 105
(ꢀT)1.5
42.75
η(ꢀT)0.5
x
I
µ ) µ0 -
µ0 +
(2)
[
]
where µ0 is the mobility at zero ionic strength, ꢀ is the dielectric
constant, T is the absolute temperature, and η is the viscosity of
the solvent. Plotting the mobility as a function of I0.5 allows
extrapolation to determine µ0 and therefore the slope predicted
by the Onsager equation. In very dilute solutions (up to about
0.02 M) the agreement is very good. At higher ionic strengths,
the mobility is generally higher than the Onsager equation would
predict.29 Because this work was done in high ionic strength
buffers, the linear relationship was observed (R2 values ranging
between 0.93 and 1), but the mobilities were much larger than
those predicted by the Onsager equation.
The effect of the gelator concentration is shown in Figure 3.
A clear nonlinear, nonmonotonic relationship is observed. At this
stage we can only speculate about the mechanistic reasons for
this dependence which may have substantial practical implications
for organogel eletrochromatography. We introduce this figure
here to illuminate the need for more extensive research in order
to comprehend organogel electrophoresis.
Capillary Electrophoresis. The separation of a mixture of
14 dansylated amino acids was carried out in both gel-filled and
(28) Porras, S. P.; Riekkola, M. L.; Kenndler, E. J. Chromatogr., A 2 0 0 1 , 924,
31-42.
(29) Castellan, G. W. Physical Chemistry, 3rd ed.; Benjamin/ Cummings Publishing
Company: Menlo Park, CA, 1983.
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Table 3. Separation Data for Gel-Filled Capillariesa
compound
tr (min) µ (10-4 cm2 V-1 s-1
)
N
1. dansyl chloride
2. aspartic acid
3. cysteine
4. threonine
5. serine
6. glycine
7. phenylalanine
8. alanine
4.3
5.0
5.2
9.4
9.6
12.1
12.7
13.2
14.3
15.0
17.1
17.7
18.2
19.2
23.5
3.02
2.60
2.47
1.38
1.35
1.07
1.02
0.980
0.905
0.866
0.759
0.734
0.712
0.676
0.551
16000
14000
16000
16000
13000
20000
22000
24000
28000
14000
24000
19000
7000
9. tyrosine
10. glutamine
11. asparagine
12. valine
13. tryptophan
14. leucine
Figure 3. Dependence of mobilities of dansylated amino acids
[serine (1), phenylalanine (2), tryptophan (3), proline (4), and histidine
(5)] on gelator concentration. Smoothed lines were added to guide
the eye, with no theoretical basis.
8000
12000
15. histidine
16. dansyl chloride
17. glutathion (glu-cys-gly)
18. gly-ser
19. glu-glu
20. ala-gln
9.3
13.0
37.1
47.9
60.6
67.5
3.10
2.22
0.777
0.602
0.475
0.427
12000
94000
85000
35000
41000
39000
buffer-filled capillaries. As stated above, the dansylated acids are
all negatively charged in this system. Since in the gel there is no
electroosmosis, and electrophoretic mobility is the only force
acting on the analytes, separations were performed in negative
polarity mode (i.e., with the detector located near the anode). In
the buffer-filled capillaries, electroosmotic flow marker mesityl
oxide was found to have a mobility of 3.4 × 10-4 cm2 V-1 s-1
toward the cathode, which is greater than the electrophoretic
mobilities of all the amino acids toward the anode, with the
exception of aspartic acid. Therefore, positive polarity was used,
and the compounds were detected in the reverse order of that in
the gel-filled capillaries. Figure 4 shows the electropherograms
without (a) and with (b) gel. The peaks were identified by
individual injection of each acid. The retention times, mobilities,
and plate counts for the gel separation are given in Table 3.
The results clearly show the advantage of the organogel.
Baseline resolution was achieved for 10 of the 14 compounds,
whereas in the buffer several peaks fell on top of one another.
Unlike polymer gel capillaries which require complicated polym-
erization procedures, these capillaries are made by a simple, one-
21. phe-ala
a
N values were calculated by the formula N ) 5.54tr2/ W2 where tr
represents the retention time and W is the half-height peak width.
step injection, and are ready in only a few minutes. The evacuation
of the capillaries is also very simple. The gel can be simply
pumped out of the capillary with a syringe pump, and after a short
rinsing with acetonitrile a new gel can be injected into the same
capillary. Organogels offer a much easier way to do capillary gel
electrophoresis.
The dependence of the mobilities on voltage was also checked
for a group of several dansylated amino acids. The dependence
was linear with zero intercept over a range of 7-20 kV with
correlation coefficients greater than 0.97. The gel-filled capillaries
show stability for long periods of time. The separation of five
amino acids was performed seven times over a period of 24 h.
The resulting separations were essentially the same with a
Figure 4. Electropherograms at 20 kV of 14 amino acids in (a) buffer-filled and (b) gel-filled capillaries. Peak numbers are identified in Table
3.
5402 Analytical Chemistry, Vol. 76, No. 18, September 15, 2004