Original

CHROMATOGRAPHY, Vol.34 No.1 (2013)

Original

Separation and determination of dopamine and epinephrine in serum bycapillary electrophoresis with inkjet introduction system
Ying Weng, Hulie Zeng, Yuri Nakagawa, Saori Ikeda,
Fengming Chen, Hizuru Nakajima, Katsumi Uchiyama
Tokyo Metropolitan University, Minamiosawa, Hachioji, Tokyo 192−0397, Japan

Abstract
A new inkjet sample introduction method was developed for capillary electrophoresis (CE). This system could control the exact volume ofejected liquid with high accuracy and precision at the picoliter level. The single droplet volume of 1×10−6 mol/L dopamine (DA) in 1×10−2
mol/L borate buffer and in the 40 times diluted serum were 240 pL and 243 pL, respectively. Sample droplets were ejected by stressing a piezoelectric slice stuck on the extrusion chamber. The pulse voltages were applied to the piezoelectric slice which made the slice deformed. Using this method, DA and epinephrine (EP) were separated and the concentration of EP in serum was determined at 5.88×10−7 mol/L, DA was
not detected. The recovery (n＝3) of EP and DA added to serum were in the range of 95.8−98.7%, and 97.5−103.4% respectively, whichshowed good accuracy and reproducibility.
Keywords : Inkjet, Exact sample introduction for CE, Dopamine, Epinephrine

for material science, nanotechnology and molecular engineering,

Introduction
Inkjet printer is one of the most commonly used printers. Inkjet

the separation efficiency of CE obtained significant progress [11−printing essentially is a technology for dispensing precise amounts13]. However, comparing the separation potential of CE with otherof liquid material with high positional resolution. The method isseparation techniques, the quantitative capacity is not so compat-

called drop−on−demand (DOD) [1,2]. Inkjet printing could be aible. It is due to the deficiency of proper sample introduction meth-promising method for droplet dispensing with high precision andods which couldn’t supply exact introduction volume for CE.

reproducibility [3]. In these years, the inkjet technology has been

DA and EP play the major roles as neurotransmitters in central

used as a tool for rapid prototyping, small volume dispensing, andand peripheral nervous systems [14]. DA is involved in the induc-the production of small sensors [4−6]. The life science is also con-tion and in the expression of behavioral sensitization by repeatedcomitantly progressing with new applications of DOD for precise

exposure to various drugs of abuse [15]. The neurobiologicaldispensing of DNA and protein molecules [7−8].

mechanism of drug abuse and addiction cannot be accommodated

CE was introduced in 1960s [9], which had rapidly evolved into

by a single, unitary hypothesis. Regarding to DA, only one among

the analysis of extensive range of targets, like DNA, peptides, pro-the many neurochemical systems are likely to be involved in drug

tein and small ions. Therefore, it was used as the one of the popularabuse, a parsimonious hypothesis to provide only a partial accountseparation technique, and made great contribution to environmentalof its role in this condition. High catecholamine levels in blood are

science and life science [10], like monitoring environmental condi-

associated with stress [16]. Extremely high levels of catecholaminetion, screening status in genetics. In accordance with the progress

can occur in central nervous system trauma due to stimulation orCorresponding author: Katsumi Uchiyama
Tel: +81−042−677−2835
Fax: +81−042−677−2821
E−mail address: uchiyama−katsumi@tmu.ac.jp―３３―

CHROMATOGRAPHY, Vol.34 No.1 (2013)

damage of nuclei in the brainstem, which can be caused bythermore, some parameters of pulse waveform could be adjusted asneuroendocrine tumors in the adrenal medulla or caused byfollows: applied voltage; 0〜120 V, the pulse duration and inter-monoamine oxidase A deficiency. It also occurs the absence ofval; 1〜100 µs.

pheochromocytoma, neuroendocrine tumor and carcinoid synInkjet introduction system

drome [17]. Nowadays, much interest has been focused not only on
researching the bioactive function of the catecholamines but also

The integrated equipment (including introduction, separation andon developing the selective methods for their detection in biologi-

detection device) has been described elsewhere [23]. The system iscal system [18−19]. DA and EP were normally detected by electro-

shown in Figure 1. Briefly, the inkjet microchip was moved by XY

chemistry analysis. A novel taurine modified glassy electrode forstage to align the center of capillary tip to the nozzle of inkjet mi-

determination of EP and DA, the linear range was 2.0×10−6 M tocrochip. In the injection process, the tip of capillary in the inlet res-

−4

−6

−4

M, respectively. And

ervoir was raised up and out of buffer solution. Then the inkjet mi-

another group reported nano Pd−Au particles modified glassy car-crochip ejected definite amount of sample droplets. The sample onbon electrode for quantitative analysis of DA. The linear range wasthe capillary tip was pushed into the capillary by Laplace pressure

from 0.5 to 6.95×10−6 M [20−21].

caused by surface tension.

6.0×10

M and 1.0×10

M to 8.0×10

In this study, the inkjet introduction system was applied to CE
Separation and detection system[22] and quantitative analysis of catecholamines in serum by accurate controlling the exact introduction volume was carried out. The

The setup for CE device was made by our laboratory. An in

inkjet microchip was used for precise placement and introduction

house built high voltage power supply (0−20 kV) was used to carryof sample for CE. They say that there is only once or no false ejec-

out CE separation. The length of capillary tube (O.D. 0.375 mm,tion out of one billion times ejections in inkjet technology. This is-

I.D. 0.050 mm, GL Science, Japan) was 45 cm, and the effective

sue also promised the consistency and reproducibility of ejection.

separation length was 25 cm. A laboratory−made LIF detection

For the accurate quantitative analysis, it is extremely important tosystem, containing a solid blue laser (HK−5517−02, 473 nm, 5.0

know the exact amount of sample introduced into the capillary. The

mW, Shimadzu, Kyoto, Japan), an incident light fluorescence mi-inkjet introduction method will be expected to solve the problem.

croscope (BX40F4 Olympus, Tokyo, Japan) with a filter set (U−MWIB2 Olympus, Tokyo, Japan), a photomultiplier tube (PMT)

Experiment(H6780−02, Hamamatsu Photonics, Shizuoka, Japan) and a labora-

Chemicalstory−made amplifier. The chromatographic data−processing system (LAS5 Soft, Chiba, Japan) was applied to collect the fluores-

All chemicals were of analytical grade except especially indi-cence signal.

cated and were used without further purification. Dopamine hydrochloride (DA) and the fluorescein isothiocyanate isomer I (FITC)

Labeling procedure

were purchased from Sigma−Aldrich Chemie GmbH (Steinheim,
Germany). L−Adrenaline Bitartrate (Epinephrine, EP) was fromTo match fluorescence detection, the derivatization of DA andTokyo Chemical Industry (Tokyo, Japan). Pyridine, Sodium

EP with FITC was implemented according to the procedures de-tetraborate decahydrate, Sodium hydroxide, Hydrochloric acid,scribed elsewhere [21]. All procedures were carried out in a 3 mL

Acetone (for spectroscopy) were bought from Kanto Chemical Co.,

brown glass. 5×10−3 mol/L of DA and EP standard solutions were

Inc. (Tokyo, Japan). The human serum used in the experiment was

prepared with water. And 5×10−3 mol/L of FITC solution was pre-purchased from Bethyl Laboratories INC (TX, US), and containing

pared in acetone containing 1% pyridine. As the instability of

0.1% sodium azide. Deionized water (Milli−Q system, Japan Milli-

FITC, the solution of FITC was freshly prepared before derivatiza-

ipore Co., Tokyo, Japan) was used throughout the experiment.

tion. And the DA and EP solutions were used within a week because both of them were easily decomposed. For the derivatization,

Inkjet microchip

100 µL of above DA or EP solutions was mixed with 500 µL FITC

Four−channel inkjet microchip was kindly gifted from Fuji Elec-solution and 400 µL 2×10−2 mol/L borate buffer solution (pH＝tronics Co. (Tokyo, Japan) that was used as a printing head of in-9.0), respectively. The mixed solution was kept at 25°
C in dark for

dustrial recorder (Fuji Electronics Co., Type CNO−1010, Tokyo,

about 12 hours.

Japan). Sample droplets were yielded via the deformation of the
extrusion chamber that was pushed by the piezoelectric slice driven

Preparation and derivatization of serum samples

by the pulse voltage. The number of ejected droplets could be con-

The labeling process of the human serum was same as above.

trolled by the number of applied pulses between 1 and 10000. Fur-

After the derivatization reaction was completed, the FITC−labeled―３
４―

CHROMATOGRAPHY, Vol.34 No.1 (2013)

Ying Weng, Hulie Zeng, Yuri Nakagawa, Saori Ikeda, Fengming Chen, Hizuru Nakajima, Katsumi Uchiyama

Figure 1. The diagram of inkjet introduction system with CE, LIF detector and the data processor.

time course variation of droplet weight.

serum was centrifuged at 1250 rpm for 5 min. The supernatant wascollected and applied to CE separation. The FITC labeled serum
was diluted for 4 times by 1×10−2 mol/L borate buffer solution

Results and discussions(pH＝9.0) before ejection to CE. The final amount of acetone in di-

The principle of inkjet injection for CE

luted serum was 12.5%, which was also an important effect inkjet

The process of liquid drop introduction in inkjet injection system

ejection and treatment of serum. All the sample solutions including

was shown in Figure 2. According to the record of high speed cam-

FITC−DA, FITC−EP solutions and diluted FITC labeled serumera (KEYENCE, VW−9000, Tokyo, Japan), all the droplets were

was filtered through 0.45 µm PDVF (Kanto Chemical Co., Inc.,

ejected through inkjet as drop−by−drop. At first, the tip of the cap-Tokyo, Japan) membrane filter before injection to the CE system.

illary was lifted from the buffer solution. Then driving pulses wereapplied to the piezoelectric slice. Consequently, the sample droplets were ejected from the nozzle of inkjet microchip and accumu-

Accurate solution gravimetric
Accurate mass measurements of micro droplets measurements oflated on the tip of capillary. Then, the accumulated droplets on the

micro droplets were carried out by the method as already reported

tip of capillary were pushed into capillary. Finally, the capillary

by Verkouteren et. al. [24]. Briefly, micro balance with Sarto_In

was immersed back into the buffer reservoir to perform CE separa-

Timer (Sartorius mechatronics, Tokyo, Japan) was used to recordtion.
Surface tension is a property of the surface of liquid that allows

the weight value. Compensation of evaporation was made from the―３５―

CHROMATOGRAPHY, Vol.34 No.1 (2013)

Figure 2. The process of liquid introduction of inkjet.
it to resist an external force, which is the important driving force tocomplete the liquid sample introduction in inkjet injection system∆p＝

for CE. This property is caused by cohesion of similar molecules,
and is responsible for many behaviors of liquids. When the liquid

2 γcosθrc(∆p＜0)(2)

Where r surface curvature radius, rc capillary internal radius, andsurface is horizontal and flat, the external pressure pex equates theθ contact angle.

internal pressure p of the liquid. When the liquid surface is curved,

As in the Figure 2 shown, the initial state ‘a’, the curvature ra-

the pex is different from p. If the surface is in the convexity, the sur-dius was large. Then when the curvature radius of sample droplet

face tension γ in the vertical direction would generate a downward

was gradually decreased from ‘a’ to ‘b’. The liquid drop was partly

pressure as shown in Figure 2. The pex is greater than p until they

introduced into the capillary. The Laplace pressure is usually very

are counterbalanced; oppositely if the surface is concave, the sur-large. For example, droplet with 10 µm of radius gives several

face tension would cause an upward pressure in the vertical direc-

thousand N/m2 of surface tension. The direction of Laplace pres-tion resulting in that pex＜p. The difference of p and pex is defined

sure at the stage ‘a’ is downward because the droplet surface is

as added pressure below curved surface ∆p. In conclusion, on theconvex. Under the Laplace pressure, the sample droplet would be

convexity ∆p＝(p−pex)＜0, and on the concave ∆p＝(p−pex)＞0.

pushed into the capillary. In this stage, with the surface tension of

And the direction of ∆p (Laplace pressure) points to the center ofdroplet gradually pressing the liquid into the capillary, the surface

the curved liquid surface.

curvature radius would be deceased; therefore the Laplace pressure
was increased. The liquid drop was more rapidly pushed into the

According to Young−Laplace equation,

capillary. Finally, the liquid drop was completely pushed into the∆p＝

2 γcosθr(∆p＞0)

capillary, and the liquid surface went to ‘c’ as flat. On the condition(1)‘c’, the Laplace pressure was 0. Then, liquid surface was pulled to―３６―

CHROMATOGRAPHY, Vol.34 No.1 (2013)

Ying Weng, Hulie Zeng, Yuri Nakagawa, Saori Ikeda, Fengming Chen, Hizuru Nakajima, Katsumi Uchiyama

form a concave surface like ‘d’ for the hydrophilic inner wall ofover 50 V, every pulse would generate one main droplet and acapillary and the gravity of water pillar inside the capillary. Thus,

small satellite droplet. Since the satellites droplet was muchthe whole liquid introduction was completed.

smaller, it was easy to be effected by the current environment togenerate high RSD. For example, the satellite droplet could be

The effect of inkjet ejection condition on the sample introduc-

strayed away easily before touching the tip of capillary. And astion reproducibility

shown in Figure 3, the optimum driving voltage of 25 V to 50 V
for inkjet was obtained, while the RSD of sample amount intro-

The driving wave form for piezoelectric slice had two parame-

duced was less than 3%.

ters, the driving pulse voltage and the duration of pulse. The speedof droplet was higher when the driving pulse voltage was higher. In

Evaluation the introduced volume

the experiment, one electric driving pulse could eject one droplet
from the inkjet. The droplets would not be ejected out, or deviate

One droplet volume could be calculated by the quotient of thefrom normal track, when the driving pulse voltage was less than 23

certain number of droplets mass and their density. So the volume

V. The reason is the driving force was not enough to push out theof sample introduced could be figured out. Comparing other injec-

droplets. In this case, the relative standard deviation (RSD) of sam-tion methods of CE, the inkjet introduction method gave absolute

ple introduction for CE was large, because it could not be guaran-

sample volume injected, which is of importance for the analyticalteed every droplet match the center of capillary on tip. On the othermethod of standardization. As the results listed in Table 1, thehand, the sample droplets would splash out from the tip of capillary

droplet volume could be obtained by dividing the mass of 10000

when the voltage was beyond 50 V, which also caused the highdrops sample with their density. Difference of the volume of single

RSD. All of the process of ejection was recorded by high speed

droplet was not obvious between DA solution and the diluted se-camera. When the driving voltage was less than 50 V, every driv-

rum. While the inkjet driving conditions were selected as follows:ing pulse could generate one droplet. Once the driving voltage wasdriving pulse voltage 50 V, the duration of pulse drive 35 µs. Here,Figure 3. The effect of driving pulse voltage on the weight of 3000 droplets of water.
Table 1. The calculation of droplet volume.

DA solution(1.00×10−6 mol/L)
40 times diluted Serum

Weight 10000 drops (g)Density (25°C) (g/mL)

1 droplet volume (pL)

0.00241

1.004

240

0.00242

0.997

243

The injection conditions for inkjet: driving voltage was 50 V, drive pulse duration was 35 µs.

―３７―CHROMATOGRAPHY, Vol.34 No.1 (2013)

the single droplet volume of 1×10−6 mol/L FITC−DA in 1×10−2

Electrophoresis separation and method validation
DA and EP were labeled by FITC in advance. And FITC is a

mol/L borate buffer solution was 240 pL, and that of 40 times di−2

mol/L borate buffer solution) was 243 pL. In

relatively large molecule comparing to DA and EP. FITC−DA andthe experiment, the original derivative solution was diluted to vari-

FITC−EP were separated on the baseline under the condition 1×ous concentrations. In order to investigate the effect of different

10−2 mol/L borate buffer solution (pH＝9.0). As shown in Figure

acetone amount (it was the main component of FITC labeled sam-4, FITC−DA and FITC−EP were separated within 350s. And weple, which probably affect the surface tension of the liquid sample,

certificated the first peak was FITC, second one was FITC−EP, andeven its droplet volume ejected form inkjet) on the droplet volume,the third was FITC−DA by adding individual standard solutions,the various concentrations of acetone solution were prepared fromrespectively. The FITC appeared in 270s, EP stayed at 292s, and

0.001% to 20%, and the droplet volumes were obtained. As a re-DA eluted at 310s.

luted serum (1×10sult, 1 droplet volume was 246±2.5 pL for the solution tested.

As Table 2 shown, the linear dynamic relationships were ob-

Variation of 1 droplet volume showed no distinct difference amongserved between the fluorescent intensity and the concentration for

the varied concentrations of acetone solutions. Therefore, the affec-

both FITC−DA and FITC−EP. The calibration plots, obtained bytion of various concentrations of acetone on the sample droplet vol-CE of the standard solutions, showed the good linear relationshipume could be ignored.

between the florescence intensity and concentration for FITC−EP
and FITC−DA. The detection of limits were 1.78×10−9 mol/L for
EP and 2.22×10−9 mol/L for DA. The regression coefficient con-Figure 4. The separation electrophoretogram of FITC, FITC−DA and FITC−EP. (a) electropherogram of FITC, FITC−DA (5.00×10−7mol/L) and FITC−EP (5.00×10−7mol/L). (b) The electrophorogram after adding FITC−DA (the final concentration of FITC−DA was 1.00×10−6
mol/L). (c) The electrophorogram after adding FITC−EP and FITC−DA (The final concentration of FITC−DA was 1.00×10−6mol/L, andFITC−EP was 1.00×10−6 mol/L). While the buffer was 1×10−2 mol/L borate buffer (pH＝9.0). The driving conditions for ink−jet: driving
pulse voltage was 20 kV.
Table 2. Regression data, LOD and RSD for FITC−EP and FITC−DA.
AnalyteFITC−EP
FITC−DA

Regression equation
y＝3335＋2203 x
y＝2672＋1665 x

Regression constant
0.998
0.999

Linear range (mol/L)−8−6

1.00×10 −2.00×10
5.00×10−8−1.00×10−6

LOD (mol/L)−9

1.78×10
2.22×10−9

RSD (%) (n=4)
1.12−2.75
1.64−2.89

y: fluorescence intensity (peak area)
x: analyte concentration
The injection conditions for inkjet: driving voltage was 50 V, drive pulse duration was 35 µs. The injected volume for FITC−EP and FITC−
DA were 2.40 nL (10 droplets).

―３８―CHROMATOGRAPHY, Vol.34 No.1 (2013)

Ying Weng, Hulie Zeng, Yuri Nakagawa, Saori Ikeda, Fengming Chen, Hizuru Nakajima, Katsumi Uchiyama

Conclusions

stants of DA and EP were 0.999 and 0.998 respectively.
The precisions were also listed in Table 2. The RSD of peak ar-

Sample introduction with inkjet was successfully applied to theeas of FITC−EP and FITC−DA were in the range of 1.12−2.75%,

determination of DA and EP in serum by CE. The exact sample in-and 1.64−2.89%, which demonstrated the good precision of thetroduction volume could be calculated accurately by this system. It

inkjet injection method for CE.

is significant for analytical method standardization. In this experiment, the single droplet volume of 1×10−6 mol/L DA solution was

Quantitative determination of DA and EP in serum

240 pL and 4 times diluted serum was 243 pL. As the inherent

The method was used for the analysis of EP and DA in humancharacter of inkjet technique, precision of quantitative analysis was

serum. A typical electropherogram is shown in Figure 5. The ex-greatly improved [24].

perimental results were summarized in Table 3. The EP content in
the serum was determined around 5.88×10−7 mol/L, which was

Acknowledgments

equal to 1.08 ng/mL. In the adult body, the concentration of EP is

This work was supported by Japan Society for the Promotion oflower than 10 ng/mL [25]. And DA could not be detected. The re-

Science and High−Tech Research Program by Tokyo Metropolitan

covery (n＝3) of FITC−EP was between 95.8−98.7%, and FITC−

Government.

DA was between 97.5−103.4 %.

Figure 5. The electrophoretograms of serum sample. Buffer solution was 1×10−2 mol/L borate buffer, pH 9.0, applied pulse voltage for inkjet was 20 kV. The insert figure was the magnified view of FITC−DA and FITC−EP peaks. Solid line was the electropherotograms of serum
sample. The dot line was the electrophoretogram added 1×10−7mol/L of FITC−DA and FITC−EP.
Table 3. Analytical results and average of DA, EP in serum.
Sample number

AnalyteAmount added (mol/L)

Amount detected (mol/L)

Recovery (%)

1

EP
DA
EP
DA
EP
DA
EP
DA

0
0
1.00×10−7
1.00×10−7
2.00×10−7
2.00×10−7
4.00×10−7
4.00×10−7

1.47×10−7NA
2.43×10−7
0.96×10−7
2.94×10−7
2.07×10−7
5.42×10−7
3.90×10−7

None
None
95.8
97.5
99.6
103.4
98.7
97.5

2
3
4

The injection conditions for ink−jet were same to Table 2. The injected volume of 4 times diluted serum was 2.43 nL(10 droplets). NA means that did not detect.

―３９―CHROMATOGRAPHY, Vol.34 No.1 (2013)

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