Analysis of low molecular weight acids by monolithic immobilized pH gradient-based capillary...

Short Communication

Analysis of low molecular weight acids bymonolithic immobilized pH gradient-basedcapillary isoelectric focusing coupled withmass spectrometry

A novel method for the separation and detection of low molecular weight (LMW) acids

was developed using monolithic immobilized pH gradient-based capillary isoelectric

focusing coupled with mass spectrometry. Two main parameters, focusing conditions and

delivery buffer conditions, which might affect separation efficiency, were optimized with

the focusing time of 7 min at 350 V/cm and the delivery buffer of 50% (v/v) acetonitrile in

10 mmol/L ammonium formate (pH 3.0). Under these conditions, the linear correlation

between the volume of delivery solvent and the pKa of the model components was

observed. In addition, the separation mechanism of LMW acids was proposed as well. We

suppose that this method may provide a useful tool for the characterization of LMW

components (e.g. natural organic matter of different origins).

Keywords: Capillary isoelectric focusing / Low molecular weight acids / Massspectrometry / Monolithic immobilized pH gradientDOI 10.1002/jssc.201000720

1 Introduction

Since the first introduction by Hjerten and Zhu [1], capillary

isoelectric focusing (CIEF) has drawn much attention due to

its high resolution, speed, concentrating effect, and the ease

in coupling with other separation modes to construct

multidimensional platforms [2, 3]. To date, more efforts

have been made for the analysis of high molecular weight

amphoteric analytes (e.g. proteins and peptides) with CIEF

and related systems. Shen et al. [4] obtained high resolution

CIEF separation of cellular lysates from Saccharomycescerevisiae, Eschericia coli, and Deinococcus radiodurans.Proteins with an isoelectric point (pI) difference of 0.004

could be separated using a linear carrier ampholyte of 3–10.

Jin et al. [5] utilized synthetic UV-detectable peptide markers

to estimate pI values of major proteins in human plasma.

Busnel et al. [6] developed a method to perform glycerol–

water CIEF in a bare silica capillary and then successfully

applied it for the separation of isoforms of horse radish

peroxidase.

Besides the analysis of peptides and proteins, CIEF can

also be a suitable tool for the characterization of amphoteric

low molecular weight (LMW) components [7–9]. Slais and

Friedl [7] proposed different amino-methylated nitrophenol

derivatives as pI markers in the pH range between 5 and 10.

Furthermore, the technique was applied for the estimation

of pIs of 12 environmentally relevant zwitterionic

triazine derivatives that corresponded to the values deter-

mined in titrations with capillary zone electrophoresis [8]. In

addition, CIEF was used for the characterization of natural

organic matter such as humic substances of different

origins [9].

Although ampholytes are indispensable for the focusing

process, the presence of which makes the hyphenation of

CIEF and MS difficult. To tackle this problem, researchers

have attempted to reduce their concentration [10, 11] or

remove them by using microdialysis [12, 13], free-flow

electrophoresis chip device [14], and reversed-phase liquid

chromatography [15, 16]. Alternatively, a promising

approach is to immobilize ampholytes onto supporting

materials (i.e. monolithic matrix) to form a stable mono-

lithic immobilized pH gradient (M-IPG) [17–19]. Compared

to the traditional CIEF, the removal of ampholytes can be

avoided in M-IPG CIEF. Moreover, minimized focused zone

broadening during mobilization can be anticipated owing to

the existence of monolithic matrix.

Tingting Wang1,2

Agnes Fekete3

Andras Gaspar3

Junfeng Ma1

Zhen Liang1

Huiming Yuan1

Lihua Zhang1

Philippe Schmitt-Kopplin3

Yukui Zhang1

1Key Laboratory of SeparationScience for AnalyticalChemistry, NationalChromatographic Research andAnalysis Center, Dalian Instituteof Chemical Physics, ChineseAcademy of Sciences, Dalian,P. R. China

2Graduate University of ChineseAcademy of Sciences, Beijing,P. R. China

3Helmholtz-Zentrum Muenchen-German Research Center forEnvironmental Health, Institutefor Ecological Chemistry,Neuherberg, Germany

Received October 12, 2010Revised November 22, 2010Accepted November 22, 2010

Abbreviations: BA, benzoic acid; HBA, 3-hydroxybenzoicacid; LMW, low molecular weight; M-IPG, monolithicimmobilized pH gradient; PHA, phthalic acid

Correspondence: Professor Lihua Zhang, Key Laboratory ofSeparation Science for Analytical Chemistry, National Chroma-tographic R. & A. Center, Dalian Institute of Chemical Physics,Chinese Academy of Science, 457 Zhongshan Road, Dalian116023, ChinaE-mail: [email protected]: 186-411-84379779

& 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com

J. Sep. Sci. 2011, 34, 422–427422

The aim of this study is to explore the feasibility of

using M-IPG CIEF-ESI/MS for the separation and char-

acterization of acid derivatives without ampholytic proper-

ties. The effects of focusing time, focusing voltage, pH, and

organic solvent content on separation were investigated.

Since the solvent volumes required to deliver the peaks to

the detector were linearly dependent on their pKa values, a

possible mechanism of separation by M-IPG CIEF-ESI/MS

was also purposed.

2 Materials and methods

2.1 Reagents and materials

Glycidyl methacrylate and 2-propanol were purchased from

Fluka (St. Gallen, Switzerland). Ampholine (pH 3.5–10.0),

3-(trimethoxysilyl) propyl methacrylate, methanol, and ACN

were from Sigma (Steinheim, Germany). PEG (molecular

weight 8000 and 10 000) was purchased from Sino-Amer-

ican Biotechnology (Shanghai, China). Acrylamide and

N,N0-methylenebisacrylamide were purchased from Acros

Organics (Geel, Belgium). Azobisisobutyronitrile was

purchased from The Fourth Shanghai Regent Plant

(Shanghai, China). Ammonium formate was purchased

from VWR (Leuven, Belgium). Formic acid and ammonia

were purchased from Merck (Darmstadt, Germany). Ultra-

pure water was supplied by Milli-Q RG unit from Millipore

(Bedford, MA, USA). Stock solutions of the target compo-

nents were prepared by dissolving each compound sepa-

rately in methanol to reach a concentration of 1 mg/mL, and

diluted with 1% (v/v) ammonia solution to the final

concentration of 33.3 mg/L.

2.2 Instrumentation

M-IPG CIEF-ESI/MS was performed with a Beckman

P/ACE 5510 CE system (Beckman Instruments, Fullerton,

CA, USA), equipped with an autosampler and a power

supply which was able to deliver up to 30 kV coupled to an

ion-trap MS Finnigan LCQDuo (Thermo Fisher Scientific,

San Jose, CA, USA) with an ESI source. The on-line

coupling of CE to MS was achieved with a home-made

sheath liquid interface, which was coaxially positioned to the

MS entrance on an XYZ stage that allowed the exact

positioning of the separation capillary. To maintain stable

electrospray, a 3 mm long portion of the polyimide coating

was removed (by burning) from the outlet edge of the

capillary. Instrumental control, data acquisition and data

processing were carried out using the Gold software

package for CE and the Xcalibur data system version 1.1

software for ESI/MS. The sheath liquid of 50%

2-propanol–49% water–1% ammonia (v/v/v) was applied at

a flow rate of 250 mL/h. The capillary voltage was set at

�4 kV to obtain high sensitivity without excessive noise

caused by electric arcing at high capillary voltage in the

negative mode. The UltiMate Plus Capillary/Nano LC

System (Dionex, California, USA) was used to drive the

compounds out from the monolithic column.

2.3 Preparation of M-IPG column

The procedure of M-IPG column preparation was similar to

our previous study [20]. Briefly, the amine groups of

ampholine and the epoxy group of glycidyl methacrylate

were reacted under the presence of formamide. Then, N,N0-methylenebisacrylamide, PEG-8000, PEG-10000, acryl-

amide, and azobisisobutyronitrile were added into the

mixture. After vortex and degassed for a few minutes, the

mixture was filled into the 3-(trimethoxysilyl) propyl

methacrylate coated capillary and focused in an electric

field. The capillary was sealed at both ends, and then put in

an oven at 701C for 20 h to immobilize pH gradient onto the

monolith.

2.4 M-IPG CIEF-ESI/MS system

The M-IPG CIEF-ESI/MS system, as shown in Fig. 1,

consists of several sections, including sampling, connecting

with CE instrument, isoelectric focusing, and solvent

delivery and MS detection. Due to the various ampholines

locating in different places according to their pIs [17], the

M-IPG column has its specified direction. The cathode of

M-IPG column was fixed into the interface and the anode

was linked up with the micropump or CE instrument. Due

to the existence of backpressure from the monolithic

column (20 cm long and 100 mm id), samples that were

hardly driven by the air pressure provided by the CE

instrument (i.e. 0.5 or 20 psi) were injected by an additional

pump. After sample injection, the anode of M-IPG column

was connected with a capillary (60 cm, 100 mm id), which

was fixed into CE instrument. The CE was operated at

normal polarization mode. The sheath liquid (50%

2-propanol–49% water–1% ammonia, v/v/v) was considered

as the catholyte solution and 1% formic acid was considered

as the anolyte solution. Then the voltage was applied for

focusing. Once the current reduced to �10% of the original

value, the focusing was considered to be complete. After-

ward the anode of M-IPG column was connected with a

micropump and the compound plugs were forced to move

toward the detector.

3 Results and discussion

For the optimization of M-IPG CIEF-ESI/MS separation of

acid derivatives, three LMW acids, benzoic acid (BA, pKa

4.20), 3-hydroxybenzoic acid (HBA, pKa 4.07), and phthalic

acid (PHA, pKa 2.95), were selected since they differ in

functional groups and pKa. BA and HBA, respectively,

contain one carboxylic acid moiety, whereas PHA has two

J. Sep. Sci. 2011, 34, 422–427 Electrodriven Separations 423


carboxylic acid groups. The difference between BA and HBA

is the presence of the phenolic group. Although these

components are not zwitterions/ampholytic components,

separation according to their pKa values was expected, and

several parameters (e.g. focusing conditions and delivery

buffer conditions) that might affect separation performance

were investigated.

3.1 Mechanism of separation

The possible mechanism of separation by M-IPG CIEF-ESI/

MS is shown in Fig. 1. Step 1, sampling: The entire M-IPG

column was initially filled with the negatively charged LMW

acids dissolved in 1% (v/v) ammonia (Fig. 1A). Step 2,

connecting with CE instrument: A 3 mm of 1% formic acid

plug was injected into the anode of monolith column while

it was connected with CE instrument (Fig. 1B). A part of

sample penetrated through to the acid plug and picked up

positive charge, resulting in neutralization of the negative

charge LMW acids, preventing them from migrating out of

the column and being missed in the ESI/MS detection

when applying a high voltage for focusing. Step 3, isoelectric

focusing: Under the influence of an electric field, pH

gradient was generated in the monolith column between the

cathode and anode (Fig. 1C). While the local pH was higher

than pKa – 2 values of the LMW acids, migration was toward

the anode. During the migration, the negatively charged

acids encountered progressively lower pH, thus picked up

more positive charge. Eventually, a zone was reached where

the net charge was zero. At this point (pKa – 2), migration

stopped and the sample was focused in a tight zone,

resulting in the separation of LMW components even if they

were not ampholytes. Step 4, solvent delivery and MS

detection: The focused components were driven by the

micropump to ESI/MS for detection (Fig. 1D).

3.2 Separation of LMW acids under different modes

The selected three model components migrated at the same

time with the absence of voltage (Fig. 2A at 0 min),

indicating notable influence of voltage on the separation

efficiency of M-IPG CIEF, and thus no difference in the

adsorption strength of the model components with the

monolith surface could be concluded. Furthermore, apply-

ing an open-tubular silica capillary instead of an M-IPG

column, the model components migrated together to the

MS under the same separation conditions as for M-IPG

CIEF model (data not shown). Although the model

components were dissolved in basic buffer, the electrolyte

of the anode was acidic where the analytes became neutral

and thus they migrated with the EOF. Presumably, EOF was

too high to enable the separation of the selected analytes. As

a whole, we can conclude that M-IPG CIEF should be

anticipated for the separation of LMW acids.

3.3 Optimization of focusing conditions

Focusing voltage and time are two important factors that

affect the separation efficiency of CIEF. It was found that

the applied voltage should be higher than 350 V/cm to

obtain baseline separation of the model compounds (data

not shown). However, the voltage should also be lower than

400 V/cm due to the high occurrence of bubble formation in

Figure 1. Instrumental setup of M-IPG CIEF-ESI/MS and the purposedmechanism for the separation ofLMW acids by M-IPG CIEF-ESI/MS.(Symbols m~� represent the threeLMW acids.)

J. Sep. Sci. 2011, 34, 422–427424 T. Wang et al.


the monolith column. The effect of focusing time on the

separation efficiency was investigated by changing the

focusing time from 0 to 10 min. The retention time did

not change significantly with the increase of the focusing

time from 5 to 10 min, but 5 min was found adequate for

the separation of BA and HBA (Fig. 2A). It can be seen from

Fig. 2B that the peak width of BA and HBA was slightly

changed with the increase in the focusing time. The peak of

PHA was relatively broader in comparison to the other

analytes, which might be ascribed to the strong interaction

with the stationary phase. The differences in the interaction

between LMW acids and the monolithic matrix rendered

easy MS detection by extracted ion chromatogram, provid-

ing additional information besides the separation character-

istics. Since the smallest peak width of PHA was observed,

the focusing time of 7 min was chosen in the following

experiments.

3.4 Optimization of delivery buffer conditions

To reduce the non-specific interaction between the analytes

and monolith, the delivery buffer condition was optimized.

As ionic attraction was assumed as the primary interaction,

the effect of pH of delivery buffer on M-IPG column was

investigated. The pH of 10 mmol/L ammonium formate

solution was adjusted with formic acid to pH 2.1, 3.0, and

6.3. It can be seen from Fig. 2C that the retention time of

PHA was increased with the increase of pH from 2.1 to 6.3,

but the retention time of BA and HBA first slightly

decreased and then increased. With the increase in pH

value, the resolutions of all the three compounds were

increased. However, the peak width of all the three

compounds became broader when the pH value was

increased from 2.1 to 6.3 (Fig. 2D), showing the strong

ionic interaction of negatively charged analytes with the

amine moiety of the solid phase. However, when the pH of

delivery buffer was 2.1, the monolith column could only be

used for two or three times due to the possible collapse of

the monolithic matrix. Therefore, the buffer with a pH value

of 3.0 was chosen as the delivery buffer of three compounds

considering both the retention time and the peak width.

Since the content of organic modifier in the delivery

buffer has a great effect on the mobilization, a water-

miscible solvent, ACN, was selected. The effect of ACN on

retention time and peak width was investigated by varying

its percentage in the mobile phase while keeping the pH

constant at 3.0. When the ACN content was increased from

0 to 50% (i.e. decreasing polarity of the mobile phase), both

retention time and peak widths of all the three compounds

were decreased (Fig. 2E and F). However, when the ACN

content was increased up to 100%, the retention time of

PHA was increased as well, and the peak width was

increased up to 22 min. In order to explain the peak

broadening of PHA with the increase in ACN content in the

mobile phase, PHA was chosen as the sample and 98%

Figure 2. Effect of focusingtime on retention time (A)and peak width (B), effect ofpH on retention time (C) andpeak width (D), and effect ofACN content on retentiontime (E) and peak width (F).Sample: BA (~, pKa 4.20);HBA (& , pKa 4.07); PHA (m,pKa 2.95); 33.3 ppm, respec-tively, were dissolved in 1%(v/v) NH3/H2O. Experimentconditions: catholyte (sheathliquid), 50% 2-propanol–49%water–1% ammonia (v/v/v);anolyte, 1% formic acid;voltage, 350 V/cm; and theflow rate of micropump,382 nL/min.



ACN in 10 mmol/L ammonium formate (pH 3.0) as the

delivery buffer. The peak width of PHA was down to

5.52 min. Theoretically, if pKa – 2 value was larger than the

pH of anolyte solution (1.8), the acid should be existing in a

neutral form. On the contrary, the acid probably existed as

two forms of neutral and ions (i.e. one or two H1 were lost).

Since pKa – 2 value of PHA (0.95) was lower than the pH of

anolyte solution (1.8), PHA might exist as C8H6O4,

C8H5O�4 , and C8H4O2�4 . When 100% ACN was used as the

delivery buffer, C8H5O�4 and C8H4O2�4 were not dissolved in

100% ACN, resulting in the severe peak broadening. With

98% ACN used as the delivery buffer, most of C8H5O�4 and

C8H4O2�4 were dissolved in 2% 10 mmol/L ammonium

formate (pH 3.0), resulting in a narrow peak in comparison

to 100% ACN. According to the mechanism mentioned

above, pKa – 2 values of BA and HBA were larger than the

pH of anolyte (1.8), both of them might exist as a neutral

molecule, thus the peak widths did not broaden as PHA

with 100% ACN as the delivery buffer. The mobile phase

with 50% ACN was selected as the eluent to achieve a

smaller peak width of PHA.

3.5 Method evaluation

With the optimal conditions, a typical electropherogram of

three LMW acids is shown in Fig. 3. Although BA and HBA

only yielded rough baseline separation (resolution factor of

1.5), the resolution was acceptable since the selective

detection of MS was additionally applied. Since partial

fragments of PHA might lose a carboxyl group even at low

capillary temperature, an additional and separated peak at

the extracted ion chromatogram of BA was observed

Figure 3. Analysis of threeLMW acids by M-IPG CIEF-ESI/MS. (A) Total ion electro-pherogram; (B) extracted ionelectropherogram of BA atm/z 121.2; (C) extracted ionelectropherogram of HBA atm/z 137.3; (D) extracted ionelectropherogram of PHAat m/z 165.5. Pump liquid,50% 10 mmol/L ammoniumformate (pH 3.0)–50% ACN(v/v). Other conditions are thesame as in Fig. 2.

Table 1. Run-to-run repeatability of the M-IPG CIEF separation

of the LMW acids (n 5 4)

Sample 1 (RT/min) 2 (RT/min) 3 (RT/min) 4 (RT/min) %RSD

BA 4.55 4.66 4.59 4.53 5.73

HBA 4.68 4.83 4.72 4.64 8.18

PHA 10.37 10.31 10.25 10.2 7.37

Figure 4. Linear correlation between the volume of the solventneeded to deliver the peak to the detector and the pKa of themodel components.

J. Sep. Sci. 2011, 34, 422–427426 T. Wang et al.


(Fig. 3B, peak a). The run-to-run repeatability of M-IPG CIEF

separation of LMW acids was assessed by the %RSD of

retention time from one monolithic column within a day

(n 5 4), ranging from 5.73 to 8.18% (Table 1). As shown in

Fig. 4, the linear correlation between the volume of the solvent

required to deliver the peak to the detector and pKa were

observed for the three LMW acids. As expected, the weakest

acid was eluted earliest and the strongest was eluted later.

4 Concluding remarks

An M-IPG CIEF-ESI/MS system was developed for the

separation of non-amphoteric LMW organic acids. Good

efficiency was achieved for the separation of the model BA

derivatives and the linear correlation of the volume of the

solvent as a function of the pKa was observed, showing that

the separation of the analytes by M-IPG CIEF was based on

the acid dissociation constant for non-amphoteric

compounds. This method could also be extended to the

analysis of acids and bases that cannot be separated well

and, therefore, might provide a suitable characterization tool

for natural organic matter of different environmental

importance (soil, sediment, water, and aerosols). The related

work concerning more organic acids, such as natural

organic matter of soil, is in process in our lab.

The main part of this work was performed at HemholtzZentrum Muenchen. The authors are grateful for the financialsupport from National Basic Research Program of China(2007CB914100), National Natural Science Foundation(20935004), Knowledge Innovation Program of Chinese Acad-emy of Sciences (KJCX2YW.H09) and Sino-German Coopera-tion Project (GZ 3164).

The authors have declared no conflict of interest.

5 References

[1] Hjerten, S., Zhu, M. D., J. Chromatogr. 1985, 346,265–270.

[2] Silvertand, L. H. H., Sastre Torano, J., van Bennekom,W. P., de Jong, G. J., J. Chromatogr. A 2008, 1204,157–170.

[3] Shimura, K., Electrophoresis 2009, 30, 11–28.

[4] Shen, Y., Xiang, F., Veenstra, T. D., Fung, E. N., Smith,R. D., Anal. Chem. 1999, 71, 5348–5353.

[5] Jin, Y., Luo, G., Oka, T., Manabe, T., Electrophoresis2002, 23, 3385–3391.

[6] Busnel, J. M., Varenne, A., Descroix, S., Peltre, G.,Gohon, Y., Gareil, P., Electrophoresis 2005, 26,3369–3379.

[7] Slais, K., Friedl, Z., J. Chromatogr. A 1994, 661, 249–256.

[8] Schmitt, P., Poiger, T., Simon, R., Freitag, D., Kettrup, A.,Garrison, A. W., Anal. Chem. 1997, 69, 2559–2566.

[9] Schmitt, P., Garrison, A. W., Freitag, D., Kettrup, A.,Water Res. 1997, 31, 2037–2049.

[10] Storms, H. F., van der Heijden, R., Tjaden, U. R., van derGreef, J., J. Chromatogr. B 2005, 824, 189–200.

[11] Storms, H. F., van der Heijden, R., Tjaden, U. R., van derGreef, J., Electrophoresis 2004, 25, 3461–3467.

[12] Lamoree, M. H., Tjaden, U. R., van der Greef, J.,J. Chromatogr. A 1997, 777, 31–39.

[13] Wu, J. Q., Pawliszyn, J., Anal. Chem. 1995, 67,2010–2014.

[14] Chartogne, A., Tjaden, U. R., van der Greef, J., RapidCommun. Mass Spectrom. 2000, 14, 1269–1274.

[15] Zhou, F., Johnston, M. V., Anal. Chem. 2004, 76,2734–2740.

[16] Wang, W. J., Guo, T., Rudnick, P. A., Song, T., Li, J.,Zhuang, Z. P., Zheng, W. X., DeVoe, D. L., Lee, C. S.,Balgley, B. M., Anal. Chem. 2007, 79, 1002–1009.

[17] Yang, C., Zhu, G. J., Zhang, L. H., Zhang, W. B., Zhang,Y. K., Electrophoresis 2004, 25, 1729–1734.

[18] Zhu, G. J., Yuan, H. M., Zhao, P., Zhang, L. H., Liang, Z.,Zhang, W. B., Zhang, Y. K., Electrophoresis 2006, 27,3578–3583.

[19] Liang, Y., Cong, Y. Z., Liang, Z., Zhang, L. H., Zhang,Y. K., Electrophoresis 2009, 30, 4034–4039.

[20] Wang, T. T., Ma, J. F., Zhu, G. J., Shan, Y. C., Liang, Z.,Zhang, L. H., Zhang, Y. K., J. Sep. Sci. 2010, 33,3194–3200.



Analysis of low molecular weight acids by monolithic immobilized pH gradient-based capillary...

Documents

Transcript of Analysis of low molecular weight acids by monolithic immobilized pH gradient-based capillary...