FTIR STUDY OF THE ON-LINE PRECONCENTRATION OF A … · Fourier transform mid-infrared spectroscopic...

INTRODUCTIONFourier transform mid-infrared spectroscopic detection is proposed as an on-line detection technique for the study of on-line preconcentration processes incapillary electrophoresis (CE). The direct molecular specific information contained in mid-IR spectra can be used to directly determine the chemical composition ofindividual zones and their boundaries. From this information detailed insight in the actual chemical composition of the individual zones governing the efficiency ofthe applied pre-concentration technique could be gained.

FTIR STUDY OF THE ON-LINE PRECONCENTRATION OF A PROTEIN IN CE

G. Quintás1, E. Núñez1, M. Vellekoop2, B. Lendl1*1Institute of Chemical Technologies and Analytics, Vienna University of Technology, Vienna, Austria.

2Institute of Sensor and Actuators Systems, Vienna University of Technology, Vienna, Austria.

Getreidemarkt 9-164, A-1060 Vienna, Austria

*: Phone: +43 1 5880115140. Fax: +43 1 5880115199. e-mail: blendl@mail.zserv.tuwien.ac.at

SI-CE-UV-FTIR MANIFOLD

1. Fully automated sequential-injection (SI) - capillary electrophoresis(CE) system.

2. Hydrodynamic injection is carried out pressurizing the whole systemusing a 100 µl syringe pump.

3. Conditioning and inter-run cleaning is performed automatically.

4. Control of the injection volume.

SI-CE hydrodinamic injection Description of the injection process(1)

1. The sample is flushed through the interface with the injection valveopen to ensure that the sample is close to the capillary inlet.

2. The injection valve is closed, the syringe can pressurize the systemand sample is injected into the capillary.

3. The interface is rinsed with the background electrolyte (BGE).

4. The voltage is applied and the separation starts while BGE is

Uncoated capillary

2-way Selection

valve

IR flow cell

UV

2-way Selection

valve

BGE Reservoir

SI Selection

valve

UV detector

FTIR ELECTROPHEROGRAMUV electropherogram

UV detection at 200 nm only allowed to detect the

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Absorbance

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8 time (min)

Myoglobin trace

(1650 cm-1)

Acetic acid trace

(1712 cm-1)

Acetate trace

(1547 cm-1)

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QUANTITATIVE

From the on-line IR data, changes in the buffer compositioncan be monitored as well as the analytes detected.

FTIR spectra

wavenumber (cm-1)

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Sample solvent

BGE

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1712 cm-1

�-C=O

(COOH group)1280 cm-1

�-C-O(COOH group)

1547 cm-1

Asymmetric stretching

COO- group

Symmetric stretching

COO- group1416 cm-1

1457 cm-1

Ammonium

Amide II

Acetic acid

4. Control of the injection volume. 4. The voltage is applied and the separation starts while BGE isreplenished at a low flow rate using the same pump.NaOH H2O

100 µL Syringe pump

CZE HV

Waste

valve

Myoglobin

BGE Reservoir

pH JUNCTION

pH 9.25pH 4.75

EOF

JUNCTIONJUNCTION

pH 9.25pH 4.75

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JUNCTIONJUNCTION

pH 9.25pH 4.75

EOF

JUNCTIONJUNCTION

1. Upon application of the voltage, all protein ions (pH<pI) migrate towards thejunction.(2)

2. The pH change at the junction modify the protein’s ionization.

3. The protein molecules are focused at the junction as zwitterions (pI=pH shift at thejunction).

FTIR

electropherogram

UV

electropherogram

CONDITIONS FOR THE CE SEPARATION

BGE: Ammonium 20 mM, pH 9.25 (adjusted with HAc). pH>pI Protein

Sample solvent: HAc 20 mM, pH 4.75 (adjusted with NH3). pH<pI Protein

Capillary 50 µm i.d. uncoated fused silica, 60 cm length; HV: 20 kV

Sample: Myoglobin 2.5 g l-1

FTIR PARAMETERS

Resolution: 8 cm-1

Flow cell pathlength: 8 µm

Scans: 50 (sample); 500 (background)

HeNe laser modulation frequency: 180 kHz

pH 9.25 pH 9.25

CONCLUSIONSFTIR spectroscopy has been successfully applied to measure on-line the compositions of boundary zones formed in a pH junction experiment in capillary electrophoresis for protein analysis. Based on the directmolecular specific information characteristic CE peak and dispersion profiles for analyte as well as sample and background electrolyte buffer can be recorded simultaneously. The sensitivity of FTIR spectrometry forthis purpose is sufficient because of the high concentrations of buffers usually employed in CE. CE-FTIR may thus be considered a promising tool for gaining in depth understanding of the working principles of alsomore advanced on-capillary sample pre-concentration techniques.

Acknowledgements: G. Quintás is grateful for a post-doctoral grant from the Ministerio de Educación y Ciencia, Secretaría de Estado de Universidades e Investigación (Spain) (EX2004-1245).

REFERENCES(1) Kulka, S., Quintás, G., Lendl, B., Analyst, 2006, 131, 739-744.

(2) Nesbitt, C.A.; Lo, J.T.-M.; Yeung, K.K.-C., J. of Chromatogr. A, 2005,1073, 175-180.

The established pH gradient, as manifested bydifferent relative intensities of acetate and aceticacid, can nicely be observed at the tailing edge ofthe sample zone.

In all performed experiments the concentration ofacetate decreases before the intensity of the aceticacid band starts to decline. This experimentalobservation shows that in addition to contributionsfrom dispersion and established pH gradients alsothe electrophoretic mobilities of the buffer speciesinvolved are responsible for their actualconcentration profile in pH junction experiments.

UV detection at 200 nm only allowed to detect theanalyte but do not provide any information on thecomposition of the surrounding zones.

Extracted traces for two myoglobin injections usingdifferent injection volumes, corresponding to 32 %(A) and 35 % (B) of the total capillary volume.

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time (min)

Myoglobin trace (1650 cm-1)

Acetate trace (1547 cm-1)

Acetic acid trace (1712 cm-1)

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UV 200 nm UV 200 nm

Inspecting the traces as well as the 3D plot it can be seen that in the leading phase boundary andprior to the protein peak which corresponds to a pH range from 9.25 to 7.2 (pI of myoglobin), aslight increase in the intensity of bands at 1547, 1416 and 1457 cm-1 can be observed. Whereasthe increase of the acetate bands (1547 and 1416 cm-1) is to be related to the dispersion of thesample peak as acetate is clearly prevailing over acetic acid in this pH range, the increase at 1457cm-1 may be assigned to the increase of ammonium concentration upon decreasing pH.

Because the employed buffer systems have maximum buffer capacities at pH 9.25 and 4.75respectively, a rather sharp zone covering the pI value of the protein may be expected where,according to theory, the protein will concentrate. It can be clearly seen that the width of the sampleplug extends over 5 minutes as judged by the spectral changes in the boundary zones whereasthe protein is clearly focused in a much more narrow zone extending over approximately 55seconds.

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wavenumber (cm-1)

B

A

D

C

Z (points)wavenumber (cm-1)

QUANTITATIVE INFORMATION

SPECTRAL INFORMATION

ABC

D

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Z

(points)

Abs

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wavenumber (cm-1)

Amide I

Myoglobin

The bands of the system acetate – aceticcan be discerned.The amide I band is free from spectralinterference of the buffer systems usedin this work. Therefore this band can beused as a marker band to determine therelative position of the protein inside thediscontinuous system.

Schematic view of the flow cell used throughout the experiments. Dimensions: Cross section (8x150 µm), channel 2 mm long.