Ultra Fast Liquid Chromatography Coupled With Graphene-Modified

Electrode For Simultaneous Determination Of Sulfonamides

Present ByMiss Nupattaranee Thammasoontaree

Faculty Of Science Chulalongkorn University

1

OUTLINE

IntroductionObjective

Experimental section

Results and discussion Conclusi

ons2

INTRODUCTIONWhat Is Antibiotic?

● A Drug that kills or slows the growth of bacteria

● One class of "antimicrobials"

● Can be used to treat infection

3

Sulfonamide

Antibacterial drugs

Sulfonamides are drugs with bacteriostatic

activity

used asfeed

additives and treating

infections 4

Toxicology

Advantages

Most widely used antibacterial agents in animal feed

● Low cost● Excellent activity against bacterial diseases ● Growth promoters in food animals

100 ng/g food

● Cancer ● Hypersensitivity

Sulfonamide

Sulfonamide advantages

5

N S

H

HN

O

O

H

R

Sulfamonomethoxine(SMM)

Sulfadoxin(SDX)

Sulfamethoxazole(SMX)

Sulfamethazine(SMZ)

Sulfonamide

The basic structure of sulfonamides

Sulfadimethoxin(SDM)

Sulfaquanidine(SG)

Sulfadiazine(SDZ)

Sulfisoxazole(SSZ)

6

GAS CHROMATOGRAPHY (GC)

THIN-LAYER CHROMATOGRAPHY (TLC)

CAPILLARY ELECTROPHORESIS (CE)

HIGH-PERFORMANCE LIQUID

CHROMATOGRAPHY (HPLC)

Sulfonamide

Methods for determination

SAs

7

Ultra Fast Performance Liquid Chromatographic

SystemsSwitch From HPLC to UFLC

Sulfonamide

Methods for determination SAs

● A new class of separation science● Improved resolution, Speed, and sensitivity ● Suitable for chromatographic applications in general 8

UFLC

Increased resolutionIncreased sensitivity

Reduced diffusion path

High flow rate

Sulfonamide

Van deemter equation

9

Electrochemical detection

Easily measurable signalExtreme sensitivityFast detectionHigh selectivity

Why ECD?

Electrochemical detection (ECD) for HPLC or

UFLC is an extremely selective and sensitive

detection technique

10

Glassy carbon

Sulfonamide

Conventional working

electrodes

Boron-doped diamond

Multi-wall carbon nanotubesSingle-wall carbon nanotubes

11

Sulfonamide

The new working electrode

Graphene (G) – a two dimensional sheet of sp2 carbon atom

an attractive materials in electrochemistry

High electrical conductivity

High mechanical strength

Low manufacturing cost

12

OUTLINE

IntroductionObjective

Experimental

Results and discussion Conclusi

ons13

OBJECTIVETO APPLY A GRAPHENE-MODIFIED

SCREEN-PRINTED CARBON ELECTRODE AS THE WORKING

ELECTRODE FOR THE DETERMINATION OF

SULFONAMIDES BY ULTRA FAST LIQUID CHROMATOGRAPHY

COUPLED WITH ELECTROCHEMICAL DETECTION

14

OUTLINE

IntroductionObjective

Experimental section

Results and discussion Conclusi

ons15

Experimental

Preparation of working electrode

PVC

A SCREEN-PRINTED TECHNIQUES

The conductive pad of Ag/AgCl

Screen-printed carbon electrodes

16

GRAPHENE-MODIFIED SCREEN-PRINTED CARBON ELECTRODES

Graphene/PANI Solution

spraying

high voltage

Graphene-modified electrode

Experimental

Preparation of working electrode

17

Experimental

Batch experiment

Cyclic Voltammetric investigation

Potential range : 0.7-1.5 V

Reference Electrode : Ag/AgCl

Counter Electrode : Pt wire

SA Concentration : 50 ppm

Electrolyte : PBS (pH 3)

Scan rate : 0.05 V/sec

Characterization of graphene-modified screen-printed carbon electrode

Batch Condition

18

Experimental

Chromatographic experiment

Inertsil C4 column 150mm X 4.6 nm i.d; partical size, 5 µm

Optimal potential of amperometry

Optimal condition of UFLC-ECD

Analytical performance &

Validation 19

OUTLINE

IntroductionObjective

Experimental

Results and discussion Conclusi

ons20

Cyclic voltammograms of [Fe(CN)6]3-/4- 1 mM for (---) Graphene-modified carbon

electrode and (---) Bare carbon electrode vs. Ag/AgCl with scan rate 100 mV/s

Results

Characterization Of Electrode

21

Results

Characterization Of The Electrodes

Fig. SEM images of G/PANI (1:1) nanocomposites, condition, No. needle; No.24 ( 0.45), voltage; 10 kV, time; 5 min

22

Cyclic voltammograms of SAs in 0.1 M phosphate solution pH 3.0 at (---)

Graphene-modified screen-printed carbon electrode and (---) Bare carbon electrode.

SMM

SDM

SG

Results

Cyclic voltammetric investigation

SDX

SMX

SSZ

23

Cyclic voltammograms of SAs in 0.1 M phosphate solution pH 3.0 at (---)

Graphene modified screen-printed carbon electrode and (---) Bare carbon electrode.

SDZ

SMZ

Results

Cyclic voltammetric investigation

24

Results

Optimal potential of amperometry

Hydrodynamic voltammetric results

1 1.1 1.2 1.3 1.4 1.5 1.60

0.0200000000000001

0.0400000000000001

0.0600000000000002

0.0800000000000002

0.1

12345678

Potential (V)

Curr

ent (

µA)

The graph of 10 ppm mixture eight standard SAs at a flow rate of 1.5 mL min-1. The detection potential at 1.3 – 1.5 V vs. Ag/AgCl using a graphene-modified screen-printed carbon electrode.

SGSDZSMZSMMSDXSMXSSZSDM

25

Results

Optimal condition of UPLC-ECD

Chromatogram of eight SAs 10 ppm in potassium hydrogen phosphate solution pH 3

Chromatogram of eight SAs 10 ppm in potassium hydrogen phosphate solution pH 4

Chromatogram of eight SAs 10 ppm in potassium hydrogen phosphate solution pH 5

26

Results

Optimal condition of UPLC-ECD

Chromatogram of eight SAs 10 ppm in potassium hydrogen phosphate solution pH 6

Chromatogram of eight SAs 10 ppm in potassium hydrogen phosphate solution pH 7

Chromatogram of eight SAs 10 ppm in potassium hydrogen phosphate solution pH 3

27

Results

Optimal condition of UFLC-ECD

PARAMETERS OPTIMAL CONDITIONS

Column Inertsil C4 (150 x 4.6 mm) Mobile phase Phosphate buffer(pH3): ACN: EtOH

Flow rate 1.5 mL/minInjection Volume 30 µL

Temperature 25 ºCDetector Amperometric detection at 1.4 V

UPLC-EC chromatogram of mixture eight SAs at 10 µg mL-1

SG SDZ

SMZ SMM

SDX SMXSSZ

SDM

28

Results

Analytical performance &

Validation

0 2 4 6 8 10 120

200400600800

1000

f(x) = 71.9109141103393 x + 71.7833557025641R² = 0.993374193014272

SG

Concentration (ppm)

Curr

ent (

nA)

0 2 4 6 8 10 120

100200300400500

f(x) = 38.9702821845309 x + 34.6030830514827R² = 0.991338678928408

SDZ

Concentration (ppm)

Curr

ent (

nA)

0 2 4 6 8 10 120

50100150200250300350

f(x) = 29.6141006826013 x + 15.3195841969671R² = 0.992155433799811

SMZ

Concentration (ppm)

Curr

ent (

nA)

0 2 4 6 8 10 120

100

200

300

400

500

f(x) = 42.0514985874679 x − 3.19580099054062R² = 0.991469517791657

SMM

Concentration (ppm)Cu

rren

t (nA

)

Linearity of eight standard SAs by UFLC-ECD using graphene-modified screen-printed carbon electrode.

y = 29.236x + 28.891R2 = 0.9954

29

Results

Analytical performance &

Validation

0 2 4 6 8 10 120

100

200

300

400

f(x) = 33.7076410257022 x + 4.57061546977971R² = 0.991152049023365

SDX

Concentration (ppm)

Curr

ent (

nA)

0 2 4 6 8 10 120

100

200

300

400

500

f(x) = 42.0241283292064 x + 13.7354292511971R² = 0.992022895883044

SMX

Concentration (ppm)

Curr

ent (

nA)

0 2 4 6 8 10 120

50100150200250300350400

f(x) = 32.4938449842891 x + 22.4527297813584R² = 0.995075519250811

SSZ

Concentration (ppm)

Curr

ent (

nA)

0 2 4 6 8 10 120

50

100

150

200

250

f(x) = 20.6565292602179 x + 15.13459281681R² = 0.994863012901876

SDM

Concentration (ppm)Cu

rren

t (nA

)

Linearity of eight standard SAs by UFLC-ECD using graphene-modified screen-printed carbon electrode.

30

Results

Analytical performance &

Validation Analy

teLinearit

y (ppm)

R2 LOD (ppb

)

LOQ (ppb)

SG 0.01-10 0.9934 1.162 3.336

SDZ 0.01-10 0.9913 1.601 5.337

SMZ 0.01-10 0.9922 2.900 9.667

SMM 0.01-10 0.9954 2.467 8.224

SDX 0.01-10 0.9912 2.995 9.983

SMX 0.01-10 0.9920 2.513 8.376

SSZ 0.01-10 0.9951 3.287 10.957

SDM 0.01-10 0.9949 6.127 20.425The limits of detection (LOD) and limits of quantitation (LOQ) were calculated from 3Sbl/S and 10Sbl /S 31

Conclusions

UFLC-ECD

This method can be applied for the determinations of the analytes in food and/or cosmetic samples in near future

Graphene-modified screen-printed carbon electrode exhibit excellent performance for the electrochemical detection of SAs

The rapid UFLC-ECD techniques were achieved within 7 minutes for eight sulfonamides

32

33

ACKNOWLEDGEMENT

34

Prof. Dr. Orawon Chailapakul

Dr. Nadnudda Rodthongkum

Electrochemistry and Optical Spectroscopy Research Unit

Chulalongkorn University’s Graduate Scholarship on the

Auspicious Occasion of His Majesty the King’s 72nd Anniversary

for Academic Year 2010

35