Ion Chromatography – Basics & Applications in Food Safety

Chetan Chaudhari
Applications – IC/SP

The world leader in serving science

 What is Chromatography?

Chromatography is a technique for separating mixtures into their

components in order to analyze, identify, purify, and/or quantify the
mixture or components.

• Analyze
Separate • Identify
• Purify

Mixture Components
• Quantify

2
 What is Ion Chromatography

1) Ion Chromatography is a liquid chromatographic technique, with

which ionic and strongly polar species can be separated and detected
2) Ions can be either Positive Charge (Cations) or Negative Charge
(Anions)
3) Separation takes place according to their affinity for Ion Exchange
Stationary Phases.
Anion
Cation

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 IC Flow path

Proprietary & Confidential 4

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 Distinguish between HPLC and IC

HPLC IC

1 The ‘hardware’ of HPLC is made up The ‘hardware’ of IC is made up of PEEK

of SS material.(Poly Ether Ether Ketone)

2 Generally non ionic mobile phase is Ionic mobile phase is required

use.
3 Stationary phase used in HPLC is Polystyrene base ion exchange resin
mostly bonded Silica copolymer with styrene and dvb is generally
used as stationary phase in IC.

4 Only molecule is determined by Charged species as well as some molecule

HPLC can be determined.
5 Absorbance is measured in HPLC Primarily Conductance is measured in IC

6 Can not used as IC. Can be used as RP- HPLC.

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 Principle of Separation on Ion Chromatography (Anions)
Anions (- ve Charge)
Eluent should give free OH-/ CO3-2/HCO3-1
Strong Bases like NaOH/ KOH and Sodium Carbonate and
Bicarbonate
Anion Column will be having Positive sites on Stationary Phase like
Quaternary Ammonium Groups with Positive Charge.
At the time of System Equilibration Negative ions from the Eluent will
get adsorbed on Positive charge of the column towards Neutrality
Negatively charged analytes will compete with Negative charged
Hydroxide or Carbonate or Bicarbonate Eluent Ions for Positive sites
on the column.
The anions will get attached and again get displaced from positive
sites of stationary phase according to their Charge and Size.
Elution Order: F < Cl < Br < SO4< PO3

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 Ion Exchange Separation – Na2CO3 and NaHCO3 Eluent

In case of NaOH or KOH Eluent “OH” will be the available ions for Interaction
CO32- SO42- CO32-
HCO3 - HCO3 - -
＋ ＋ ＋ HCO3
＋ CO 2- ＋ COSO32- ＋ SO 2-
Equilibrium ＋
3
＋
4
2-
＋
4

＋ HCO3- CO32- ＋ HCO3- CO32- ＋ HCO3- CO32-

＋ 2- ＋ 2- ＋ 2-
CO3 CO3 CO3
＋ HCO3- ＋ Cl- ＋ HCO3-
＋ HCO3 - ＋ HCO3 - ＋ HCO3 -
＋ HCO - ＋ HCO
Cl- 3- ＋ Cl-
3
＋ HCO - ＋ HCO - ＋ HCO -
3 3 3
＋ ＋ ＋ HCO3-
＋ CO 2-
HCO3- ＋ CO 2-
HCO3- ＋ CO 2-
3 3 3

HCO3- CO32- HCO3- CO32-

＋ ＋
＋ SO 2- ＋ SO 2-
4 32-
CO
4
＋ ＋
＋ HCO3- CO32- ＋ HCO3- SO42-
＋ ＋ 2-
2-
CO3 CO3
＋ ＋
HCO3- HCO3-
＋ HCO3- ＋ HCO3-
CO
＋ Cl - 2- ＋ HCO3-
3
＋ HCO - ＋ HCO -
3 3
＋ Cl- ＋
＋ CO 2- ＋ CO32- HCO3-
3

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 Principle of Separation on Ion Chromatography (Cations)

Cations (+ ve Charge)

Eluent should give free H+

Strong Acids like Methane Sulfonic acid or Sulfuric Acid
Cation Column will be having Negative sites on Stationary Phase like
Sulfonates and Carbonates Groups with Negative Charge.
At the time of System Equilibration Positive ions from the Eluent (H+)
will get adsorbed on Negative charge of the column towards
Neutrality.
Positively charged analytes (NaCl, KCl) will compete with Positive
charged Hydrogen Ions for Negative sites on the column.
The Cations will get attached and again get displaced from Negative
sites of stationary phase according to their Charge and Size.
Elution Order: Li < Na < K < Mg< Ca

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 Retention Determining Parameters

Charge Size Polarizability

-1 --2 -3
-1 -2
-1
-1-1 -2
-3-3
-1
-1

-1

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 Instrumentation: Pump, Autosampler

I) Eluent Reservoir – It is nothing but mobile phase container. This can

be made up of good quality of glass or polypropylene depending upon
the nature of mobile phase.

II) Pump – For optimal system performance it is essential that the pump
provide smooth, accurate, and precise eluent delivery .The pump
material as well as fluid path in the system is made up of Peek (Poly
ether ether ketone) material. This peek material is compatible with 0 to
14 PH and with most of organic solvents.

III)Sample Injector – This can be manual or auto to load the fixed amount
of standard as well as sample in to the column.

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 Instrumentation: Guard and Separator Column
• I) Guard Column – generally guard column is made up of same material as analytical
column. Guard column prevents direct shock to main analytical column. AG –Anion Guard.

• II) Analytical Column – The column is the heart of ion chromatography. Physically, it
consists of a chemically inert tube packed with a polymeric resin. IC columns are available
in different sizes and packed with different resins depending upon the application and
desired mode of separation. AS – Anion Separator

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 Working Principle of Suppressor

Power cable

REGN OUT
From Eluent OUT To
Column Detector

Eluent IN
REGN IN

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 Chemistry and Ion Movement in an Anion SRS ®

REGN REGN
Eluent IN
Anode Cathode
Waste/Vent Waste/Vent
Na+, Cl- , F-,OH-
in NaOH Eluant

H2O, O2 NaOH, H2
-
OH
H+ Na+
+ -
H + O2 H+ + OH H2 O H2 + OH-
+ -
H , Cl
H2 O in H2O H2 O

H2 O To Detector H 2O

H2 O
+
2H + ½ O2 + Cation- 2H2O + 2e
- -
2OH +
2e
- Exchange H2
Membranes

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Chemistry and Ion Movement in Cation SRS

Anode Cathode
Waste/Vent Waste/Vent
Na + in H+MSA-
Eluent

H2O + O2 H2O,H2

H+ MSA- MSA-
4H+ +O2 OH-
H+ + OH- H2 O
H2 + 2 OH -

2 H2 O Na+ OH- in
2 H2 O
H2 O

To Detector
H2 O H 2O

Anion
Exchange
Membranes
13696-
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 Equivalent Conductance's of Common Ions

Anions Cations

Ion l0(µS cm-1) Ion l0(µS cm-1)

-
OH 198.6 H+ 349.8
-
CI 76.4 Na+ 50.1

SO42- 80.0 K+ 73.5

-
NO3 71.5
-
MSA * 48.8

*MSA-: Methanesulfonate

8145-
04
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 Advantages of Suppressor

1. It reduces the background signal of Conductivity Detector. Increases the sensitivity of analysis.

2. It converts analyte in to highly conducting dilute acid and base and hence increases the response of
the analyte and hence the sensitivity.

3. It removes all the counter ions from Eluent as well as standard salts and hence the analysis become
more selective.

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 3) Detection mode :- Conductivity Detector

• The conductivity of a solution is measured by applying an alternating voltage between two electrodes in a conductivity cell.

• At any instant in time, negatively charged anions migrate towards positive electrode and positively charged cations migrate
towards negative electrode

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 4) Data Mode :- Chromeleon Software

• Signal generated by detector is transmitted to Recorder or directly displayed as a chromatogram on

computer. The concentration of ionic analytes are automatically determined and tabulated.

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 Ion Chromatography Instruments (Gradient Compatible)

1. Aquion System

1. Isocratic Pump

2. Max Pressure limit

5000 psi

3. Only Conductivity
and UV detector

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 Ion Chromatography Instruments (Gradient Compatible)

2. Integrion System

1. Isocratic Pump

2. Max pressure limit

5000 psi

3. Conductivity,
Amperometry and
UV detector (One at
a time)

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 Ion Chromatography Instruments (Gradient Compatible)

3. ICS 6000 System

High End System
1. Quaternary gradient Pump

2. Max. pressure 6000 psi

3. Consumable Monitoring

3. Conductivity,
Amperometry and UV
detector (in series)

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 Ion-Exchange Chromatography Offers Simple, Direct, and Interference-Free Analyses

• Sensitive Detection
Samples can be diluted to decrease the concentration of matrix components

• Specific Detection
Low abundant analytes can be detected in presence of large concentration of
matrix components

• Analyte-Specific Separations
The selectivity of the separator columns is tailored for the specific requirements
of the various analyte classes

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 Advantages of Ion Chromatography / 1

Speed
- Complete anion and cation profiles in about 15-20 minutes

Sensitivity
- Analyses in the lowest µg/L-range without pre-concentration
- Analyses in the lowest ng/L-range after pre-concentration

Selectivity
- Huge variety of stationary phases
- Specific detection (suppression, UV, fluorescence, MS, ICP)

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 Advantages of Ion Chromatography / 2

Simultaneity
- Simultaneous analysis of many sample components (In contrast to AAS, photometry, titration, etc.)

Costs
- Cost effective as Mobile phase like coustic or Acids in diluted form is required for analysis.

Robustness
- pH and solvent compatible separators allow a variety of applications
- Analysis of complex matrices such as wastewater, foods, body fluids, etc.

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 Example: Retention Time Change as per capacity

• Capacity

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 Anion Standard on IonPac AS12A

10 Column: IonPac AS12A

Eluent: 0.3 mM Sodium bicarbonate
8 1 2.7 mM Sodium carbonate
Flow Rate: 1.5 mL/min.
2 Inj. Volume: 25 µL
6 5
10 Detection: Suppressed Conductivity
4 Peaks: 1. Fluoride 3
4
µS mg/L
3
6 8 2. Chlorite 10
7 3. Bromate 10
2
9 4. Chloride 4
5. Nitrite 10
0
6. Bromide 10
7. Chlorate 10
-2 8. Nitrate 10
9. Orthophosphate 10
0 2 4 6 8 10 12 14
10. Sulfate 20
Minutes
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10495
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 Separation of Alkali and Alkaline-Earth Metals and Ammonium

Separator: IonPac CS12A

20 4 Eluant: 18 mmol/L MSA
Flow rate: 1 mL/min
1 Inj. volume: 25 µL
2 Detection: Suppressed conductivity,
Recycle Mode
7 8
Peaks: 1. Lithium 1 mg/L
µS 2. Sodium 4
5 3. Ammonium 5
4. Potassium 10
3 6 5. Rubidium 10
10 6. Cesium 10
9
7. Magnesium 5
8. Calcium 10
0
9. Strontium 10
25 10. Barium 10
0 5 10 15 20
Minutes
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 ELECTRO CHEMICAL
DETECTION

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ELECTROCHEMICAL DETECTION

The electrochemical detector A

requires three electrodes:
V

 Working Electrode (where the

oxidation or reduction takes place)

 Auxiliary Electrode (counter-

REFERENCE

AUXILIARY
WORKING
electrode, cell body)

 Reference Electrode (compensates

for any changes in the background
signal of the mobile phase)

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 Design of Amperometirc Detector

Ampermetic Detector Consist of three part :

[Link] electrode
[Link] electrode
[Link] electrode

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 Principle of Separation on Ion Chromatography (Carbohydrates)
Carbohydrates (with neutral charge)
Eluent comprises of High Strength Hydroxide Phase with sodium acetate for highly retaining
analytes
The carboPac series of columns with Pellicular Anion exchange resin bed.

Carbohydrates molecules get ionized at High pH of Eluent forms Oxy-Anions.

These Oxy-Anions retains on Anion exchange columns according to their pKa value.

Higher the pKa value, Lesser the retention

Sodium acetate accelerates the elution of strongly bound species without compromising selectivity
and without interfering with pulsed ampereometric detection.

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Principle
Principleof of
Separation on Ion
Separation on Chromatography (Carbohydrates)
Ion Chromatography (Carbohydrates)

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Principle of Detection on Ion Chromatography (Carbohydrates)
Carbohydrates
Single Potential (DC) or Waveform (series of potential) runs continuously on Working
electrode.
After eluting out from column, the analyte comes in contact with Working electrode and get
either oxidized or reduced at the detection potential.

After give and take electrons the current will be generated (recorded as peak), and it will
be directly proportional to Analyte concentration.
Constant potential is always maintained between Working and Auxiliary electrode; while
Reference electrode whose potential is already known measures and controls the
constant potential among them.

In three electrode system, when working electrode works as cathode (Oxidative potential)
similar magnitude reductive potential will be generated at other half cell i.e. Auxiliary
electrode.

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Principle of Detection on Ion Chromatography (Carbohydrates)

Eluent and analyte coming out from column and going to the working electrode surface where it get either
oxidizes or reduced and current generated which shows the peak.

Column Eluent and Analytes coming out

of column & going to Detector

1 mm Working Electrode where

Ox/Red of analyte takes place

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 Direct-Current (DC) Amperometry

How it works: • Constant Potential is applied to an electrode

• Redox of analyte generates current which is measured.

 Catecholamines
Applications:  Some inorganic anions

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 Example: Analysis of Sulfide and Cyanide

1
HS-

Column: CarboPac PA1 with Guard

Eluent: 0.1 mol/L NaOH + 0.5 mol/L
NaOAc + 0.5 % (v/v)
ethylenediamine
µA Flow rate: 1 mL/min
Detection: DC Amperometry,
Ag electrode, 0 V
Inj. volume: 50 µL
CN-
Sample
preparation: Samples have been acidified,
distilled acc. DIN 38 405 D13
and absorbed in 0.1-1 mol/L
0 NaOH.

0 5 10
Minutes

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 Integrated Amperometry

• Rapidly repeating pattern of potentials (“waveform”)

How it works: applied to electrode
• Current integrated at analytical potential
• Electrode continuously reconditioned

Applications:
 Carbohydrates
 Amino acids
 Alcohols
 Sulfur-containing compounds
 Many electroactive inorganic ions

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 Standard Quadruple Waveform for Carbohydrates

1.00 Time Potential Integration

E3 (t3)
(s) (V)
0.50
Integrate 0.00 0.10
Potential (V vs. Ag/AgCl)

E1 (t1)
0.00 0.20 0.10 Start
E4 (t4)
-0.50 0.40 0.10 End

0.41 -2.0
-1.00
0.42 -2.0
-1.50
0.43 0.60
E2 (t2)
-2.00 0.44 -0.10
0.00 0.10 0.20 0.30 0.40 0.50
0.50 -0.10
Time (Seconds)

1. Detection potential (E1) 2. Duration time of E1 (t1)

3. Integration period (Integrate)
4. Reductive cleaning potential (E2) 5. Duration time of E2 (t2)
6. Oxidative cleaning potential (E3) 7. Duration time of E3 (t3)
8. Pre-detection (oxide reduction) potential (E4) 9. Duration time of E4 (t4)
Dionex (now part of Thermo Scientific) Technical Note 21 Optimal Settings for Pulsed Amperometric
Detection of Carbohydrates Using the Dionex ED40 Electochemical Detector. Sunnyvale, CA, 1998.

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 Chromatogram : Example

Detection of food sugars and sugar alcohols

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 UV-VIS DETECTOR

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 Flow Chart for PCR + UV-Vis analysis – Transition Metals and Chromium

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Principle of Separation on Ion Chromatography (Transition metals)

Metals (with net Positive charge)

Eluent Contains Chelating agents like carboxylic acid (Pyridine Dicarboxylic acid-PDCA) or
Organic acid (Oxalic acid) acid with pH modifiers
The column CS 5 A will be having both Anion as well as cation exchangers
The metal ions will form Complexes with Chelating agents and these complexes will bear
particular charge (either positive or Negative)
With Carboxylic acid Eluent the charges will be negative; while with Oxalic acid eluent the
charges will be both positive and Negative.
Metal complexes has different charges and Stability Constants (Log k) on which elution order
depends.
This eluate comes in contact with PAR indicator in PCR; where these metal complexes again
reacts with PAR indicator to form chromophore groups which further get detected on visible
wavelength (530 nm)

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Principle of Separation on Ion Chromatography (Transition metals)

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Principle of Separation on Ion Chromatography (Transition metals)

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 Species Detected by Ion Chromatography

Conductivity DC Amperometry Integrated UV-Vis

Amperometry
Inorganic anions Catecholamines Carbohydrates Silicate

Inorganic Cations Phenols Aliphatic amines 10, 20, 30

Bromate
Carboxylic acids Aromatic amines Amino acids Chromate

Sulfonic acids Thiols Alcohols Transition metals

Phosphonic acids Cyanide Aldehydes Lanthanides

Amines, 10, 20, 30 Sulfide S species Nitrite, Nitrate

(except S[VI])
Iodide Sulfite
Sulfite Iodide

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 APPLICATIONS

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Water: Bromate in Drinking water
In Disinfection process of water, Ozonation causes formation of Bromate in water which has been proven to be
carcinogenic.

Column Ion Pac AS 19 (250 X 4mm) + Guard

Eluent 20 mM NaOH
Flow 1.0 ml/Min
Injection 250 µl
Vol
detection Conductivity with Anion Suppressor
(Current 50 mA)
LOD 1 µg/L

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Water: Perchlorate in Drinking water
Perchlorate analysis:
Commonly used Oxidizing agent in Solid Propellants, Rockets, fireworks etc. Adverse Health effect because of its
presence in ground water:

Column Ion Pac AS 16 (250 X 2mm) 4 µ +

Guard
Eluent 65 mM NaOH
Flow 0.38 ml/Min
Injection 250 µl
Vol
detection Conductivity with Anion Suppressor
(Current 62 mA)
LOQ 1 µg/L

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Water: Inorganic anions in Drinking water (EPA Method 300 and 300.1)

Column Ion Pac AS 22 (250 X 4mm) Guard

Eluent 4.5 mM Carbonate+1.4 mM

Bicarbonate
Flow 1.2 ml/Min
Injection 10 µl
Vol
detection Conductivity with Anion Suppressor
(Current 31 mA)

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Water: Cyanide in Ground water and Drinking water

Cyanide in ground water may be the source. Cyanide is toxic even at low concentrations

1
HS-
Column Ion Pac AS 7 (250 X 4mm) + Guard

Eluent 0.1 mol/L NaOH+0.5 mol/L

NaOAC+0.5 % v/v Ethylenediamine µA
Flow 1.0 ml/Min
Injection 50 µl CN-
Vol
detection DC Amperometry (Gold working
electrode) OR Cyanide Waveform 0

LOQ 1 µg/L 0 5 10
Minutes

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Water: Total and Hexavalent Chromium in Drinking water

Hexavalent Chromium can enter in ground water through paints, dyes, wood preservatives etc.

Column Ion Pac CS 5A (250 X 4mm) + Guard

Eluent PDCA Eluent

Flow 1.0 ml/Min
Injection 50 µl Std
Vol
Sample
detection UV Detector at 365 nm

LOQ 50 µg/L

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Food: Bromate and iodate in Bread and its floor.
Potassium Bromate and Iodate are added in Bread to make it spongier and improve its size. But Bromate is
Carcinogenic.

Column Ion Pac AS 19 (250 X 4mm) + Guard

Eluent 20 mM NaOH
PCR 0.3 % Promethazine in 5M HCl (0.5
ml/min)
Eluent Flow 1.0 ml/Min

Injection Vol 25 µl Bread Samples

detection UV detector 515 nm

Standard

LOD 0.03 mg/L

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Food: Choline in Infant Milk
Choline is required additive in infant formulas and hence need to be evaluated

Standard
Column Ion Pac CS 19 (250 X 2mm) + Guard

Eluent 6.5 mM MSA

Eluent Flow 0.25 ml/Min

Injection Vol 5 µl

detection Conductivity Detector with cation

suppressor (Current 4 mA)

Sample

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Food: Iodide in Infant Milk
Excess Iodide may lead to Thyroid disorders in babies

Column Ion Pac AS 15 (150 X 3 mm) + Guard

Eluent 0.25 M NaOH+1 M NaOAC

Eluent Flow 0.5 ml/Min

Injection Vol 25 µl

detection Pulsed Amperometry with Silver

working electrode

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Food: Sugars in Dairy Products
Sugar contents need to be Specified on Nutritional fact labels of dairy products.

Standard
Column CarboPac PA 1 Analytical + Guard

Eluent Hydroxide Gradient

Eluent Flow 0.25 ml/Min

PCR 0.3 M NaOH at 0.3 ml/min Flow rate

Injection Vol 5 µl Milk Sample

Detection Pulsed Amperometry with Gold

working electrode and Ag/AgCl
Reference electrode.

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Food: Polyphosphates in Cheese samples

A) Bad Quality
Column AS 11 (250 X 2 mm) + AG 11 (50 X 2
mm)
Eluent Hydroxide Gradient
Eluent Flow 0.3 ml/Min

Injection Vol 10 µl
A) Good Quality
detection Suppressed Conductivity (Anion
Suppressor)
Peaks: 1. PO4
2. P2O7
3. P3O9
4. P3O10
5. P4O12
6. P4O13

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Monosaccharides in Soluble Coffee
Profile of Monosaccharides in coffee sample evaluates its quality 35
Standard (1-9 µg/mL)

Column CarboPac PA 10 with Guard nC

1
3 6 8 11
2 5
4 9 10
Eluent 1 mM KOH 7
20
Eluent Flow 1.0 ml/min (Post Column Addition of
0 5 10 15 20 25 30 35 40 45 50
0.3 M NaOH)
3
Injection Vol 5 µl 35
Instant Coffee
5 (OnGuard RP Treatment)
1
detection Pulsed Amperometry with Gold
nC
working electrode 2
9
6
Peaks : 4 7 8 10 11
1. Mannitol 7. Sucrose 20
2. Fucose 8. Xylose
3. Arabinose 9. Mannose 0 5 10 15 20 25 30 35 40 45 50
4. Rhamnose 10. Fructose
Minutes
5. Galactose 11. Ribose
6. Glucose

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Food: Fluoride in Tea
Fluoride may cause dental fluorosis, Bone fractures etc. better Alternative for current Ion selective electrode
method NY/T 838-2004

Column Ion Pac AS 18 (150 X 4 mm) + Guard

Eluent 20 mM KOH
Eluent Flow 1.0 ml/min

Injection Vol 100 µl

detection Suppressed Conductivity with Anion

Suppressor

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Beverages: Citrate and Phosphate in Soft Drinks
Phosphoric acid and Citric acid used in Cola Syrup as a Stabilizer and Taste maker. Their concentrations are
monitored in Manufacturing of Syrup and bottling of Cola Product.

Column Ion Pac Anion III A (150 X 3 mm) +

Guard Anion Standard
Eluent 22 mM KOH
Eluent Flow 0.5 ml/Min

Injection Vol 25 µl

detection Suppressed Conductivity

Cola sample

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Beverages: Sugars in Soft Drinks
Sugars should be monitored in Soft drinks for quality purpose.
1. Glucose
2. Fructose
Column Carbopac PA 20 Analytical + Guard 3. Sucrose

Eluent 33 mM KOH Soft Drink 2

Eluent Flow 0.5 ml/Min
Diet Soft Drink 1
Injection Vol 10 µl
Soft Drink 1
Detection Pulsed Amperometry with Ag/AgCl Ref.
electrode Quadrapole Waveform Standard

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Beverages: High Conc Sugars in Scotch Liqueur Sample
In Scotch Liqueur samples Sugars are monitored for Quality testing purpose
1. Glucose
2. Fructose
3. Sucrose
Column Carbopac PA 20 Analytical + Guard

Eluent 35 mM KOH with 100 mM KOH wash

Eluent Flow 0.5 ml/Min

Injection Vol 0.4 µl

Detection Pulsed Amperometry with Ag/AgCl Ref. Scotch Liqueur

electrode Quadrapole Waveform sample

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Beverages: Organic acid Content in Beer samples
Organic acids are end products of yeast fermentation critical to the flavor of beer, but are also products of
bacterial fermentation that introduce a sour flavor, either purposely or unintentionally due to spoilage.

Column Ion Pac AS 11 HC Column l + Guard

Eluent Hydroxide gradient

Eluent Flow 0.38 ml/Min

Injection Vol 2.5 µl

Detection Suppressed Conductivity

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Beverages: Organic acid Content in Beer samples

Standard

63
Beverages: Organic acid Content in Beer samples

Beer Samples

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 For Any Queries Contact :
Chanakya Thaker : 9769109963 ([Link]@[Link])
Chetan Chaudhari : 7738714447 ([Link]@[Link])
Ganesh N : 9892048258 (ganesh.n@[Link])
Santosh Gunaga : 9880175960 ([Link]@[Link])

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