CHROMATOGRAPHY- GAS LIQUID CHROMATOGRAPHY(GLC) AND HIGH PERFPORMANCE LIQUID CHROMATOGRAPHY (HPLC).BY ASHIM CHOWDHURY

Defi nition of chromatography

“ Chromatography is a physical method of separation, in which the components to be separated are distributed between two phases, one of these phases constituting a stationery bed of a large surface area, the other being a fluid that percolates through or along the stationary bed.” Keulemans (1959)

Chromatographic separations involve interactions between three components:

the mixture to be separated,

a solid stationary phase and

a mobile fluid (liquid or gas) phase.

Classification of chromatographic processes

Based on the solute properties involved in separation, chromatographic processes may be classified accordingly. The properties of solutes mainly used for chromatographic separations are:

adsorption

partition between two phases based on their solubility or volatility

ionization and

Molecular size and shape

Chromatographic Separations

Solute property

used

Chromatographic process with application technique

Stationary phase Mobile phase

Adsorption

Adsorption chromatography,

eg. TLC

Column chromatography

Thin layer of silica gel and glass plate.

Silica gel, florisil, celite, charcoal,

etc. in glass column

Organic solvents

Organic solvents

Solubility Partition chromatography,

e.g.i) Paper chromatography

Adsorbed water in cellulose fibres

Organic solvents

ii) HPLC Liquid phase held by the inert

solids in HPLC column

Organic, solvents, water etc

Chromatographic Separations contd …

Solute property

used

Chromatographic process with application technique

Stationary phase

Mobile phase

Volatility Partition chromatography,

GLC

Liquid phase held by the

inert solid in GC column

Gas

Ionization

Ion exchange chromatography

Matrix with ionized groups (e.g. synthetic

resins)

Aqueous buffer

solutions

Size and Shape

Gel filtration/GPC Hydrated gel (e.g., sephadex)

Aqueous buffer

solution

Samples must be volatile (significant vapour pressure below 250oC).

Derivatisation increase analyte volatility (but

cumbersome and introduces possible quantitative errors).

Most GC analytes are under 500 Da Molecular Weight for volatility purposes.

Samples that are thermally unstable may decompose.

Many GC detectors such as (FID) are destructive

GC samples are prepared in organic solvents and extraction of analytes from aqueous samples will be necessary.

Sample size is usually between 1 and 5 μl with typical

detector sensitivity between nanograms (ng) and

picograms (pg) on column.

Advantages of HPLC over GLC

GLC

HPLC analysis has no volatility issues

(however the analyte must be soluble in the mobile phase.)

It can analyse samples over a wide polarity range

HPLC has no real upper molecular weight limit

HPLC

Schematic Diagram of HPLC

Components of HPLC

The components are :

Solvent delivery system which includes a pump, associated pressure and flow controls and a filter on the inlet side.

Sample injection system

The Column

The detector

Strip chart recorder

Data handling device and microprocessor control

COMPOUNDS ANALYZED BY HPLC

HPLC – UV Detector.

Column: C18 (Shandon Hypersil 250 x 4.6 mm ODS 2 5μ ).

Mobile Phase: Methanol: Water (97:3).

Flow Rate: 1mL/min.

C.T. : 25°C.

Wave-length : 282nm.

Retention time: 3.75 ± 0.25 min

Detector: : UV-VIS detector

Data Processor : Chemito-5000

Solvent : acetonitile: water (9: 1)

Column : thermo Hypersil® ODS 5

Flow : 1 ml/min

Wave length (λmax.)

: 280 nm

Operating Conditions of HPLC (Model –Jasco, Japan)

HPLC Chromatogram of Analytical Chlorpyrifos and Its Two Metabolites

Peak no.

1 (RT: 2.133 mins) =TCP

2 (RT: 3.305 mins) =CHLORPYRIPHOS

3 (RT: 4.675 mins) =TMP

GAS LIQUID CHROMATOGRAPHY

GC ColumnsTYPES: Conventional packed columnOpen tubular column – WCOT (Wall Coated Open Tubular) PLOT (Porous Layer Open Tubular)

Parameters Capillary Packed

Length (mt.) 25 – 200 1.5 - 6

Diameter (mm.) 0.2 – 0.5 2 – 6

Film thickness (µm)

0.05 – 1 1 – 10

Total effective plates

1,50,000 (50 meters

length)

5000 (2 meter length)

Stationary Liquid Phase

Application Stationary Liquid PhaseHerbicides 50% Phenyl Polysiphenylene-

siloxane

OC Pesticides 5% Phenylor 95% Dimethyl Polysiloxane 50% Phenyl Polysiphenylene-siloxane

OP Pesticides 5% Phenyl or 95% Dimethyl Polysiloxane 50% Phenyl Polysiphenylene-siloxane

Pyrethroids 5% Phenyl or 95% Dimethyl Polysiloxane35% Phenyl Polysiphenylene-siloxane

CLEAN-UP

COMPOUNDS ANALYZED BY GLC

• GLC – Ni63 ECD

• Column: Glass column (6 m X 2 mm packed with 3% ov-101 on chromasorb with W.H.P. (80-100 mesh).

• O.T. : 255°C.• I.T.: 275°C and D.T.: 275°C.• Carrier gas: N2 at 60mL/min flow rate.

• Retention time: 5.86min

Dissipattion Pattern of Deltamethrin in Paddy Plant with Treatment

-0.5

0

0.5

1

1.5

2

2.5

3

3.5

4

0 2 4 6 8 10 12

Period of Incubation (Days)

Log

10 (R

esid

ue x

100

0)

T1 = 12.5g a.i./ ha

T2 = 25.0 g a.i./ ha

T10 = 125 g a.i./ ha

Dissipattion Pattern of Deltamethrin in Paddy Soil with Treatment

-1.5

-1

-0.5

0

0.5

1

1.5

2

2.5

3

3.5

0 2 4 6 8 10 12

Period of Incubation (Days)

Log

10 (R

esid

ue x

100

0)

T1 = 12.5g a.i./ ha

T2 = 25.0 g a.i./ ha

T10 = 125 g a.i./ ha

Dissipattion Pattern of Deltamethrin in Paddy Water with Treatment

-1

-0.5

0

0.5

1

1.5

2

2.5

3

3.5

0 2 4 6 8

Period of Incubation (Days)

Log

10 (R

esid

ue x

100

0)

T1 = 12.5g a.i./ ha

T2 = 25.0 g a.i./ ha

T10 = 125 g a.i./ ha

GLC – FPD in “p” mode.

Column: Megabore Column (DB-5).

O.T. : 220°C.

I.T.: 250°C.

D.T.: 275°C.

Carrier gas: N2 at 50mL/min flow rate. Air: 20mL/min, H2: 15mL/min.

Retention time: 5.86min .

Dissipation Pattern of Triazophos with treatment in Paddy Plant in 1st Season

0

0.5

1

1.5

2

2.5

3

3.5

4

0 10 20 30 40 50

Period of Incubation (Days)

Log 1

0 (R

esid

ue

x 10

00)

T1 = 400.0 g a.i./ ha

T2 = 800.0 g a.i./ ha

T10 = 4000.0 g a.i./ ha

Dissipation Pattern of Triazophos with treatment in Paddy Soil in 1st Season

-1

-0.5

0

0.5

1

1.5

2

2.5

3

3.5

4

0 10 20 30 40

Period of Incubation (Days)

Log

10(R

esid

ue

x 10

00)

T1 = 400.0 g a.i./ ha

T2 = 800.0 g a.i./ ha

T10 = 4000.0 g a.i./ ha

Dissipation Pattern of Triazophos with treatment in Paddy Water in 1st Season

0

0.5

1

1.5

2

2.5

3

3.5

0 2 4 6 8 10 12

Period of Incubation (Days)

Log 1

0(R

esid

ue

x 10

00)

T1 = 400.0 g a.i./ ha

T2 = 800.0 g a.i./ ha

T10 = 4000.0 g a.i./ ha

GLC - FID.

Column: Elite – 5 capillary column (30 m X 0.53 mm i.d., 0.50 µm thickness).

O.T. : 110°C for 1 min, to 250°C at 30°C / min, held for 1.0 min, @ 25°C /min to 285°C, held for 2.0 min.

I.T.: 285°C and D.T.: 290°C.

Carrier gas: Helium at 3.0 cm/s flow rate.

Injection volume was 1 µL.

Fatty Acid Methyl Esters were identified by comparison of retention times with known standards.

Table : 3 Results of Methomyl 40 SP residues and dissipation in/on pigeon pea seeds sprayed @ 300 g a . i. / ha (T1)

Days Season IMean Residues in ppm + SD (% Dissipation)

Season IIMeans residues in ppm

+ SD (% Dissipation)

0 3.90 + 0.08 3.50 + 0.28

1 1.39 + 2.02 (64.4) 1.38 + 0.06 (60.6)

3 0.45 + 0.02 (88.5) 0.28 + 0.03 (92.0)

5 Not Detected Not Detected

7 Not Detected Not Detected

10 Not Detected Not Detected

Y = 3.36 – 0.41 X Y = 3.29 – 0.41 xT1/2 = 0.73 day T1/2 = 0.73 day

TMRL = 4.07 days TMRL = 3.90 days

Table : 4 Results of Methomyl 40 SP residues and dissipation in/on pigeon pea seeds sprayed @ 450 g a . i. /ha (T2)

Days Season IMean Residues in ppm + SD (%

Dissipation)

Season IIMeans residues in ppm

+ SD (% Dissipation)

0 6.89 + 0.08 6.03 + 0.05

1 2.45 + 0.03 (64.4) 1.84 + 0.07 (69.5)

3 0.65 + 0.02 (90.6) 0.54 + 0.04) (91.0)

5 0.35 + 0.02 (94.9) 0.32 + 0.02 ( 94.7)

7 Not Detected Not Detected

10 Not Detected Not Detected

Y = 3.92 – 0.42 X Y = 3.82 – 0.41 xT1/2 = 0.72 day T1/2 = 0.73 day

TMRL = 5.31 days TMRL = 5.19 days

Courtesy : Dr. Ashim Chowdhury

University Of Calcutta

Uploaded by : Swayambhu Ghosh