Gas Chromatograp

By

By

K Rakesh gupta

  INTRODUCTIONINTRODUCTION   PRINCIPLEPRINCIPLE   THEORIESTHEORIES   PARAMETERSPARAMETERS   INSTRUMENTATIONINSTRUMENTATION   APPLICATIONSAPPLICATIONS   REFERENCEREFERENCE

  INTRODUCTIONINTRODUCTION   PRINCIPLEPRINCIPLE   THEORIESTHEORIES   PARAMETERSPARAMETERS   INSTRUMENTATIONINSTRUMENTATION   APPLICATIONSAPPLICATIONS   REFERENCEREFERENCE





CHROMATOGRAPHY 

CHROMATOGRAPHY 

: : SEPSEPARAARATION TION TECHNIQUETECHNIQUE

:

: TWO TWO PHASES PHASES are are usedused

:

: MIKHAIL TSWETTMIKHAIL TSWETT _{inventedinvented}





GAS CHROMATOGRAPHY: 

GAS CHROMATOGRAPHY: 

MARTIN AND JAMESMARTIN AND JAMES

: 

: 

GAS as M.phase alwaysGAS as M.phase always

: solid or liquid S.Phase

: solid or liquid S.Phase



 Choice forChoice for

THERMALLY STABLE 

THERMALLY STABLE 

andand

VOLATILE 

VOLATILE 

compounds

compounds





TWO TYPES BASED ON STATIONARY PHASE 

TWO TYPES BASED ON STATIONARY PHASE 

GSC: 

GSC: 

Stationary phase isStationary phase is

SOLID 

SOLID 

GLC: 

 GSC

: ADSORPTION 

:RELATIVE AFFINITY 

 GLC

:

PARTITION 

: SOLUBILITY 

 Mainly three theories are involved in GC

1.

PLATE THEORY 

2.

BAND BROADENING THEORY 

 Concept compared counter-current distribution  Plates are hypothetical lines where equilibration occur  Plates analogous to tubes in CCD (Catalytic Combustion

Detector)system

 In plate theory two main terms are used as quantitative

measures of chromaographic column efficiency

PLATE HEIGHT( 

HETP)

CALCULATION OF THE DISTRIBUTION  THROUGH 4 TRANSFERS: Transfer no ’n’ Tube no. r = 0 Tube no r=1 Tube no: r=2 Tube no: r=4 Tube no: r=3 n=0 B A 0/1 p/q n=1 B A 0/q Pq/q2 p/0 p2 _/pq n=2 B A 0/q2 Pq2 _/q3 pq/pq 2p2 _q/2pq2 p2 _/0 p3 _/p2 _q n=3 B A 0/q3 Pq3 _/q4 pq2 _/2pq2 3p2_q/3p2 _q3 2p2 _q/p2 _q 3p3_q/3p2 _q2 p3 _/0 p4 _/p3 _q n=4 B 0/q4 _Pq3 _/3pq3 _3p2_q2 _/3p2_q2 _3p3_q/p3 _{q p/0} Total after 4 transfers q4 _4pq3 _6p2_q2 _4p3_{q p}4

 The expansion of the function (p+q)n is laborious

for large „n‟, and an easier calculation is available.

 The binomial expansion may be written

(p+q)n _{= q}n _{+ nq}n-1_{p + n(n-1) q}n-2 _q2 _{+……+ p}n

2 which can be expressed (p+q)n ₌ n_∑

r=0 n! prq(n-r)

 Binominal distribution expression for the fraction of total

solute in „rth‟ plate after „n‟ mobile phase volumes have

passed into the column

p

r

q

(n-r)

r! (n-r)!

n = no.of mobile phase volumes passed into the column

r = plate number( 0,1,2,3,…..r)

p = 1/(KU+1) = fraction of solute per plate in M.P at equilibrium q = KU/(KU+1)= fraction of solute per plate in S.P at equilibria

Tnr =

n!

p

r

_q

(n-r)

r! (n-r)!

 When „n‟ is large and „p‟ is not small (as in CCDS), binomial

distribution approaches normal (Gaussian) distribution

  According to statistic‟s MEAN is given as “ų”

 Standard deviation “σ”

ų = np

ELUTION CHROMATOGRAM

Concentration

| Tr |

CALCULATION FOR NO.OF PLATES 

( efficiency ) 

 Length= velocity. time

= Rvt_r

 Standard deviation „σ‟ = Rvז

Where „ז„ is zone-standard deviation

 Combining above equations

σ = L ז

t_r

 t_r2 = L ז2 / H since: σ2 = HL

 t_r = זr ½ since: ז= L/H

 Random walk

 Reflects loss of efficiency

 Rate process controls zone width  From plate theory : σ2 = HL

H is a measure of zone spreading and column efficiency

 ( height equivalent to theoretical plates)

HETP = Length of the column no.of theoretical plates

1.

LONGITUDINAL MOLECULAR 

DIFFUSION 

2.

MASS TRANSFER(SORPTION- 

DESORPTION KINETICS) 

1.LONGITUDINAL MOLECULAR 

DIFFUSION 

L= vt

σ²=2D_m t

σ²=2D_m L/v

H_diff =2γD_m /v γ is empirical factor of value 0.6

H_diff = 2γD_m + 2γ_s D_S (1-R)/R v

This is in the form of H_diff = B/v

Where „B‟ is the function of molecular and chromatographic

2.MASS TRANSFER(SORPTION- 

DESORPTION KINETICS) 

 No.of random steps „n‟ = 2L/vt_a

 True step length „L‟ =vt_a - Rvt_a or (1-R)vt_a  According to random walk theory σ²=L²n

σ²=2(1-R)²vt_a mm

3.EDDY DIFFUSION 

 Flow and diffusion mechanism are coupled

 Plate height contribution through flow and diffusion is not

additive ,but found to be

H_eddy = 1

1/H_F + 1/H_D

 H is independent of velocity and H is dependent on

average velocity

H_eddy = 1

1/A + 1/Ev

CALCULATION FOR HETP 

 Total plate height contribution from 3 process

H = H_eddy + H_diff + H_(s-d)

 Van Deemeter equation

H = A + B/v + Cv

H = 1 + B + Cv

 Random walk Fronting tailing  Quantitative measure detector signal time  Fronting- saturation of S.P

 RETENTION TIME ( R _t )

 RETENTION VOLUMN (R  _v )

  ADJUSTED RETENTION VOLUMN (V _R 1)  SELECTIVITY ( )

 RESOLUTION ( R _s)

 EFFICIENCY(NUMBER OF PLATES)’n’  HETP (H)

RETENTION TIME 

_(R 

_t 

₎ 

 Retention time is the difference in the time between the

point of injection and appearance of peak maxima

 Rt is the time required for 50% of a component to be

eluted from the column

 Unites : min or sec

 Retention time is also proportional to the distance moved

RETENTION VOLUMN 

RETENTION VOLUMN 

(V 

(V 

_R 

_R 

) 

) 



 Retention volume is the volume of carrier gasRetention volume is the volume of carrier gas

required to elute 50% of the component from

required to elute 50% of the component from thethe

column

column



 Corrected retention volumeCorrected retention volume



 Where „j‟ is pressure drop correction factor Where „j‟ is pressure drop correction factor 



 PP_ii and Pand P_oo are inlet and outlet pressuresare inlet and outlet pressures

Retention

Retention volume volume = = Retention Retention time time flow flow raterate

V V_RR00 _{= = j j VV} R R  j  j = = 3 . ( P3 . ( P_ii / P/ P_oo ))22 _{- 1- 1} 2 . (P 2 . (P_ii / P/ P_oo ))33 _{- 1- 1}

ADJUSTED RETENTION 

ADJUSTED RETENTION 

VOLUMN 

VOLUMN 

(V 

(V 

_R 

_R 

! 

! 

₎ 

₎ 



 Adjusted retention volume is calculated asAdjusted retention volume is calculated as

Where V

Where V_MM is is DEAD DEAD VOLUME VOLUME of of mobile mobile phasephase



 Applying pressure drop correction to VApplying pressure drop correction to V_RR!! GivesGives

“

“ net retention  net retention volume ”volume ”

V V_RR‟‟ _{= V= V} R R - V- VMM V V_NN = j = j VV_RR‟‟

SELECTIVITY 

SELECTIVITY 

(

(

) 

) 



 A A way of improving resolution way of improving resolution is to change the selectivityis to change the selectivity

of the column

of the column – – by changing stationary and mobileby changing stationary and mobile

phases

phases



 Selectivity is the rSelectivity is the ratio of partition coefficientsatio of partition coefficients 

 Selectivity term can be Selectivity term can be evaluated from the chromatogramevaluated from the chromatogram

=

= VV_R,2R,2 – – VV_{M M (or)(or)} = = trtr₂₂ - t- t_mm

V

RESOLUTION (R 

_s 

) 

 The degree of disengagement of two bands is resolution.  In terms of width and diameter

 In terms of time and width  In terms of zone of migration  In terms of , k ,N

where k is capacity factor k=nS/nM R_S = d_A –d_B W R_S = 2 Rt₁ -Rt₂ w_A + w_B R_S = L . R 16H R R_S = N k -1 4 k+1

EFFICIANCY;NO.OF PLATES(n) 

 Efficiency of column is expressed by the no. of theoretical

plates

 If the no.of theoretical plates is high, the column is said to

be highly efficient

 If the no.of theoretical plates is low , the column is said to

be less efficient

 For GC columns, a value of 600/meter is sufficient

 But for HPLC , high values like 40,000 to 70,000/meter

are recommended

HETP(H) 

 Decides the efficiency of separation

 If HETP is less, the column is more efficient  If HETP is more, the column is less efficient  HETP = Length of column

no.of theoretical plates

 HETP calculated by using Van Deemeter equation

HETP = A + B + Cv v

HETP 1

 INSTRUMENTATION



Carrier Gas



Flow regulators and meters



Sample injection system



Columns & ovens



Detectors

30

Gas Chromatograph

Components

Flame

Ionization Detector Column Oven Injection Port top view

front view

Carrier gas

32



The mobile phase gas is called the carrier gas and

must be chemically inert.

Sample component



column



detector



mobile phase gas

Helium ,argon ,nitrogen , carbon dioxide and

hydrogen also used.



Selection of the best carrier gas very important ,

because it effects both the column separation and

detector performance .



The ratio of viscosity of diffusion coefficient

should be minimum for rapid analysis that’s why

H, He are prepared for a carrier gas .



Impurities in the carrier gas such as air water vapour

and trace gaseous hydrocarbons can cause sample

reaction, column character and affect the detector

performance.



The carrier gas system should contains a molecular

sieve to remove water and other impurities.



These gases are available in pressurized tanks.

pressure regulators and flow meters are required to

control the flow rate of the gas.



The gases are supplied from the high pressure gas

cylinder , being stored at pressure up to 300psi



carrier gas should be better then 99.99%and 99.999%

Air inlet (detector))

H₂inlet (detector)

N₂inlet(make-up gas)

He inlet (carrier gas)

Process Flow Schematic

Carrier gas (nitrogen or helium) Sample injection

Long Column (30 m)

Detector (flame

ionization

detector or FID) Hydrogen

Air

Carrier Gas(mobile phase)



Requirements:



It should be inert and available at low cost



High purity



Easily available



Less risk of explosion or fire hazards



Pressure:

-Inlet



10 to 50 psi

-packed column

_

25 to 150 mL/min.

- capillary column



1 to 25 mL/min.

Flow regulators & meters



Flow regulators are used to deliver the gas

with uniform pressure or flow rate



Flow rates of carrier gas:

 –

Linear flow rate (cm/s): u = L/tr

 –

Volumetric flow rate (mL

/min): u (π r2)

L is length of column, it is retention time, r is the internal radius of column 

Flow rate depends on type of column

 –

Packed column: 25-100 mL/min

 –

Capillary column: 1 to 25 mL/min



Flow rate will

decrease 

as column T increases

Soap bubble flow meter Aqueous solution of soap or detergent 39



soap bubbles formed

indicates the flow rate.



Glass tube with a inlet tube

at the bottom.



Rubber bulb---store soap

solution



When the bulb is gently

pressed of soap solution is

converted into a bubble by

the pressure of a carrier gas

&travel up.

40

INJECTO

R

Sample injection port

 Calibrated Micro syringes are used to

inject liquid sample

 Purge :volatile components are

removed from sample by gentle heating

 Rubber or silicone diaphragm(septum)  Sample port Temp: 50°C

 Packed Column: sample sizes-1 to 20

μL

 Capillary Column : 10 to 30 mL

splitter is used to deliver a fraction of injection(1:50 to 1:500)

  Avoid over loading

 Slow injection & oversized samples

44

1. Wash a syringe with acetone by filling

the syringe completely and ejecting the

waste acetone onto a paper

towel. Wash 2-3 times.

2. Remove air bubbles in the syringe by

rapidly moving the plunger up and down

while the needle is in the sample.

3.Usually 1-2 mL of sample is injected

into the GC.

47



Column temperature is very important in GC



The column is ordinarily housed in a

thermostatic oven.



they are usually formed as coils having

diameters of 10 to 30 cm.



The optimum column temperature depends

upon the boiling point of the sample and the

degree of separation required.



Roughly, a temperature equal to or slightly

above the average boiling point of a sample

results in a reasonable elution time (2 to 30

min).

Columns

49



Two types of columns are used in gas

chromatography,

packed

and

open

tubular or capillary.



Packed column length from less than 2

m to 5 m



Capillary columns from few m to 100 m



They are constructed of stainless steel,

Column



Types of columns

1.packed columns

2. Open tubular or capillary.

50

Capillary column- 30m Packed column-3m

Packed columns



Packed columns are fabricated from glass,

metal (stainless steel, copper, aluminum), or

teflon tubes that typically have



Lengths--- 2m to 3 m



Internal diameters --- 2 to 4 mm.



These tubes are densely packed with a

uniform, finely divided packing material, or

solid support, that is coated with a thin layer

(0.05



m) of the stationary liquid phase.



In order to fit in a thermostatic oven, the tubes

are formed as coils having diameters of

Capillary (or)Open tubular

Columns

1.Wall-coated open tubular (WCOT)



Capillary tubes coated with a thin layer of

stationary phase



Old: stainless steel, Al, Cu, plastic, glass.

2.Support-coated open tubular (SCOT)



Inner surface of the capillary is lined with a thin

film (~30

μm)

of a support materials, like

diatomaceous earth



Lower efficiency than WCOT, higher than packed

column

3.Fused-silica open tubular column (FSOT):



Physical strength, low reactivity, flexibility. 0.32 to

0.25 mm

Column Stationary Phases:



Packed



liquid coated silica particles (<100-300 mm

diameter) in glass tube



best for large scale but slow and inefficient



Capillary/Open Tubular



wall-coated (WCOT) <1 mm thick liquid coating

on inside of silica tube



support-coated (SCOT) 30 mm thick coating of

liquid coated support on inside of silica tube



best for speed and efficiency but only small

The Stationary Phase



requirements for stationary phase



Low vapor pressure



Thermal stability



Low viscosity (for fast mass transfer)



High selectivity for compounds of interest

DETECTOR

S



Ideal characters of

detector

 High sensitivity to even small concentrtion

 linerity, ie, less response to low concentration

&proportional response to high concentration

 Large linear dynamic range

 Useful at a range of temperatures

 Good stability and reproducibility

 Rapid response time

 Easy to use  Stable, Predictable response  Inexpensive  operation from RT to 400 oC 57

Types of detectors

1.

Thermal Conductivity Detector(TCD)

2.

Flame Ionization Detector(FID)

3.

Atomic Emission Detector(AED)

4.

Electron Capture Detector(ECD)

5.

Nitrogen Phosphoroes Detector(NPD)

6.

Photo Ionization Detector(PID)

7.

Flame Photometric detector(FPD)

8.

Electrolytic conductivity detector (Hall

detector)

9.

Absolute Mass Detector(AMD)

10.

Thermionic Detector(TD)

ame on za on

Detector(FID)

Most widely used, Air-H2 flame

Number of ions depends on number of reduced (methylene) carbons in a molecule

The positive ions will be attracted to the cylindrical cathode.

Negative ions and electrons will be attracted to the jet anode.

Organic compounds  Produces ions and electrons  pyrolyzed(temp of flame)  burner tip and

electrode.(fhv power)

Ions &electrons move toward the collector

less sensitive for non-hydrocarbon groups

 Insensitive to noncombustible gases(CO2, SO2, NO2 and H2O)

Thermal Conductivity Detector(TCD)

Thermal conductivity detector cell

Arrangement of the twin detectors

Element(platinum, gold or tungsten wire) is electrically heated at constant power

 – Temperature depends on thermal

conductivity (He & H)of surrounding gas.

Hydrogen and helium have higher thermal conductivity and carrier gas provide best sensitivity

Six times greater than the Organic compounds

Poorer sensitivity than FID, but more universal

Advantages: simplicity, large range, inexpensive, linearity is excellent.

Electron Capture Detectors (ECD)

The sample elute from a column is passed over a radioactive β

-emitter(nickel-63)

 Selectively to halogen-containing organic sample ,like pesticides and, polychlorinated biphenyls

Ni-63: radioactive β-emitter electron --ionization of carrier gas (N2)

High electronegative group (halogen, peroxide, quinones and nitro group) in the sample capture the electron

Highly selective and sensitive, nondestructive

Insensitive to amines, alcohols and hydrocarbons

AD: High sensitivity, analyse the polyhalogenated organic compounds

Thermionic detector(nitrogen phosphorus detector)

N or P containing organic compounds

 phosphorus atom is approximately ten times greater than to a nitrogen atom and 104 _{to 10}6 _{larger than a carbon atom.}

Compared with the FID , the thermionic detector is approximately 500 times more sensitive to phosphorus-containing

compounds and 50 times more sensitive to nitrogen bearing species.

Column effluant + H2 +air(hot gas)electrically hearted Rb2SiO4

(rubidium silicate)bead at 180 V  plasma (600 – 800°C ) ions  to determine

compounds

useful for detecting and determining the many phosphorus-containing pesticides.

Eluent(column)  helium(carrier)  water cooled microwave cavity

 helium plasma(high temp)  characteristic atomic emission spectra  grating  diode array optical emission spectrometer 

detect the element .

Six elements detect simultaneously .

Determine the hetero atoms(H,P,S,O),silicon , heavy metals(Pb , Hg),tin, arsenic ,copper ,iron.



UV light (10.2 e

V

H2

or 11.7 e

V

Ar lamp)



photo ionization of

molecular



current

to flow between

based electrodes



Most sensitive for

Aromatic and S, P

easily

photoionized

molecules



Linear range is high

PHOTO IONIZATION

DETECTOR(PID)

Flame photometric detector

(FPD):



S and P

 –

compounds



photomultiplier to view light of 394

nm for sulphur (H

2

+ air



S

2

)

measurement or 526 nm for

phosphorus (H

2

+ air



HPO

species)



Filteres are used to isolate the

appropriate bands



Intensity is recorded

photometrically



X-, N-, Sn , Cr, Se and Ge

66 filteres photomultiplier H2+ air Column effluent

67

Qualitative analysis:



Retention time data should be useful for

identification of mixtures



Comparing the retention time of the

sample as well as the standard



Checking the purity of a compound:

compar the standard and sample



Additional peaks are

obtained…..impurities are

present….compound is not pure

Quantitative

analysis:

 Direct comparison method:

-comparing the area of the peak, peak height, width of peak.

 Calibration curves:

-standards of varying concentration are used determine peak areas .

o Internal standard method:

-A known concentration of the internal standard is added separately to the standard solution

-The peak area ratio of sample and internal

standard….unknown concentration is easily

determined .

Elemental analysis



Determination of C,H ,O ,S and N .



Determination of mixture of drugs



Isolation and identification of drugs



Isolation and identification of mixture of

components(amino acids ,plant extracts

,volatile oils)

 Instrumental Analysis by Douglas A.Skoog , F.James

Holler & Stanley R.Crouch.

 Text book of Pharmaceutical Analysis by Kenneth

A.Connor

 www.google .com

 Text book of Pharmaceutical analysis by Dr.S.Ravi Sankar  Introduction to instrumental analysis by Robert D.Braun  Chromatograhic methods by Smith