Gas Chromatograp

By K Rakesh gupta

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INTRODUCTION

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PRINCIPLE

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THEORIES

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PARAMETERS

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INSTRUMENTATION

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APPLICATIONS

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REFERENCE

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INTRODUCTION

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PRINCIPLE

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THEORIES

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PARAMETERS

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INSTRUMENTATION

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APPLICATIONS

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REFERENCE

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CHROMATOGRAPHY : SEP SEPARA ARATION TION TECHNIQUE : TWO PHASES are used : MIKHAIL TSWETT invented

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GAS CHROMATOGRAPHY:  MARTIN AND JAMES : GAS as M.phase always : solid or liquid S.Phase Choice for THERMALLY STABLE and VOLATILE  compounds

TWO TYPES BASED ON STATIONARY PHASE  GSC: Stationary phase is SOLID  GLC: Stationary phase is LIQUID 

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GSC

: ADSORPTION  :RELATIVE AFFINITY  : More affinity towards S.P travels slowly-slow elution

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GLC :PARTITION 

: SOLUBILITY  : more partition coefficient travels slowly  – slow elution

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1. 2. 3.

Mainly three theories are involved in GC

PLATE THEORY  BAND BROADENING THEORY  RATE THEORY 

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Concept compared counter-current distribution Plates are hypothetical lines where equilibration occur

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Plates analogous to tubes in CCD (Catalytic Combustion Detector)system

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In plate theory two main terms are used as quantitative measures of chromaographic column efficiency PLATE HEIGHT( HETP) PLATE COUNT or no. of PLATES( N)

CALCULATION OF THE DISTRIBUTION  THROUGH 4 TRANSFERS: Transfer no Tube no. ’n’ 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 3p2q/3p2 q3

2p2 q/p2 q 3p3q/3p2 q2

p3 /0 p4 /p3 q

n=4

B

0/q4

Pq3 /3pq3

3p2q2 /3p2q2

3p3q/p3 q

p/0

q4

4pq3

6p2q2

4p3q

p4

Total after 4 transfers

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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 = qn + nqn-1p + n(n-1) qn-2 q2 +……+ pn 2 which can be expressed (p+q)n = n∑r=0

n! r!(n-r)!

prq(n-r)

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Binominal distribution expression for the fraction of total solute in „rth‟ plate after „n‟ mobile phase volumes have passed into the column r q(n-r)= pTnr

n! pr q(n-r) r! (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

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When „n‟ is large and „p‟ is not small (as in CCDS), binomial distribution approaches normal (Gaussian) distribution

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

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Standard deviation “σ” σ = np

ELUTION CHROMATOGRAM

Concentration

| time

Tr |

| width

|

CALCULATION FOR NO.OF PLATES  ( efficiency )  

Length= velocity. time = Rvtr

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Standard deviation „σ‟ = Rv‫ז‬ Where „‫ „ז‬is zone-standard deviation

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Combining above equations σ =L‫ז‬ tr since: σ2 = HL tr2 = L ‫ז‬2 / H since: ‫ =ז‬L/H tr = ‫ז‬r ½ 2 r = 16(t /w) r

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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

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LONGITUDINAL MOLECULAR  DIFFUSION 

2.

MASS TRANSFER(SORPTION-  DESORPTION KINETICS) 

3.

EDDY DIFFUSION 

1.LONGITUDINAL MOLECULAR  DIFFUSION  L= vt σ²=2Dm t σ²=2Dm L/v

Hdiff =2γDm /v

γ is empirical factor of value 0.6

Hdiff = 2γDm + 2γs DS (1-R)/R v This is in the form of Hdiff = B/v Where „B‟ is the function of molecular and chromatographic properties

2.MASS TRANSFER(SORPTION-  DESORPTION KINETICS)  

No.of random steps „n‟ = 2L/vt a

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True step length „L‟ =vta - Rvta or (1-R)vta

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According to random walk theory σ²=L²n σ²=2(1-R)²vta mm

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Hs-d = Cv

3.EDDY DIFFUSION  Flow and diffusion mechanism are coupled  Plate height contribution through flow and diffusion is not additive ,but found to be Heddy = 1 1/HF + 1/HD  H is independent of velocity and H is dependent on average velocity Heddy = 1 1/A + 1/Ev Where A and E include the partical diameter 

CALCULATION FOR HETP  

Total plate height contribution from 3 process H = Heddy + Hdiff + H(s-d)

H=

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1 + B + Cv 1/A + 1/Ev v

Van Deemeter equation H = A + B/v + Cv

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Random walk

Fronting

Quantitative measure

detector signal

time  

Fronting- saturation of S.P Tailing-more active adsorption

tailing

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RETENTION TIME ( R t )

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RETENTION VOLUMN (R  v )

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 ADJUSTED RETENTION VOLUMN (V R 1)

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SELECTIVITY (

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RESOLUTION ( R s)

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EFFICIENCY(NUMBER OF PLATES)’n’

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HETP (H)

RETENTION TIME (R t )  

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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 on a chart paper, which can be measured in cm or mm

RETENTION VOLUMN (V  VOLUMN (V R )  

Retention volume is the volume of carrier gas required to elute 50% of the component from the column Retention volume = Retention time

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Corrected retention volume VR0 = j

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flow rate

VR

Where „j‟ is pressure drop correction factor   j = 3 . ( Pi / Po )2 - 1 2 . (Pi / Po )3 - 1

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Pi and Po are inlet and outlet pressures

ADJUSTED RETENTION  VOLUMN (V  VOLUMN  (V R ! )  

Adjusted retention volume is calculated as VR‟ = VR - VM

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Where VM is DEAD VOLUME of mobile phase Applying pressure drop correction to V R! Gives “ net retention volume ” VN = j VR‟

SELECTIVITY (( SELECTIVITY  

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A way of improving resolution is to change the selectivity of the column – by changing stationary and mobile phases Selectivity is the ratio r atio of partition coefficients Selectivity term can be evaluated from the chromatogram = VR,2 – VM VR,1 – VM

(or)

= tr2 - tm tr1 - tm

RESOLUTION (R s )   

The degree of disengagement of two bands is resolution. In terms of width and diameter RS = dA –dB W

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In terms of time and width

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In terms of zone of migration

In terms of , k ,N where k is capacity factor k=nS/nM

RS = 2 Rt 1 -Rt2 wA + wB RS = L . 16H

R R

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RS =

N 4

k k+1

-1

EFFICIANCY;NO.OF PLATES(n)  

Efficiency of column is expressed by the no. of theoretical plates No.of theoretical plates

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α

efficiency

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 HETP

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1 EFFICIENCY

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

 INSTRUMENTATION     

Carrier Gas Flow regulators and meters Sample injection system Columns & ovens Detectors

SCHEMATIC DIAGRAM OF GAS CHROMATOGRAPH

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Gas Chromatograph Components top view Injection Port

Column Oven

front view

Flame Ionization Detector

Carrier gas 

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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 .

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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.

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The carrier gas system should contains a molecular sieve to remove water and other impurities.

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These gases are available in pressurized tanks. pressure regulators and flow meters are required to control the flow rate of the gas.

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The gases are supplied from the high pressure gas cylinder , being stored at pressure up to 300psi

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carrier gas should be better then 99.99%and 99.999% is often used

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H2 inlet (detector) Air inlet (detector))

He inlet (carrier gas)

N2 inlet(make-up gas)

Process Flow Schematic Sample injection Carrier gas (nitrogen or helium)

Long Column (30 m)

Detector (flame ionization detector or FID)

Air Hydrogen

Carrier Gas(mobile phase) 

Requirements:

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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.

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Flow regulators & meters 

Flow regulators are used to deliver the gas with uniform pressure or flow rate

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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

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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 37

FLOW REGULATORS

Soap bubble meter 

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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 Aqueous solution of the pressure of a carrier gas soap or detergent &travel up. 39

Soap bubble flow meter

inlet tube

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INJECTO R

Sample injection port 

Calibrated Micro syringes are used to inject liquid sample

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Purge :volatile components are removed from sample by gentle heating

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Rubber or silicone diaphragm(septum)

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Sample port Temp: 50°C

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Packed Column: sample sizes-1 to 20 μL

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Capillary Column : 10 to 30 mL splitter is used to deliver a fraction of injection(1:50 to 1:500)

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 Avoid over loading Slow injection & oversized samples cause band spreading & poor 

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Micro syringe

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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. 45

COLUMN OVENS

Column ovens  

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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).

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COLUMNS

Columns 

Two types of columns are used in gas chromatography, packed and open tubular or capillary.

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Packed column length from less than 2 m to 5 m

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Capillary columns from few m to 100 m

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They are constructed of stainless steel, glass, fused silica, or Teflon. 49

Column 

Types of columns

1.packed columns 2. Open tubular or capillary.

Packed column-3m

Capillary column- 30m 50

Packed columns 

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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 roughly 15 cm.

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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 (~30μm) of a support materials, like diatomaceous earth  Lower efficiency than WCOT, higher than packed column

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

0.25 mm

strength, low reactivity, flexibility. 0.32 to

Column Stationary Phases: 

Packed  

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liquid coated silica particles (