1.
PRESENTED BY
SUBODH S SATHEESH
MPHARM
PHARMACEUTICS
ECPS
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PHARMACEUTICAL ANALYSIS-GAS
CHROMATOGRAPHY

2.
 It is expressed by the number of theoretical plates
 It is determined by the formula
 The number of theoretical plates is a measure of the
“goodness” of the column
 If the retention time is high and peak width is narrow then it
shows excellent chromatograms
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PHARMACEUTICAL ANALYSIS-GAS
CHROMATOGRAPHY

3.
where tr is the retention time measured from the instant of injection
w is the peak width
W is determined by SD= σ ie w=4σ
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PHARMACEUTICAL ANALYSIS-GAS
CHROMATOGRAPHY

4.
 Resolution is the ability to seperate two signals
 In chromatography its the ability to seperate two peaks.ie
seperation of constituents
where tr1 and tr2 and w1 and w2 are the times and widths,
respectively, of the two immediately adjacent peaks.
 If the peaks are sufficiently close w is nearly the same for both
peaks and resolution may be expressed as

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PHARMACEUTICAL ANALYSIS-GAS
CHROMATOGRAPHY

5.
Greater the distance more resolution and vice versa.
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PHARMACEUTICAL ANALYSIS-GAS
CHROMATOGRAPHY

6.
 The rates of migration of substances in chromatographic
procedures depend on the relative affinity of the substances for
the stationary and the mobile phases
 Its the difference in time between the point of injection and the
time of emergence of separation of component from the
column.
 It is actually the time required for 50% of the component to
get eluted.
 It is measure in minutes or seconds
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PHARMACEUTICAL ANALYSIS-GAS
CHROMATOGRAPHY

7.
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PHARMACEUTICAL ANALYSIS-GAS
CHROMATOGRAPHY

8.
 It is the volume of carrier gas required to elute
components from the column to the time the peak
maximum is obtained.
 Retention volume depends upon flowrate and retention
time
 VR= tR-FC
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PHARMACEUTICAL ANALYSIS-GAS
CHROMATOGRAPHY

9.
 It is the ratio of partition coefficient of two components to be
separated.
 S= Kb/Ka = K’a/k’b= (tb-to)/(ta-to)
If peaks are far apart ie there is more difference in partition coefficient
between compounds hence more seperation factor and viceversa
Less seperation
factor
More seperation
factor
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PHARMACEUTICAL ANALYSIS-GAS
CHROMATOGRAPHY

10.
 HETP is numerically equal to the column length divided by
the number of theoretical plates in the column
 It varies from to one column to another as well as one solute to
other
 The more efficient the column the better the resolution and the
smaller the HETP.
 HETP=Length of column / no of theoretical plates
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PHARMACEUTICAL ANALYSIS-GAS
CHROMATOGRAPHY

11.
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PHARMACEUTICAL ANALYSIS-GAS
CHROMATOGRAPHY

12.
 A chromatographic peak should be symmetrical about its
centre to follow gaussian distribution
 Asymetric factor is the measure of peak tailing or fronting.
 It is defined as the distance from the centre line of the peak to
the back slope divided by the distance from the centre line of
the peak to the front slope.
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PHARMACEUTICAL ANALYSIS-GAS
CHROMATOGRAPHY

13.
The chromatographic peak in (a) is an example of tailing, which
occurs when some sites on the stationary phase retain the solute
more strongly than other sites.
The peak in (b) is an example of fronting, which most often is the
result of overloading the column with sample. For both (a) and (b)
the green chromatogram is the asymmetric peak and the red dashed
chromatogram shows the ideal, Gaussian peak shape.
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PHARMACEUTICAL ANALYSIS-GAS
CHROMATOGRAPHY

14.
 Its function is to separate sample components to
discrete peaks
 It should have reasonable chemical and thermal
stability
 There are a lot liquid stationary phases available for gas
chromatography. But there is no solvent that meet all
the requirements of a perfect stationary phase
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PHARMACEUTICAL ANALYSIS-GAS
CHROMATOGRAPHY

15.
 Liquid phase should not permeate too deeply into the fine
pores of the support structure as slow diffusion in and out of
pores affects column efficiency
 It should be chemically inert
 It should be a good solvent for sample component
 Liquid phase should have low volatility and high stability at
elevated temperatures otherwise they can contribute to
interference in analysis
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PHARMACEUTICAL ANALYSIS-GAS
CHROMATOGRAPHY

16.

Stationary Phase Trade Name Max Temp Common
Applications
Dimethyl Polysiloxane OV – 1, SE – 30 350oC Hydrocarbons,
Polynuclear
aromatics, PCB’s
Poly(phenyl methyl)
siloxane
OV – 17 250oC Steroids, Pesticides,
Glycols
Poly (Trifluoro propyl
dimethyl) siloxane
OV – 210 200oC Chlorinated
Aromatics, Nitro
Aromatics, Alkyl
substituted Benzenes
Polyethylene Glycol Carbowax 20 M 250oC Free acids, Alcohols,
Essential Oils, Glycols
5% Diphenyl – 95%
Dimethyl polysiloxane
DB – 5 325oC Flavors,
environmental
samples and aromatic
hydrocarbons
Typical liquid stationary phases
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PHARMACEUTICAL ANALYSIS-GAS
CHROMATOGRAPHY

17.
 Nonpolar solutes like pentane butane etc can be effectively
separated by nonpolar solvents like squalene. And polar
solutes can be easily separated by polar solvents eg; PEG.
 Boiling point is also a factor of consideration. among solutes
with similar polarity if there is sufficient difference in BP
effective separation can take place.
 Eg. Squalene min/max temp= 293/423 SE4 423/573
 A solvent that could generate different partition ratios
among solvents can only be useful in GLC
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PHARMACEUTICAL ANALYSIS-GAS
CHROMATOGRAPHY

18.
 H KAUR Instrumental methods of chemical analysis
ninth edition 2013 ;1091-1092
 Skoog holler crouch Instrumental analysis 2012 841-
846
 B K sharma Instrumental methods of chemical analysis
twenty fourth edition 2005 c188-191
 En.wikipedia.org gas chromatography
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CHROMATOGRAPHY

19.
MANY THANKS
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CHROMATOGRAPHY