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PRICIPLE & APPLICATION OFISOTHERMAL
PROGRAMMING &PTGCMODERN PHARMACEUTICAL
ANALYSIS
PADODARA RUCHIT K.1ST M.PHARMA
DEPARTMENT OF PHARMACHEMISTRY NGSMIPS
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INTRODUCTIONGAS CHROMATOGRAPHY
It is a technique for the separation of thermally stable and volatile organic and inorganic compounds.
The principle of separation in gas liquid
chromatography is partition, where as in gas solid
chromatography principle of separation is adsorption.
Partition coefficient and solubility of a solute depends
upon temperature therefore temperature
maintenance in a column is highly essential for
efficient separation.
Hence the column as well as injecting devices should
be maintained at a particular temperature.
ISOTHERMAL PROGRAMMING
Isothermal programming in which the same
temperature is maintained through out the process of
separation.
Gas chromatograms are usually obtained with the
column kept at a constant temperature.
Many samples have components with a very wide
range of volatility. The temperature directly affects the
tendency of organic compounds to enter the gas phase
and therefore affects k, the distribution coefficient.
At low temperature, the higher boiling point
compounds will spend most of its time in the
stationary phase and emerge from the column only
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after a prolonged time period. So the GC peak is very
much broadened and the data is not very useful.
At higher temperature, the more volatile i.e; low boiling
components may not be resolved.Isothermal OperationIn general, retention times become shorter as the column temperature
increases,
primarily as a result of increasing solute vapor pressures. The column
temperature
also influences solute-specific interactions, such as polarizability,
hydrogen
bonding, and steric hindrance, which gives rise to differential effects
and causessolute relative retentions to change with temperature.
Figure 4.8 illustrates both effects for a capillary column test mixture.
At 90C,
dodecane is eluted last at around 11 min. As the temperature increases
in 10increments, all of the peaks¶ retention times decrease, and the entire
separation
takes only 3.6 min at 120C. The solutes¶ retention times decrease
by about
half for every 15±20C increase in column temperature. However,
the last two
peaks merge at 100±110C, and naphthalene becomes the last peak
at 120C.
Thus, careful attention must be paid to unambiguous peak
identification during
a separation optimization that includes the column temperature.
We can illustrate the various peaks¶ retention behavior as a functionof temperature
by making a plot such as shown in Figure 4.9, which presents the log
of the retention factor as a function of the reciprocal of the (absolute)
column.
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DISADVANTAGES OF ISOTHERMAL PROGRAMMING
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Early peaks are sharp and closely spaced that is
resolution is relatively poor.
Late peaks tend to be low, broad and widely spaced i.e;
resolution is excessive.
Higher boiling point compounds are often
undetected.
PROGRAMMED TEMPERATURE GAS
CHROMATOGRAPHY
PTGC was developed by Steve dal Nogare.
The temperature of the whole column is raised at a
controlled rate during the sample analysis.
The mobile phase is gas and solute components are
separated as vapours.
The components in the mixture have wide range of boiling
point so the temperature programming helps in the
increasing the resolution by reducing the width of thepeaks and also reducing the retention time of the sample
with very high retention time.
PRINCIPLE
The variation in temperature may cause change in
retention time.
Elevated temperature causes decreased mobile phase viscosity, increased mass transfer & increased sample
solubility resulting in better resolution & faster analysis.
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In this a lower initial temperature is used & the
temperature of the column is maintained at a suitable low
temperature such as 50o C during injection.
The column temperature is than increased at a controlledrate i.e;20o C per min up to a maximum temperature as
high as 300o C.
As the temperature increases the vapour pressure of the
middle and higher boiling components increases & they in
turn emerge from the column and are resolved and
analyzed.
METHODS TO INCREACE COLUMN TEMPERATURE
DURING ELUTION PROCESS:
Temperature is increaced immediately after sample
injection & brought to programmed level & kept constant
untill high boiling component have eluted out & then
returned to normal.
Initial column temperature is maintained for few minutes
after sample injection & then increasing the temperature to
a predetermined level.
In this, increasing the column temperature in several steps
before reaching the final temperature
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ADVANTAGES
It permits the separation of compounds of very wide boiling
range more rapidly than isothermal operation.
Nicely shaped peaks are obtained.
Total analysis time is shorter than isothermal operation.
DISADVANTAGES
As the temperature of the column is increased the bleed
rate of liquid phase of some column increases, causing an
upward slope in base line which interfere with desired
analysis.
INSTRUMENTATION
The essential features for PTGC operation are:
1) Separate heaters for injection port, column oven &detector.
2) A temperature programmer.
3) A low mass oven.
4) A liquid phase.
5) Differential flow controller.
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6) Pure, dry carrier gas
SEPARATE HEATERS
The injection port, column oven & detector should be
controlled by separate heaters & well insulated from others.
The change in temperature of any one of these during the
analysis is not desirable, particularly TCDs are effected by
changes in temperature while FID is not sensitive to
temperature changes.
TEMPERATURE PROGRAMMER
A mechanism which can precisely reproduce a range of
programming rates 0.25oC to 20oC per minute is essential
for identification by retention time & for quantitation by
peak height.
The initial temperature chosen is normally less than the
boiling point of the low boiling components.
The heating rate is chosen is compromise betweenresolution & speed of analysis.
At lower rates analysis time is too long for high boiling &
band determination will take place.
At high rates severe loss of resolution occurs.
The typical rates used for 6-10 feet columns are 1oC to 4oC
per min.
The final temperature chosen should be near the boiling
point at the highest boiling component present in the
mixture.
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LOW MASS OVEN
This is required to allow rapid heating
& cooling of the column.
A thin walled short columns are used.
Stainless steel oven with tight lid & high
speed circulating air fans seems to be
best used.
LIQUID PHASE
It must be stable at maximum operating temperature.
V aporization for liquid phase is referred as bleeding, it
produces noise, shifting of base line & changes in column
characteristics.
LIQUID PHASE MAXIMUM COLUMNTEMPERATURE
A)NON POLAR PHASES
Methyl silicone gum rubber 350
o
C
Flouro silicone 250o
C
Methyl silicone 350o
C
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B)POLAR PHASES
Versamid 900 250o
C
Methyl phenyl silicone 200o
C
Steroid analysis phase 250o
C
FLOW CONTROLLER
Differential flow controller with increase in inlet pressure
with respect to increase in column temperature are
employed.
Is required to provide a constant carrier gas flow rate
during programming.
PURE DRY CARRIER GAS
A molecular sieve filter is used to remove traces of water
which produce ghost peak under programmed condition.
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Pr r mm d T mp r t r GC (PTGC)
Low t mp r t r
Weakly retained component (low boilin point ) resol v ed
Stron ly retained components (hi h boilin points) not
eluted in desired time
Hi h temperature
Stron ly retained components eluted and detected
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W eakly retained components poorly resolved
Programmed temperature
Temperature is increased during run
Good retention and resolution for wide range of boiling
points
APPLICATION
Analysis of mixture containing components with a wide
range of molecular weight:
1) Alcohols from CH3OH to C20H41OH
2) Paraffins from CH4 to C40H80
The following class of drugs can be analysed:
1) Diuretics:- Acetazolamide, Benzthiazide.
2) Laxatives:- Aloe emodin, biscodyl.
3) NSAIDS:- Aceclofenac, Acetanilide
4) Antibiotics:-penicillins, gentamycin,neomycin
It is used to determine the purity of drugs,
eg,atropine sulphate, fenfluramine tablet
Isolation and identification of mixtures of components like
amino acids, plants extracts, volatile oils.
Isolation and identification of drugs or metabolites in
urine, plasma, serum etc
CONCLUSION
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The complex multi component samples that cover a wide
temperature range of boiling point can not be analyzed by a
single isothermal run.
So this disadvantage can be overcome by the use of PTGC.This technique has extended the use of gas
chromatography to the analysis of mixtures containing
components with a wide range of molecular weights.
REFERENCES
A.V.Kasture & K.R.Mahadik, Pharmaceutical
analysis(instrumental methods),2nd edition page no 95-99
H obart H .Willard, Instrumental methods of analysis, 7 th
edition Page no 562-565
Robert L. Grob & Eugene F. Barry, Modern practice of Gas
chromatography, 4th edition