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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

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