CHNS ANALYSIS AND ACCELERATED STABILITY STUDIES

1. KARNATAKA COLLEGE OF PHARMACY
PHARMACEUTICAL ANALYSIS
SEMINAR TOPIC:
“C,H,N,S ANALYSIS AND ACCELERATED
STABILTY STUDIES”
GUIDED BY-
Dr. C. SREEDHAR (H.O.D)
(PH. ANALYSIS)
PREPARED BY-
JOSEF YAKIN
M.PHARM
1ST YEAR

2. C,H,N,S ANALYSIS

3. CONTENTS
 Introduction
 Methods
 Combustion analysis
 CHNS analyzer
 Experimentation
 Reference

4. Introduction
 Percent Composition is a process where a sample
of some material is analyzed for its elemental and
sometimes isotopic composition.
 Elemental analysis can be qualitative (determining
what elements are present), and it can be
quantitative (determining how much of each are
present).
 Elemental analysis falls within the ambit of
analytical chemistry.

5.  For organic chemists, elemental analysis or "EA"
almost always refers to CHNX analysis—the
determination of the mass fractions of carbon,
hydrogen, nitrogen, and heteroatoms (X) (halogens,
sulfur) of a sample.
 This information is important to help determine the
structure of an unknown compound.
 As well as it helps to determine the purity of a
synthesized compound.

6. Methods
 The most common form of elemental analysis, CHN
analysis, is accomplished by combustion analysis.
 In this technique, a sample is burned in an excess of
oxygen, and various traps collect the combustion
products—carbon dioxide, water, and nitric oxide.
 The masses of these combustion products can be
used to calculate the composition of the unknown
sample

7. Combustion analysis
 The method was invented by Joseph Louis Gay-
Lussac.
 Justus von Liebig studied the method while
working with Gay-Lussac between 1822 and 1824
and improved the method in the following years to
a level that it could used as standard procedure for
organic analysis.
 Combustion analysis is a method used in both
organic chemistry and analytical chemistry.

8.  It’s use to determine the elemental composition of a
pure organic compound by combusting the sample
under suitable conditions .
 where the resulting combustion products can be
quantitatively analyzed.
 Once the number of moles of each combustion
product has been determined the empirical formula
or a partial empirical formula of the original
compound can be calculated.
 A combustion train is an analytical tool for the
determination of elemental composition of a
chemical compound
 With knowledge of elemental composition a chemical
formula can be derived.

9.  The combustion train allows the determination of carbon
and hydrogen in a succession of steps:
 combustion of the sample at high temperatures with
copper oxide as the oxidizing agent,
 collection of the resulting gas in an anhydrous agent
(magnesium perchlorate or calcium chloride) to trap
generated water,
 collection of the remainder gas in a strong base (for
instance potassium hydroxide) to trap generated
carbon dioxide.
 Analytical determination of the amounts of water
and carbon dioxide produced from a known
amount of sample gives the empirical formula.

10.  For every hydrogen atom in the compound
1/2 equivalent of water is produced.
 And for every carbon atom in the compound
1 equivalent of carbon dioxide is produced.

11. CHNS Analyzer
 A CHN Analyzer is a scientific instrument which can
determine the elemental composition of a sample.
 The analyzer uses a combustion process to break
down substances into simple compounds which are
then measured.
 In this technique the substance under study is
combusted under oxygen stream in a furnace at high
temperatures.
 The end products of the combustion would be mostly
the oxides of the concerned elements in the form of
gases.

12.  These are then separated and carried to the
detector using inert gases like helium or argon.
 It is one of the few analytical techniques that give a
clear quantitative measurement of the carbon,
hydrogen, nitrogen and sulphur.
 CHN microanalysis is a powerful and relatively
straight forward method.
 Which is use for determining whether or not a
sample is pure by providing a precise and accurate
analysis of a sample's percentage carbon,
hydrogen and nitrogen content.

13.  The main drawbacks of CHN analysis are the
requirements for accurate weighing of 1-2 mgs of
sample (that is consumed in the analysis)
 And also the fact that samples are usually run as
an automated batch rather than on an individual
sample basis.
 The analysis of results is performed by
determining the ratio of elements from within the
sample, and working out a chemical formula that
fits with those results.

14.  The accepted deviation of elemental analysis
results from the calculated is 0.4%.
 The method for working out the ratio of elements
from the results is shown below:
 1. Take the percentage of each element found
and divide by the element's mass. Do this for all
the elements for which you have results
 2. Find the smallest value from step 1 and divide
every value obtained in step 1 by this smallest
value

15.  3. Multiply the results in step 2 by a factor to obtain
reasonable values for either carbon or nitrogen and
then compare to what was expected from a pure
sample of the compound that was thought to be
submitted.
 This process is tedious to perform by hand, and
automated tools have been released to simplify
with this process.

16. Experiment
 The original analysis
method is based on the
complete and
instantaneous oxidation
of the sample by "flash
combustion" which
converts all organic and
inorganic substances into
combustion products
Figure 6.1: C-H-N-S-O Analyzer

17.  Model: Perkin Elmer, U.S.A 2400 Series II
 CHNS/O Elemental Analyzer - Perkin Elmer PE 2400
 Analyzed Elements: Carbon, Hydrogen, Nitrogen, Sulfur
and Oxygen
 Operating Mode: CHN, CHNS and OXYGEN
 Accuracy: 0.3% abs
 Analysis Time: 6 to 8 minutes per sample
 Automatic Weight Transfer
 Sample Size: 1 to 200 mg
 Multiple Sample Mode if required: 60 samples
 Sample Combustion Temperature: Around 2000 º c.
SPECIFICATION

18. HOW IT WORKS
 From the combustion product the resulting combustion
gases pass through a reduction furnace and are swept into
the chromatographic column by the carrier gas which is
helium.
 The gases are separated in the column and detected by the
thermal conductivity detector which gives an output signal
proportional to the concentration of the individual
components of the mixture.
 The results are comparable to those obtained by traditional
methods, such as Kjeldahl and Dumas, but it offers faster
analysis time with greater reproducibility and accuracy.

19. REFERENCE
 Shedlovsky T. (1960)
Conductometry, in A. Weissberger (ed.), "Technique of Organic
Chemistry," vol. 1, "Physical Methods of Organic Chemistry," 3d
ed., pt. 4, chap. 45, Interscience Publishers, Inc., New York.
 Geary W.J.(1971)
The use of conductivity measurements in organic solvents for the
characterization of coordination compounds‖ Coord.Chem. Rev., 7,
81.
 Bhim B. Prasad, Anil Kumar, Meenakshi Singh, Sandhya
Singh(1995) Electrolytic Conductivity of Crystal Violet-Based
Quaternary Ammonium Polyelectrolytes in Propylene Carbonate
and Sulfolane J. Chem. Eng. Data, 40 (1), pp 79–82

20. ACCELERATED STABILITY
STUDY

21. CONTENTS
 Introduction
 Objective
 Need of Accelerated stability testing
 Determination of stabilty
 Limitations of accelerated stability testing
 ICH guidelines
 References

22. INTRODUCTION
 Stability:
Stability of pharmaceutical product may be
defined as the capability of a particular
formulation in a specific container/closure
system to remain within its physical,
chemical, microbiological, therapeutic and
toxicological specification.

23. Objective
 The objective of accelerated stability testing is
to predict shelf life of the product by
accelerating the rate of degradation, preferably
by increasing the temperature.
 Accelerated stability studies are the
experimental designs.

24.  Need for stability testing:
1. Provide evidence as to how the quality of the
drug product varies with time.
2. Establish shelf life for the drug product.
3. Determine recommended storage conditions.
4. Determine container closure system suitability.
5. Safety point of view of patient.
6. Prevention of economical repercussion.
7. Essential quality attribute.

25. According to USP Types
of Stability

26. TYPES OF STABILITY TESTS:
 Long term stability tests
 Field test
 Accelerated stability tests
Accelerated stability studies:
 In accelerated stability studies, a product is
stored at elevated stress condition (such as temp,
humidity and PH).
 Degradation at the recommended storage
conditions can be predicted using known
relationship between the acceleration factor and
the degradation rate.

27.  The concept of accelerated stability
testing is based upon the Arrhenius
equation and modified Arrhenius
equation:
LogK = LogA+ΔE/RT
Where K= degradation rate/s
A=frequency factor/s
ΔE= activation energy(KJ/mol)
R=universal gas constant (0.00831KJ/mol)
T=absolute temperature

28. ARRHENIUS EQUATION
 The drug’s stability at room temp is of primary
interest, a stability study at room temperature
would take too long to be useful as a screening
procedure for new formulations.
 Therefore, such screening studies are
conducted at elevated temp in accordance with
Arrhenius equation

29. Tests at elevated temperature:
 Drug liquid preparation stored at 50, 60, 70,85,100
and 121˚c.
 Also performed at room temp and refrigerator temp.
 Sampling:
First year- 3 month interval
Second year- 6 month interval
 Four climatic zones:
Temperate zone 21˚c/45%RH
Mediterranean zone 25˚c/60%RH
Tropical zone 30˚c/70%RH
Desert zone 30˚c/35%RH

30. Tests at high intensity of
light:
 Drug substances fade or darken on exposing to
light, can be controlled by using amber glass or
opaque container.
 By exposing drug substance to 400 & 900 (FC)of
illumination for 4 & 2 weeks to light and another
sample examined protected from light .
 Results found on appearance and chemical loss
may be recorded.

31.  Comparing color or using diffused
reflectance spectroscopy for
examination.
 e.g. cycloprofen becomes very yellow
after five days under 900 foot candles
of light.

32. Tests at high partial pressure
of oxygen:
 Sensitivity of the drugs to atmospheric oxygen
must be evaluated from which it should be packed
in inert atmospheric condition with antioxidants is
decided .
 Here, high oxygen tension plays important role to
investigate stability Usually ,40% of oxygen
atmosphere allows for rapid evaluation.
 Results were correlated with inert & without inert
condition .

33. Tests at high relative humidity:
 Presence of moisture may cause hydrolysis and
oxidation.
 These reactions may accelerated by exposing
the drug to different relative humidities.
 Control humidity by Lab desiccators
 Closed desiccator are placed in an oven to
provide constant temperature.

34. Microbiological Stability Test
 Microbial contaminants usually come from
different origins
 During production and filling
 During the use of product by the consumer
 It is necessary to carry out routine
microbiological analysis of each of the finished
product.
 Main potential pathogens in cosmetic products
are
 Staphylococcus aureus
 Candida albicans

35. Common test procedure
 Screening test
 Quantitative test
Screening test (dip slide or count plate
method)
 This method is used to detect aerobic bacteria in
aqueous samples.
 Dip slide is coated on both side with a solid culture
medium like agar gels
 A small quantity of dye triphenyl tetra zolium
chloride is added to detect the bacteria in the
sample

36.  Dip the slide into the aqueous sample for 10
seconds. Drain off the excess liquid from the
slide.
 Incubate at 35-37°C for 18-48 hours.
 The slide appearance is compared to calibration
charts.
 Aerobic bacteria species grow on this medium
and it can be detected by their ability to reduce
TTC to a red colored formozan dye.

37. LIMITATIONS OF ACCELERATED
STABILITY TESTING
 Valid only when the break down depends on
temperature.
 It is not useful when degradation is due to:
• Microbial contamination
• Photochemical reactions
• Diffusion
• Excessive agitation
 When the product looses its physical integrity at
higher temperatures.
 When the order changes at elevated temperatures.

38. ICH GUIDELINES ON STRESS
TESTING:

39. Predicting shelf life
 ICH RECOMMENDED EVALUATION
 The shelf life of a commercial drug product must be
determined in the commercial container closure at
defined storage condition.
 ICH requires at least 12 months stability data at the
time of NDA submission
 The acceptance criteria for the test have a lower and
an upper limit of 95% and 105% respectively.

40. REFERENCES
 Patrick J.Sinko , Martin’s Physical Pharmacy and
Pharmaceutical Sciences.
 Remington, the science and practice of pharmacy,vol-
1,21st edition, pg no. 1032-1034
 Theory and practice of Industrial Pharmacy – Lachman
 International Stability Testing Drug stability- Cartensen
 C.V.S. Subrahmanyam
 www.ich.org

41. THANK YOU…