Validation is the process of proving that analytical methods, equipment, or processes work as expected to consistently provide accurate results. It establishes documented evidence that a specific process will produce a product meeting predetermined specifications. Method validation involves establishing the performance characteristics and limitations of an analytical method and identifying factors that could affect the method's characteristics. Calibration of analytical equipment like pH meters, UV-Vis spectrophotometers, and other instruments is an important part of the validation process to ensure accurate measurement results.

1. Prepared by
P. Jagadeesh
M.Pharm I year

2. WHAT IS VALIDATION?
Definition:
 Validation is the documented act of proving that any procedure,
process, equipment, material, activity or system actually leads to the
expected result.
ISO definition:
 Validation is the confirmation by examination and the provision of
objective evidence that the particular requirement for a specific
intended use are fulfilled.

3.  According to the Food and Drug Administration (FDA)
the goal of validation is to establish documented
evidence which provides a high degree of assurance that a
specific process will consistently produce a product
meeting its predetermined specifications and quality
attributes.

4. METHOD OF VALIDATION
 Method validation is the process of establishing the
performance characteristics and limitations of a method and the
identification of the influences which may change these
characteristics and to what extent. It is also the process of
verifying that a method is fit for purpose

5. When validation is needed?
 Before introduction of a new method into routine use.
 whenever the conditions changes for which a method has
been validated, e.g, instrument with different
characteristics
 Whenever the method is changed and the change is
outside the original scope of the method.

7. What is a pH Meter?
 An instrument that measures the H+ ion concentration
(pH) of a solution using an ion sensitive electrode which
will ideally respond to one specific ion, in this case H+ .
 The pH of a solution is the negative logarithm of the H+
ion concentration .
 A typical modern pH meter has a glass and reference
electrode in one tube.

8. Calibrating a pH Meter
 Make sure the meter is in pH mode
 For a 3 point calibration, use high pH (--),
pH 7.0 and low (-.0) solution.
 Before calibration, rinse probe thoroughly
with de-ionized water or a rinse solution
 Immerse the end of the probe completely in
the calibration solution .
 Stir the probe gently to create a homogenous
sample.

9. Measuring the pH of a Solution
 Always rinse electrodes with de-ionized
water prior to placing in a solution for pH
measurement
 Allow meter to stabilize for 30 seconds or a
minute, then read
 Remove electrodes and rinse with de-ionized
solution
 The pH bulb should always be stored wet
preferably in pH 4.0 buffer with 1/100 KCl
 Other buffers or tap water can be used for
storage

10. Maintenance of pH Meter
 Wipe off exterior of pH meter with a damp Wipe off
exterior of pH meter with a damp cloth after use cloth
after use .
 The outside of the meter is made of polyester The outside
of the meter is made of polyester and is not affected by
most solvents but is and is not affected by most solvents
but is affected by some organic solvents affected by some
organic solvents .
 pH the “power of hydrogen”.

11. The pH Scale
 The pH measures the concentration
of H+ in a solution.
 The lower the number, the more
acidic the solution.
 The higher the number, the more
basic the solution .

12. The pH Scale – Another
Description

13. CALIBRATION OF UV VISIBLE SPECTROPHOTOMETER

14.  Calibration is the act of ensuring that a method or
instrument used in measurement will produce accurate
results.
 Calibration is performed using primary reference standard
WHAT IS CALIBRATION?

15.  When a specific time period is elapsed.
 When a specific operating hours has elapsed.
 With a new instrument.
 When observations appear questionable.
NEED OF CALIBRATION

16. UV APPARATUS

18. 1. Control of wavelength
2. Control of absorbance
For ultraviolet region For
visible region Limit of
stray light resolution
power
3. Maintenance
4. Precautions
5. Abbrevations
PROCEDURE OF CALIBRATION

19. CHEMICALS: 1.4M
perchloric acid,
holmium
perchlorate
solution
STEP 1: Dissolve 1
gram of holmium
oxide in 1.4M
perchloric acid
with the aid of
heating on water
bath, cool and
dilute to 25ml with
same solvent.
STEP 2: Record the
spectrum holmium
perchlorate
solution from
200nmto 600nm
using 1.4M
perchloric acid as
reference solution
CONTROL OF WAVELENGTH

20.  Note down the maxima observed at wavelength
against the acceptance criteria given below:

21.  Deviation of wavelength
reading at an absorbance band
WAVELENGTH ACCURACY

22. STEP 1:Weigh 57 to 63mg of potassium
dichromate primary standard and
transfer to 1000ml volumetric flask.
Dissolve in 0.005m sulphuric acid and
make up to the mark with the same acid.
Step 2: Measure the absorbance at 235
nm, 257nm, 313nm and 350nm using
0.005M sulphuric acid as reference
CONTROL OF ABSORBANCE

23.  FOR VISIBLE REGION: Whole procedure is
same as UV region but at the end measure the
absorbance at 430nm.
 CALCULATION: Value of A(1%,1cm)
A(1%,1cm)= absorbance x 100
weight in gm x 100

24. STEP 1: Prepare the solution of
1.2%v/v potassium chloride and
dissolve with 50ml distilled water.
STEP 2:Determine the absorbance
using path length of 1cm at 200nm
against purified water as blank
LIMIT OF STRAY LIGHT

25. STEP 1: Prepare a solution 0.02%v/v toluene in hexane
STEP 2: Record the spectrum of 0.02%v/v toluene in hexane from
250nm – 300nm using hexane as reference
STEP 3: record the absorbance at 269nm (max) and 266nm (min)n
STEP 4: Calculate the ration of absorbance by dividing the absorbance
at maxima and minima
RESOLUTION POWER

26.  When the instrument not produce calibrated results it
should be labeled “FAUL TY” and should be repaired by
service engineer of instrument.
MAINTENANCE

27.  The instrument should be always free from dust
 Place the instrument as far as possible from any
strong magnetic or electric field or any electric apparatus
generating a high frequency.
 Avoid places where vibrations exist or direct
sunlight.
 To extent the life of source lamp have them
powered only when required.
 The temp. Of all solution used in the test should not
differ by more then 0.5ºC.
PRECAUTIONS