WHAT IS REGULATION ?

A regulation is a binding instruction issued by an agency (in our case, the FDA) that tells you
how to interpret and comply with law. Regulations are MUST FOLLOWS that is, if you fail to
follow a regulation, and you have an inspection, the FDA inspector must write up your failure;
failures to follow regulation usually end up in the “issued warning letter” section of the FDA
Web site, not a good place to be.
The most important regulations applying to validation are good manufacturing practices(GMP),
good clinical practices (GCPs) and good laboratory practices (GLP) .
The best –known quality standards are the ISO 9000 series, which provide generic standards for
development, manufacturing and service. The most frequently used quality and accreditation
standard in chemical testing laboratories is the ISO17025 std.
HISTORICAL BACKGROUND FOR THE REGULATORY BASIS
The emphasis on validation began in the late 1970s, the requirement has been set at 1963 as
cGMP regulations for finished pharmaceuticals. Validation is an integral part of Quality
Assurance & its meaning is “Action of providing an evidence”. Validation is necessarily include
process qualification (qualification of raw materials, equipment, system) under the section 21
CFR 211.100 which states: “There shall be written procedures for production and process control
designed to assure that the drug products have the identity, strength, quality, and purity”.

HISTORY : The Kefauver-Harris Amendments to the FD&C Act were approved in1962 with
Section 501(a)(2)(B) as an amendment. The result of the amendments Provided an additional
powerful regulatory tool to FDA to stop particular manufacturing process when the drug product
is deemed to adulteration. The Drug Product Quality Assurance Program of the 1960s and 1970s
involved first conducting a massive sampling and testing program of finished batches. The
investigation of clinical failures of several products(including Digoxin, Digitoxin, Prednisolone,
and Prednisone) by FDA found significant content uniformity problems that were the result of
poorly controlled manufacturing processes
REGULATORY BASIS : The regulatory basis validation program of process validation is
embodied within the regulations & guidelines provided by cGMP & FDA. The ultimate legal
authority is Sec501(a)(2)(B) by the FD&C Act, which states “Drug is deemed to be adulterated
due to the methods/ facilities used for the manufacturing, processing, packing/holding fails to
administer in conformity – cGMP” . Validation-Process validation is not just an FDA or U.S.
requirement. Similar requirements included in the World Health Organization (WHO), the
Pharmaceutical Inspection Co-operation Scheme (PIC/S), and the European Union(EU).
REGULATION FOR VALIDATION UNDER USFDA
Section211.100(a): Written procedures/deviations. “There shall be written procedures for
production and process control designed to assure that the drug products have the identity,
strength, quality, and purity.”
Section 211.110: Sampling and testing of in-process materials and drug products "....control
procedures shall be established to monitor the output and Validate the performance of those
manufacturing processes that may be responsible for causing variability in the characteristics of
in-process material and the drug product”
21CFR211.133: Control of Microbiological Contamination " Appropriate written procedures,
designed to prevent microbiological contamination of drug products purporting to be sterile,
shall be established and followed. Such procedures shall include Validation of any sterilization
process.“
FDA must inspect every drug manufacturing establishment at least once every 2 years .
REGULATION FOR VALIDATION UNDER CGMP
Owing to the dynamic nature of the GMPs, In the year 1976, the term cGMP came into
existence. The next revision of the cGMP took place in 1978, and these are in effect at present.
In fact , the word “current” in reference to suggest that they are dynamic and the regulatory
agency constantly updates and maintains them in relation to the current state of the art and
science of drug manufacturing practice in the revision .
Regulation defined the “GMP as a system for ensuring that product are consistently produced
and controlled according to quality standard. It is designed to minimize the risk involved in any
pharmaceutical production that cannot be eliminated through testing the final product.”
The first cGMP regulations, based largely on the Pharmaceutical Manufacturers Association’s-
manufacturing control guidelines .
The Medicines Act (1968) covers most aspects of cGMP in what is commonly referred to as
"The Orange Guide" Validation under document of cGMP covers procedure, process
qualification, equipment,& facilities.
Validation under document of cGMP covers procedure, process qualification, equipment and
facilities. The current regulations are covered in Code of Federal Regulations(CFR)
211.68 :validation of automated process.
211.84(d)(2): validation of supplier’s test results for components.
211.84(d)(3): validation of supplier’s test results for container and closures.
211.110(a) : validation of manufacturing process to ensure content uniformity& integrity.
211.1113(b): validation of sterilization process.
211.165 : validation of analytical methods. By June 2010, the same GLP/GMP Validation
requirements will apply to all manufacturers of dietary supplements.

PLANS OF FDA CGMP :

 The FDA plans to oversee 591 national GMP inspections in 2014 and 2015, reduced from
967 performed last year.
 Consequently the agency plans to perform 30 percent more foreign GMP inspections,
increasing last year’s total of 604 to a new grand total of 843 inspections.
 Companies will now be chosen for inspection using the agency’s risk-based inspection
model that equates inspection periodicity to company quality practices and procedures.
 This risk based model develop specifically for FDA GMP PLANS use, takes into account
risk factors; such as, Class I recalls, adverse events, as well as compliance history as it
assigns an appropriate inspection cycle.

VALIDATION REQUIREMENT FOR WHO : WHO (World Health Organization), cGMP

Guidelines state Validation studies are an essential part of current good manufacturing practice
(cGMP) and should be conducted in accordance with predefined protocols.
WHO validation definition: “The documented act of proving any procedure, process, equipment,
material, activity or system which actually leads to the expected results.”
DQ: Design Qualification
IQ: Installation Qualification
OQ : Operational qualification.
PQ: Performance qualification
 DQ: The compliance of the basic design (location plan) with the user requirements &
regulatory requirements should be submitted & documented.
IQ: Documentary evidence to prove that the premises & equipment have been built & installed in
compliance with their specifications. IQ include: 1.Preventive maintenance. 2. Equipment info.
3. Calibration. 4.Verification of the equipment.
OQ: A series of tests to measure the performance capability of equipment. The OQ for HPLC
system is the operation of pump, injector & detector will be tested at this stage.
PQ: Process to verify that the system is repeatable & capable for consistently producing a quality
product.

VALIDATION REQUIREMENT FOR EU: The European Union requirements for validation
is an extract from ICH Q8, Q9 and Q10 documented guidelines and helps to study continuous
process verification
EU Validation Definition: “Documented evidence that the process, operated within established
parameters, can perform effectively and reproducibly, To produce a medicinal product meeting
its predetermined specifications and quality attributes.”
Strategies of validation under EU includes:
1. Traditional process verification : process validation should focus on the control strategy,
which primarily includes critical process parameters and other relevant studies demonstrating
that the process is capable of delivering the desired product quality.
2. Continuous process validation(CPV): an alternative approach to process validation in which
manufacturing process performance is continuously monitored & evaluated.
3. Critical process parameter(CPP): a process parameter whose variability has an impact on a
critical quality attribute and therefore should be controlled to ensure the process produce the
desired quality.
4. Critical quality attributes.(CQA): a physical ,chemical, biological or microbiological
property should be within an appropriate limit, range to ensure product quality.

VALIDATION REQUIREMENT UNDER PIC/S (PHARMACEUTICAL INSPECTION

CONVENTION PHARMACEUTICAL INSPECTION CO-OPERATION SCHEME) :
According the EU Guidelines to Good Manufacturing Practice for Medicinal Products in Annex
15 the principles of qualification & validation of the PIC/S is given under document PIC/S PI
006-3:

“ Doc states: GMP for medicinal products Recommendations on Validation Master Plan
Installation and Operational Qualification( Non-Sterile Process Validation Cleaning Validation)
can assist with the interpretation and the implementation.”

This document applies primarily to inspectorates of the PIC/S member for whom it is intended as
instruction for preparing an inspection, and as an advanced training aid for
qualification/validation .
 User Requirements Specification (URS), is the most critical of documents and yet, the most
often bungled. Whether the system is purely mechanical, or a mix of electro-mechanical, or
solely a software program, the successful compilation and execution of the Installation
Qualification (IQ) (for installation), Operational Qualification (OQ) (for functionality) and the
Performance / Product Qualification (PQ) (for operability), is dependent on an User
Requirements Specification (URS) containing clear, concise and testable requirements.
Once the end user requirements specification is documented, agreed and approved they form the
basic URS Level-1 document. The engineers (or vendor) can then commence the preliminary
design to establish exactly what functions are required for each of the items specified in the user
requirements specification, the end user has listed. Once this functionality is documented and
approved it forms URS Level-2 document. This is the final level of the URS unless software is
used.

If software is to be used, the URS Level-2 document, is passed to the code writers. As the code is
written, lines, or groups of lines, of code must be attributed to the individual functions that
necessitate their presence. The completion of this task results in the completion of the URS
Level-3 document
Developing the URS to this level is unique in most industries, but is, standard practice in strictly
regulated industries, as it is a major building block in the creation of quality software. The URS
Level-3 document, contains all the traceability which is deemed mandatory for software assessed
to be critical to product quality, in the pharmaceutical regulated industries.

Structure and content of URS

The URS can contain a large number of requirements and should therefore be structured in a
way that will permit easy access to information.
The requirement specification must be formally reviewed and approved by the pharmaceutical
manufacturer. The following guidelines should be followed during the production of the URS :
1. Each requirement statement to be uniquely referenced, and no longer than 250 words.
2. Requirement statements should not be duplicated nor contradicted.
3. The URS should express requirements and not design solutions.
4. Each requirement should be testable.
5. The URS must be understood by both user and supplier; ambiguity and jargon should be
avoided.
6. The use of diagrams is often useful.
7. The scope for readers to make assumptions or misinterpret should be minimized.
8. Wherever possible, the URS should distinguish between mandatory/regulatory requirements
and desirable features.
URS FORMAT:
The URS for a GMP computer control system application will typically address the following:
 Scope of system supply
 Project objectives
 Regulatory requirements
 Process overview
 System boundaries
  Operational considerations
 Manufacturing design data
 Instrument application data
 Data records
 System functions
 System software
 System hardware and peripherals
 System interfaces
 Environmental conditions
 Access security
 Diagnostics
 System Availability
 Safety Test and calibration
 Quality procedures
 Software development
 life cycle
 Documentation requirements
 Training Engineering/installation standards
 Ongoing support
 Warranty
 Delivery/commercial requirements
Newly sanctioned systems will require compliance with regulations for GMP electronic records
and electronic signatures, and definition of the functionality required will need to be included.

The structure of the URS be used as the basis for the presentation format of the FDS and
hardware and software design specifications; this helps ensure design decisions are auditable
back to the source requirement.

Once reviewed and approved internally, the URS is issued to prospective suppliers as part of the
tender document set so that detailed quotations for the system application can be obtained.

URS provides the following key benefits for the validation program:
1. Clarifies technical, quality, and documentation requirements to the vendor( s).
2. Enables the pharmaceutical manufacturer to assess the technical, regulatory, and commercial
compliance (or otherwise) of submitted bids against a formal specification.
3. Ensures the basis of a structured approach to the presentation of information.
4. Provides a basis for testing and test acceptance criteria.
5. Provide a baseline for validation and verification..

User Requirement Specifications

 They must be comprehensive. Each and every requirement relating to product safety, identity,
strength, purity, and quality must be identified. Hence, Quality Assurance (QA) must have a
significant role in reviewing and approving the final set of requirements, and must be an
approver of changes to any requirement that can affect the above product or process attributes
(e.g., cGMP’s).
 Given a comprehensive User Requirements Specification that has been approved by QA and is
under project change management, the Design Qualification(DQ) process then can be reduced to
two key objectives:
a) Documented verification that the overall design appears to address, by some means, each
and every requirement affecting the product and performance of the manufacturing process
(or, in the case of unknown product or multi-product manufacturing facility, the required
equipment/ system performance capabilities).
b) Identification (and documentation) of the critical individual physical components,
attributes, and operational features that directly support meeting each requirement.

PHASES OF VALIDATION:

Validation is broken down into 5 main phases, 1) Design qualification (DQ). 2) Installation
qualification (IQ). 3) Operational qualification (OQ). 4) Performance qualification (PQ).
5) Maintenance Qualification (MQ) 6) Component qualification (CQ).

Validation Time Line

 19.  Validation: Refers to the total life cycle of a product from development through use and
maintenance. Owners are responsible for Validating Their Processes (personnel, equipment,
methods, SOPs) to ensure compliance to cGMP/GLP regulations.
Qualification: (Inspection, functional testing and documentation review) Is a part of the
validation process which verifies module and system functional performance prior to being
placed on-line and thereafter according to a standard operating procedure.
Equipment Qualification
It is a basic requirement of good analytical chemistry that balances and other analytical
instruments must be suitable for the purpose for which they are used and that they must be
appropriately calibrated. As a consequence, Equipment Qualification is gaining more and more
importance in ensuring the validity of results. Regulatory bodies also seem to be turning their
attention increasingly to this area, and manufacturers of analytical equipment are forced to play a
significant role in the various steps of Equipment Qualification.
Step 1: Design Qualification (DQ): defines the functional and operational specifications of a
balance or instrument.
Step 2 :Installation Qualification (IQ) ensures that a balance or instrument is received as
designed and specified. It documents the installation in the selected user environment.
Step 3: Operational Qualification (OQ) demonstrates that a balance or instrument will function
according to its operational specification in the selected environment.
Step 4: Performance Qualification (PQ) demonstrates that a balance or instrument consistently
performs according to a specification appropriate to its routine use.
Step 5: Maintenance Qualification (MQ) describes and documents any maintenance required on
the equipment.
Design qualification (DQ) is the process of completing and documenting design reviews to
illustrate that all quality aspects have been fully considered at the design stage. The purpose is to
ensure that all the requirements for the final systems have been clearly defined at the start.
Design Qualification (DQ) defines the functional and operational specifications of the instrument
and details the conscious decisions made in the selection of the supplier. DQ should ensure that
instruments have all the necessary functions and performance criteria that will enable them to be
successfully implemented for the intended application and to meet user requirements.
The list below shows the recommended steps that should be considered for inclusion in a Design
Qualification.
- Description of the analysis problem
- Description of the intended use for the equipment
- Description of the intended environment
- Preliminary selection of the functional and performance specifications (technical,
environmental, safety)
- Preliminary selection of the supplier
- Final selection of the supplier and equipment
- Development and documentation of final functional and operational specifications
Vendor Qualification as the part of DQ
As part of the design qualification process, the vendor should be qualified; the question is how
should this be done? Is an established and documented quality system enough (e.g. ISO 9001), or
should there be a direct audit?
The answer is that there may be situations where a vendor audit is recommended: for example,
when complex computer systems are being developed for a specific user. However, this is rarely
the case for balances and analytical instruments.
If equipment does not include a computer system, a good reputation, one's own experience or
good references from other users - together with ISO 9001 certification - can be sufficient.

Installation qualification (IQ) is the process of checking the installation, to ensure that the
components meet the approved specification and are installed correctly, and to see how that
information is recorded. The purpose is to ensure that all aspects (static attributes) of the facility
or equipment are installed correctly and comply with the original design. all of the
instrumentation components are identified and checked against the manufacturer’s component
listing. The working environment conditions are documented and checked to ensure that they are
suitable for the operation of the instrument.

Installation Qualification establishes that the instrument is received as designed and specified,
that it is properly installed in the selected environment, and that this environment is suitable for
the operation and use of the instrument.
Before installation:
- Obtain manufacturer's recommendations for installation site requirements.
- Check the site for the fulfillment of the manufacturer's recommendations (utilities such as
electricity, water and gases plus environmental conditions such as humidity, temperature,
vibration level and dust).
- Allow sufficient shelf space for the equipment itself, related SOPs, operating manuals,
logbooks and software.
Operational qualification (OQ) is the process of testing to ensure that the individual and
combined systems function to meet agreed performance criteria and to check how the result of
testing is recorded. The purpose is to ensure that all the dynamic attributes comply with the
original design. Each of the instrument’s function are checked to ensure that they conform to the
manufacturer’s specifications.
This includes the use of certified, traceable electrical simulators and standards to verify that the
equipment is processing input signals correctly.

Performance qualification (PQ), also called process qualification, is the process of testing to
ensure that the individual and combined systems function to meet agreed performance criteria on
a consistent basis and to check how the result of testing is recorded. The purpose is to ensure that
the criteria specified can be achieved on a reliable basis over a period of time.
The performance of the equipment for its routine analytical use is checked to ensure that this
complies with its specification.
The temperature sensor readings are compared with a certified reference thermometer. After
calibration, the conductivity sensor readings are compared using certified, traceable control
standards.

Control Standards of similar values to the intended test samples must be used for PQ.
Performance Qualification (PQ) is the process of demonstrating that an instrument consistently
performs according to a specification appropriate to its routine use.
Important here is the word consistently. The test frequency is much higher than for OQ. Another
difference is that PQ should always be performed under conditions that are similar to routine
sample analysis.

PQ should be performed on a daily (or at least a weekly) basis, or whenever the instrument is
used. The test frequency depends not only on the stability of the equipment but also on
everything in the system that may contribute to the analysis results.
1. Define the performance criteria and test procedures.
2. Select critical parameters.
3. Define the test intervals.

Maintenance Qualification (MQ)

The MQ describes and documents any maintenance required on the equipment. This includes
routine servicing and any repairs necessary. Details of any maintenance contracts are also
documented in this section, together with a list of authorized service engineers. In addition, the
MQ includes the routine cleaning of the equipment and also its ultimate disposal.

Component qualification (CQ) – is a relatively new term developed in 2005. This term refers
to the manufacturing of auxiliary components to ensure that they are manufactured to the correct
design criteria. This could include packaging components such as folding cartons, shipping
cases, labels or even phase change material. All of these components must have some type of
random inspection to ensure that the third party manufacturer's process is consistently producing
components that are used in the world of GMP at drug or biologic manufacturer.
Calibration and validation of equipment as per ICH guidelines:

ICH GUIDELINE ICH Q2 defines typical analytical validation characteristics, to which tests
to apply them and examples on the “how to”.
It include:
1. SPECIFICITY is the ability to assess unequivocally the analytes in the presence of
components which may be expected to be present.
2. DETECTION LIMIT of an individual analytical procedure is the lowest amount of analyte
in a sample which can be detected but not necessarily quantitated as an exact value.
3. ACCURACY of an analytical procedure expresses the closeness of agreement between the
value which is accepted either as a conventional true value or an accepted reference value and
the value found. This is sometimes termed trueness.
4. PRECISION expresses the closeness of agreement (degree of scatter) between a series of
measurements obtained from multiple sampling of the same homogeneous sample under the
prescribed conditions. Precision may be considered at three levels: repeatability, intermediate
precision and reproducibility.
5. LINEARITY of an analytical procedure is its ability (within a given range) to obtain test
results which are directly proportional to the concentration (amount) of analyte in the sample.
E.g. Theoretical value =37 Practical value A= 35, 35.5,34.5 ACURATE Practical value B=34,
38,40 PRECISE
6. ROBUSTNESS is a measure of its capacity to remain unaffected by small, but deliberate
variations in method parameters and provides an indication of its reliability during normal usage.
1- Change in pH of mobile phase. 2- Change of column 3- A little bit change of temperature
7. RANGE of an analytical procedure is the interval between the upper and lower concentration
(amounts) of analyte in the sample (including these concentrations) for which it has been
demonstrated that the analytical procedure has a suitable level of precision, accuracy and
linearity.
8. QUANTITATION LIMIT of an individual analytical procedure is the lowest amount of
analyte in a sample which can be quantitatively determined with suitable precision and accuracy.
The quantitation limit is a parameter of quantitative assays for low levels of compounds in
sample matrices, and is used particularly for the determination of impurities and/or degradation
products.
Difference between calibration and validation

Calibration Validation
It is measurement of critical value of Validation is establishing documented
equipments function falling with in the evidence which provides a high degree of
optimum range of its working assurance that a specific process will
consistently produce a product meeting its
predetermined specifications and quality
attributes
It is applicable mainly for measuring and test It is applicable mainly for procedure, process
equipment or method.
Principal is comparison of it’s critical value Principal is comparison of it’s derived value
with known national/international standard with established range
having fixed value.
Example of equipment requiring calibration are Example of process/procedure /method
Q.C instruments, pressure, temp./vacuum requiring validation are batch manufacturing
gauges, weighing balance etc. process. Aseptic fill procedure, cleaning
procedure, Analytical method.

WHO Guideline Equipment validation/qualification (EQ) include: ◦ Design qualification

(DQ) ◦ Installation qualification (IQ) ◦ Operational qualification (OQ) ◦ Performance
qualification (PQ)

CALIBRATION OF HPLC: Various Calibration parameters are:

 Flow rate accuracy

 Injector accuracy
 System Precision
 Wavelength accuracy
 Detector linearity
 Injector linearity
 Gradient Performance Check
 Column oven temperature accuracy

Flow Rate Accuracy: 1. Prime all the solvent lines with Milli Q water. 2. Set the flow rate to
0.500 ml/m. 3. Wait for about 15 m to stabilize the system and ensure that the pressure is stable.
4. Insert the outlet tubing into a 10 ml volumetric flask and start the stop watch simultaneously.
5. Stop the stopwatch when the lower meniscus reaches the 10 ml mark on the flask. 6. Record
the elapsed time. 7. Similarly check the flow for 1.0 ml/m and 2.0 ml/m. Acceptance criteria:
The time taken to collect the water should be with in ± 2.0% of the actual value.

Injector Accuracy: 1. Connect the pump and detector inlet with union. 2. Prepare mobile phase
consisting of a mixture of water and Methanol (70:30 v/v) 3. Set a flow rate of 0.5 ml/m and a
run time of 1 m. 4. Set the column temperature at 25± 2°C. 5. Fill a standard HPLC vial to 2/3rd
with Milli-Q water. Seal the vial properly with a cap. 6. Weigh the vial and record the weight as
W1 grams. 7. Place the vial in the chromatographic system and perform 6 injections of 50µl
volume from this vial. 8. Weigh the vial again and note the weigh after the injections as W2
grams. Calculate the mean volume injected per injection as follows: Mean injected volume (µl) =
(W1 – W2) ×100/6 Acceptance criteria: The mean injected volume should be 50.0±1.0 µl.

System Precision: Standard Preparation: Accurately weigh and transfer about 60mg of
Caffeine into a 100ml volumetric flask. Dissolve and dilute to the volume with mobile phase.
Transfer 10ml of this solution into a 100ml volumetric flask and dilute to the volume with
mobile phase. Procedure: Inject blank, followed by standard preparation in 6 replicates. Note
down the areas and retention times. Now calculate the %RSD of retention time and peak areas
for 6 replicates injections. Acceptance criteria: The %RSD of retention time & peak area should
be <1.0%.

Wavelength Accuracy: Procedure: Create and instrument method with a wavelength in nm and
inject blank, followed by Standard preparation and note down the height or absorbance.
Acceptance criteria: The maximum absorbance should be ±2nm. PDA Detector Accuracy:
Select 3D mode and set the wavelength range as 200-400nm.Inject 20 µl of standard preparation
once into the chromatographic system. Extract and record the chromatograms at wavelengths of
202 to 208nm with an interval of 1nm and at 269 to 275 nm with an interval of 1nm.Note down
the height or absorbance. Acceptance criteria: The maximum absorbance should be at 205±2nm
and 272±2nm.

Detector Linearity: Standard Preparation: Accurately weigh and transfer about 60mg of
Caffeine into a 100ml volumetric flask. Dissolve and dilute to the volume with mobile phase.
Detector linearity solution 1(0.06 mg/ml): Transfer 10ml of Standard Preparation into a 100ml
volumetric flask and dilute to the volume with mobile phase. Detector linearity solution
2(0.048 mg/ml): Transfer 8ml of Standard Preparation into a 100ml volumetric flask and dilute
to the volume with mobile phase. Detector linearity solution 3(0.03 mg/ml): Transfer 5ml of
Standard Preparation into a 100ml volumetric flask and dilute to the volume with mobile phase.
Detector linearity solution 4(0.24 mg/ml): Transfer 4ml of Standard Preparation into a 100ml
volumetric flask and dilute to the volume with mobile phase. Detector linearity solution
5(0.012 mg/ml): Transfer 2ml of Standard Preparation into a 100ml volumetric flask and dilute
to the volume with mobile phase.

Procedure: Inject blank, followed by Detector linearity solutions and record the peak responses
of Caffeine standard plot between the concentration vs the peak responses. Acceptance criteria:
The plot should be linear and regression coefficient (R2 ) should not be less than 0.99.

Injector Linearity: Standard Preparation: Accurately weigh and transfer about 60mg of
Caffeine into a 100ml volumetric flask. Dissolve and dilute to the volume with mobile phase.
Transfer 10ml of Standard Preparation into a 100ml volumetric flask and dilute to the volume
with mobile phase. Procedure: Inject 5 µl of the mobile phase as blank injection. Inject 5 µl, 10
µl, 20 µl, 50 µl and 80 µl of the Standard Preparation and record the peak areas. Plot a curve for
the volume injected Vs peak area. Acceptance criteria: The plot should be linear and regression
coefficient (R2 ) should not be less than 0.99.

Gradient performance check: Add 5ml of acetone to 1000ml of methanol filter and degas.
Connect the pump and detector inlet with union. Set the detector wave length at 254 nm. Place
Channels A and C in methanol and channel B and D in 0.5% acetone in methanol. Set binary
gradient with a total flow rate of 2.0ml/m. Set gradient program as shown below for channels A,
B and C, D individually. Purge all the channels at a flow rate of 2ml/m for about 5 m. Set the
flow rate at 2.0ml/m and wait until the base line is stable. Set the gradient profile for A and B
and run the gradient profile by injecting “0.0”volume of methanol. Record the height of the
peaks. Consider the height of the peak resulting from B at 100% concentration as 100 and
calculate the percentage height of other peaks. Perform the gradient performance check similarly
for channels C and D. Consider the height of the peak resulting from D at 100% concentration as
100 and calculate the percentage height of other peaks.

Calculations: Height (%) of B/D =Height of B/D peak ×100/Height of full scale peak

Acceptance criteria: The calculated percentage composition (Height (%) should be with
in±1.0% of the set composition.

Column Oven Temperature Accuracy: It is evaluated with a calibrated digital thermometer at

30°Cand 60°C .Place the thermometer probe in the column oven and set the column oven
temperature at 30°C.Wait till the temperature stabilizes. Record the temperature displayed on the
thermometer. Similarly performs the column oven temperature accuracy test at 60°C.
Acceptance criteria: The resulting oven temperature from the thermometer display should be
within ±2°C of the set temperature

Calibration of UV-Visible spectrophotometer:

Spectral calibration: visible spectral region

 Ensure-the socket of the power cord of the instrument is inserted properly -cuvettes are clean

 Switch ON the instrument. Allow 15m to warm up.

 Keep dummy cuvette in position of sample holder.

 Set the λ to 485 nm and press %T button.

 Press 0%T in appropriate direction to adjust 0.00 reading on read out.

 Now remove dummy cuvette from sample holder. Close the lid.
 By adjusting coarse and fine control set a reading of around 80.0 on read out

 Now set the value of wavelengths in increments of 0.1 nm up to λ of 487 nm and read the value
of %T at each increment of λ

 Draw a curve %T Vs λ.

 If the peak value of %T is occurring at a λ 486.1 ± 0.5 nm, the spectral calibration of the
instrument in the visible spectral regionis proper.

 This can be confirmed by repeating the above steps with a maximum value of %T of around
30.0 on the read out and λ setting from 655 to 657 nm.

 If the maximum %T is obtained at a λ 656.2 ± 0.5 nm, the spectral calibration of the instrument
in the visible spectral region is confirmed to be proper.

Spectral calibration: U.Vspectral region:

 Keep blank (distilled water) filled cuvette and sample (benzene vapor) filled cuvette.

 Set the λ to 253 nm and press absorbance button.

 Adjust blank to 0.000 on the read out by using coarse and fine adjustment

 Now place the sample into optical path, value of Absorbance of sample at the λ set appears on
the read out.

 Again set the values of wavelengths increments of 0.1nm up to a λ of 255nm.Measure the A at

each increment. If maximum A is obtained at λ 253.9 ± 0.5nm the “spectral calibration” of the
instrument in U.V region is confirmed to be proper.

Photometric Calibration: Absorbance: Visible region

 Place dummy cuvette in sample holder and set %T to “zero”. Now remove dummy cuvette, by
using fine & coarse control set a reading exactly 40.0 on the read out.

 Press Absorbance push button. If the maximum absorbance obtained at λ of 485nm is 0.398 ±
0.002, the photometric calibration of instrument is confirmed to be proper.

 To confirm, repeat above steps, and set 10.00 on read out

 Press Absorbance botton. If the λ at 485 nm is 1.000±0.002 then it is confirmed the

photometric performance in the visible region is proper. Absorbance: U.V region

 Place blank0.1N H2SO4 cuvette and 60ppm K2Cr2O7 as sample

 Set λ exactly to 257 nm, if the value of Absorbance of sample at the set λ is 0.864±0.005, the
instrument is measuring Absorbance properly.

%Transmittance: As the value of %T is delivered from Absorbance itself, if the instrument is

measuring Absorbance properly it is deemed that it measures %T properly.

Concentration:

 Place blank 0.1N H2SO4 cuvette and 60 ppm K2Cr2O7 (0.06006g/l of 0.1NH2SO4) as
standard and 20 ppm (0.02002 g/l of 0.1N H2SO4) as sample.

 Press “Concentration” push button and adjust Concentration control to 600 for standard on read
out.

 Now place sample holder into optical path, if the value o f Concentration appearing on the read
out for sample is 200±5, the instrument is measuring “concentration” properly.

Calibration of Wavelength: a) Holmium filter: For routine calibrations, holmium filter is

satisfactory. Record the absorption spectrum from 500 to 230 nm using slowest scan speed and
narrowest slit setting. Identify 3 fused absorption bandscentered on 452.2nm and single band at
around 360.9nm.Instruments with accurately calibrated λ scales will show λ max at 453.2, 418.4,
360.9, 287.5, 279.4 and 241.5nm.

b) Holmium per chlorate solution: Prepare a solution of Holmium (III) per chlorate by
dissolving 0.5g of holmium oxide in 2.4 ml perchloric acid (72% AR grade) by warming gently
and diluting to 10ml with water. Record the absorption spectrum from 500- 230nm.The
wavelengths of principal bands (Absorbance- 0.4) should be 485.8, 450.8, 416.3, 361.5, 287.1,
278.7, 241.1 nm.

c) Discharge lamps: A low pressure discharge lamp is suitable. Record the transmission
spectrum from 600 to 240 nm of Mercury lamp place near the entrance to monochromator, using
minimum slit setting and slowest scan speed. The principal emission lines of Mercury are at
579.0, 576.9, 546.1, 435.8, 404.5, 364.9 and 253.7 nm.

d) Prepare standard solution by dissolve 100 mg of Potassium dichromate in 0.05N Potassium

hydroxide solution in 100ml volumetric flask. Make up to volume with the same. From the
standard solution take 20ml and make up to 500ml with 0.05N Potassium hydroxide solution.
Now scan the wave length from 340 to 400nm using blank 0.05N Potassium hydroxide solution.
The maximum wave length is observed at 370 nm.

Limit of stray light: Weigh accurately 1.2g of dried Potassium chloride in 100 ml volumetric
flask and makeup to mark with Double distilled water. Measure the absorbance at 200 nm.
Acceptance criteria: Tolerance limit NLT 2.0
Resolution: Prepare 0.02%v/v solution of Toluene and make up with Hexane. Scan the
wavelength from 250 to 280nm.Maximum absorbance is 269 nm and Minimum absorbance is
266nm Acceptance criteria: Ratio limit NLT 1.5

Photometric linearity:

 Weigh accurately 100mg of Potassium chromate in 100ml volumetric flask and dissolve in
0.05N Potassium hydroxide solution. Make up with the same solvent.

 From the above solution take 20ml and make up to 500ml with 0.05N Potassium hydroxide
solution.

 Now prepare dilution of 4,8,16,24,32 µg/ml

 Measure the absorbance at 370nm using blank. Acceptance criteria: The plot should be linear
and regression coefficient (R2) should NLT 0.999.