Analytical Methods Validation Guide

Module 2
Presentation
Submitted by
Shereen Naguib Abdel-hamid
Samah Madbouly
El-NASR – Company

Analytical Methods Validation
As Per Ich And Usp
Contents
1. Introduction
2. Objectives
3. Types of Analytical Procedures To be Validated
4. Validation Parameters As Per Ich And Usp
5. Conclusion
6. References

Introduction
01
02
03
04
Validation
Is the documented act of proving that any
procedure , process equipment , material , activity
,or system actually leads to the expected results
A pharmaceutical drug product must meet all it’s
specifications through out it’s shelf life
Analytical methods validation
Is a process of documenting proving that an analytical
method provide analytical data acceptable for the
intended use
the method of analysis used must be validated
this is require to insure the products safety and
efficacy through out all phases of it’s shelf life

Objective
The main objective of analytical validation is to insure that a selected
analytical procedure will give reproducible and relible results . That
are adequate for the intended purpose
This is applicable to all the procedure either pharmacopeial or non
pharmacopeial
01

Types of Analytical procedure to be
validated
The required Validation Parameters also termed analytical Performance
characteristics depends up on the type of analytical method. Pharmaceutical
analytical methods are characterized into 5 general.
 Identification tests
 Potency assays.
 Limit tests for the control of impurities
 Impurity tests . Qualitative
 Specific test

Validation
parameteres as per ich
/ usp
USP
Specificity
Linearity or Range
Accuracy
Precision
Limit of detection
Limit of quantitation
Ruggedness
Robustness
ICH
Specificity
Linearity
Range
Accuracy
Precision
Limit of detection
Limit of quantitation
Robustness

Accuracy
Definition :- The Accuracy of an analytical procedure is the
closeness of agreement between the values that are accepted
either as conventional true values or and accepted reference
value and the value found

Determination
Assay
Drug substance
Drug product
Impurities (quantitation)

Recommended data :
Assessed by 9 determinations over a minimum of
3 conentration levels covering a specified range
limit
1-Typical accuracy of the recovery of the drug
substance is expected to be about 99-101%
2-Typical accuracy of the recovery of the drug
product is expected to be about 98 – 102%

Precision :-
definition :- the closeness of agreement (degree
of scatter ) between a series of measurement
abstained from multiple sampling of the same
homogenous sample .
Precision includes :-
Repeatability
intermediate precision
Re Producibility

Repeatability :- Repeatability expressed the precision under the
same operating conditions over a short interval of time .
-Repeatability should be assessed using a minimum of 9
determinations covering the specific range

Intermediate precision :-
Intermediate precision expressed variation with in
laboratories such as different days , different
analysts , different equipment etc. .
Reproducibility :-
Reproducibility expresses the precision between
laboratories .
Following parameters should be reported .
Standard deviation
Relative standard deviation

Limit of detection :-
It is the lowest amount of analyte in a sample which can be detected but not necessarily
quantitated
Limit of Quantitation :-
It is the lowest amount of analyte in a sample which can be quantitively determined with
suitable precision and accuracy .

Determination of LOD and LOQ
Limit of Detection Method
• - Based in visual examination
• -Based on standard deviation of
response and slope
• Signal to noise ratio 3:1
Limit of quantitaion
• - Based in visual examination
• -Based on standard deviation of
response and slope
• Signal to noise ratio 10:1

Specificity
Definition :- specificity is the ability to assess unequivocally the analyte in presence of
component which may be expected to be present
Determination :-
Identification tests
Assay and impurity tests
Impurities are available
Impurities are not available

Linearity
Definition :- the ability of the method to abtain results
That are directly proportional to concentration with a given range
Method :- dilution of stock solution / separate weightings
Minimum 5 concentrations are used .

Range
Definition :- the interval between the upper and lower concentration of
analyte in the sample that have been demonstrate to have a suitable Level
of precision , accuracy and linearity
- Established by confirming that method provides acceptable degree of
linearity , accuracy and precision
- Specific range dependent upon intended application of the procedure

ASSAY :- 80 to 120 % of test concentration
content uniformity :- 70-130% of test concentration
Dissolution :- 20 to 120 %
impurities reporting level :- 120% of specification limit with respect to test
concentration of API

Ruggedness
Definition :- the ruggedness of an analytical method is the degree of
resproducibility of test results obtained by the analysis of the same
samples under a variety of coniditions such a different laboratories ,
different analysts , different instruments , different days
Certain may include :-
• Source
• Concentration
• Heating rate
• Column temperature
• Humidity

Robustness
Definition :- the robustness of analytical Procedure is a measure of it’s
capacity to remain unaffected by small but dliberate cariation in methd
parameters and provide and indication of it’s reliablility during normal
usage
Determination :-
The evaluation of rubstness should be considered during the development
phae and depnds on the type of precedure under study

Variations may include :-
• Stability of analytical solution
• Variation of PH in a mobile phase
• Different column (LOT / Supplier )
• Tempreature
• Flowrate

System suitablity
-System suitablity testing is an integral part of many analytical procedure
The tests are based on the concept that the equibment , electronics
analytical operations and samples to be Analyzed constitute an integral
system that can be evaluated as such .
- System suitability testing has been recommended by USP in HPIC
procedure

Revaliation may be necessary in the following circumstamces
1-Changes in the synthisis of the drug substances
2-Changes in the composition of the finished proudct
3-Changes in the analytical procedure the degree of revalidation
Required depend on the nature of the change . Certain other changes may
require validation as well

When do you validate a method :-
During method development
Before usig any method for samples
• Verify own ability to match puplished data
• Verify own suitability for analytical Requirements
Change of application / working environment / analyst
Following period of non use

Method development
• Method development life cycle

WHO validates a methods ?
 The analyst :-
• in house development and validation of new methods
• Verfication of the perforamance of previously validated
methods
 The laboratory :-
 Method development and validation section .
 Sectoral / professional / stanrdization body
• Validation of methods via in the laporatory study .

How do you validate a method ?
 Define the analytical requirements
 Develop / identify candidate method
 Plan validation experiment
 Carry out experiment
 Use data to assess fitness – for – purpose
 Analytical requirement met ?
No
Develop / identify
yes
Statement of validation

Summary
Method validation is required to preoduce meaningful data
Both in the house and standard methods require validation / verfication .
Validation should be planned activity .
Parameters require will vary with application .
Vailidation is not complete without a statement of fitness for purpose

Atomic absorption
Content :-
1-introduction
2-principle
3-instruments
4-interferences
5-applications
6-classification
7-best practices and trouble shooting

Introduction
1-atomic absorption spectroscopy is a very common technique for detecting
metals and metalloids in samples
2-it’s very reliable and simple to use .
3-It also measure the concentration of metals in the sample .
4-Atomic absorption spectroscopy is an analytical technique that measure
the concentration of an element by measuring the amount of light that is
absorbed at a characteristic wave length passes through cloud of atoms .
5-As the number of atoms in the light path increase , the amount of light
absorbed increase

Principle of atomic absorption spectroscopy
1-the technique uses basically the principle that free atoms generated in
an atomizer can absorb radiation at specific frequency.
2- atomic absorption spectroscopy qualifies the absorption of ground state
atoms in the gaseous state .
3-The atoms absorb UV or visible light and make transition to higher
electronic energy level .
4-the analyte concentration is determined from the amount of absorption
5-concentration measurements are usually determined from a working
curve after the instrument with standards of known concentration

Instrumentation
Parts of atomic absorption spectrophotometer
1-Light Source
2-Vebulizer
3-Atomizer
4-monochrometer
5-Detector and Amplifier
6-Read out System

Schematic diagram of atomic absorption
spectroscopy

Light source
1-Hollow Cathode lamp are the most common radiation source in AAS
2- in contain a tungsten a mode and a hollow cylindrical cathode
These are sealed in a glass tube filled with an inert gas
(mainly neon or argon) .
3-Each element has it’s own unique lamp which must be used for that
analysis

Nebulizer :-
- Nebulizer suck up liquid samples at controlled rate
- Create a fine aerosol spray for introduction into the flame
- Mix the aerosol and fuel and oxidant Throughly for introduction into flame

Atomizer :-
1-Element to be analyzed needs to in atomic state and this is done by means of
atomizer .
2-Atomization is separation of particles into individual molecules and breaking
molecules into atoms .
This done by exposing the analyte to high temperature in a flame or graphite
furnace .
The atomizes most commonly used now adays are (spectroscopic) flames and
electrothermal (graphite tube ) atomizer .

Flame atomization
Nebulizer suck up liquid sample at controlled rate and creates a fine aerosol spray
for introduction into flame
to create flame , we need to mix an oxidant gas and a fuel gas
in most of the cases air - acetylene flame or nitrous oxide acetylene flame is used
liquid or dissolve samples are typically used with flame atomizer
STEPS
Sample
Nebulization
Mist Solid / gas / aerosol
De solvation
Gaseous /molecule Atoms
Dissociation
Volatilization

Electrothermal Atomization ( Graphite furnace atomic )
-it uses a graphite coated furnace to vaporize the sample
- samples are deposited in a small graphite coated tube which then heated to
vaporize and atomize the analyte .
-The graphite tube are heated using a high current power supply .
-Steps in electro thermal atomization :-
Drying – pyrolysis – atomization – cleaning

Mono Chromator
This is very important part in an AAS
it’s used to separate out all of the thousand of lines
A mono chromator is used to select the specific wave length of
light which is absorbed by the specific wave lengths .
The selection of the specific light allows the determination of the
selected element in the presence of others .

Detector and Amplifier :-
The light selected by the mono chromator is directed on to a
detector whose function is convert the light signal into an elcectrical
signal .
Photomultiplier tube detector is mainly used
the processing of electrical signal is fulfilled by a signal amplifier
the amplified signal is then display on read out system or fed into a
data station for printout by the requested formate .

Calibration curve
A calibration curve is used to determine the unknown concentration
of an element in a sample
the instrument is calibrated using several solutions of known
concentrations
the absorbance of each known solution is plotted
the sample solution is fed into instrument and the absorbance of he
element in the solution is measured
the un known concentration of element is then calculated from the
calibrated curve

Interferences in atomic absorption spectroscopy .
Interferences is a phenomenon that leads to change in intensity of analyte signal in
spectroscopy
interferences in AAS fall into two basic categories .
1-NON – spectral interferences
* affect the formation of analyte items
2- spectral interferences
high light absorption due to presence of absorbing species
1- Matrix interference
2 - Chemical interference
3 -lanization interference

Non – Spectral interferences :-
1- matrix interferences :-
- when a sample is more viscouse or has different surface tension than the
standard it result in different sample uptake rate due to change in nebulization
efficiency
- such interferences are minimized by matching the matrix composition of
standard and sample

Chemical interferences :-
if a sample contains a species which forms a thermally stable compound
with analyte that isn’t completely decomposed by the flame energy then
chemical interferences exist
such interferences are minimized by using higher flame temp to provide
higher dissociation energy

Lanization interference :-
it’s more common in hot flames
the dissociation process dosen’t stop at formation of ground state atoms
excess energy of the flame lead to excitation of ground state atom to ionic state
atoms
ionization interferences is eliminated by an excess of an element of electrons in
the flame suppressing the ionization of the analyte

Spectral interferences
spectral interference are caused by presence of another atomic
absorption line or amolecular absorbance band close to the spectral line
of element of interest
most of these interference are due to molecular emission from oxides of
other elements is a sample

Applications of atomic absorption spectroscopy
1-Determiniation of small amount of metals (lead , mercury , calcium , magnesium )
2-AAS is widely used in metallurgy , alloys and in morganic analysis
3- Biochemical analysis : A number of elements present in biological samples can be analysis by AAS
. These include estimated of sodium , calcium , potassium , zinc , iron , lead , mercury , etc .
4- pharmaceutical analysis :- Estimation of zinc in insulin preparation calcium salt is done by using
AAS
5- sodium , potassium , calcium , in saline and ringer solution are estimated by AAS
6-analysis of ash for determining the content of sodium , potassium calcium and iron is done by AAS
7-atomic absorption spectroscopy is used in assay of
A-intraperitoneal dialysis of fluid for calcium and magnesium
B-Actrivated charcoal for zing
C- cisplastin for liver

Classification :- spectroscopy is abroad field
with many subdiscipline , which can be classified by the type of
material being analyzed
Atomic spectroscopy :-
Atoms
Atomic spectroscopy
AAS
MP-AES
ICP – OES
ICP – MS
Molecules
Molecule spectroscopy
UV – VIS
UV – VIS – NIR
FTIR
Floure sence
Crystals
X- RAY
crystallography
Nuclear
Nuclear
Magnetic
Resonance

Atomic spectroscopy identification based on :-
Electro magnetic spectrum Mass spectrum
Atomic absorption
Flame AAS
Graphite furnance AAS
Vapor (hydride) Generation AAS
Atomic Emission
MP – AES
ICP – OES
X-Ray fluore scence
ICP – MS
Atomic interference
X- Ray diffraction (XRD)

What’s measured
1- Absorption of energy cause an electron to move to a higher energy level
(E2) ----- Atomic absorption (AA)
2-The excited electron will eventually drop back to the ground state and
emit light at a particular wave length (emission) MP – ES – ICP – OES
3-if there is enough energy the electron will leave behind a positivity
charged ion (ionization) >> ICP – MS

Atomic absorption spectroscopy other atomizer
Hydride generation technique
suitable for elements forming volatile hydrides (As- Sn-Bi-Sb-Te- Ge and Se) when
reached with a reducing agent , such as sodium borohydride
Advantages
-Separation of specific elements as hydrides which can eliminate matrix interference
- good sensitivity due to 100% sampling efficiency
-good precision
-faster than graphite furnace AA
Limitations :-
-Limited to specific elements
-same chemical interferences
-Require specific sample preparation lanalyte must be converted to a specific oxidation
state

Plasma Chemistry
Microwave plasma atomic
Emission spectroscopy
MP – AES
inductively couples plasma optical
emission spectroscopy
(ICP – OES )
Advantage :-
-safe (no flammable gas)
-Low operating costs as nitrogen
-Can be extracted from compressed air
using a nitrogen generator
-no lamps required for analysis
-Identification and quantitation of virtually
all metals
-Better performance than AAS
Limitation :-
-Higher initial cost than AA
-including spectral interference
-not an productive ICP – OES
No iso top determination
Is capable of measuring both atomic and
ionic emission so more wave lengths can
be monitored
Advantages :-
-fastest sample throughput
-simultaneous multi – element
-analysis (up to 73 element )
-wide dynamic range
-low argon gas consumption
-safe (no flammable gas )
Limitation :-
Higher initial costs than AAS or MP ,
AES
More spectral interference compared
with MP – AES

Import notes and common mistakes OQ of
AAS and GFAAS
-Should be done following repairs or maintenance
-it’s recommended to be done periodically
- check the recommendation of the manufacture

Important notes and common mistakes
1- Check the waste and ventilation system
2-Check the pressure and remaining volume of acetylene
3-Check the burner alignment
4-Check the cook – book ( for the recommended condition )
5-Flow a blank solution for at lease 5 min – for flame stabilization
6-After calibration curve , remeasure a standard to check system stability
7-Always check the absorbance of the recommended concentration
8-for the analysis of the volatile elements (AS – SE – Hg ) closed vessel system
(EG – HW )
9-Standard solutions should be retained for NMT 24h unless stability is
demonstrated experimentally 1≥10 ppm stable
10- always use freshly prepared solutions
11-after you finish the analysis of CA and MG
12-add Lacl3 during the analysis of CA and MG
13- Standard addition methods is commonly used in GFAAS
14-adding KCL incase of VA analysis

Cold vapor technique
use specifically for mercury (has a large enough vapor pressure at ambient
temperature ) which can be reduced to atomic state by a strong reducing
agent such as sodium , borohydride , Tin (11) chloride
Advantages :-
-Eliminates many matrix interferences
-Good sensitivity due to 100% sampling efficiency
-Good precision
-Faster than graphite furnace AA
Limitations :-
Limited to mercury only .
Mercury must be stabilized in solution

Graphite furnance AAS Atomizer
-Dissolution of sample into a liquid form is required in most cases
-sample is injected into a graphite tube and electrothermally heated in different
stages to atomize the analyte .
-In graphite furnance atomic absorption (GFAAS) the atomization happens in
three stages (Drying – Ashing – Atomization )
Graphite furnace operation is a complementary technique to conventional Flam
AA and adds some advantages some advantages to the analysis .
Graphite furnace
Advantages :-
High sensitivity due to
-entire sample is atomized at one time
-free atoms remain in the optical path longer
-Reduced sample volume
-ultra trace analysis possible
-can run unattended , even overnight

Limitations :-
-Very slow
-Fewer elements can be analyzed
-poorer precision
-More chemical interferences (compared to
flame AA)
-Method development requires skills .
- standard additions calibration required
more frequently
-Expensive cansumables (graphite tubes )

Mid infra spectroscopy (USP)
IR spectroscopy (introduction)
1-used to identify organic compounds
-IR spectroscopy provides a 100% identification if the spectrum is
matched if not , IR at least provides information about the types of
bonds present
2-Easy to use
-liquid analyzed between salt plates
-Solids in a KBR pallet
-Small amounts of unknowns via an fTIR microscope
3-in expensive
IR spectrophotometers are found in most labs

IR spectroscopy
uses
Structure investigation
Identification
Poly marphism /pseudo
poly marphism
Interactions (drugs – excipients )
Patent of antibiotics
Quantitative measurements

What type Technique is IR spectroscopy
Non destructive technique
IR spectroscopy is
Non invasive technique
-that requires minimal sample preparation and provides a real- time response
- There are two main regions in the infrared spectrum employed in various analysis applications based on the
type of interaction with the molecules

What’s is the principle of IR ?
- The basic principle of infrared (IR)
spectroscopy is the absorption of infrared
light by a sample .
Infrared light is a type of electromagnetic
radiation with longer wave length than
visible light .
It’s absorbed by certain vibrational modes
of the bonds between atos in a molecule

Practice IR spectroscopy
-the infrared spectrum of a sample is recoded by passing a beam of
infrared light through the sample
-when the frequency of the IR is the same as the vibrational frequency
of a bond or collection of bonds , absorption occurs
- Examination of the transmitted light reveals how much energy was
absorbed at each frequency (or wave length )
- this measurement can be achieved by scanning the wave length range
a mono chromator
-alternatively , the entire wave length range is measure using a fourier
transform instrument and then a transmittance or absorbance spectrum
is generated using dedicated procedure
this technique is commonly used for analyzing samples with covalent
bonds
-Simple spectra are obtained from samples with few IR active bonds and
high levels of purity . More complex molecular structures lead to more
absorption bonds and more complex spectra

Following IR
- care is important to ensure that the film is not too thick other wise
light can’t pass through this technique is suitable for qualitative
analysis
- the final method is to use microtomy to cut a thin (20-100mm) film
from a solid sample this is one of the most important ways of
analyzing failed plastic product for example because the integrity
of the solid is preserved

In a photoacoustic spectroscopy the need for ample treatment is
minimal the sample , liquid or solid , is placed into the sample cup
which is interested into photoacoustic cell which is then sealed for
the measurement
the sample may be are solid piece , powder or basically in any form
for the measurement for example a piece of rock can be inserted
into the sample cup and spectrum measured from it

A useful way of analyzing sold samples without the need for cutting
samples uses (ATR or attenuated total reflections) spectroscopy
using this approach , samples are pressed against the face of a single
crystal ..
This infrared radiation passes through the crystal and only interacts with
the sample at the interface between the two materials

FIIR
fourier transform infrared spectroscopy (FIIR) is :-
-a measurement technique that allows are to record infrared spectra
infrared light is guided through and interferometer and then through the
sample (or vice versa )
- A moving mirror inside the alter the distribution of infrared light that
passes through the interferometer . The signal directly called and
“ interferogram “ represent light out put as a function of mirror position
- A data – processing technique called transform turn this raw ata into the
desired result the samples spectrum
light out put as a function of infrared wave length or equivalently wave
number
As described above the samples spectrum is always compared to a reference

Absorption bonds regions
a spectrograph is often interpreted as having two regions
-functional group region ≥ 1500 cm-1
in the fingerprint region there are many troughs per functional
group
-finger print region < 1500 cm -1
in the finger print region there are many trughs which form an
inticate pattern which can be used like a finger print to determine
the compounds
infrared spectroscopy correlation table

Typical infrared absorption regions :-
wave length

Survey of spectra
hydro carbons (Ch absorption)
Alcohols
Acids
(OH-Absorption)
Amines ( N – H absorption)

Molecular vibrations
Two major type
Bending
Stretching

Dipole momento
only bonds which have signficiant dipole
momento will absorb infrared radiation
Bonds which don’t absorb infrared include
1-Symmetricaly substituted alkenes and
alkynes
2- many types of c-c bonds
3- symmetric diatomic molecules H-H / Cl-Cl

Strong absorbers
the carbonyl group is one of the strongest absorbers also o-H and C-O bonds
OH C-O

Best practice and trouble shooting
important notes and common mistakes
1-qualfifcations  icp oq pQ
2- verification  periodically , maintenance , change location
3-polystyrene film - covered and stroed in desiccator don’t touch the surface or
blow off
use clean dry air
4- all accessories should be stored in desiccator (pellets preparation kit alakli
halid windows  ETC .
5-KBR should be pure IR – grade , dried before use (dry at 110 degree 2 to 3
hours)

Importance notes and common mistakes
avoid rapid heating of KBR to prevent oxidation (KBro3)
-KBr could be contaminated with Kvo3 (band at 1640)
while grinding , KBr is added portion wise
if not dry abroad band at 3400 due to water will appear measure background and
Atm correction

Weighning give better spectrum intensity
extraction organic and equeous mix and drying are very critical steps
clean kits or holders with (iso propanol ) ccl4 , Chcl3)
Don’t ever use water or toluene
after switching on the instrument , leave it for about 30 min to stabilize
don’t pull the electric cable by the end of analysis (dehunidifier should be always
on )

For viscous and non- volatile samples use demontable cell
for volatile samples use fixed cell (quantitative analysis )
for aqueous solution use ATR
use ATR pellets or ATR instead of KBR pellets methods in case of
(hydrochlorides) due to an exchange

HPIC
High performance liquid chromatography
introduction
Types of HPIC technique
principle
instrumentation
parameters used in HPIC
advantages of HPIC
Derivatisation in HPIC
Applications

Introduction
HPIC is a form of liquid chromotography used to separate compounds that
are dissolved in solution
HPIC is characterized by the use of high pressure to push a mobile phase
solution through a column of stationary phase allowing separation of
complex mixtures with higher resolution
compound are separated by injecting on to the column
the mobile phase must be degassed to eliminate the formation of air bubbles

Types of HPIC Technique
A- Based on modes of chromatography
1-normal phase mode
2-reversed phase mode
B-Based on principle of separation
1-absorption chromatography
2-ion exchange chromatography
3-ion pair chromatography
4-size exclusion gel permeation
5-chiral phase chromatography
6-Affinity chromatography

C-Based on elution technique
1-isocrotic separation
2-Gradient separation
D- Based on the scale of separation
1-analytical HPIC
2-Preparative HPIC
E- Based on the types of analysis
1-qualtitative analysis
2-Quanitative analysis

PRINCIPLE
The principle of separation in normal phase mode and reverse phase mode is adsorption.
When a mixture of components are introduced into a HPLC column, they travel
according to their relative affinities towards the stationary phase.
The component which has more affinity towards the adsorbent, travels slower.
The component which has less affinity towards the stationary phase travels faster. Since
no 2 components have the same affinity towards the stationary phase, the components are
separated

Normal Phase.
-Polar-Stationary phase
-Nonpolar- Solvent(Mobile phase)
Reverse Phase.
- Non-polar- Stationary phase
- Polar - Mobile phase (solvent).

Methanol
• Acetonitrile
• Tetrahydrofuran
• Water
CH3OH
CH3CN
H2O

Solid Support - Backbone for bonded phases.
◦ Usually 10µ, 5µ or 3µ silica or polymeric particles.
Bonded Phases - Functional groups firmly linked (chemically bound) to
the solid support.
◦ Extremely stable
◦ Reproducible
Guard - Protects the analytical column:
◦ Particles
◦ Interferences
◦ Prolongs the life of the analytical column
• Analytical - Performs the separation.

C-2 Ethyl Silyl -Si-CH2-CH3
•CN Cyanopropyl Silyl -Si-(CH2)3-CN
• C-18 Octadecyl Silyl -Si-(CH2)17-CH3
•C-8 Octyl Silyl -Si-(CH2)7-CH3

Chromatography Stationary Phases
O
|
O
|
O
|
O Si O Si O Si O
H |
O
|
|
O
|
|
O
|
O Si O Si O Si O
H |
O
|
O
|
O
bulk (SiO2)x surface
Silica Gel
O
|
O
|
O
|
O Si O Si O Si
O R |
O
|
|
O
|
|
O
|
O Si O Si O Si
O R|
O
|
O
|
O
x
bulk (SiO2) su rface
Derivatized Silica Gel

Instrumentation
Gradient
Controller
Mobile Phases
Pump
Injector
Column Detector

Solvent Reservoir
Degasser
Solvent Delivery System
(Pump)
Injector
Column &oven
Detectors
Recorder (Data Collection)
HPLC system

Instrumentation
1. Solvent delivery system
2. Pumps
3. Sample injection system
4. Column
5. Detectors
6. Recorders and Integrators
SOLVENT DELIVERY SYSTEM:
The solvents or mobile phases used must be passed through the column at high pressure at
about 1000 to 3000 psi. this is because as the particle size of stationary phase is few µ (5-10µ), the
resistance to the flow of solvent is high. Hence such high pressure is recommended.
The choice of mobile phase is very important in HPLC and the eluting power of the mobile
phase is determined by its overall polarity, the polarity of the stationary phase and the nature of the
sample components.

Mixing unit is used to mix the solvents in different proportions and pass through
the column. There are 2 types of mixing units.
They are low pressure mixing chamber and high pressure mixing chamber.
Mixing of solvents is done either with a static mixer or a dynamic mixer.
In an isocratic separation, mobile phase is prepared by using solvent of
same eluting power or polarity.
But in gradient elution technique, the polarity of the solvent is
gradually increased and hence the solvent composition has to be changed. Hence
a gradient controller is used when 2 or more solvent pumps are used for such
separations.

Several gases are soluble in organic solvents.
When solvents are pumped under high pressure, gas bubbles are formed which will
interfere with the separation process, steady base line and the shape of the peak.
Hence degassing of solvent is important. This can be done b
yusing Vacuum
filtration, Helium purging, Ultrasonication.
In normal phase chromatography hexane, iso octane, di ethyl etherare the mobile phases. In
reverse phase chromatography water, methanol, acetonitrile are the mobile phases.

Degasser
In order to avoid causing the problems, mobile
phase should be degassed.
◦ vacuum pumping systems
◦ distillation system
◦ a system for heating and stirring the solvents
◦ sparging system - bubbles an inert gas of low solubility through
the solvent
Problems caused by dissolved air(O2, N2)in mobile phase
◦ Unstable delivery in pump
◦ Bigger noise and large baseline-drift in detector cell

Compnents
Three basic types of LC Pumps are:
Pneumatic pumps
Motor driven syringe type pumps
Reciprocating pumps
Pumping systems:
• Requirement: high Pressure (6kpsi), Pulse-free,
• Constant Flow(0.1 10mL/min.),
Reproducibility(0.5%), Resistant to corrosion

Reciprocating Pumps
Advantages
◦ small internal volume
◦ high output pressures (up to 10,000 psi)
◦ readily adaptable to gradient elution
◦ “unlimited” solvent reservoir
Disadvantages
◦ produces a pulsed flow
◦ expensive

Solvent Delivery System
Requirements
◦ ability to mix solvents and vary polarity of mobile phase during run
◦ “unlimited” solvent reservoir
◦ generation of pressures up to 6000 psi
◦ flow rates ranging from 0.1 to 10 mL/min
◦ flow reproducibility’s of 0.5 % or better
◦ resistance to corrosion by a variety of solvents
◦ pulse-free output

Injectors
Sample Injection System
◦ sample valve
◦ syringe
Sample Injection Systems
●
●
●
●
●
●
For injecting the solvent through the column
Minimize possible flow disturbances
Limiting factor in precision of liquid chromatographic measurement
Volumes must be small
.1-500 L
Sampling loops
● interchangeable loops (5-500 L at pressures up to 7000 psi)

Precolumn
◦ remove impurities from solvent
◦ saturates mobile phase with liquid of stationary phase before
the analytical column
Column
◦ straight, 15 to 150 cm in
length; 2 to 3 mm i.d.
◦ packing - silica gel,
alumina, Celite

COLUMNS:
– Stainless steel tubing for high pressure
– Heavy-wall glass or PEEK tubing for low P (< 600 psi)
– Analytical column: straight, L(5 ~ 25 cm), dc(3 ~ 5 mm), dp(35 μm). N (40 k
~ 70 k plates/m)
-Micro column: L (3 ~ 7.5 cm), d (1 ~ 5 mm), dp: 3 ~ 5 μm, N: ~100k plates/m,high speed and
minimum solvent consumption
– Guard column: remove particulate matter and contamination protect analytical column, similar
packing
– T control: < 150 °C, 0.1 °C
-Column packing: silica, alumina, a polystyrene-di vinyl benzene synthetic or an ion-
exchange resin
– Pellicular particle: original, Spherical, nonporous beads, proteins and large biomolecules
separation (dp: 5 μm)
– Porous particle: common used, dp: 3 ~ 10 μm. Narrow size distribution, porous micro particle

Detectors
UV
◦ Single wavelength (filter)]
◦ Variable wavelength (monochromator)
◦ Multiple wavelengths (PDA)
Fluorescence
Electrochemical
Mass Spectrometric

DETECTORS
Detectors used depends upon the property of the compounds
to be separated. Different detectors available are:
1. Refractive index detectors
2. U.V detectors
3. Fluorescence detectors
4. Electro chemical detectors
5. Evaporative light scattering detectors
6. IR detectors
7. Photo diode array detector:

RECORDERS AND INTEGRATORS:
Recorders are used to record the responses obtained from detectors after amplification.
They record the base line and all the peaks obtained, with respect to time. Retention time for all the peaks
can be found out from such recordings, but the area of individual peaks cannot be known.
Integrators are improved version of recorders with some data processing capabilities. They can record the
individual peaks with retention time, height and width of peaks, peak area, percentage of area, etc. Integrators provide
more information on peaks than recorders. Now a days computers and printers are used for recording and
processing the obtained data and for controlling several operations.
5. PARAMETERS USED IN HPLC:
1.Retention time
2.Retention volume
3.Seperation factor
4. Resolution
5. Height Equivalent to a Theoretical Plate (HETP)
6. Efficiency
7. Asymmetry factor

Advantages of HPLC
Rapid and precise quantitative analysis
■ Typical analysis time of 5-20 min, precision <0.5-1% RSD
Automated analysis
■ Using autosampler and data system for unattended analysis and report
generation
High sensitivity detection
■ Detection limits of ng to pg
Quantitative sample recovery
■ Preparative technique from g to kg quantities
Amenable to diverse samples
■ Can handle >60% of all existing compounds vs. 15% for GC
■ Can analyze samples with little or minimal preparation

DERIVATISATION IN HPLC:
In order to increase the detectability of various classes of compounds (
for which sensitive detectors are not available ) derivatisation is carried out in HPLC.
A good amount of work has been performed on the labelling of
compounds with chromophores and flurophores for detection using UV
spectrometers and fluorimeters respectively.
There are 2 important types of derivatisation.
These are
1. Pre column derivatisation
2. Post column derivatisation

PRE COLUMN DERIVATISATION:
In pre column derivatisation there are no restrictions on the solvents, reagents, or
reaction rates chosen and excess of reagents can be removed before the injection. However, artifact
formation, if present, can be checked by positive identification of the eluted peaks. For example, in
the derivatisation of a triketone with more than one functional group capable of being derivatised
there is a possibility of range of derivatives being formed from one solute. It is clearly
necessary to check that the derivatisation reactions are quantitative or the sample derivatisations
proceed in a manner analogues to the derivatisation of standards.
Examples of pre column derivatisation to form UV chromophores include the treatment of
ketosteroids with 2,4, DNP and the benzoylation of hydroxy steroids or the esterification of
fatty acids. Similarly, fluorophores have been introduced into amino acids, biogenic amines, and
alkaloids by treatment with dansyl chloride.
POST COLUMN DERIVATISATION:
It is carried out on the separated solutes as they emerge from the chromatographic
column. In HPLC, this places serious restriction on the derivatisation reactions, because
dilution of the eluent peak must be minimized. Consequently, very fast reactions must be used and
the reagents and mobile phase must be compatible.

Examples of post column derivatization reactions for use with UV detectors include:
A. Reaction of amino acids with ninhydrin and fluorescamine.
B. Reaction of fatty acid with ortho nitro phenol.
C. Reaction of ketones with 2, 4, DNP.
D. Thermal or acid treatment of carbohydrates.
An oxidation detector for the fluorimetric analysis of carbohydrates in body fluids using Ce (III) flourescence
has also been reported

How to deal with solvents
• Use clean bottles only.
• Use borosilicate glass bottle only.
• Rinse bottle with desired solvent before refilling it.
• Bottles can get contaminated with detergents form the dishwasher.
• Exchange water-based solvents daily.
• Algae growth may block the degasser or filters.
• Precipitation of insoluble salts may block filters or capillaries.
• Select solvent volume to be used up within 1 – 2 days.
• Use only HPLC-grade solvents and water filtered through 0.2 µm
filters.
• Residues or contaminations may block filters or capillaries.
• Label bottles correctly with bottle content, and filling date / expiry
date.
• Use solvent inlet filters to protect the system from incoming particles.
• Reduce risk of algae growth: use brown bottles for aqueous solvents,
avoid direct sunlight or wrap the bottles in aluminium foil.

CAUTION
Contaminatedseal wash solvent
➔Donot recycle sealwash solvent to avoid contamination
➔Weeklyexchangeseal wash solvent
➔Useof SealWashBottle HeadKit (5067-6131)is strongly
recommended
NOTE
ExtrameasureswithAcetonitrile (ACN)
• Filter ACN using a 0.45 µm nylon filter.
Filtering through nylon filters is not recommendedfor High
Sensitivity LCMS.
NOTE • Fill ACN in brown bottles and keep amount to minimum to prevent
photochemical reactions and oxidation.
Add5– 10%water to ACN,especially for LCMSapplications when 0.1%formic acid is
present(if possible).
• Flush the system monthly with warm water (60 – 70 °C (140 – 158
°F)) - 1 L at 2 mL/min to dissolve traces of ACN reaction products.

Howtoprepare samples
Possible sampleprecipitation
➔T
akecare that the sampleis completesoluble in both, the usedsample
solvent andthe mobile phaseat starting conditions.
➔Match the sample solvent matches andthe proposedmobile phaseasclosely
as possibleto preventprecipitation.
CAUTION
• Filter, decant, or centrifuge sample to separate from
insoluble solid.
• Take care that the sample solvent is free of particles.

Daily/ Weekly tasks
• Replace solvents and solvent bottles for mobile phases based on
water/buffer.
• Replace solvents and solvent bottles for organic mobile phase latest every
second day.
• Check presence of seal wash solvent.
• Purge each channel with fresh solvent at 2.5 – 3 mL/min for 5 min.
• Equilibrate your system with composition of your application for 15
min. Use conditioning for 1290 systems.
Weeklytasks
• Change seal wash solvent (10 %/ 90 %isopropanol/water) and bottle.
• Flush all channels with water at 2.5 – 3 mL/min for 5 min to remove salt
deposits if buffer applications were used.
• Inspect solvent filters for dirt or blockages. Clean or exchange if no flow is
coming out of the solvent line when removed from the degasser inlet.

Powerup/ Shut-downthesystem
Power-upthesystem
Powerupthe pump
• Use new or different mobile phase (as required).
• Purge each channel with 2.5 – 3 mL/min for 5 min. Open the purge valve
(1260) or use the purge command (1290).
• Equilibrate your system with composition of your application for 15 min.
Use conditioning for 1290 systems.
Powerupthe sampler
• Purge the autosampler daily, and before and after sample analysis, especially if
you are using buffers.
• Set flow to required value of your application and close the purge valve.
• Pump for approximately 10 min.
• Use fresh needle wash and/or needle seat backflush solvents like methanol
or acetonitrile and water mixtures without buffer.
• Ensure that the vials contain enough sample solution for all injections.
Powerupthe detector
• Warmup the lamp for at least 1 h.
• For RI detectors only: flush the reference and sample side with fresh solvent
used for the current application.

Shut-downthesystem
NOTE Use5050 MethanolWater or 2-propanolWaterwithout additions to store system.
Long-term storage of the column
• Flush the column with appropriate solvent found in the column manual.
• Remove and seal column, and store according to good laboratory practice if
needed.
Long-term shut-down of the system
• Flush system with water to remove buffer.
• Remove all samples from the sampler and store according to good
laboratory practice.
• Use recommended solvents to store the system.
• Power off the system.

Recommendationsfordegassers
CAUTION Condensationof vaporinsidethedegasser
If aninternal orhighperformancedegasseris usedwith low boilingsolvents,thesolvent vapors
cancondensate insidethe degasser chamberwhenthe vacuumpumpis turned off.
➔ Purgeall solvent channels with 2-propanol andlet the degasser pumpfor two morehours.
• Check compatibility of solvent with degasser and application
• Use internal or high performance degassers for standard applications
• Use the standard degasser (G1322A or G7122A) for RI applications
• Use the standard degasser for high volatile solvents with vapor pressure below
100 mbar at room temperatur.
• Use the EvacuationMode if degassing performance of internal degassers is not
optimal. Access it in the degasser control from the instrument control screen in the
Agilent LabAdvisor.

Recommendationsforpumps
• Check pumps performance on regular basis.
• Perform preventive maintenance in the recommended usage
interval.
• Prepare the pump as recommended like described in the
power up section to ensure optimal performance and best
life time.
• Use the seal wash function as recommended to ensure
optimal performance and best life time, see below.
RecommendationsforPumpswith MCGV
Selectchannelsfor Multi-Channel Gradient V
alve (MCGV)
• Use lower channels (A and/or D) for buffer solutions.
• Regularly flush all MCGV channels with 200 mL of warm water to remove possible
salt deposits.
• Check compatibility of buffers and organic solvents to avoid precipitation in the
MCGVs mixing chamber.
NOTE Whenmixing incompatible solvents, salts canprecipitate at the point of mixing blocking the
downstreamflow pathanddamagingparts.

SealWash(usage mandatorywheninstalled)
SealWash(G4204A, G4220A, all 1260Pumps)
CAUTION
Contaminatedseal wash solvent
➔ Donot recycle sealwashsolvent to avoid contamination
➔ Weeklyexchangeseal washsolvent
➔ Useof SealWashBottle HeadKit (5067-6131)is strongly recommended
Using the seal wash function is strongly recommended when using water or water based solvents like buffer, other non-volatile
solvents or additives that could deposit on pistons and seals. The seal wash function regularly cleans these parts automatically.
BenefitsofSeal WashOperation:
• Removal of particles, salt crystals and other non-volatile residues from the pistons and seals, which have the potential to
damage the piston and piston seals
• Lubrication of seal/piston interface
• Cooling of pistons
Seal WashDialog in yourCDS
The dialog can be found under the control screen, it is recommended to use the settings displayed in Figure 2 on page 10.
Be aware that:
• The seal wash settings are NOT method parameters
• The seal wash has to be turned on again manually after:
• An ERROR has been cleared
• Power on

Seal Wash Operation:
• PERIODIC operation, for example 0.5 min every 7
min
• Setting can be changed in the Control screen,
see Figure 2 on page 10. The settings are
available via the context menu, see Figure 4
on page 10.
• Typical solvent flow is 0.7 mL/min what
corresponds to an approximate consumption
of 3 mL/h of or 0.5 L/week at constant
operation
• Use 10 %2- Propanol in water
• 100 %2- Propanol for normal phase applications
• Position wash solvent bottle above and waste bottle
below instrument
• DO NOT recycle the seal wash solvent
• Usage of the Seal Wash Bottle Head Kit is
recommended

Recommendations for samplers
• Purge the autosampler after sample analysis.
• Remove buffer with HPLC grade water.
• Remove contaminating substances with a strong solvent, for example pure
acetonitrile.
• Toggle the injection valve between Mainpass and Bypass while purging.
• Always use fresh wash solvent for the needle or seat wash function.
• Remove buffer with HPLC grade water.
• Remove contaminating substances with a strong solvent, for example pure
acetonitrile.
• Place the wash solvent reservoir for needle wash (optional: needle seat flush) into
the solvent cabinet.
• Use an appropriate solvent based on the sample and mobile phase properties.
NOTE The composition of the needle-wash solvent should be the most solubilizing compatible solvent
(your strongest diluent). Selecting it is part of the method development. A mixture of 50 %up to
100%organic solvent in distilled water is agoodchoice formanyapplications.

• Check the drainage routing of the washport outlet into a waste container.
• Fill each vial with enough sample solution for all injections.
• Use Agilent recommended vials only.
• Do not overfill the vials, that is to say fill each vial up to 90 %only.
• Use pre-slitted septa when drawing large volumes or multiple times from the same
vial.
• Filter, decant, or centrifuge sample to separate from insoluble solid.
NOTE Samplesolvent should befreeof particles.
• Take care that the sample solvents match the proposed mobile phase as
closely as possible.

Recommendations for columns
• Use columns only in the marked direction.
• Always use suitable fittings for your specific column.
• Different vendor columns require different fitting dimensions.
• Using an unappropriate fitting may result in peak dispersion or
even terminal damage to the column.
• Agilent recommends using A-Line fittings to overcome fitting
incompatibilities when using different vendor columns.
• Always adhere to operating and application limits, as put forth in the
column user guide.
• Equilibrate the column with 10 – 20 column volumes before use.
• It is advisable to do an intermediate flush with a mobile phase of
the correct composition without additives before equilibrating to
the final solvent with additives.
• The use of a guard column is recommended to protect your column
and increase its lifetime.
NOTE Long-term storageof columns shouldalways bein the appropriate storing solvent, for moredetails
onthe columnin use,seethe User Guideinserted in the columnpackage.

Recommendationsfordetectors
CAUTION
Frequentlampon/off Reducedlifetime of the lamp
➔ Avoid unnecessarily switching on/off thelamp.
NOTE Thereis asafetyperiod/wait time beforealamp can bere-ignitedafter it has been turned off.
• Warm-up the lamp at least 1 h.
• Keep environment and ambient temperature stable.
• Do not expose the detector to direct sun light.
• Do not expose the detector to too much air current from the HVAC.
• Install pressure relieve valve when connecting a second detector after the Max-light cartridge cell.
• Use the recommended waste lines for each detector type. Avoid pinching the waste tube after the cell outlet.
• Ensure that the detector flow cell is bubble free.
• For RI detectors only: flush the reference and sample side with fresh solvent used for the current
application.
• Flush the flow cell after use.
• Use HPLC grade water to remove salts.
• Use isopropanol to remove organic solvents.
• Before removing an unused flowcell for storage fill it with isopropanol to prevent algae growth.

Outline
• Potential Sources
• Where to Begin?
• Baseline Troubleshooting
• Column Troubleshooting
• Other Issues
HPLC Troubleshooting

Potential Sources of Chromatographic
Problems
• Mobile
Phase
• Injector
• In -Line
Filter
• Column
• Detector
• Pump
• Guard Column
• Connecting
Tubing and
Fittings
• Integrator/Reco
rder
The
Scientist/Analyst

Whereto Begin?
• Check for little or no back
pressure
• Inject blank – No baseline
problems
• Compare chromatograms
• Inspect baseline

BASELINETROUBLESHOOTING
Noisybaseline Cyclic
Synchronous noise
Asynchronousnoise
Spikes
Nopeaks

Baseline Troubleshooting
Noisy baseline
• Gas in mobile phase
• Degas mobile phase
• Leaks
• Find leak and repair
• Electronic noise
• Remove source. Shield cables
• Weak detector lamp
• Replace lamp
• Sensitivity too high
• Lower sensitivity
• Detector cell dirty
• Flush with 6N nitric acid

SynchronousNoise
ALMOST ALWAYS CAUSED BY THE PUMP
• Air in the pump head
• Prime pump and degas solvent
• Check valve problem
• Clean or replace
• Broken plunger
• Replace
• Mixing Problem
• Increase system volume
• Electrical noise
• Remove source

AsynchronousNoise
• Bubbles
• Gas caught in detector
• Degas mobile phase. Put backpressure on cell
• Leaks
• Find leak and repair
• Mixing problems
• Plugged lines
• Remove plug – flush system
• Electrical problems
• Remove source

BaselineDrift
• Gradient – Solvent B absorbs more than Solvent A
• Try new mobile phase. Use baseline subtraction
• Compounds eluting off column
• Run strong solvent until baseline is stable
• Solvent compostion change (e.g. Evaporation)
• Ensure solvents are enclosed
• Solvent leaks
• Tighten, replace fittings
• Backpressure changes
• Filter solvents and samples. Samples may be too viscous
• Mixing problems

Cyclic
Baseline
• Temperature fluctuations
• Thermally insulate. Move away from ventilation.
Increase cell temperature
• Mixing problems
• Gas in mobile phase
• Degas solvents
• Electrical problems
• Remove source
• Erratic pump
• Repair
• Plug
• Remove obstruction – flush system

Spikes
• Bubbles
• Degas solvent
• Poor electrical connection, loose wiring
• Clean and tighten detector leads, check wiring
• Lamp relay trying to fire a dead lamp
• Replace lamp
• Electrical noise
• Remove source. Common sources – Switching valves, compressors,
muffle furnaces, fraction collectors, power conditioners, lighting

NoPeaks
• Injector not injecting
• Pump not pumping
• Dead detector
• Integrator/recorder/PC not connected correctly
• Gain setting too low
• Leaks
• Column retaining all compounds
• Bad or incorrect mobile phase
• Bad or incorrect standard or sample
• Incorrect guard column
INJECT ACETONE SOLUTION
TO MAKE A PEAK

Negative&Positive
Peaks
• Some eluting compounds absorb less
than solvent
• Use a different or cleaner solvent
• Air bubbles passing through cell
• All peaks are negative
• Change detector polarity
• Negative peaks with RI detector
• May be normal (peak direction is function of RI
differential from mobile phase

PoorPeak
Shape
• Contaminated in-line filter
• Replace frit
• Column dying
• Replace column
• System void volume
• Check system tubing
• Contaminated guard column
• Replace
• Incorrect or wrong solvent
• Make new mobile phase. Consider ion
pairing/suppression
• Column destroyed
• pH <2 washes off functional group
• pH >8 dissolves silica base

ColumnTroubleshooting
CommonProblems
• Peak shape
• Retention
time
• Other

Chromatographic problems may be
related to:
• Instrument
• Sample
• Column
Two Major Problems are:
• Peak Shape/Width
• Retention Time Changes

PeakShape Problems
Most Common Problem in HPLC:
Distorted peaks will cause integration or resolution
problem
Indication that optimal column performance is not
being attained

PeakShape Problems
• Column Destroyed
• Secondary
Interactions
• Incorrect Sample
Solvent
• Column
Overload
• Other Extra-Column
Effects
• Mass
Overload
• Volume
Overload

All PeaksAffected
• COLUMN
• Connection
• Replace frit
• Regenerate or replace
column
• COLUMN DESTROYED
• pH <2 washes off functional
group
• pH >8 dissolves silica base

Column Protection
Major cause of column deterioration is contamination.
Use of guard columns may increase column life-time to >
10,000 analyses

ColumnProtection
Guard column should be regarded as a cost-effective sacrifice to extend
analytical column life-time
Should contain IDENTICAL packing material as the analytical column
e.g. using a different C18, with different retention properties could actually
destroy the separation or impair protection
Well designed, well packed guard columns will actually IMPROVE the
analytical separation efficiency

Column Protection
Other Techniques to Protect the Column:
• In-line Filter between the Injector and Column
• Filtering of the Sample ( Doesn't Protect against
Seal Shedding)
• Sample Cleanup through Solid Phase Extraction
(SPE)

Column Storage
Store in Mobile Phase for Short Periods of Time
( <72hrs.) Store in Shipping Solvent for Longer
Periods of Time

ColumnStorage
• Column should be stored in solvent which manufacturer recommends
• For bonded phases, use organic solvent
• (eg. MeOH or ACN) Using non aqueous solvents minimizes
hydrolysis.
• Some bonded phases (CN) become unstable in polar organic mobile phases.
• Storage in water or buffer is then okay.
• Worst mobile phase for CN column is CH3CN (Acetonitrile)

ColumnStorage
Columns which may be stored in Water or Buffered Solvents:
• Ion exchangers
• Aqueous SEC packings
However:
Prevent microbial growth by using 0.05% sodium azide in mobile phase
OR
Small quantity of organic solvent (acetonitrile 5% or methanol 10%)

Column
Storage
Columns which should be
stored in Mobile Phase:
• Normal Phase
• Organic SEC (GPC)

OtherIssues
Tailing
What Causes
Tailing? Mixed
mode retention
Hydrophobic – interaction with
bonded phase Ion exchange –
interaction with charged sites

OtherIssues
Column/VolumeOverload

OtherIssues
Column/VolumeOverload
A good approach to a loading study is to increase the amount
injected by a
factor of 2
This is fine enough to find the point of maximum load but course
enough to cover a large concentration quickly
It is often not necessary to perform measurements on the
chromatogram. A visual inspection is often
sufficient

OtherIssues
MassOverload
How to identify?
• Indicated by a rapid detector response
How to remedy the problem?
• Dilute the sample by 50% then re-inject
One can observe better resolution and slightly rounded leading
edge of the peak We then dilute the sample by 50% again until
we observe a much more pronounced
leading edge of the peak of interest.

Summary
• Assess all potential sources
• What is the baseline telling you?
• Treat your columns well
• Assess the suitability of your
method
• Mobile phase composition etc.
• Ensure injection volume and
sample concentration are suitable

Instrumental Analytical Techniques
Most common Analyticaltechniques
Chromatography
Spectroscopic
Titration
Electrochemical
analysis
Thermal analysis

Chromatographic techniques
Thin layer chromatography (TLC /
HPTLC)
CAMAG® Automatic
TLC Sampler 4 (ATS
4)
CAMAG® TLC Scanner 4

High Performance Liquid Chromatography
(HPLC)
Gas Chromatography (GC)
Powerful separation technique for detection of
volatile organic compounds.
Used for assay of drugs
Determination of residual solvents
Important tool for analysis of impurities of
pharmaceuticals

Spectroscopic techniques
Spectrophotometry
• Based on natural UV absorption and chemical reactions
• Spectrophotometry is the quantitative measurement of the absorption,
reflection or transmission properties of a material as a function of
wavelength.
• The advantages of these methods are low time and labor consumption.

Spectrophotometry
Based on natural UV absorption and chemical reactions
Spectrophotometry is the quantitative measurement of the
absorption, reflection or transmission properties of a material as a
function of wavelength.
The advantages of these methods are low time and labor
consumption.

Spectrophotometry
• The colorimetric methods are usually based on the
following aspects:
1. Complex-formation reaction.
2. Oxidation-reduction process.
3. A catalyticeffect.

Fourier Transform Infrared spectroscopy (FTIR)
It is preferred method of infraredSpectroscopy. In infrared spectroscopy, IR
radiation is passed through a sample. Some of the infrared radiation is absorbed
by the sample and some of it is passed through(transmitted).
The resulting spectrum represents the molecular absorption and transmission,
creating a molecular fingerprint of the sample.

Fourier Transform Infrared spectroscopy (FTIR)
Like a fingerprint no two unique molecular structures produce the
same infrared spectrum.
FTIR is used for identifying organic compounds, both quantitatively
and qualitatively.
In quantitative research, FTIR is used to figure out an analyte
concentration in a sample. In qualitative research, FTIR is used for
identifying the functional groups that make up a compound.

Atomic Absorption Spectroscopy (AAS)
This is the most widely used technique for the quantitative
Determination of metals at trace levels(0.1to100ppm), which present
in various materials. The sample is vaporized by aspiration of solution
into a flame or
evaporation from electrically heated surface. At this condition where
the individual atoms co-exist, a beam of light is passed through them.

Atomic Absorption Spectroscopy
(AAS)
The atoms will absorb in the visible and ultraviolet region resulting in
changes in electronic structure (excited state). So, the resultant light
beam coming out of the sample will be missing the light in the
corresponding wave length, which is a measure of the
characteristics of the sample.
Plasma Emission Spectroscopy

Sunscreen analyzer
• SPF Study for sunscreen
• Water resistance study

Electrochemical analysis
There are several techniques under this title like Potentiometry, Amperometry,
Conductometry, Voltametry, etc., Potentiometry
We use a potentiometer to determine the difference between the potential of two
electrodes.The potential of one electrode—the working or indicator electrode—
responds to the analyte’s activity, and the other electrode—the counter or reference
electrode—has a known, fixed potential

Electrochemical analysis
Applications Determination of chloride It is usedfor the analysis
of cyanide, ammoniaetc.,in water or wastewater. It is used in
agriculture for the detection of different elements in soils,
fertilizers etc. It is used in detergent manufacturing, food
processing etc.

Thermal
analysis
Substance
Thermal
change
DSC
Differential
scanning
calorimeter
DTA
Differential
thermal
Analysis
Weight
Change
TGA
Thermo
Gravemetric
Analyzer
Dimensional
Change
TMA
Thermo
Mechanical
Analyzer
Cooling
Heating

Good Weighing Practices At Laboratory
Content
• Significance of balances in the QC Laboratories
• Types of Balances
• Definition
• Minimum weight
• Location for installation Balances
• USP requirement
• Performances test
• Calibration
– Repeatability
– Linearity
– Eccentricity
– Sensitivity
• Factor influence the accuracy of weight
• Types of samples and handling
• Precaution to be taken while weighing

Impact of Weighing in Analysis
• Weighing is a one of key activities in all the QC laboratories
• Most of the time, our understanding is not at sufficient level
• Its importance or complexity is underestimated.
• Quality of weighing determines the Quality & Accuracy of final test result.
• The USP specifically requires highly accurate results when weighing
analytes for quantitative measures
• Right choice of balances (Analytical/semi-micro/micro ) with desired
resolution, accuracy & repeatability is essential to reduce the error and meet
the compliance

Type of balances
Balance name Resolution Quantity of
decimal digits (gm)
Ultra-microbalances 0.1 µg 0.0000001
Microbalances 1 µg 0.000001
Semi-microbalances 0.01mg 0.00001
Analytical balances 0.1mg 0.0001
Precision balances 1g ÷ 1mg 1g ÷ 1mg

What is minimum weight ?
• Minimum weight is the minimum sample quantity required to perform
an accurate quantitative analysis is based on the measurement
error of the balance used
• In order to satisfy the required weighing tolerance, when samples are
weighed the amount of sample mass (i.e., the net weight) must be
equal to or larger than the minimum weight.
• The minimum weight applies to the sample weight, not to the tare or
gross weight.
• If the sample quantity is too small, the measurement error will be huge and
result of the analysis will be unreliable.

Definition:
Accuracy
• Closeness of agreement between a measured quantity value and a true quantity value
of a measurand. VIM *
• Difference between measurements average value and the real value according to USP
Precision
• Closeness of agreement between indications or measured quantity values obtained by
replicate measurements on the same or similar objects under specified conditions. VIM
Trueness
closeness of agreement between the average of an infinite number of replicate measured
quantity values and a reference quantity value
(*) VIM – International Vocabulary of Terms in Legal Metrology
(**) USP – United States Pharmacopeia

USP General Chapters
• Measurement is stated to be 'accurately
measured' or 'accurately weighed',
– (41) Balances
– (1251) Weighing On An Analytical Balance

Understand the USP Requirement
USP General Chapter <1251>:
"In order to satisfy the required weighing tolerance,
when samples are weighed, the amount of sample
mass (i.e., the net weight) must be equal or larger
than the minimum weight.
The minimum weight applies to the sample weight,
not to the tare or gross weight.

Requirement of Balances
Select the appropriate balance based on the accuracy,
repeatability, stability, access control, printout or connect to other
instrument or LIMS etc.
 URS
 Select Correct accuracy and repeatability
 Qualification
 Installation at right place / location
 Operation qualification
 Performance qualification / Calibration
 Password protection, Access control , printer, etc

Location : installation of Balances
• Install the balance on anti- vibration table &
• non-magnetic surface and grounded to prevent static electricity
• Room should be temperature and humidity controlled vibration, air currents,
• Should be free of drafts and away from air conditioner or fans or windows to
avoid strong air current or direct sunlight
• Away from magnetic fields (magnetic stirrer), electromagnetic radiation eg RF
generators/communication devices and electric motors, Away from Corrosive
materials are used nearby.

Operational Qualification
OQ has to cover following, but not limited
to;
• Control of stable indication
• Mechanical mobility of all moveable parts
• Manually triggered or automatic adjustment by
means of
built-in weights.
– Automatic adjustment reduce the drift of the balance.
• Operation of ancillary equipment
• Tare function
• Calibration part of OQ

Accuracy <41>:
Accuracy:
• The accuracy is satisfactory if its weighing value, is
within 0.10% of the test weight value.
• A test weight is suitable if it has a mass between 5%
and 100% of the balance's capacity.
• maximum permissible error (mpe) or uncertinity shall
1/3 of the applied test ie 0.03%. (ASTM E617)
Note: A readability of 0.1 mg of balance is believed as “accurate to
0.1 mg” as misconception.

Balance Calibration
According to USP General Chapter <41> “Balances”, for substances to be
accurately weighed, the balance used must be calibrated over the operating
range
The most important are ;
 Repeatability (RP),
 Eccentricity (EC),
 Linearity (L) and
 Sensitivity (SE),

Repeatability
• Why Repeatability is so important?
• It will have significant impact on all the quantitative
analysis
• Ability of a weighing instrument to display identical
measurement values for repeated weighings of the
same objects under the same conditions, e.g., the
same measurement procedure, same operator,
same measuring system, same operating
conditions, and same location over a short period
of time.
• Systematic deviations normally can
be prevented if Repeatability is
performed.

Repeatability :
Repeatability :
Individual measurement deviation
from average value does not exceed
standard deviation, that is ~ 0,0003g
with probability 68,5%.
Individual measurement deviation does not
exceed three standard deviations, that is ~
0,0009g with probability higher than 99,7%,
so very close to certainty.

Linearity test
• Linearity: To ensure that balance
is accurate at the desired level
in the operating range
• Ability of a balance to follow the
linear relationship between a load
and the indicated weighing value.
• Non-linearity usually is expressed as the
largest magnitude of any linearity deviation
within the test interval.
• Perform 3 to 6 points over the range of the
balance.
• Limit: NMT 0.05% deviation where 〈41〉 is
applicable. For other uses, respective
tolerance requirement divided by 2.
It is a deviation of balance real curve from straight lin
joining two points A:B – ideal weight.

Eccentricity Test
• Deviation in the measurement value caused by eccentric loading— in other words,
the asymmetrical placement of the center of gravity of the load
• Eccentricity usually is expressed as the largest magnitude of any of the deviations
between an off-center reading and the center reading for a given test load.
• In practice, a difference is defined between indication when mass standard is put at
central point of weighing pan and indication when the same mass standard is located at
another place on the weighing pan.
• Performed in the center of gravity and the four quadrants
• Test load usually should be 30% of the capacity of the balance or higher.
Limit: NMT 0.05% Deviation where (41) is applicable.

Sensitivity Test
• Change in weighing value divided by the change
in load, usually measured between zero and the
capacity of the balance.
• Use certified weights with an
appropriate weight class
• Perform as performed for repeatability test
• Limit: NMT 0.05% deviation where 〈41〉 is applicable.
For other uses, respective tolerance requirement
divided by 2.

Testing of Balance parameters
• How often balance parameters should be tested?
Intervals defining balance calibration/testing shall be
based on range of operation, their intensity, balance
stability in time and expected weighing process
measurement
precision.
• Assuming that external conditions are stable,
following balance parameters control periods can
be fixed:
– Calibration annually
– Repeatability & centricity monthly

Factors affects the weighing Accuracy
There are several external factors influence the accuracy of
weighing;
• Ambient area and people influence result of weight
measurement;
• Major balance external factors are ;
– Oscillations, vibrations
– Breeze of air
– Temperature drifts
– Electrostatics
– Evaporation and absorption phenomena (hygroscopicity)
– Magnetism
• Other factors is nature of sample ,
• Both balance and the sample will influence the accuracy of

Impact of Vibration & Strong Air
• Oscillations – vibrations are transmitted by the ground and walls that
are generates and affects weighing balance
– Effect is longer measurement time and higher indication dispersion.
– Prevent vibrations by keep away from vibration area
– Keep on anti-vibration table. Anti vibration double rubber console, to
suppress vibrations
• Breeze of air influence to instability and long weighing time.
– Balance workstation should not be located close to doors or windows.
– Closeness to devices such as air-conditioning, fans, should be avoided.

Impact due to Temperature
How Temperature affect the
weighing;
• Weighing room temperature should be maintained at
constant level. Eg variation must not more than
0.5°C/hour.
• Equilibrate the sample to room temperature
before
weighing , will give error due to heat convection ( hot sample
will be less than true value
than true value)
• Precaution/ aspects for weighing process:
 pick up the samples with use of tweezers or other holders
 should not put their hands into weighing chamber
 Touched with hands, samples –may change their temperature

Hygroscopic samples
Sample nature influence the weighing accuracy:
 Hygroscopic samples absorbs the moisture from
ambient air and steadily gain the weight
 Measured weight will have higher mass than actual
 Weighed promptly
 Use the hermetic vessels or a gas-tight enclosure.
 Weighing vessel should be clean & dry and
easily transferable
 Add the desired amount of sample, and replace
the enclosure

Volatile or hygroscopic samples
Sample nature influence the weighing accuracy:
Volatile liquids (low boiling solvents or solid with volatile solvents ) can
undergo evaporation during weighing
Balance indicates fluctuation / drift; ie weighing continuously decrease while
measurement
Use appropriate weighing vessels, like bulbs with narrow necks or vessels with
top cover.
Weighing of Volatile samples:
 Weighing of low boiling liquid point in a vessel with a gas-tight enclosure of
small diameter.
 Close immediately after transfer of material
 After the balance display stabilizes, the analyst records the specimen
weight

Corrosive and Bio-hazardous Samples
Aseptic or Biohazardous Samples
• Weighing the samples in the confines area / bio-safety
cabinet
/isolator, or similar containment device.
• Care should be taken if airflow in the hood may cause
balance instability
Corrosive Materials
• Extra care is essential when materials of this
nature are weighed.
• Use sealed containers such as weighing bottles or
syringes

Samples with Electrostatics
Effect of electrostatic presence.
o Slow drift of weighing result,
o Large dispersion of weighing results in a series of
measurements, and
o No return to zero if a load is taken off the weighing pan.
Possible source:
– Dry, finely divided powders may be charged with static
electricity
– The static charge may develop due to low relative
humidity, clothing worn and gloves used
How to prevent:
 Antistatic weigh boats, antistatic guns, and antistatic
screens
 Placing the container in a metal holder
 Balances with a built-in antistatic device is available
(piezoelectric components or low amount of a

Magnetism as interfering factor
• If magnetic load is measured, electromagnetic field
of a balance is disturbed or weighed sample is
influenced by magnet installed in a balance.
• It will lead to incorrect mass reading of a
weighed sample.
• High resolution balances are constructed on
basis of electromagnetic sets which include a
force-motor and magnet.
• How to avoid: Increasing a distance between a
sample and balance mechanism. Use under-hook
weighing with application of special racks or hooks
made of aluminum.

• All receivers must be clean, dry, and inert.
weighing uncertainty for small samples, i.e., net weights with a mass
determined around repeatability.
Receivers should be nonmagnetic used at ambient temperature
Weighing dishes should be polymer or aluminum. Antistatic and
compatible with the liquid sample.
Safety measures:
Use proper PPEs gloves , mask, goggles etc during a weighing to avoidexposure
Hazardous materials should be handled in an enclosure that has appropriateair filtration.
Many toxic— and possibly allergenic— substances are present as liquids orfinely divided
particles.
Precaution while
weighing

• US Pharmacopeia- 42
General Chapter <41> Balances,
General Chapter <1251> Weighing on an Analytical Balance,
Reichmuth, A., Weighing Small Samples on Laboratory Balances, 13th
International Metrology Congress, Lille (F), 2007
Reference

Appendix III - Evaluation of uncertainty of calibration of a balance
This appendix gives an example to demonstrate the procedure for evaluating uncertainty of
measurement in calibration of the balance.
When using the balance for normal usage, the conditions (i.e. weighing process, environment,
repeatability, loads, etc.) are different than during calibration. Therefore, the uncertainty of the daily
usage of the balance (uncertainty of the weighing result) is different than the uncertainty of
calibration of the balance and it is estimated using the uncertainty of calibration of the balance and
other relevant contributors: uncertainty of environmental conditions and uncertainty from operation
of the instrument (weight to weight variability, taken from repeated weighing - repeatability). An
example of calculation of uncertainty of the weighing result is described in Annex 1 of the guideline
Uncertainty of measurement [8].
Example:
Calibration of electronic balance (maximum weighing capacity 220 g/scale interval, d = 0.0001 g)
Conditions specific for calibration:
Temperature: at the beginning of calibration: 20°C ( T = 0.4°C)
Relative humidity: 42.7 ±1.4 % RH
Barometric pressure: 992 ±6 hPa
Standard weight (E2): Nominal value of the weight: 100 g, Conventional mass: 100.000066 g, 𝑈𝑈𝑚
𝑚
𝑐
𝑐
= 0.000050 g (k = 2, for approximately 95% level of confidence).
The obtained calibration results are given in Table 1 (Appendix III). Please note that the value of
the average below reported is rounded according to the standard deviation obtained.

1. Repeatability
Test load 100 g
Result (indication) (g)
1 100.0001
2 100.0001
3 100.0002
4 100.0001
5 100.0001
6 100.0001
7 100.0001
8 100.0001
9 99.9999
10 100.0001
Average 100.00009
Standard deviation 𝑆
𝑆
(
𝐼
𝐼
𝐼
𝐼
) 7.37865E-05
2. Eccentricity
Position Indication (g)
Error (∆Iecc)(g)
1. centre 100.0000 0.0000
2. left forward 100.0000 0.0000
3. left back 100.0003 0.0003
4. right back 100.0002 0.0002
5. right forward 100.0003 0.0003
∆Iecc max 0.0003

PA/PH/OMCL (12) 77 R12 – Qualification of Balances
1. Step 1. Specification of a measurand
The measurand is error of indication (Ei):
𝑀
𝑀
𝑖
𝑖= 𝐼
𝐼−𝑚𝑚𝑟𝑟𝑏𝑏𝑟
𝑟
where,
I is the measured value (Indication)
𝑚𝑚𝑟𝑟𝑏𝑏𝑟
𝑟is the reference value of the mass of the weight used (nominal value
or conventional value).
2. Step 2. Identification of uncertainty sources

Fig. 1 Cause and effect diagram
3.Step 3. Quantification of uncertainty components
1. Component 1. Uncertainty of Repeatability of the instrument, 𝒖𝒖(𝒓𝒓𝒓𝒓𝒓𝒓)
The repeatability test can be performed by reading/recording of results (indications, 𝐼
𝐼
𝐼
𝐼
)
of repeated depositions of a single test load or using different test loads. The result
(indication, 𝐼
𝐼
𝐼
𝐼
)may be recorded (one value) for each individual deposition of the load
obtained during the repeatability test or the mean value (repeated measurements) for
each individual deposition of the load obtained during the error of indication test.
Where only one repeatability test is performed, using a single test load, the uncertainty
of repeatability can be considered as representative for the whole range of the
instrument (normal
distribution is assumed):
𝑢𝑢(𝑟𝑟𝑏𝑏𝑟𝑟) = 𝑆
𝑆
(
𝐼
𝐼
𝐼
𝐼
)
Where, 𝑆
𝑆
𝐼
𝐼
𝐼
𝐼= Standard deviation calculated from repeatability test of
the balance Therefore:
𝑢𝑢(𝑟𝑟𝑏𝑏𝑟𝑟) = 0.0000737865𝑔
𝑔
If the result (indication) is the mean of repeated measurements (n) for each individual
deposition of the same test load, during the error of indication test, the corresponding
standard uncertainty is:

Measuring pH Correctly
 Introduction
 Sensor
 Sample
 FAQ Examples

Introduction
Good practice needed for consistent accurate results
= Widely used technique and appears to be straight forward…..
But: many possibilities for errors in the whole system
- Meter
- Environment
- People
- Documentation
- Electrode
- Sample
Only correct usage and maintenance of the system
guarantees reliable results

Introduction
Calibration of sensor inputs
Once to twice annually as part of operational qualification
In case of problems: first make sure meter is not the cause
Sometimes the meter or electrode cable has a short
- No signal due to working electrode
- Ease to verify (other cable, resistors)
Get certificates for prove
Calibration service can be included in
service contracts

Introduction
Environment
 During installation qualification, the suitability of the
environment must be verified
Appropriate temperature
Minimal temperature fluctuations
Humidity appropriate
Avoid strong air flow close to the meter
Have enough space to work (accidents)

Introduction
Training
 The more you aware of possible risks, the better you can minimize them
 Ensures correct handling of the system
 Saves time troubleshooting
 Helps understanding possibilities and limits of the measuring system
Documentation
 If is not documented, it is not done
 Ensures traceability
 Supports legal requirements

Sensor
Daily tasks
 Electrode preparation
 Electrode storage
 Electrode cleaning
 Electrode calibration
To get
 Longer lifetime (saves money)
 Constantly reliable results
 Fewer problems (saves time)

Sensor
Electrode lifetime
The pH sensitivity of the gel layer sinks with age The
aging process is temperature dependent
Example lifetimes at different temperatures (same usage) assuming :
Application of good practices with measurement in aqueous solutions and pH range 1 to
12

Sensor
Electrode lifetime
 Electrodes don’t last forever and have to be replaced sometimes
 Aging depends also on handling, sample, frequency of usage
 Indication of too old electrode
X slower response times
X higher membrane resistance X
smaller slope
X bigger offset

Electrode preparation – reference electrolyte
Replace reference electrolyte regularly (e.g. once per months)
Less crystallization at the diaphragm
Less impurities in the electrolyte
Constant high ion concentration
! Don't fill up, empty it completely
Fill it again using fresh electrolyte
Electrolyte level in electrode must be higher than the sample
Avoid the reflux of sample into the electrode (contaminations)
No air bubbles behind junction
Vertical shaking of electrode to get rid of them

Sensor
Electrode preparation – reference electrolyte
Replace reference electrolyte regularly (e.g. once per months)
 Less crystallization at the diaphragm
 Less impurities in the electrolyte
 Constant high ion concentration
! Don't fill up, empty it completely
 Fill it again using fresh electrolyte
Electrolyte level in electrode must be higher than the sample
 Avoid the reflux of sample into the electrode (contaminations)
No air bubbles behind junction
 Vertical shaking of electrode to get rid of them

Sensor
Electrode preparation – dry membrane
Cause
 Measuring in non-aqueous or ion deficient media
 Wrong storage
Effect
 Reduced sensitivity of glass (gel layer “washed out”)
 Unstable signal
Action
 Conditioning in 0.1 mol/L HCl during 12 hours

Sensor
Electrode storage
Objective  Ensure that the pH sensitive gel layer which forms on the pH glass
membrane remains hydrated and ion rich
Always store
 In inner electrolyte (e.g 3 mol/L KCI)
 In buffer solutions (e.g pH 4 or 7)
 In HCl diluted (approx. 0.1 mol/L)
 Together with sample (same conditions)
Never store
Dry, in distilled water or non-aqueous solutions
Reduces lifetime
 Needs conditioning before use (costs time)

Sensor
Electrode cleaning
Aqueous sample
 Rinse with distilled water after every measurement (contamination of next
sample)
 Dip it dry with paper towel
! Never wipe it with paper towel (electrostatics)
Non-aqueous or dirty sample
 First rinse with solvent to get rid of dirt which is not water soluble
 Rinse with distilled water, dip it dry
 Condition in aqueous solution

Sensor
Electrode cleaning - diaphragm
Blocked with silver chloride (AgCl)
 With concentrated ammonia
Blocked with silver sulfide (Ag2S)
 With 8 % thiourea in 0.1 molar HCl
Blocked with proteins
 With 5 % pepsin in 0.1 molar HCl
Other junction blockages
 In ultrasonic bath with water or 0.1 molar HCl

Sensor
Electrode calibration
Possible errors
X Not done frequently enough
X Done at a different temperature than subsequent measurement X Wrong or contaminated
buffers used
Actions
 Calibrate at least once per day, more if high temperature fluctuations
 Make sure conditions at calibration and measurement (temperature, stirring etc.) are equal
 Always use fresh buffers – if buffers not accurate, pH calibration is not accurate and the
measurement will not be accurate
 Perform at least a two point calibration
 Make sure the calibration points frame the expected sample pH

Sensor
Electrode calibration – buffer handling
 Buffers have expiry date – don’t order in bulk
 Store well sealed at room temperature
 Take out needed amount and close bottle immediately
again
 Never calibrate electrode directly in the bottle
 Never re-use already used buffer solution
 Take single-use buffer sachets

Sensor
Electrode calibration – indicating electrode
condition
 “Offset” value (mV) – indicates the age of electrode and
provides an estimation when the electrode need to be
changed.
 Calibration Slope (%) – indicates the sensitivity of the glass
membrane
 Recommended offset range at pH 7.00 is ± 30mV.
 Recommended calibration slope range is 95% - 105%.
(DIN 19263 requirements: Offset 0 ± 30 mV; Zero Point: pH0 = 7 ±0.5
pH)

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