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Dear Reader
G. Reutemann H. Huber
Manager Market Support Regional Market Manager
North East Asia
Preface .................................................................................................................. 4
1 Summary .............................................................................................................. 5
2 Principle of Validation ......................................................................................... 5
3 Steps of Validation and Recommended Limits ................................................. 6
3.1 Definition of Accuracy .......................................................................................... 6
3.2 Definition of Precision .......................................................................................... 6
3.3 Systematic Errors and Linearity ............................................................................ 7
3.3.1 Definition of Systematic Errors ............................................................................. 7
3.3.2 Definition of Linearity ........................................................................................... 7
3.4 Definition of Robustness and Ruggedness ............................................................ 8
3.5 Definition of Determination Limit ........................................................................ 9
4 Practical Hints ................................................................................................... 10
4.1 Preparations and Precautions ............................................................................... 10
4.2 Titration Control Parameters ............................................................................... 10
4.3 Titration Evaluation Parameters .......................................................................... 10
4.4 Titration ............................................................................................................... 11
5 Possible Sources of Error .................................................................................. 11
6 Recommendations for Troubleshooting ........................................................... 12
7 Results not Conforming to Specifications ....................................................... 13
8 Examples ............................................................................................................ 14
8.1 Determination of Sulphuric Acid ........................................................................ 14
8.2 Titer Determination ............................................................................................. 15
8.3 Precision and Accuracy ....................................................................................... 16
8.4 Systematic Errors, Linearity ................................................................................ 17
8.5 Robustness and Ruggedness ................................................................................ 18
8.6 Determination Limit ............................................................................................ 20
8.7 Closing Remarks ................................................................................................. 21
9 Appendix 1 ......................................................................................................... 22
10 Appendix 2 ......................................................................................................... 24
10.1 Assessment of Results ......................................................................................... 24
10.2 Precision versus Accuracy ................................................................................... 24
11 Glossary .............................................................................................................. 25
12 Literature ........................................................................................................... 26
The method must be simple yet accurate. Serious consideration must be given to the possibil-
ity of the method being automated. This strategy eliminates human errors, increases produc-
tivity and reduces the time needed to release the product to consumers without compromising
method accuracy and reproducibility hence, product quality. Of course along with all the
methods simplification comes analysis cost-saving. In addition, the method must be environ-
ment-friendly via reduction, if not elimination, of the conventional use of organic solvents,
especially non-biodegradable and toxic chemical reagents.
Accepted protocols/SOP's followed during method validation work are generally universal,
irrespective of country, industry, company or product category. The only thing that is differ-
ent between them is the accuracy required, with the strictest limits applied to those products
prepared for human consumption, e.g., food and medicine.
In this brochure, METTLER TOLEDO summarizes the general method development protocols.
These include accuracy, reproducibility, linearity, ruggedness and limit of determination. Each
method must pass all these tests, just like the consumer products for which the method will be
used.
A detailed method is applied to obtain correct results. This method describes all the different
steps from the sampling to the result. Whether correct results can be obtained or not with a
certain method has to be validated. Validation of a method comprises tests for accuracy,
precision, linearity, systematic errors, robustness/ruggedness and detection limit/determina-
tion limit. So the validation of a method proves, whether or not the instruments used for this
purpose fulfill the specific requirements.
In the context of validation a variety of expressions is used. Please refer to the glossary
(chapter 11) for a short definition of the expressions used in this brochure.
2 Principle of Validation
Accuracy, Precision, Linearity, Systematic Errors, Robustness/Ruggedness and Determina-
tion limit are checked, considering the complete analytical procedure from taking the sample
to result calculation and documentation.
The following application serves as a guideline, showing how a titration method can be vali-
dated. As an example, the method for the determination of sulphuric acid was validated.
Recommended limits for accuracy, reproducibility and linearity are subject to the tested method.
Other methods e.g. analysis of foods and drugs may require much stricter limits.
Recommendation:
Results obtained should not deviate from the true value by more than 0.3%.
Systematic errors of a titration are for example disturbing influences due to the method itself
or to solvent blank values.
In the linear regression according to chapter 3.3 systematic errors show up as a significant
deviation of the y-axis intercept a of the regression line from the zero point coordinates (see
graph 1), i.e. asys is clearly different from zero.
graph 1:
volume
asys
sample size
Recommendation:
The systematic error asys should be smaller than 15 L. If it can not be eliminated by optimizing
the method or the reagents, it has to be corrected for in the results calculation of the titration
method.
Linearity expresses whether a certain method produces correct results over the interesting
concentration range [4]. In titration the analyte concentration depends on the sample concen-
tration, on the sample size and on the solvent volume added for the analysis. By varying the
sample size and thereby the analyte concentration, the linearity of a titration method may be
detected in the range of interest.
A) The regression coefficient (R2) of the linear regression described in graph 1 must be
better than a given limit, depending on the demanded accuracy for the specific determi-
nation:
i.e. R2 > 0.995
B) A significant positive or negative slope b (resp R/V) of the regression line in graph
2 (results of the titration versus sample size) indicates a non-linearity of the titration
method, meaning that the result depends on the sample size.
graph 2:
result
result
non linear
linear
Recommendation:
If R/V is greater than 0.1%, a systematic non-linearity has to be assumed.
The RUGGEDNESS describes the correctness of the results obtained under disturbed experi-
mental (analytical) conditions such as different matrices (e.g. solutions, reagents etc.), other
temperatures of the analyte solution or elsewise deviating conditions. To determine rugged-
ness, the same sample is titrated with and without exposure to relevant disturbances. If the
results are the same, the method is considered to be rugged against this specific influence.
The determination limit is determined by titrating sample series, each with a continuously
reduced amount of sample. The determination limit is the smallest amount of substance (mmol)
or sample, which can be titrated with a good precision (RSD) of 0.3%. It can be evaluated
by intrapolation of the graph amount of substance versus relative standard deviation.
determination limit
rel. stand. dev. [RSD]
0.3%
graph 3:
amount of substance [mmol]
In normal routine titration the determination limit is not a problem, since one can simply
enlarge the sample size to get a better response from the electrode. However, this can be
different, if the analyte has a very low concentration in the sample.
The primary standard must be dried in a drying oven (e.g. 2 h at 105 C, depending on the
type of primary standard) and cooled to ambient temperature in a desiccator for at least 1
hour. It should always be stored in a desiccator.
For acid/base endpoint titrations, it is necessary to calibrate the pH electrode. Certified
buffers from METTLER TOLEDO may be used for this purpose.
The experimental setup must be protected from direct sunlight and should be in thermal
equilibrium with the environment.
The analytical balance must have a vibration free standing and should be calibrated regu-
larly. METTLER TOLEDO balances of the MT, AT and PR series offer FACT (Fully
Automatic Calibration Technology), which automatically executes a calibration whenever
needed. All steps to ensure proper weighing must be observed [5].
Samples should be titrated immediately after weighing and dissolution. Enough solvent
must be added to cover the sensor.
When performing a series of titrations, the interval time between samples should be kept
to a minimum.
In sample series, the electrode as well as stirrer and temperature sensor should be rinsed
between two measurements.
Temperature compensation is essential for pH endpoint titrations.
Sample size/Balance balance not accurate, air humidity too high or too low, contami-
nated balance, temperature changes or gradient from titration ves-
sel to balance, careless weighing, sample weight, concentration or
volume too low or too high, sample inhomogeneous, improper
sampling.
Dispensing unit tube connections not tight, contaminated burette cylinder (visible
corrosion marks), leaky piston (liquid film or crystals below the
piston), leaking burette tip, air in tubing system, three-way stop-
cock leaking.
Solvent impure (blank value), poor solubilising power, not stable, contami-
nated (e.g. by CO2, O2), wrong pH value or ionic strength.
Titration parameters unsuitable titration mode, wrong measure mode parameters, titra-
tion rate too fast or too slow, unsuitable evaluation procedure.
All non-conforming values must be reported and commented on in the validation record and
the subsequent procedure noted and explained.
If relevant deviations are found, the sections Possible Sources of Error and Recommen-
dations for Troubleshooting must be checked carefully and the disturbing influences elimi-
nated. It is essential to repeat the validation afterwards.
The titrators of METTLER TOLEDO have undergone various tests during development and
manufacturing. Furthermore, they have been time tested by numerous users in different ap-
plications all over the world and considered to be okay. If irregular results are obtained,
primary consideration should be given to the working technique of the operator or to wrong
or accidentally altered titration parameters.
Titrant . . . . . . . . . . . . . NaOH
Printer HP Deskjet Concentration [mol/L] . . . . . . 1.0
Sensor . . . . . . . . . . . . . . DG111-SC
Unit of meas. . . . . . . . . . . As installed
Accessories: DT120 T-sensor Titration mode . . . . . . . . . . EQP
Predispensing 1 . . . . . . . . mL
Volume [mL] . . . . . . . . 2
Indication: DG111-SC Titrant addition . . . .
E(set) [mV] . . . . .
.
.
.
.
.
.
DYN
8.0
Limits V . . . . . . . . . Absolute
V(min) [mL] . . . . . . 0.05
V(max) [mL] . . . . . . 0.3
Measure mode . . . . . . . . . EQU
E [mV] . . . . . . . . . . 1.0
t [s] . . . . . . . . . . . 1.0
t(min) [s] . . . . . . . . . 3.0
t(max) [s] . . . . . . . . . 15.0
Threshold . . . . . . . . . . . 3.0
Maximum volume [mL] . . . . . . 10.0
Termination after n EQPs . . . Yes
n = . . . . . . . . . . . . 1
Evaluation procedure . . . . . Standard
Rinse
Auxiliary reagent . . . . . . . . H2O
Volume [mL] . . . . . . . . . . . 10.0
Calculation
Result name . . . . . . . . . . . H2SO4 Conc.
Formula . . . . . . . . . . . . . R=Q*C/U
Constant . . . . . . . . . . . . . C=M/z
Result unit . . . . . . . . . . . g/L
Decimal places . . . . . . . . . . 5
Record
Output unit . . . . . . . . . . . Printer
Raw results last sample . . . . . Yes
Table of values . . . . . . . . . Yes
E - V curve . . . . . . . . . . . Yes
Conditioning
Interval . . . . . . . . . . . . . 1
Time [s] . . . . . . . . . . . . . 10
Rinse . . . . . . . . . . . . . . Yes
Auxiliary reagent . . . . . . . H2O
Volume [mL] . . . . . . . . . . 10.0
Statistics
Ri (i=index) . . . . . . . . . . . R1
Standard deviation s . . . . . . . Yes
Rel. standard deviation srel . . . Yes
Outlier test . . . . . . . . . . . Yes
Record
Output unit . . . . . . . . . . . Printer
All results . . . . . . . . . . . Yes
The titer of this NaOH (1.0 mol/L) was determined against the primary standard potassium
hydrogen phthalate (dried for 2 h at 150 C). The following results were obtained:
Results:
Titer
9 1.73847 1.0024 1.010
10 1.32274 1.0011
1.005
11 1.63289 1.0008
1.000
12 1.17670 1.0011
0.995
13 0.64051 1.0018
14 1.52071 1.0017 0.990
Number of samples: 22
Mean value x: 1.0012
Standard deviation: 9.06 10-4
Relative Standard Deviation: 0.0905 %
Comment:
The standardization is highly reproducible and linear. Results do not depend on the sample
weight.
945
Number Size Result Temperature:
y = 4.9106 - 1.030e-4x R =21
0.43 C
940 Theoretical Content: 4.9030 g/L
[mL] [g/L] 935 No. of Samples: 12
Mean Value: 4.90529 g/L
930
Stand. Dev.: 0.004752 g/L
1 75 4.90143 925
Rel. Stand. Dev.: 0.0968%
920
2 34 4.90828
H2SO4 [g/L]
1234567890123456789012345678
915
3 44 4.89803 1234567890123456789012345678
910
1234567890123456789012345678
4 60 4.90046 905 1234567890123456789012345678
1234567890123456789012345678
1234567890123456789012345678
5 35 4.90870 900
895
6 44 4.90672
890
7 77 4.89912 885
8 32 4.90273 880
Number of samples: 12
Theoretical value: 4.9030 g/L
Mean value found: 4.90529 g/l
Deviation to theoretical: 0.00229 g/L
Relative deviation to theoretical: 0.0467%
Standard deviation: 0.04752 g/L
Relative standard deviation: 0.0968%
Comment:
Precision as well as accuracy are excellent. The requirements are easily met.
43 4.3972 4.90672
4
77 7.6831 4.89912
32 3.1953 4.90273 3
52 5.2001 4.90995
2
91 9.0925 4.90584
31 3.1008 4.91109 1
42 4.2011 4.91117
0
0 20 40 60 80 100
4.90
The second way to determine the linearity is to
plot the results of the titration (H2SO4 in g/L) 4.89
against the sample size, as one can see in the graph
nearby. Then a linear regression is performed on 4.88
The results show a systematic error (SE) and non-linearity (NL). Presumable causes are
pipetting errors when preparing the samples.
SE as well as NL are very small and well below the recommended limits.
The uptake of carbon dioxide CO2 from ambient air is the major threat of alkaline titrants.
CO2 reacts to CO32-. Carbonate precipitation and reduction of the strength of the titrant are
the consequences.
Other analytical influences as well as the robustness (see chap. 3.4) can be checked in a
similar way.
The ruggedness of the sulphuric acid method was evaluated by exposing the titrant to air and
thereby also to CO2. Batches of NaOH titrants were exposed to air for 1, 2, 3, 4, 5, 6, 7 days.
The CO32- content of each sample was determined by titration with sulphuric acid.
Result CO32- [mg/L]
CO32--Content 30000
1 2526
2 5026
3 8793 10000
4 14422
6 20684
7 24568
0
0 2 4 6 8
Air exposure [days]
Each NaOH batch was then standardised against potassium hydrogen phthalate. Then it was
used as the titrant to determine the H2SO4 concentration. The following table shows the cor-
responding results.
Comment:
The method was found not to be rugged at all against exposure to air. Even the NaOH sample
exposed to air for only one day did not allow correct determinations any more.
Reason: When titrating strong acids with NaOH that contains CO32-, a typical double
jump of the titration curve is found, caused by the following reactions:
However, this double jump does not occur when titrating weak acids such as potassium hy-
drogen phthalate, which is mainly used for the titer determination. Therefore, the carbonate
error cannot be compensated for by frequent standardization of the titrant. It is advisable to
periodically check the carbonate content by a specific titration and dispose of the titrant if a
significant amount of carbonate is found.
The determination limit was examined using 0.005 mol/L NaOH. Series of 5 to 6 samples
were run, in order to check the reproducibility (RSD) with a low amount of sample.
The results show, that in the sample with less than 0.01 mmol sulphuric acid the Relative
Standard Deviation RSD increases drastically and continuously, while the absolute standard
deviation s remains more or less constant. The uptake of CO2 from the air is very severe in
this concentration range. Therefore the titrant has to be protected from air intake with an
absorption tube filled with NaOH on a carrier. Even then, it remains usable only for one day.
Comment: 2
The limits for different parameters or the set up of priorities in the validation process have to
be adapted by the user depending on the method and the specifications for a given task (e.g.
other tests for the determination of ruggedness).
Anyway the basic course of a method validation remains the same. Thus, this example may
well serve as a guideline for further validations.
Iodine di-Arsenic trioxide 100120 Deion. water daily Keep bottle in dark.
c(1 /2 I2) = 0.1 mol/L As 2O 3; M = 197.84 NaHCO3 Keep in PE bottles.
Keep cool.
Cerium sulfate di-Sodium oxalate 106556 Deion. water biweekly
c(Ce(SO4)2) = 0.1 mol/L C2Na2O 4; M=134.00 Sulfuric acid
5%
Potassium permanganate di-Sodium oxalate 106556 Sulfuric acid biweekly Keep bottle in dark.
c(1 /5 KMnO4) = 0.1 mol/L C2Na2O 4; M=134.00 5%; 70 C
Sodium nitrite Sulfanilic acid 100686 HBr weekly
c(NaNO2) = 0.1 mol/L C 6H 7NO 3S; M = 173.19 * 1) 0.5 mol/L
Fehling solution Glucose 1% in water 108337 Deion. water weekly Prepare Glucose
C6H 12O 6; M = 180.16 * 1) solution daily.
2,6-Dichlorophenol-indo- Ascorbic acid 100127 Deion. water daily Keep bottle in dark.
phenol sodium salt C 6H 8O 6; M = 176.13 * 1) Keep in PE bottles.
c(DPI) = 0.01 mol/L Keep cool.
Turbidimetric Titrations
Sodium dodecylsulfate N-Cetylpyridinium chlo- 102340 Deion. water biweekly Rinse bottle and
c(SDS) = 0.01 mol/L ride [CPC] monohydrate; * 1) beakers with deion.
M = 358.01 water before use.
Hyamine Sodium dodecylsulfate 112012 Deion. water biweekly Rinse bottle and
c(Hyamine) = 0.01 mol/L [SDS]; M = 288.4 * 1) beakers with deion.
water before use.
N-Cetylpyridinium chloride Sodium dodecylsulfate 112012 Deion. water biweekly Rinse bottle and
c(CPC) = 0.01 mol/L [SDS]; M = 288.4 * 1) beakers with deion.
water before use.
. * 1) These substances can not be acchieved as guaranteed primary standard substances from MERCK.
So the highest aviable quality is indicated.
Errors
Deviations from the correct or expected value
Correctness
high precision
(small RSD)
low precision
(big RSD)
This application bulletin represents selected, possible application examples. These have been tested with all possible
care in our lab with the analytical instrument mentioned in the bulletin. The experiments were conducted and the
resulting data evaluated based on our current state of knowledge.
However, the application bulletin does not absolve you from personally testing its suitability for your intended methods,
instruments and purposes. As the use and transfer of an application example are beyond our control, we cannot accept
responsibility therefore.
When chemicals and solvents are used, the general safety rules and the directions of the producer must be
observed.