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Comparison Relation To Retention Time Relative Retention Resolution

This document discusses various chromatographic methods and their parameters. It compares the retention times, relative retention, and resolution of two chromatographic systems with different gradient profiles and dwell volumes. Different gradient profiles and dwell volumes can affect the selectivity. The document also discusses the sensitivity, precision, and system suitability criteria for chromatographic methods. It provides examples of precision criteria used in pharmacopoeias and how number of injections affects the acceptable relative standard deviation.

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124 views13 pages

Comparison Relation To Retention Time Relative Retention Resolution

This document discusses various chromatographic methods and their parameters. It compares the retention times, relative retention, and resolution of two chromatographic systems with different gradient profiles and dwell volumes. Different gradient profiles and dwell volumes can affect the selectivity. The document also discusses the sensitivity, precision, and system suitability criteria for chromatographic methods. It provides examples of precision criteria used in pharmacopoeias and how number of injections affects the acceptable relative standard deviation.

Uploaded by

andreas hp
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as PPTX, PDF, TXT or read online on Scribd
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A comparison of the two chromatographic systems

has been made with relation to retention time,


relative retention and resolution, the results are
presented in Table 2.8-8 from which it can be seen
that:
The steeper gradient greater difference in retention
time, relative retention and resolution when
comparing chromatographic gradient systems with
markedly different dwell volumes.
The greater dwell volumes the greater resolution.
The more gradual gradient the less different are the
retention times, relative retentions and resolution.
The introduction of an initial isocratic step and
correction for the differences in dwell volume between
the two systems similar values for the three
parameters determined.

Comparison of different chromatographic gradients on


selectivity for chromatographs with different dwell
volumes.
Gradient A: Acetonitrile (70 : 30, v/v) to (90 : 10, v/v)
in 5 mins (linear gradient).
Gradient B: Acetonitrile in water (70 : 30, v/v) to (90 :
10, v/v) in 20 mins (linear gradient).
Gradient C: Acetonitrile in water (60 : 40, v/v) to (80 :
20, v/v) in 20 mins (linear gradient).
Gradient D: Acetonitrile in water (70 : 30, v/v) in 5
mins (isocratic), then acetonitrile in water (70 : 30,
v/v) to (90 : 10, v/v) in 10 mins (linear gradient),
then acetonitrile in water (90 : 10, v/v) in 10 mins
(isocratic).
Column: 4.6 250 mm Hypersil ODS (5 mm), flow rate:
1.0 ml/min, detection wavelength: 240 nm.

a comparison of two systems has been


made with the test for related
substances in the monograph of
trimethazidine hydrochloride as an
example of where a minimum peak-tovalley ration of 3 is required.
The superimposed chromatograms are
shown in Figure 2.8-8.

The contribution of the dwell volume of an


analytical gradient chromatographic system
to variation in retention times when changing
from one system to another is very small,
and a more important factor is probably the
performance of the stationary phase
employed.
The greatest effect of differing dwell volumes
on retention times was for those substances
which were not strongly retained.
Thus, gradient systems should be conceived
in such a way that analytes should be
strongly retained at the beginning of the
gradient.

It is best if less strongly retained


components are eluted with an initial
isocratic phase followed by a
gradient for elution of the more
strongly retained analytes.
By such a strategy, the effect of
differences in dwell volumes is
minimised.

Sensitivity

The limit of detection of the peak


(corresponding to a signal-to-noise
ratio of three) is below the disregard
limit (reporting threshold) in the test
for related substances.
It follows that the limit of
quantitation of the peak
(corresponding to a signal-to-noise
ratio of ten) is equal or less than the
disregard limit in the test for related
substances.

Contd Sensitivity
The reporting threshold depends on the
maximum daily dose. For drug substances where
the maximum daily dose exceeds 2 g the
reporting threshold is 0.03 percent, otherwise the
reporting threshold is 0.05 percent (see Table 2.89)

Precision
System suitability criteria for system
precision are included in the description of
the assay procedures prescribed in
specifications or in individual monographs.
Often the test requires six replicate
injections of the same solution and the
RSD should not exceed 1.0 or 2.0 %.
The European Pharmacopoeia introduced
system precision criteria which were
dependent on the number of replicate
injections performed and the
reproducibility of the method.

Contd Precision
a maximum RSD of 0.6 after six replicate injections
was required to set a limit of 1.0 percent for
direct methods such as volumetric titration.
For comparative assay methods this value is to be
divided by 2.

B = the upper limit of the assay (provided it


represents the reproducibility of the method minus
100 percent.
n = number of replicate injections
t90 %,n1 = student- t value at the 90 percent
probability level.

Contd Precision
The relationship between the number of
replicate injections, the maximum permitted
relative standard deviation and the upper
content limit is given in Table 2.8-10.

Contd Precision

Any decision to accept or reject


analytical results should include an
assessment of the system suitability
criteria to ensure that adequate
precision is achieved.
The maximum number of replicate
injections to be performed is six but
fewer may be performed provided the
system precision RSD is equal to or less
than RSDmax given in the table for the
appropriate number of injections.

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