Transcript Slide 1

Optimization and uncertainty budgets of externally
standardised NMR purity assays
Fahmina Fardus1,2 John Warren1 Adam Le Gresley2 Jean-Marie Peron2
1- LGC Ltd, Queens Rd, Teddington TW11 0LY
2- Kingston University, Penrhyn Rd, Kingston-Upon-Thames KT1 2EE
[email protected]
Introduction
Receiver Gain Compensation
Environmental Factors
The use of qNMR for purity measurement has been
steadily growing in recent years. The assessment
of the purity of calibration materials and reference
standards requires a detailed understanding of the
uncertainties associated with the measurements on
which the analysis is based. In addition, the
measurement results should be traceable directly
to SI units and it is here that qNMR offers
advantages over chromatographic techniques.
Direct purity determination using qNMR is normally
achieved by adding an internal standard of known
purity to the analyte. In cases where an internal
standard is undesirable, purity by qNMR is
achieved using external standards, however this
method introduces additional sources of errors
which need to be quantified before they can be
applied to reference materials. The aim of this
project is to optimize conditions on an Avance III
600 spectrometer equipped with a 5 mm PATXI
1H/D-13C/15N Z-gradients probe to give accurate
purity assessment by external standards and to
derive a full uncertainty budget from the results.
Experiments to quantify three of these potential
sources of uncertainty are described in this poster.
The receiver gain (RG) is a measure of the
amplification of the analogue signal coming from the
NMR probe but has been shown to deviate
significantly from linearity1. A series of experiments
were run to calibrate a range of RG values and
derive an uncertainty budget. Solutions at different
concentrations (0.2-30 mg/g) were prepared and
run at RG series 2n from 1 to 256 each sample was
analysed 5 times at each receiver gain (excluding
those that overloaded ADC).
Figure 2 shows the integration results from the
replicate runs normalised against the receiver gain.
As anticipated, each receiver gain gave a consistent
but distinct deviation from mean.
It is well known that NMR instruments are very
sensitive to building vibrations which cause
deleterious effects on the spectra. For quantitative
work this poses a problem as the artefacts around
the base of the resonances may interfere with the
accuracy and reproducibility of the NMR
measurements. The 600 MHz NMR facility at
Kingston University sits on an anti vibration
system with dampening equipment integrated into
the magnet legs. The laboratory’s air conditioning
unit cycles on and off during the day and so the
impact of this on the uncertainty budget has been
investigated. A series of 10 replicate experiments
were performed during times with the air
conditioning either exclusively on or exclusively
off. Figure 4a shows the visual difference of the
base of the resonance peaks when two spectra
are overlaid, the red peak shows the air con off
and the blue shows it on. Figure 4b shows the
variation of the absolute integral plotted against
number of replicates. As it can be seen the
standard deviation of replicates is large compared
to when the air con is off shown in red in Figure 4.
Figure 2
Figure 4
a)
Sample Depth
b)
9.45
Compensation factors for the series of receiver
gains have been determined along with the
associated uncertainties.
Figure 3 compares mean normalised integrals, both
before (red) and after (blue) calibration along with
the standard deviations of the measurements.
9.40
Absolute integral (x10^7)
Whilst the dimensions of the NMR tube are clearly
vital to the externally standardised assay, it is often
assumed that the NMR solution outside the coils
has no impact on the spectrum. To a single NMR
tube, 4cm depth of a stock solution of dioxane was
added and 5 replicate analysis performed. Further
stock solution was added to give a depth of 5cm
and then 6cm with replicates performed in each
case. A final set of replicates was performed with
the 6cm deep sample displaced upwards by 3mm
in the depth gauge.
9.35
Air con on
9.30
Air Con off
9.25
9.20
9.15
1
2
3
4
5
6
7
8
9
10
Experiment number
Figure 3
Clearly the air conditioning is contributing a
significant burden on the uncertainty budget with
an increase in the relative standard deviation from
0.09% without air con to 0.48% with it on as
shown in Table 2.
Figure 1
Sample Depth Experiments
21.7
Table 2
21.6
Absolute integral (x10^8)
21.5
4ml
5ml
6ml
6ml displaced
21.3
Air con On
93124827.73
0.48
Air con Off
92733917.27
0.09
21.2
21.1
21
20.9
1
2
3
4
5
No of replicate
Figure 1 shows significant differences between
sample heights with about 2% variation in the mean
integrations between volumes. This equates to
0.1% per mm over the normal range of sample
depth and thus the reproducibility of sample depth
will impact on the uncertainty. Further experiments
are required to determine how accurately this can
be controlled in practise.
1
Average Integration
% RSD
21.4
Huaping Mo et al, Magn. Reson. Chem. 2010, 48, 235–238
The expanded uncertainties were calculated
three cases:
RG uncorrected – optimal but different RGs
reference and analyte – no compensation
RG corrected - optimal but different RGs
reference and analyte - compensated
Same RG - non optimal but identical RGs
reference and analyte
for
Conclusion
for
The three factors discussed in this poster are all
shown to impact on the uncertainty budget and
highlight where steps can be taken to minimize
their impact. These factors are just some of the
experimental parameters being investigated in
this study that will allow a comprehensive
understanding to the uncertainties associated with
qNMR and improve it’s methodology.
for
for
Table 1
Expanded U
RG uncorrected
15
RG corrected
0.74
same RG used
0.52
The expanded uncertainties for %w/w purity in are
given at the 95% confidence level in Table 1.