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5 Pda
5 Pda
5 Pda
Detector (PDA)
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Photodiode Array Detector
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Instrumentation of PDA Detector
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Comparison between UV-Vis and
PDA Detector
UV-Vis Detector
" Measures only one wavelength or at maximum two at the same time.
" Wider linear range and slightly better sensitivity compared to PDA.
PDA Detector
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Lambert-Beer’s Law
Lambert-Beer’s Law
Ideal
Actual A : absorbance
2.5
ge
ε : molar absorptivity
an
rr
C : analyte concentration
ea
lin
Spectra
Chromatogram
Absorbance
th
ng
e le
av
W
Time
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Display of UV-Vis Spectrum &
Extraction of Chromatogram
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Example of PDA Application (1)
- Identification by UV-Vis Spectrum
ACN/H2O=15/85
min
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Example of PDA Application (1)
- Identification by UV-Vis Spectrum
Elution sequence
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Example of PDA Application (2)
- Checking Peak Purity
UV-Vis Spectrum of
Acetyl Salicylic Acid
Sample
Standard
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Peak Purity
S 1
θ S 2
" Similarity Index (SI) is a technique to evaluate & compare spectral shapes.
" The technique converts each spectrum into a vector.
" Similar spectra will produce vectors that point in the same direction.
" Spectra that have different shapes will produce vectors that point in different
directions.
" Threshold is the uncertainty angle θ and all spectral changes that are
greater than the threshold indicate the presence of spectrally different
compounds.
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Peak Purity Function
(Total Peak Method)
Peak Purity Function using All Peak Method, compares the similarity
of the spectra at the Peak Top with every points within the peak,
from the start of the peak to the end of the peak.
Peak Top
Absorbance
Peak Peak
Start End
Elution Time
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Peak Purity Function
(Total Peak Method)
" Impurity : Indicates the retention time when impurity is detected, at the
point when the spectrum is most different from the rest of the points in
the peak.
" Peak Purity Index : Indicates the similarity index of the point when the
spectrum is most different from other points in the peak. When the
value is 1, the peak is pure. Purity Index of less than 1 indicates the
presence of impurity.
" Single Point Threshold : Indicates the threshold value of the point when
the spectrum is most different from other points in the peak. Threshold
is the uncertainty contributed by the noise.
• Minimum Peak Purity Index : (Peak Purity Index – Single Point
Threshold) x 106. Positive value indicates that no impurity is detected.
Negative value indicates that impurity is present.
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Peak Purity Function
(3 Point Method)
Peak Top
3 Point Peak Purity Method :
Average of up-slope similarity
and dow-slope similarity
Absorbance
Index :
Similarity– Threshold. Positive
Peak Peak value indicates that no impurity
Start End
is detected.
Elution Time
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Peak Purity Function
" When the target compound and the interference have similar
spectrum, the peak purity calculation may not be accurate.
" When the concentration of the interference is very small
compared to target compound, the impurity when the sample is
coeluting may not be easily detected.
" The absorption should be within the linear range of the PDA
detector to produce good UV-Vis Spectra. Sample with very
high concentration above the linear range of the PDA Detector
will produce distorted spectrum.
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PDA Detector
Advantages:
• PDA Detector could analyze a sample simultaneously at many
different wavelengths.
• UV Visible spectra are useful for compound identification,
checking peak purity, as well as finding the optimum
absorbance for the compounds.
• UV Visible spectra of many compounds could be stored in the
spectrum libraries, which are useful for compound
identification.
• Relatively robust to temperature and flow rate fluctuations
• Compatible with gradient elution.
Disadvantages:
• Slightly less sensitive than UV-Visible detector.
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