Photodiode Array

Detector (PDA)

SPINCO BIOTECH PVT LTD

HYDERABAD
 Photodiode Array Detector

• Principles of PDA Detector and Comparison with UV-

Vis Detector

• Application of PDA Detector

" Display of UV-Vis Spectra and Extraction of

Chromatogram
" UV-Vis Spectra Library
" Peak Purity Function

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 Photodiode Array Detector

" 3D UV-Vis Detector with the ability to record the

LC Chromatogram at different wavelengths
simultaneously
" Record the UV Visible spectrum of each peak as it
elute from the column, as well as other points in
the chromatogram.

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 Instrumentation of PDA Detector

Sample Cell Grating

One element detects

D2 / W lamp one absorbance at
one wavelength.

512 Elements Photodiode Array

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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

" Measures at many different wavelengths at the same time and

extraction of chromatogram from the memory is possible.
" Less linear ranger and slightly less sensitivity compared to UV-Vis.
" UV-Vis Spectrum is obtained for every point in the chromatogram
" UV-Vis Library can be compiled and used for compound identification
" Peak purity function can detect coeluting peaks.

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 Lambert-Beer’s Law

Lambert-Beer’s Law

A = ε C L = - log (Eout / Ein)

Absorbance

Ideal

Actual A : absorbance
2.5
ge

ε : molar absorptivity
an
rr

C : analyte concentration
ea
lin

L : path length of the flow cell

E : energy
Backgroud absorbance
Concentration
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 Photodiode Array Detector
(3-D Data)

Spectra

Chromatogram
Absorbance

th
ng
e le
av
W
Time
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 Display of UV-Vis Spectrum &
Extraction of Chromatogram

" Every point in the chromatogram has a UV-Vis Spectrum.

" Extraction of Chromatogram at different wavelengths,

other than data acquisition wavelength is possible by

retrieval from the memory.

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 Example of PDA Application (1)
- Identification by UV-Vis Spectrum

ACN/H2O=30/70 1. Azelaic acid

2. Benzoic acid
1 2
3. Nitrobenzoic acid

ACN/H2O=15/85

min

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 Example of PDA Application (1)
- Identification by UV-Vis Spectrum

Elution sequence

Peak 1 Peak 2 Peak 3

Azelaic acid Benzoic acid Nitrobenzoic acid

ACN/H2O
=30/70

Benzoic acid Azelaic acid Nitrobenzoic acid

ACN/H2O
=15/85

Peak 1 Peak 2 Peak 3

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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

Under optimally defined conditions for separations and

detection :
" A pure compound will produce a peak with spectrum that
have the same shape across the peak
" Interference from co-eluting analytes will produce composite
spectra with various degrees of spectral dissimilarity across
the peak

Peak Purity Function is useful to detect if a peak is only

from one compound or if it contains coeluting peaks. It is
done by comparing the similarity of the spectra at different
points on the peak
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 Similarity Index (SI)

ˆ
S 1
ˆ
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 Purity Function using 3 Point Method compares the similarity of

the spectrum at the Peak Top with the half point between the Peak
Start and the Peak Top (up slope similarity) and the the two third point
between the Peak Top and the Peak End (down slope similarity).

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

Things to take note when doing 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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