UPLC
van Deemter plot, illustrating the evolution of particle sizes over the last three decades
Swartz. Journal of Liquid Chromatography & Related Technologies 28(2005) 1253–1263
Spacil et al. Talanta 76 (2008) 189–199
Waters ACQUITY UPLC Photodiode Array (PDA) Detector
Flow Cell10 mm (500 nL) analytical cell
high acquisition rate (20–80 points s-1)
Series 200 Diode Array Detector, PerkinElmer, Inc.
Flow Cells10 mm path (12 mL) standard4.5 mm path (5 mL) analytical2 mm path (4 mL) preparative
Diminuição do volume da cela do detectorLongo caminho optico
Picos cromatográficos com largura a altura meia menores de 1 sAltas taxas de amostragem
Comprobação da pureza de pico em HPLC e CE usando a tecnologia de arranjo de diodos
Introdução
• A confirmação da pureza de pico debe ser realizada antes de proceder com ás análises quantitativas.
• As metodologias analíticas validadas usualmente incluem a comprobação de pureza de pico como um dos itens mais importantes na lista dos critérios de validação metodológica.
Impurity detection with a singlewavelength UV-visible detector
Gausian Tailing Valley Shoulder
Coelution of three compounds A, B, C, in the chromatographic peak. No shoulders, valleys, or excessive tailing are seen
SIGNAL OVERLAY FOR PURITY ANALYSIS
Normalized signals for (a) pure and (b) impure peaks
SIGNAL OVERLAY FOR PURITY ANALYSIS: RATIOGRAMS
Ratiograms taken from (a) an impure and (b) a pure peak
• Generally limited to instances for which the spectra of both analytes and impurities are well known, a requirement for selecting the wavelengths best suited for comparison.
PEAK PURITY USING SPECTRAL DATA
• A comparação dos espectros é o método mais popular na determinação de pureza de pico
• A principal vantagem dessa abordagem é que o conhecimento prévio do componente de espectros não é necessária
Selection of Spectra for Comparison
• Traditionally, spectra have been sampled up-slope, at the apex and down-slope of the eluted peak.
• This selection pattern may overlook the presence of impurities near the peak extremities.
• On the other hand, acquisition of many spectra may increase calculation and display time without adding significant information.
Background Correction
Spectrum Normalization
• Os espectros utilizados para a comparação durante a determinação da pureza do pico deven ser normalizados para compensar as diferenças de concentração
Absorbance ThresholdWavelength range
• Definir um limite de absorção aumenta a precisão da comparação espectral ao nao pegar espectros perto da linha de base do sinal.
• O rando de comprimento de onda para os espectros pode ser seleccionados de modo a que apenas a área espectral significativo seja sob observação
PEAKS PURITY DETERMINATION: 1. Comparing the peak spectra
Normalized spectra and randomly distributed residual spectra resulting from spectral noise
Systematic trends of different spectra indicating spectral impurity
PEAKS PURITY DETERMINATION: 2. The similarity factor
• Ai and Bi: measured absorbances in the first and second spectrum respectively at the same wavelength;
• n: number of data points • Aav and Bav: average absorbance of
the first and second spectrum respectively
Graphical display of similarity factors for different pairs of normalized spectra
similarity factor of 0: no match 1000: indentical spectra. +995: very similar-990: some similarity
As a rule of thumb, impurity concentrations in the 0.1–1% range may be detected when the spectra are dissimilar. However, if the spectra of the different components are highly similar and the HPLC peaks are not well resolved, the impurity detection limit is of the order of 5%.
PEAKS PURITY DETERMINATIONADVANCED TECHNIQUES
3. Similarity curves
PEAKS PURITY DETERMINATIONADVANCED TECHNIQUES
4. The threshold curve
Modes to display the similarity and threshold curves
Similarity & Threshold (with out any transformation)Similarity & Threshold as the natural logarithmSimilarity / Threshold ratioPurity ratio: The purity value of each single spectrum is displayed as the logarithm of the difference from the threshold value
Peak purity analysis of a pure peak and a peak containing an impurity.