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.