UPLC

van Deemter plot, illustrating the evolution of particle sizes over the last three decades
Swartz. Journal of Liquid Chromatography & Related Technologies 28(2005) 12531263

Spacil et al. Talanta 76 (2008) 189199

Waters ACQUITY UPLC Photodiode Array (PDA) Detector Flow Cell 10 mm (500 nL) analytical cell high acquisition rate (2080 points s-1) Diminuio do volume da cela do detector Longo caminho optico Picos cromatogrficos com largura a altura meia menores de 1 s Altas taxas de amostragem

Series 200 Diode Array Detector, PerkinElmer, Inc. Flow Cells 10 mm path (12 mL) standard 4.5 mm path (5 mL) analytical 2 mm path (4 mL) preparative

Comprobao da pureza de pico em HPLC e CE usando a tecnologia de arranjo de diodos

Introduo
A confirmao da pureza de pico debe ser realizada antes de proceder com s anlises quantitativas. As metodologias analticas validadas usualmente incluem a comprobao de pureza de pico como um dos itens mais importantes na lista dos critrios de validao metodolgica.

Impurity detection with a single wavelength 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 comparao dos espectros o mtodo mais popular na determinao de pureza de pico A principal vantagem dessa abordagem que o conhecimento prvio do componente de espectros no necessria

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 comparao durante a determinao da pureza do pico deven ser normalizados para compensar as diferenas de concentrao

Absorbance Threshold Wavelength range

Definir um limite de absoro aumenta a preciso da comparao 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 rea espectral significativo seja sob observao

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

similarity factor of

0: 1000: +995: -990:

no match indentical spectra. very similar some similarity

Graphical display of similarity factors for different pairs of normalized spectra

As a rule of thumb, impurity concentrations in the 0.11% 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 DETERMINATION ADVANCED TECHNIQUES 3. Similarity curves

PEAKS PURITY DETERMINATION ADVANCED TECHNIQUES 4. The threshold curve

Modes to display the similarity and threshold curves

Similarity & Threshold (with out any transformation) Similarity & Threshold as the natural logarithm Similarity / Threshold ratio Purity 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.