THE DIODE ARRAY ADVANTAGE Agilent Cary 8454 UV-Visible Spectroscopy System

There are specifi c benefi ts to using a UV-Vis spectrophotometer with diode array detector technology compared to those offered when using traditional scanning UV-Vis instruments. Within the molecular spectroscopy group, Agilent is able to offer both types of UV-Vis instruments. The Agilent Cary 60 UV-Vis, based on Xenon fl ash lamp technology, is the ideal scanning instrument for many routine UV-Vis applications. The Agilent Cary 8454 UV-Visible, and its predecessor the Agilent 8453 UV-Vis are diode array instruments that lead the market in regulatory Pharma QA/QC applications, and can acquire a complete spectrum in less than 1 second. This technical note outlines both applications-based and technical considerations for choosing a diode array UV-Vis.

Introduction to the Agilent Cary 8454 UV-Visible Spectrophotometer

The Agilent Cary 8454 UV-Visible spectrophotometer is the successor to the Agilent 8453 UV-Vis, which was released in 1995 and is the market leader in diode array technology. The diode array spectrophotometer uses “reverse” optics confi guration where the dispersion device, the grating, comes after the sample (Figure 1).

Grating

1024 element diode array

SlitLens

Cell

Shutter

Lens Tungsten lamp

Deuterium lamp

Figure 1. Schematic of Agilent Cary 8454 optics confi guration.

In the Cary 8454, the source is a deuterium arc lamp, which gives good intensity in the UV region and a tungsten halogen lamp that has good intensity over the visible region of the spectrum with very low noise and drift. Light hits the sample and is then dispersed by the grating before falling on the diode array. As light of all wavelengths falls on the array it is measured simultaneously, that is, data acquisition is done in parallel. A spectrum is obtained by electronically scanning the array.

1. Open sample areaAn advantage of the reversed optics confi guration used by the Cary 8454 is that only light traveling along the axis from the source to the inlet slit before the dispersion device can reach the detector. Light from any other angle cannot reach the detector, effectively making the instrument immune to interference from ambient “stray” light. For this reason, the Cary 8454 design has an open sample area, which has many benefi ts for the user (Figure 2).

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The open sampling area makes sample introduction very easy, so sample handling is faster and less prone to user error which can occur with conventional instruments due to improper closure of the sample area cover. It also allows for quicker access, which is a benefi t for kinetics applications where prompt introduction of reaction components is required. The open sampling area also makes it more straightforward to install and uninstal accessories such as the multi-cell transport and Peltier thermostatted cell holder.

2. Speed of data acquisitionThe Agilent Cary 8454 UV-Visible spectrophotometer can measure a complete spectrum from 190 nm to 1100 nm in as little as 0.1 seconds. Data is acquired in parallel, where each wavelength is acquired simultaneously. The detectors are read-out by electronic scanning and microprocessors are used to process the data. Therefore, for kinetic measurements, particularly multi-wavelength kinetics measurements, the Agilent Cary 8454 UV-Visible spectrophotometer is ideal as each wavelength of interest can be monitored simultaneously.

Figure 2. Open sample area of the Agilent Cary 8454 UV-Visible spectrophotometer

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The benefi ts of a fast spectrophotometer include increased productivity, because the user is not waiting for a result for extended periods of time. Also, each result is a complete spectrum so no data is missed and is available if required in the future. For applications such as quality control and quality assurance, being able to quickly and completely assess a sample enhances productivity.

An additional advantage is the fast presentation of data and results from the analysis features within UV-Vis ChemStation. A method can be set up to include the analysis of the spectra that are acquired, so the fi nal result is displayed at completion of data collection.

An example of a fast time-collect is shown in Figure 3. The hydrolysis reaction was monitored over time with spectra acquired at three second intervals during the course of the reaction. The high quality of data

Figure 3. Spectra measured during the hydrolysis of a common indicator dye

Wavelength (nm)

Abso

rban

ce (A

U)

2500.0

0.4

0.6

0.8

1.0

1.2

0.2

3000 350 400 450 500

collected can be seen through the clear isosbestic points and the superior S/N of the spectra.

3. Simultaneous multi wavelength analysisBy design, a diode array spectrophotometer collects the entire spectrum simultaneously. This is one of the key differences of a diode array compared to conventional scanning instruments which offers benefi ts for specifi c applications.

An application advantage for multi wavelength measurements is that they can be used for Confi rmation Analysis. In the example below, caffeine would be conventionally quantifi ed by calibrating with a standard and measuring unknowns at 274 nm. As long as the unknowns are pure, good results are obtained. But if an impurity is present, for example salicylic acid, the quantitative result is wrong because the impurity also absorbs at 274 nm and worse, there is no way of knowing that the result is wrong.

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With a diode array, as you collect the entire wavelength spectrum by design, you can quickly confi rm whether impurities are effecting your result. Standards and unknowns are measured at multiple wavelengths at the same time. Thus a quantitative analysis and a purity check can be done simultaneously (Figure 4).

Another benefi t of the capability to collect multiple wavelengths simultaneously is that all data is included with the fi le. As part of the method, you can specify that all data should be automatically stored as it is acquired to ensure traceability of the results. Raw data is stored with all the annotation information described above. In addition, even if only one wavelength is used for the

analysis, full spectral acquisition and storage can be specifi ed so that all sample information is available for review.

4. Wavelength reproducibilityAn important advantage of the Agilent Cary 8454 UV-Visible diode-array spectrophotometer is wavelength reproducibility. The optical design of the instrument is such that there are no moving parts that affect the measurement of a sample. This fi xed optical system is the reason behind the excellent wavelength reproducibility seen with the Agilent Cary 8454 UV-Visible spectrophotometer .

Figure 4. Confi rmation analysis for caffeine in the presence of salicylic acid. The absorbance of salicylic acid can be detected at 284 nm so its contribution to the absorbance at 274 nm can be determined.

Wavelength (nm)

Caffeine

Salicylic acid

Abso

rban

ce (A

U)

2500.0

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

0.1

260

264 nm

274 nm

284 nm

270 280 290 300 310 320

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The importance of wavelength reproducibility is shown in Figure 5. Quantitative spectrophotometry best practice is to measure a sample at the absorbance maximum. At the absorbance maximum there is the best signal-to-noise ratio, but also, the rate of change of absorbance with wavelength is close to zero at the peak.

Thus if there is any wavelength irreproducibility there will be little effect on the measured absorbance. On the side of an absorbance band there is a rapid change of absorbance with wavelength, and any wavelength irreproducibility will result in signifi cant absorbance errors. Diode array instruments can measure reproducibly even on the side of absorbance bands, not only from measurement-to-measurement, but also over weeks, months and even years.

The benefi t of wavelength reproducibility is that there are no longer any limitations in the choice of analytical wavelength. A wavelength on the side of an absorbance band can be used if it gives better results because it avoids interference from an impurity which occurs at the absorbance maximum. There is confi dence in data at all points in the spectrum, which is important for techniques such as least squares multi-component analysis where data from a spectral range is used. Also, because of long term reproducibility, “electronic standards” stored on disk, are practical. Diffi cult-to-obtain, unstable or toxic compounds need only be measured as standards once, and can be used from fi le with high confi dence.

Figure 5. Effects of wavelength reproducibility on absorbance error.

Abso

rban

ce (A

U)

0.00

0.10

0.15

0.05

Wavelength (nm)

Error = 0.0 AU

Error = 0.01 AU (10%)

250 260 270 280 290 300230 240

Agilent Technologies, Inc.www.agilent.com

© Agilent Technologies, Inc. 2014This information is subject to change without notice.

Published on March 13, 20145991-4272EN

5. Measurement statisticsFor the Agilent Cary 8454 UV-Visible system, parallel detection gives high speed data acquisition. Because of this, measurement times of 0.5 or 1.0 sec are used, and 5 or 10 spectra are measured and averaged together for each measurement that is performed. In addition, the standard deviation for each data point is calculated; this standard deviation is a measure of the reliability of the data point.

This listing of absorbance values and standard deviations for a perchlorate standard in the range 190 - 226 nm in Table 1 shows how the reliability of the data can change signifi cantly. Problems of poorer reproducibility in the low UV region are well known (for example, because of ozone formation in the lamp housing).

Table 1. An example of the measurement statistics available with each spectrum acquired on an Agilent Cary 8454 UV-Visible system

Wavelength Absorbance Std. Dev. %RSD

190 1.82904 ±0.00478 0.2613192 1.83295 ±0.00450 0.2455194 1.74550 ±0.00251 0.1438196 1.47369 ±0.00152 0.1031198 1.12149 ±0.00065 0.0580200 0.83278 ±0.00033 0.0396202 0.63210 ±0.00016 0.0253204 0.49852 ±0.00017 0.0341206 0.41089 ±0.00011 0.0268208 0.34927 ±0.00010 0.0286210 0.30312 ±0.00008 0.0264212 0.26969 ±0.00009 0.0335214 0.24016 ±0.00009 0.0375216 0.21777 ±0.00010 0.0459218 0.20207 ±0.00007 0.0346220 0.18304 ±0.00008 0.0437222 0.16895 ±0.00008 0.0474224 0.15639 ±0.00008 0.0512226 0.14497 ±0.00009 0.0621

With a diode array spectrophotometer, the uncertainties in all parts of the spectrum are clearly and quantifi ably measured.

The statistical information on the reliability of data points is important for chemometric techniques such as multi-component analysis using least squares with maximum likelihood. The statistical information can signifi cantly improve the accuracy of analytical results. In addition, by monitoring statistical values, sample or instrumental problems can often be detected. This is especially important for automated systems.

Summary

The Agilent 8454 UV-Visible diode array spectrophotometer has been designed for fast and accurate liquid sample analysis. For multi wavelength measurements, such as kinetics experiments or multi-component analysis of liquid samples, a Cary 8454 diode array is the ideal instrument. The release of the Agilent Cary 8454 UV-Visible system continues the tradition of reliable, rugged diode array based UV-Vis spectrophotometry. With the minimum of moving parts, and a short and rigid optical bench, the Agilent Cary 8454 UV-Visible spectrophotometer is exceptionally reliable, and requires very little maintenance.