Agilent 8453 UV/Visible Diode Array Sp e c t r o p h o t o m e t e r
Optical Specifications Wavelength range 190–1100 nm, Slit width 1 nm EP resolution test >1.6 spectrum of a 0.02% v/v solution of toluene in hexane, ratio abs. at 269 nm/266 nm; Stray light <0.03% at 340 nm; <0.05% at 220 nm ;<1% at 200 nm; Wavelength accuracy <±0.5 nm <±0.2 nm at 486.0 and 656.1nm Wavelength reproducibility <±0.02 nm ten consecutive scans; Photometric accuracy <±0.005 A at 1 A; <±0.01A; Photometric noise <0.0002 A sixty 0.5-s scans at 0 A, 500 nm, rms; Photometric stability <0.001 A/h at 0 A, 340 nm, after 1-hour warm-up, measured over 1 hour, every 5 seconds, constant ambient temperature Baseline flatness <0.001 A 0.5-second blank, 0.5-second scan, rms Typical scan time 1.5 second full range
Optical System The optical system of the spectrophotometer is shown at the right. The radiation source is a combination of a deuterium-discharge lamp for the ultraviolet (UV) wavelength range and a tungsten lamp for the visible (VIS) and short wave near-infrared (SWNIR) wavelength range. The image of the filament of the tungsten lamp is focused on the discharge aperture of the deuterium lamp by means of a special rear-access lamp design which allows both light sources to be optically combined and share a common axis to the source lens. The source lens forms a single, collimated beam of light. The beam passes through the shutter/stray-light correction filter area then through the sample to the spectrograph lens and slit. In the spectrograph light is dispersed onto the diode array by a holographic grating. This allows simultaneous access to all wavelength information. The result is a fundamental increase in the rate at which spectra can be acquired.
Lamps The light source for the UV wavelength range is a deuterium lamp with a shine-through aperture. As a result of plasma discharge in a low pressure deuterium gas, the lamp emits light over the 190 nm to approximately 800 nm wavelength range. The light source for the VIS and NIR wavelength range is a low-noise tungsten lamp. This lamp emits light over the 370 nm to 1100 nm wavelength range.
Spectrograph Lens The spectrograph lens is the first of the parts which are collectively known as the spectrograph. It is mounted on the housing of the spectrograph. The spectrograph lens refocuses the collimated light beam after it has passed through the sample. Slit The slit is a narrow aperture in a plate located at the focus of the spectrograph lens. It is exactly the size of one of the photodiodes in the photodiode array. By limiting the size of the incoming light beam, the slit makes sure that each band of wavelengths is projected onto only the appropriate photodiode. Grating The combination of dispersion and spectral imaging is accomplished by using a concave holographic grating. The grating disperses the light onto the diode array at an angle proportional to the wavelength.
Stray-Light Correction Filter In a standard measurement sequence, reference or sample intensity spectra are measured without and then with the stray-light filter in the light beam. Without the filter the intensity spectrum over the whole wavelength range from 190–1100 nm is measured. The stray-light filter is a blocking filter with 50 % blocking at 420 nm. With this filter in place any light measured below 400 nm is stray light only. This stray-light intensity is then subtracted from the first spectrum to give a stray-light corrected spectrum. The stray light correction can be turned off for very fast repetitive scans.
Sample Compartment The spectrophotometer has an open sample compartment for easier access to sample cells. Because of the optical design a cover for the sample area is not required. The spectrophotometer is supplied with a single-cell holder already installed in the sample compartment. This can be replaced with the Peltier temperature control accessory, the thermostattable cell holder, the long path cell holder or the multicell transport. All of these optional cell holders mount in the sample compartment using the same quick, simple mounting system. An optical filter wheel is also available for use with the spectrophotometer and most of the accessories.
Spectrograph The spectrograph housing material is ceramic to reduce thermal effects to a minimum. It main components of the spectrograph are the lens, the slit, the grating and the photo diode array with front-end electronics. The mean sampling interval of the diode array is about 0.9 nm over the wavelength range 190 nm to 1100 nm. The nominal spectral slit width is 1 nm.
Solvents The choice of solvents should be based primarily on the solvent’s absorbance characteristics over the wavelengths of interest, its suitability as a solvent for the analyte, and on experimental conditions. The Table at the right lists common solvents and the lower limit of their useful wavelength range.When using volatile solvents such as acetone or methylene chloride, make sure that the sample cell is stoppered. Evaporation of a solvent does change the solute concentration and can cause solution noise due to solute convection currents. Both of these will affect the accuracy. Also when using water as solvent we recommend using UV grade or HPLC grade water to reduce unwanted absorbance from impurities in the water.
Diode Array The photodiode array is the heart of the spectrograph. It is a series of 1024 individual photodiodes and control circuits etched onto a semiconductor chip. With a wavelength range from 190 nm to 1100 nm the sampling interval is nominal 0.9 nm. The diode-array detector acquires spectral data at all wavelengths simultaneously. These data are available for the analysis of the standard or sample. Either use data at a single wavelength, or define several wavelengths or wavelength ranges for inclusion in the calculation of an analytical function. The result of the analytical function calculation is the function result. If you select a single wavelength, the function result is equal to the absorbance at that wavelength.
The analytical wavelength is the primary wavelength that selected by the user to analyzing a sample. The analytical wavelength is typically the wavelength at the absorbance maximum, although the full-spectrum acquisition and virtually absolute wavelength reproducibility of the diode-array spectrophotometer allow selection of any wavelength in the absorbance band. If the maximum absorbance is high (for example, greater than 2 Absorbance Units), selecting wavelengths at the side of an absorbance band, where the absorbance is lower, can avoid non-linearity due to stray light. Measurements on the side of absorbance bands are usually also included when measuring over a wavelength range, and in multicomponent analysis, when measurements over a wide spectral range are used.
Confirmation Analysis -- The analysis of pure caffeine, the analytical wavelength is set to 274 nm, the absorbance maximum. The absorbance measurements produce accurate results for caffeine when no impurities are present. If, however, there is a low level of salicylic acid impurity in the caffeine sample, the absorbance value measured at 274 nm is too high for the concentration of caffeine in the sample because of the additional absorbance due to salicylic acid.
Instrument Description The spectrophotometer is easy to use. It has a line power indicator, a status indicator and some push buttons. All electrical connections are made at the rear of the instrument. At the left is the front view, notice that the sample compartment is open. Unlike conventional instruments the Agilent 8453 does not suffer from ambient stray light. The open sample area makes it easier to access for cuvette handling and to connect tubing to a flow cell or thermostattable cell holder. The spectrophotometer has a standard single-cell cell holder. Standard and accessory cell holders can be removed and replaced in seconds with few or no tools. The line power switch is located at the lower-left part of the instrument. Pressing it in turns on the instrument. It stays pressed in and shows a green light when the instrument is turned on. When the line power switch stands out and the green light is off, the instrument is turned off. On the front panel of the spectrophotometer is a status indicator which will display different colors depending of the actual condition of the instrument.
• Green—the instrument is ready to measure. • Green, blinking—the instrument is measuring. • Yellow—the instrument is in not-ready state, for example, turning one of the lamps on or if both lamps are switched off. • Red—error condition, that is, the spectrophotometer does not pass one of the self-tests which are run when the spectrophotometer is turned on or an error occurred during operation. In this case the UV-Visible operating software gives a detailed error message. • Red, blinking—error condition of the spectrophotometer processor system. Because in this case there is no communication with the computer there will be no error message.
Agilent 8453 Self Test 1. Select the Verification and Diagnostics mode. The mode is indicated on the tool bar of your Agilent ChemStation session. 2. Select the Self-Test task in the analysis panel’s selection box.
3 Choose Self-Test, Start from the Task menu or click Start to start the self-test. NOTE: The spectrophotometer must be stable before initiating the test. If the instrument is not properly warmed up a warning message is shown.
4. The self-test results are displayed with pass/fail criteria. Note: Self-test results can be stored with the spectrophotometer. Storing the self-test results with the spectrophotometer allows the performance to be monitor as a function of time. Graphical representations of the self-test histories can be generated.
Computer Description
HP Comuter with with Microsoft Windows XP professional with a 17 inch thin sceen monitor and HP DeskJet color printer. The compact computer has a Pentium III processor (3.4 GHz), 1 GB RAM, 150 GB Hard drive partitioned into sections (C: 20 GB and D: 130 GB for data), CD-ROM drive, a 3.5" floppy drive, and USB access. It has SVGA graphics with an instrument control LAN card. No GPIB cable is required.
