Diode Array UV Visible Spectrophotometer

Overview

This software is used to acquire data from an 8452A HP diode-array spectrophotometer. It is likely that it can be readily adapted to other HP diode array spectrophotometers. The software includes extensive analytical capabilities including both single wavelength and spectral analysis. The analytical wavelength for single wavelength analysis and the wavelength range for spectral analysis are software selected as post processing steps. Although the spectrophotometer can be used to collect data at a single wavelength this feature is not used. Instead the entire spectra is always measured to facilitate further analysis.

The software communicates with the spectrophotometer over the GPIB. The software is also fully functional for post processing even when not connected to a spectrophotometer.

The software creates two types of files. Standards files contain spectra and concentration information for standards and Sample files contain spectra and descriptions for samples. Both file types can only be opened with the Spectrophotometer software.   Both standards and samples files also contain information about whether the measured absorbance at each wavelength is less than the maximum measurable absorbance.

Both file types can be exported as tab-delimited text for further spreadsheet analysis.

Start up

Open the Spectrophotometer program. If the Spectrophotometer window is open but it does not respond to your commands it is open but not running. Within LabVIEW the arrow button (or command R) can be used to run the program.

  1. If none of the indicator lights are lit on the top back right corner of the spectrophotometer the instrument is turned off. Turn the spectrophotometer on with the rocker switch that is located on the back panel at the bottom of the left side. Locate the switch by feel and push the top of the switch in.

  2. If the instrument is running and the lamp is out, then you will need to turn the lamp on using the Virtual Instrument control palette. The lamp turning on procedure takes more than a minute during which time a checking lamp display will appear on the computer screen. The lamp button will change to bright yellow when the lamp is lit.

Analysis

Analysis can either be done using a sipper cell or sample cuvettes. If the sipper cell is used a rinse step can be included between samples to minimize sample carryover.

  1. The first sample needs to be a reference sample. This may be distilled water or a reagent blank. Select "Measure Reference" from the Spectrophotometer control palette. Reference measurements are also used to determine the effective dynamic range of the instrument. The dynamic range is a function of the lamp intensity and the absorbance of the reference sample. The maximum  measurable absorbance (MMA) is a function of wavelength. All subsequent absorbance measurements that exceed the MMA are flagged and are not used for analysis. Although flagged values aren't used for analysis they are displayed on the graphs.

  2. Select "Measure Standards". Enter the concentration of the standards that you will be using. The software will prompt you for the different standards. (It is also possible to load previously saved standards.)

  3. Select "Measure Samples". Enter the sample description information. The software will prompt you for the different samples. (It is also possible to load previously saved samples.)

  4. After both samples and standards are analyzed or loaded from disk it is possible to determinethe concentrations of the samples. The top graph is the standards, the middle graph is the samples and the bottom graph is the calibration curve. The top graph (standards) is the master. The standards graph cursor is used to choose the analytical wavelength. The wavelength can also be entered in the digital control at the right of the standards graph.

Maximum Absorbance Calculation

The maximum absorbance is obtained by taking 

0.1*log( light intensity at fixed gain - the dark current at fixed gain)

where the light intensity at fixed gain is measured with the reference sample in place. This criteria eliminates some potentially useable data, however, it is more appropriate to use wavelengths with lower absorbance.

Shut down

Select "Quit" from the Spectrophotometer application control palette. If the Spectrophotometer will not be used for over 5 hours turn off the lamp when prompted. This will extend the lamp life and save energy. If the Spectrophotometer will not be used for several days you may turn it off using the rocker switch on the back panel.

Spectral Analysis

Compounds that absorb ultraviolet and/or visible light have characteristic absorbance curves as a function of wavelength. Absorbance of different wavelengths of light occurs as the molecules move to higher energy states.

From Beer’s law, the absorbance is proportional to the concentration of the species.

                         A(g) = b e(g) c                               (1)

where A(g) is the absorbance at some wavelength g and e(g) is the extinction coefficient at some wavelength g, b is the path length of the cell and c is the concentration. Absorbance is dimensionless and e is the absorbance per unit concentration per unit path length. We can rewrite this as

                          [A] = b [e] c                                (2)

where [A] is the absorbance vector and [e] is the vector of extinction coefficients. Each element in the vectors corresponds to a particular wavelength of light.

For a given excitation process, a molecule absorbs only one discrete amount of energy, and thus absorbs radiation of only one wavelength. If all molecules of a compound were in exactly the same state then a plot of the extinction vector would have very narrow absorption lines. However, molecules have different vibrational and rotational states with each state at slightly different energy levels. Thus the base state is variable and the amount of energy required for a transition to a higher energy state will be a function of the base state. Thus, an ensemble of molecules absorbs radiation at slightly different wavelengths as the individual molecules move from their various base states to higher energy states and thus the result is a broad absorption band.

Mixtures

If several species are present, the absorbance is simply the addition of the absorbances from the species.

   (3)

The result of the linear addition of two compounds can be seen in Figure 1.

Figure 1. Mixture of methylene blue and nitrate.

The diode array spectrophotometer (HP 8452A) measures the absorbance in the range 190 to 820 nm. Each diode covers a wavelength range of 2 nm. Thus there is a bank of 316 diodes and the vectors [A] and [e] have 316 elements.

The ability to resolve a mixture into the component species is a function of the shape of the extinction vector and the relative concentrations of the species in the mixture. Extinction vectors tend to have peaks that are many nm wide and spectra from different compounds may have peaks that are only slightly different. When the extinction vectors of species are similar, the ability to resolve the species is poor.

Spectral analysis can be used to measure a species over many orders of magnitude. Beer's Law, however, isn't obeyed perfectly as interactions between molecules become significant at higher concentrations. This nonlinearity produces a complication for spectral analysis. The potential for interspecies interactions exists and it is not possible to know the actual value of the extinction coefficient matrix. In practice the nonlinearities are not expected to be very significant. Although the interspecies interactions cannot be resolved, the effect of concentration of individual species on their extinction coefficient matrices can be resolved.

Solution Scheme

In matrix notation equation 3 can be written as

                         [A] = b{e}[c]                             (4)

where {e} is a m x n matrix of extinction coefficients for measurements at m wavelengths for n compounds and [c] is the n element concentration vector. Equation 4 can be used to resolve a mixture of a few components by solving for the concentration vector.

As a first step the extinction coefficient is determined at each wavelength based on linear regression of the available standards. It is assumed that the intercept of the linear regression is small and only the slope of the regression line is used. Standards that exceed the maximum absorbance criteria at a particular wavelength are not used for the regression analysis at that wavelength. The advantage of using some standards at very high concentrations is that the extinction coefficient far from the peak absorbance wavelengths can be determined more accurately. The linear-regression based extinction coefficient array is assembled for each available standard.

A least squares linear fit algorithm is used to obtain a first estimate of the concentration vector. The algorithm seeks to minimize the difference between the measured absorbance vector, [A], and b{e}[c].   After the first estimate of the concentration vector a second set of extinction coefficient arrays is assembled based on linear interpolation between the two adjacent standards. If the concentration is less than the lowest concentration standard or greater than the highest concentration standard then the closest standard is used to obtain the extinction coefficient matrix. The least squares linear fit algorithm is used again to obtain a refined concentration vector.

Limitations to Spectral Analysis

Number of Components

It is mathematically possible to separate m components of a mixture given measurements at m wavelengths and given extinction coefficient vectors for m compounds. In practice this is not feasible because the extinction vectors, [e], from different species are not sufficiently distinct to allow such high resolution.

Negative Concentrations

It is mathematically possible for a solution to equation 11 to include negative concentrations. At present, the remedy is to remove the compound’s extinction coefficient vector from the extinction coefficient matrix, {e}.

Maximum Absorbance Values

High absorbance values are suspect because they indicate that most of the incident light has been absorbed by the sample and the remaining transmitted light intensity is very weak. In practice, absorbance values greater than 2.5 are of limited value. This is illustrated in Figure 2 where a 10-fold increase in methylene blue concentration did not result in a 10-fold increase in absorbance because the maximum measurable absorbance is approximately 2.5.

Figure 2.   Effect of maximum measurable absorbance on absorbance spectra.

The spectral analysis software does not use absorbance values that exceed a criteria based on diode signal strength measured when the reference sample is analyzed. This makes it possible for the spectral analysis software to measure species over many orders of magnitude by automatically eliminating wavelengths that do not transmit sufficient light to be used for analysis.

Spectral Analysis Software

Spectral analysis is available as a subroutine in the Spectrophotometer program. Spectral analysis is post-processing analysis that is done after measuring the absorbance of the relevant standards and samples. Each of the components of the mixture must be analyzed as a “standard” and the resulting absorbance spectra saved to disk. Ideally, a broad range of concentrations should be analyzed for each “standard.”

Spectral analysis requires the operator to select the relevant standards and samples. The concentrations of each of the standards that would result in the observed absorbance are then calculated.

Continuous Sampling

The sipper cell can be used to continuously monitor a time-varying process. The spectral analysis capabilities can be used in real-time to measure the concentrations of multiple components. By combining these two features it is possible to continuously monitor multiple time-varying components. The Continuous Sampling option requires the selection of a set of standards as well as sample rate and the analytical wavelength range. Continuous sampling processes the spectra and logs (saves to disk in real time)   the concentrations of the various components. The actual spectra are not saved when using continuous sampling.

References

Dyer, J. R. Applications of Absorption Spectroscopy of Organic Compounds. Prentice-Hall, Inc. 1965