Matched cells

For matched cells (i.e. I constant) the Beer-Lambert Law may be written ... 

A gas-washing bottle (Figure 4.7B) may also be used for trapping. This technique is especially useful in conjunction with infrared analysis. The sample is simply bubbled through the anhydrous solvent as it exits the chromatographic column. The solution is then placed in a liquid sample infrared cell. A matching cell containing only the solvent is placed in the reference beam. An infrared spectrum of the sample may then be recorded. 

For accurate work a pair of matched cells should be used and each should be placed in the instrument so that the incident radiation enters via the same optical face every time. 

There are applications, where best results can be obtained without using a baseline (McClure, 1987). If solvent and sample bands overlap, the solvent bands can be compensated for by placing a matching cell in the reference beam, filled with pure solvent only. Using modem computer assisted instrumentation, this compensation can also be performed mathematically by subtracting the solvent spectrum from that of the dissolved sample. 

Therefore, to observe only the effect caused by the substance of interest, all other factors have to be eliminated. This is normally achieved by the use of two matched cells. By comparison of the energy of the incident and emergent beams from these matched cells, the extent of light absorbed by the sample of interest (or analyte) can be expressed in terms of a quantity called absorbance. ... 

In turbid or cloudy solutions, in which absorbance is affected by scattering of the sample, two absorbance measurements are made one at and the other at a different wavelength where the analyte does not absorb. Then, the difference being proportional to concentration of the analyte, the analyte concentration can be calculated. Difference spectrophotometry is used in the determination of constituents in tablets, complex pharmaceutical preparations, plant extracts, syrups, biological matrices such as blood and serum, injectable oil preparations, and the like. For this technique, in addition to other general requirements such as well-matched cells that are positioned accurately, the solution should be made homogeneous, and the instrumental stray radiation at the wavelength of interest should be extremely small. 

Another almost trivial, but important, deviation from adherence to Beer s law is caused by mismatched ceils. If the cells holding the analyte and blank solutions are not of equal path length and equivalent in optical characteristics, an intercept k will occur in the calibration curve and A F.hc r k will be the actual equation instead of Equation 1.3-1. This error ean be avoided by using either carefully matched cells or a linear regres-.sion procedure to calculate both the slope and intercept of the calibration curve. In most cases linear regression is the best strategy because an intercept can also occur if the blank solution does not totally compensate for interferences. Another way to avoid the... 

Accurate spectrophotometric analysis requires the use of good quality, matched cells. These should be regularly calibrated against one another to detect differ-... 

Turn on the recording spectrophotometer and the UV lamp Allow the mstm-ment to warm up for about 15 minutes. Set the wavelength to 260 nm. During the warm-up period, prepare samples by transferring 1 or 3 mL of Tris buffer into a quartz cuvette and an equal volume of purified a-lactalbumin solution into a matched cell. Place the cuvette with buffer only m the sample beam and adjust the absorbance to zero. Remove the blank cuvette and replace with the cuvette containing a-lactalbumin Read and record the absorbance at 260 nm If the is greater than 1 0, dilute the sample with a known volume of Tris buffer and repeat the reading... 

Usually, the quantities T and Ti are nearly the same because cells are made of materials that will not appreciably absorb or scatter the radiation used. Any slight difference can be minimized by using matched cells, one containing the sample and the other the reagent blank (a solution containing all the components except the compound of interest). If T is set at 100% for the blank, a measurement of T for the sample gives 71. 

The absorbance for just the second cell is A = —log 0.50 = 0.30. The two cells are identical in their absorbance of light. Identical or optically-matched cells are required for accurate quantitative analysis using spectroscopy in many cases. 

---