Qualitative anlaysis of an analyte may be deduced by comparing the absorption spectrum of the unknown substance with the spectra

of known substances. Quantitative analysis of an analyte can be determined by measuring the absorbance at one or more wavelengths and using the Beer-Lambert law and the molar absorption coefficient to calculate its amount concentration. Analysis of a mixture consisting of two absorbing analytes is possible in accord with the Beer-Lambert Law, by measuring the absorbance at two different suitable wavelengths. For each wavelength, the total absorbance is given by = + = cA + cB , where a is the molar absorption coefficient of analyte at the wavelength of analysis, b is the absorption path length, and c is the concentration of the analayte. The precision of each method is limited by the signal-to-noise ratio of the UV-Vis , given in absolute error terms as and in relative error terms as or .

Random errors affect and limit the precision of any analysis, especially significant for very small absrbances. To obtain better absorption absorption and decrease by expanding the scale of analysis, several techniques may be employed, such as high precision absorbance method, where the 100%T on the instrument is adjusted with a solution of the analyte of interest and not with the pure solvent. The high precision absorbance method technique will be utilized to measure a set of analyte solutions of similar concentrations analysed with respect to a reference solution standard of similar composition.

Q2) Determine the absolute error in concentration (equation 5) as a function of transmittance (equation2) for the two sets of data obtained, using the K value on the print out.

Sample calculation of absolute error in concentration, c: Conventional Absorbance Method High Absorbance Method

Tabulated absorbance and concentration values for the six standards ranging from 0.200mM to 3.000mM obtained under ordinary conventional absorbance method.

Unknown K2Cr2O7 Concentration (mM) 1.4094 Absorbance 0.8425

Diluted Unknown Solution (B)

Tabulated absorbance and concentration values for the diluted unknown K2Cr2O7

Tabulated absorbance and concentration values for the six standards ranging from 0.200mM to 3.000mM obtained under high precision absorbance method.

Conventional Absorbance Method Measured Standard Concentration No. (mM) 0 0 1 0.200 2 0.400 3 1.000 4 1.400 5 2.000 6 3.000 Slope (K) Intercept (B) R factor (R)

Absorbance 0.093 0.184 0.456 0.627 0.922 1.339 1.348 2.0171 -0.2900 0.9141

dC -11.661 -7.2678 -5.486 -5.915 -7.934 -14.270 -14.472

Question 3) Determine, using equation 7 or 8, tabulate and plot the relative percentage error of the concentration for the two sets of data. Indicate from these plots the optimum percentage Transmittance. for the Schimadzu UV-240 is constant and is rated as T. Sample calculation for relative concentration per error in %T: Conventional Absorbance Method High Absorbance Method

Conventional Absorbance Method High precision Absorbance Method Measured Standard Concentration No. (mM) 1 1.000 2 1.400 3 2.000 4 3.000 Slope (K) Measured Standard Concentration Intercept No. (mM) (B) 0 0 R factor 1 0.200 (R)

Absorbance 0.178 0.419 0.936 0.936 2.0136 Absorbance 0.6060 0.093 0.7781 0.184

dC -7.39678 -5.4733 -8.05725 -8.05725

0.867 0.718

2 3 4 5 6

0.400 1.000 1.400 2.000 3.000

0.456 0.627 0.922 1.339 1.348

0.677 0.766 1.07 1.98 2.0

The best precision absorbance of about 0.456 occurs when the relative concentration error for conventional absorbance method is minimum, 0.677%. ,at 0.400mM.
Relative concentration uncertainty, % 2.5 2 1.5 1 0.5 0 0 0.2 0.4 0.6 0.8 Absorbance 1 1.2 1.4 1.6

Relative Concentration Uncertainty for Conventional Absorbance Method

Relative concentration uncertainty, %, arising from various categories of instrumental noise.

High Precision Absorbance Method Measured Standard Concentration No. (mM) 1 1.000 2 1.400 3 2.000 4 3.000

Absorbance 0.178 0.419 0.936 0.936 1.1 0.815 1.2 1.6

The relative concentration error for high precision absorbance method is minimum 1.400mM, 0.419absorbance, 0.815%.

1.8 Relative concentration uncertainty, % 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 0

Relative Concentration Uncertainty for High Precision Absorbance Method

0.2

0.4

0.6 Absorbance

0.8

1.2

Question 4) Explain the differences in the HIGH ABSORBANCE method error curve relative to the ordinary colorimetric error curve. For a 0.002 M Cr2O72- solution, what would be the difference in precision associated with the two methods? The high absorbance method did not require a large number of standard solutions to calibrate over a wide range of absorption. The wide range required for analysis d in ordinary conventional method was compensated by better linearity in the calibration curve. The high absorbance method matched the samples absorption with the absorption of a standard reference solution of similar composition, 1.000mM. As a result, the errors arising from incorrect adjustments of the instrument controls are inferred to be smaller, since the calibration curve is tied to that standard reference solution. The advantage in high precision method of measuring only a small difference between the concentrations of solutions should have suggested that errors caused by temperature fluctuations are minimized, since the densities of the samples analyzed vary in a similar manner and the light beam traverses absorbants of similar concentration. For the 0.002mM Cr2O72- , the difference in precision between the ordinary absorbance method and the high absorbance method was 1.98 - 1.2=0.78%. Question 5) Report the molarity of your original unknown Cr2O72- solution (A), using the appropriate number of significant figures. Cr2O72- in solution A Question 6) Do the cuvettes in this experiment need to be optically matched? Why or why not? Would switching the cuvettes during the experiment affect the results? Optical matching of cells is a procedure where the concentration of a known analyte may be determined by diluting the sample with solvent until the absorbance matches the absorbance of a known

concentration concentration of analyte in the referene cell, giving similar absorbance readings. It was not required in this case since all the measurements were made from a single cuvette, which was rinsed between samples and the analysis was conducted in order of increasing concentration. The differences in path length between two cuvettes would not lead to appreciable differences in absorbance measurements as long as one cuvette is dedicated for the reference and the other for the sample. However, if the two cuvettes are interchanged during use, the differences in absorbance readings will cause noticeable discrepancies in absorbance readings. Part II. Question 8) Examine the Cr2O72- spectra and select to be the wavelength of the absorption peak near 440 nm. Examine the Mn2+ spectra and select to be the wavelength of the absorption peak near 545nm. Concentration Cr2O72- spectra MnO4- spectra (mM) Absorbance at Absorbance at 0.400 1.000 2.000 Unknown 0.250 0.810 2.000 1.895 0.795 1.198 1.592 1.198 and

Tabulate the absorbances of all the standards and unknown solutions at both

Question 9) Plot graphs of absorbance vs. concentration of the Cr2O72- and Mn2+ standards at Determine and report the values for the molar absorptivities for Cr2O72- and Mn2+ standards

and

1.8 1.6 1.4 Absorbance 1.2 1 0.8 0.6 0.4 0.2 0 0

Absorbance vs. Concentration of Dichromate

0.5

1.5

2.5

Concentration, mM

The molar absorptivity of the Cr2O72- corresponds to the slope, m, fro mthe equation of the linear regression, 1.1036.
1.8 1.6 1.4 Absorbance 1.2 1 0.8 0.6 0.4 0.2 0 0 0.5 1 1.5 2 2.5 Concentration, mM y = 0.4875x + 0.6425 R = 0.9777

Absorbance vs. Concentration for Mn2+

The molar absorptivity of the Mn2+ corresponds to the slope, m, from the equation of the linear regression, 0.4875. Question10) Determine the concentration of Cr2O72- and Mn2+ in unknown mixture (D) by using the ata to solve equations (9) and equations (10). Discuss the sources of error in this determination.

A highly concentrated abalyte in the analyticl sample can be determined with better precision by replacing the blank (reference) cell by one containing a solution of the analyte of known concentration.