Spectrophotometry Uranine Determination 10-point-calibration for creating an own measurement method with photolab 7600 UV-VIS Uranine Uranine is the sodium salt of the fluoresceine. The pure material is a red-brown crystalline powder. In contrast to fluoresceine uranine has a good water solubility and dissolves in a yellow-green fluorescent dye. Uranine is used as dye for various materials, as a tracer to locate leakages in pipe system or for the investigation of flow paths and flow velocity in natural waterways. Figure 1: Usage of Uranine as a tracer at the entrance of a karst cave.(kit University of Baden-Württemberg and national research center of the Helmholtz-Society). User defined methods Programming an own measurement method gives the user a possibility to create or adapt an individual photometric measurement routine, using this method alike commercial available test kits in measurement and calculation of the result. In other words, setting up a calibration curve. 2015 WTW GmbH. [Monat 201X]
Preparations For photometric measurements a calibration function describes the dependency between the absorbance as the basic indicator and a calculated concentration as the result. The knowledge of this dependency is a requirement for the development of a new method. Usually the calibration function will be determined by a series of measurements of known standard concentrations, for example with a so called 10-point-calibration. The software of the spectrophotomerters photolab 6000 and 7000 series supports user-defined method data programming. Spectrum Selecting the wavelength The spectrum of Uranine shows the highest sensitivity in a range from 450 to 480 nm. Here, lowest concentrations can be detected. This wavelengths can be set for spectrophotometers. Since Filter- or LED meters such as photoflex Series have restricted wavelengths availabilities, here 436 nm has been choosen to be the suitable wavelengths, The slightly lower sensitivity of the rising edge does not affect the measurement quality and allows calibration meters other than a spectrophotometer. Figure 2: Spectrum of Uranine in the range 350 550 nm. Calculation of Concentration Assuming the characteristics curve of the method has a linear behaviour with positive slope, the calculation of concentrations follows the formula: c E E 0 F S concentration measured absorbance zero point of the method (also reagent blank) factor slope Method data There are different options to establish the photometric method data: 1. Direct measurement of the absorbance-/concentrationpair of variates and automatic calculation of the method data by the photometer including mean values for parallel standard samples. 2. Manual entry of the absorbance-/concentrationpair of variates and calculation of the method data by the photometer 3. Manual entry of already known method data in a predefined calculation formula 4. Entry of a known slope of the curve?! This document describes the procedure along point 2 via manual entry. 2
Standard solutions Fixing the measurement range on: 1 10 mg/l Uranine Preparation of 10 standard solution with 1 to 10 mg/l Uranine in ascending order with 1 mg/l steps. Figure 3: line of 10 standard solution in 100 ml graduated flasks. Absorbance measurements of the respective 10 standard solution at 436 nm. Performing a double or triple set of calibration standards increases the accuracy. To program the method, the mean value will be entered. concentration (mg/l) absorbance (A) 1 0,053 2 0,097 3 0,144 4 0,204 5 0,255 6 0,305 7 0,336 8 0,383 9 0,462 10 0,513 Programming of the new method Switch the meter on; waiting the heat up phase for best accuracy From the main menu of the photometer, selecteable with the [HOME] key, choose the [Concentration] measurement mode. Confirm with [START * ENTER] key. 3
Select [New Method] with the [F4] function key. In the [Edit method] display input data for characterization of the method. Essential information of the method is inserted here. Input is possible via the keypad of the photometer or via an external keyboard connected at the USB-A interface. Number Designation Version Wavelength Cell Citation form Unit Resolution Calibration curve Unique numbering in the method list. Next free will be offered automatically, but a manual selection between 1001 and 1100 is possible too. Name of the method within the method list. Version number. Wellenlänge für die Messung der Extinktionswerte für die Konzentrationsberechnung Type of cuvette for the measurement. Labelling of the result. Type of the dimension of the result. Decimal places of the result display. Different ways to calculate or input the method data are possible: - [Measurement of standard soluitons] - [Input of absorbance-/concentration pairs] - [Input a formula] [Measurement of standard solutions] For the calculation of method data required calibration points are measured and stored by the photometer as absorbances-/concentration pairs. [Input of absorbance-/concentration - pairs] Already known absorbance-/concentration pairs are inputtet manually. [Input a formula] Alreasy known coefficients can be insierted in the formula of a predefinied polynomial of maximum 5. grade. 4
Continue with The required absorbance-/concentration pairs will be determined from standard solutions of the concerning parameter. The concentration of the standard solutions are prepared constantly ascending in the desired measurement rang. To achieve a sufficient accuracy a minimum of 3 calibration points are required better 5. To follow standard methods, at least a 10-point-calibration should be performed. For an expected linear behaviour of the characteristics curve the choice of the concentration of the standard solutions can look like this: 3 calibration points: 1 5 10 mg/l 5 calibration points: 1 3 5 8 10 mg/l 10 calibration points: 1 2 3 4 5 6 7 8 9 10 mg/l At this moment information belonging to the AQA are not required. The method can be edited later again. Manually input of absorbance-/concentration pairs of the 10-point-calibration via keypad. Function key [F4] assumes the entered data, in the next display the photometer shows the calculated method properties in a graphical presentation, which can be stored or printed to a PDF. (KOMMENTAR: CHECK) Several adjustments can be set in the last display. Alternatively, considering an optional sample blank value for later measurements, the option [reagent blank value required] can be set to yes. Key F4 complete the programming: 5
After completing the new method can be selected over the method list. Measurement performed: 6