Published on August 4, 2014

Author: parasvirani



CALIBRATION OF UV/VISIBLE SPECTROPHOTOMETER: CALIBRATION OF UV/VISIBLE SPECTROPHOTOMETER PARAS VIRANI DEPARTMENT OF QUALITY ASSURANCE SHREE DHANVANTARY PHARMACY COLLEGE KIM, SURAT Content: Content INTRODUCTION PURPOSE COMPEDIAL TEST PERFORMANCE QUALIFICATION REFERENCE Introduction: Introduction UV–Visible (UV–Vis) spectrophotometer is commonly used in analytical laboratories for qualitative and quantitative analyses. The performance requirements of spectrophotometers vary according to the nature of the tests and the design of the instrument. It is important for accuracy and reproducibility in measurement that this instrument is correctly set up and calibrated. The primary objective of GLP is to ensure the generation of high quality data. Essential to this is the ability to produce estimates for the measurement uncertainty associated with calibrations Purpose: Purpose Compedial calibration: Compedial calibration Performance Tests Standards USP 26. BP 2001 EP 4th Ed. JP (14) IP Wavelength accuracy Deuterium lamp Y Y Y Y Y Mercury vapour lamp Y Y Y Y - Holmium oxide glass filter Y - - Y - Didymium glass filter Y - - Y - Holmium oxide in HClO4 Y Y Y Y Y Stray light Potassium chloride Solution(200 nm) - Y Y - Y Potassium chloride Solution(220 nm) Y - - - - Resolution Toluene in hexane Solution - Y Y - Y Photometric accuracy Potassium dichromate solution Y Y Y Y Y Noise   - - - - - Baseline flatness   - - - - - Stability   - - - - - Linearity   - - - - - Performance qualification: Performance qualification Following eight criteria should be varified with help of standard referance in calibration of uv spectrophotometer . Wavelength Accuracy Stray Light Resolution Noise Baseline Flatness Stability Photometric Accuracy Linearity Reference standards: Reference standards Wavelength Accuracy : Wavelength Accuracy Wavelength accuracy is defined as the deviation of the wavelength reading at an absorption band or emission band from the known wavelength of the band. The wavelength deviation can cause significant errors in the qualitative and quantitative results of the UV–Vis measurement.It is quite obvious that if the spectrophotometer is not able to maintain an accurate wavelength scale, the UV absorption profile of the sample measured by the instrument will be inaccurate. The true λ max and λ min of the analyte cannot be characterized accurately . PowerPoint Presentation: PROTOCOL: Wavelength accuracy verification is checked by measuring a known wave- length reference standard with well-characterized absorption or emission peaks and comparing the recorded wavelength of the peak(s) against the value (s) listed in the certificate of that reference standard. There are many standards that are commonly used to verify the wavelength accuracy of a spectrophotometer. Spectra of some commonly used wavelength standards such as a deuterium lamp, mercury vapour lamp, holmium oxide filter, and holmium oxide solution (4% holmium oxide in 10% perchloric acid in a 1-cm cell ). Acceptance. ±1 nm in the UV range (200 to 380 nm) and ±3 nm in the visible range (380 to 800 nm). Three repeated scans of the same peak should be within ±0.5nm . Stray Light : Stray Light Stray light is defined as the detected light of any wavelength that is outside the bandwidth of the wavelength selected. The causes for stray light are scattering, higher-order diffraction of the monochromator , or poor instrument design. Stray light causes a decrease in absorbance and reduces the linear range of the instrument. High-absorbance measurements are affected more severely by stray light . The stray light problem causes a deviation from linearity at high absorbance PROTOCOL: For the stray light test, various cut-off filters or solutions can be used to estimate the stray light contribution, depending on the wavelengths is used. PowerPoint Presentation: Table provides information on three test solutions( aqueous KCl , NaI , and NaNO2 ) for stray light measurement at 200, 220, and 340 nm. Scan the stray light testing solution in a 1-cm cell using air as the reference. Acceptance . : The absorbance value of 1- cm cell should be greater than 2 Spectral Range (nm) Solution Measurement Wavelength (nm) 175 – 200 Aq. KCl (12 g/L) 200 210 – 260 Aq. NaI (10 g/L) 220 300-385 Aq. NaNO2 (50 g/L) 340 Resolution : Resolution The resolution of a UV-Vis spectrophotometer is related to its spectral bandwidth (SBW). The smaller bandwidth, the finer the resolution. The SBW depends on the slit width and the dispersive power of the monochrometer . Typically, only spectrophotometers designed for high-resolution work have a variable slit width. Spectrophotometers for routine analysis usually have a fixed slit width. The resolution of the absorbance measurement depends on the ratio of the spectral bandwidth (SBW) of the spectrophotometer to the natural bandwidth (NBW) of the spectral band to be measured PowerPoint Presentation: PROTOCOL : The resolution of the absorbance measurement depends on the ratio of the spectral bandwidth (SBW) of the spectrophotometer to the natural bandwidth (NBW) of the spectral band to be measured Acceptance : The ratio of the absorbance at λ max (269 nm) and absorbance at λmin (266 nm) should be greater than 1.5. Noise : Noise Noise in the UV-Vis measurement originates primarily from the light source and electronic components. Noise in the measurement affects the accuracy at both ends of the absorbance scale. Photon noise from the light source affects the accuracy of the measurements at low absorbance. Electronic noise from the electronic components affects the accuracy of the measurements at high absorbance. A high noise level affects the precision of the measurements and reduces the limit of detection, thereby rendering the instrument less sensitive. PowerPoint Presentation: PROTOCOL: Air is scanned in the absorbance mode for 10 min. peak-to-peak noise is recorded at 500 nm. The root mean square (RMS) noise is then calculated. Modern spectrophotometers are usually equipped with the noise estimation function. Acceptance: The RMS noise should typically be less than 0.001 AU Baseline Flatness : Baseline Flatness The intensity of radiation coming from the light source varies over the entire UV-Vis range. Most UV-Vis spectrophotometers have dual light sources. A deuterium lamp is used for the UV range and a tungsten lamp is used for the visible range. The response of the detector also varies over the spectral range. The flat baseline test demonstrates the ability of the instrument to normalize the light intensity measurement and the spectral output at different wavelengths throughout the spectral range . PROTOCOL: Air is scanned in the absorbance mode. The highest and lowest deflections in the absorbance unit are recorded. Acceptance: The deflection is typically less than 0.01 AU. Stability: Stability Variations in lamp intensity and electronic output between the measurements of the reference and the sample result in instrument drift. The lamp intensity is a function of the age of the lamp, temperature fluctuation, and wavelength of the measurement. These changes can lead to errors in the value of the measurements, especially over an extended period of time. The resulting error in the measurement may be positive or negative. The stability test checks the ability of the instrument to maintain a steady state over time so that the effect of the drift on the accuracy of the measurements is insignificant. PowerPoint Presentation: PROTOCOL: Air is scanned in the absorbance mode for 60 min at a specific wavelength (typically, 340 nm). The highest and lowest deflections in the absorbance unit are recorded. Acceptance: The deflection is typically less than 0.002 AU/h . Photometric Accuracy : Photometric Accuracy Photometric accuracy is determined by comparing the difference between the measured absorbance of the reference standard materials and the established standard value. An optically neutral material with little wavelength dependency for its transmittance/absorbance is desirable because it eliminates the spectral bandwidth dependency of measurements. PowerPoint Presentation: PROTOCOL In that 0.006 % w/v solution of potassium dichromate (60.06 mg/L) is prepared in 0.005 M sulphuric acid. A 0.005 M sulphuric acid solution as the reference is scanned and then the potassium dichromate solutions are scanned at 235, 257, 313, and 350 nm. The standard values for the BP/EP potassium dichromate solutions are given in Table Acceptance : Six replicate measurements of the 0.006% w/v potassium dichromate solution at 235, 257, 313, and 350 nm should be less than 0.5% RSD. Linearity : Linearity The linear dynamic range of the measurement is limited by stray light at high absorbance and by noise at low absorbance. For routine measurements involving samples and the related reference chemical standards, the accuracy of the quantification of the sample depends on the precision and linearity of the measurements PROTOCOL: A series of potassium dichromate solutions of concentration 20, 40, 60, 80, and 100 mg/L in 0.005 M sulphuric acid can be used to test the linearity of the system. First, 0.005 M sulphuric acid as reference is scanned and then the potassium dichromate solutions at 235, 257, 313, and 350 nm. PowerPoint Presentation: The absorbance values at various wavelengths are plotted against the concentration of the solutions and the correlation coefficients are calculated. Acceptance: Correlation coefficient r = 0.999 PowerPoint Presentation: Maintenance / repairs When the instrument does not comply with the requirement / tolerance range specified above, the instrument should be labeled “OUT OF CALIBRATION” and should be repaired / serviced. After repair / maintenance, again calibrate the instrument. Referance: Referance INDIAN PHARMACOPOEIA , 2007 www_pharmaguideline_com_2010_05_calibration-of-uv-visible spectroscopy.htm Operation and Calibration of the UV-VIS Spectrophotometer by tiear pharmaceuticals. PowerPoint Presentation: THANKS

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