Application of UV/Vis Spetrophotometry Methode:

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1 Application of UV/Vis Spetrophotometry Methode:
SMK Negeri 13 Bandung

2 UV-Vis area Near UV area : 200-400 nm Vacuum UV area : 100-200 nm
Visible area : nm

3 UV/Vis usage Purity and identity test in farmacopeia monography
Content determination (main use): (1) One-point method: external standard, standar addition (2) Multi-point method, calibration curve: external standard, standar addition (3) Single substance or multi component sample As detector: HPLC, CE

4 Purity and identity test
Measurement of UV/Vis spectrum of a substance refer to reference spectrum or literature Maximum wavelength Determination of absorptivity at maximum wavelength

5 Determination of content (Quantitative analysis)
Relative method needs reference standard Preparation of standard solution (Lambert-Beer‘s law, about 10 ug/ml) For one-point method only needed 1 standard solution, the concentration should be near the sample concentration For multi-point method (calibration curve ): differential dilution, calibration curve concentration vs absorbance For multi-point method (standard addition): constant sample, differential standard addition, linear regretion of standard concentration added vs absorbance

6 Solvent Absorption below (nm) water

7 One Point Method Cs = ( As / Ab) x Cb Cs = Sample concentration
As = Sample Absorbance Ab = Standard Absorbance Cb = Standard concentration

8 Multi Point Method: External Calibration
Sample Absorbansi c

9 Multi Point Method: Standard Addition
Analit concentration in sample solution

10 UV Spectrum

11 Lambert-Beer‘s Law A = Absorbance e(l) = molar absorptivity
C = concentration [ mole / l] c = concentration[g/ 100 ml] b = Cell length (cuvette length [cm]) A1%, 1 cm = Absorptivity jenis e(l) is absorbance (A) of a solution (C= 1 mol/ l) whwn cell length is 1 cm (b= 1) and the wave length is l.

12 Lambert-Beer‘s validity area
0,2 - 0,8 absorbance unit (A) 1 mg/ 100 ml equivalent to 0,2 - 0,8 absorbance unit (A) A > 0,8 : interference A < 0,2 : worse precision and accuracy Smallest fotometric error when A = 0,434

13 Experiment and calculation
Prepare 2 standard solution of substance 1 and 2 Measure the absorbance of substance 1at 1 and 2, count the a1(1) and a1(2) Measure the absorbance of substance at 1 and 2, tcount the a2(1) and a2(2) Measure the absorbance of sample solution (mixture of substance1 and 2) at 1 (A1) and 2 (A2) This equation is valid : A1 = a1(1) c1 + a2(1) c2 A2 = a1(2) c1 + a2(2) c2 c1 and c2 could be determined

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15 Influence of pH to spectrum

16 Spectrum of Methyl and Propilparaben
Methyl Paraben Propil Paraben

17 CALIBRATION CURVE OF METHYL PARABEN
Concentration of methyl paraben (µg/mL) Absorbance (A) at 256 nm 0,6 0,8 1,0 1,2 1,4 1,6 1,8 2,0 0,2841 0,3061 0,3196 0,3785 0,4216 0,4607 0,4987 0,5504

18 Calibration curve of Methyl paraben at 256 nm
Regretion equation: y = 0,1960 x + 0,1476; koefisien korelasi (r) = 0,99; batas deteksi (BD) = 0,3306 mg/mL; batas kuantisasi (BK) = 1,1020 mg/mL dan koefisien variasi fungsi regresi (V xo) = 0,85%

19 Kalibrasi Spektrofotometer
Perlu dilakukan untuk mencegah kesalahan pembacaan panjang gelombang dan absorban Kalibrasi skala panjang gelombang: larutan holmium dioksid dalam asam perklorat. Perbedaan penunjukkan skala panjang gelombang pada alat dengan panjang gelombang seharusnya digunakan untuk mengoreksi pembacaan alat. Kalibrasi skala fotometrik: larutan kalium bikromat dalam asam sulfat. Berhubungan dengan intensitas sumber radiasi (life time sumber radiasi)

20 Kalibrasi Skala Fotometrik
Kontrolle: Messung von A bzw A(1%, 1 cm). im Bereich von 200 bis 400 nm mit schwefelsaurer K2Cr2O7-Lösung. Für den Bereich von 400 bis 800 nm gibt das DAB keine Kontrollsubstanz an. Geeignet wären z.B. Nickelsulfat, Cobaltsulfat, Kaliumpermanganat.

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