1. Basic Analytical Techniques in Analytical Chemistry Laboratory Abdul Rohman Laboratory of Analytical Chemistry, Department of Pharmaceutical Chemistry, Gadjah Mada University, Indonesia

2. Contents Weighing Procedures Preparation of standard solution Basic techniques in spectroscopic and Chromatographic Method Validation

3. Weighing Procedure The accurate results of our research are dependent to how appropriate we use an analytical balance to weigh the standards There are two weighing techniques:  Weighing by difference  Weighing by addition

4. 4 YO G- 08. Gra v. TEKNIK MENIMBANG Weighing by difference: Wadah + zat=A g Wadah + sisa= B g Berat zat= A - B g Weighing by addition: Wadah ditimbang= Cg Wadah + zat= D g Berat zat= D - C g

5. 5 YO G- 08. Gra v. N E R A C A Jenis neracaSensitivitas (mg) Makro/analitik 0,1 Semimikro 0,01 Mikro 0,001

6. Sometimes, we find this term in paper Weigh accurately of approximately 1.0 g samples equivalent to…… What is the meaning of the word accurately and approximately in the above sentence

7. Sensitivity of Analytical balance 0.1 mg The balance sensitivity must be taken into account before performing a weighing How much samples which are allowed to be weighed with a analytical balance having a sensitivity of 0.1 mg????? In the procedure: approximately of 100 mg of samples is accurately weighed Which balance (in term of sensitivity) must be used for weighing??????

8. Preparation of standard solutions Molarity (M)= moles/liter or mmoles/mL Normality(N) = equivalence/liter or meq/mL Formality(F)= is identical to molarity Molality(m) = moles/1000g solvent

9. Expression of Analytical Results So Many Ways %(wt/wt) = (wt analyte/wt sample) %(v/wt) = (volume analyte/wt sample) % (v/v) = (volume/volume) Sometimes we must prepare standard solutions in molarity, normality or percentages How to prepare one litre of sulfuric acid 0.1 N from sulfuric acid 96 % How to prepare sulfuric acid 1 % (b/v) from sulfuric acid 98 %

10. Density Calculations How do we convert to Molarity Density = mass solute /unit volume Specific Gravity = D solute /D H20 D H2O = 1.00000 g/mL @ 4 o C D H2O = 0.99821 g/mL @ 20 o C D HCl = 1.19 g/mL D H2SO4 = 1.84 g/mL

11. The units ppm or ppb are used to express trace concentrations. These are weigh or volume based, rather than mole based. The units ppm or ppb are used to express trace concentrations. These are weigh or volume based, rather than mole based.

12. . Volumetric flask. Volumetric flasks are calibrated to contain an accurate volume. See the inside back cover of the text for tolerances of Class A volumetric glassware ©Gary Christian, Analytical Chemistry, 6 th Ed. (Wiley)

13. Transfer or volumetric pipets. Volumetric pipets accurately deliver a fixed volume. A small volume remains in the tip. Volumetric pipets accurately deliver a fixed volume. A small volume remains in the tip. ©Gary Christian, Analytical Chemistry, 6 th Ed. (Wiley)

14. Fig. 2.10. Measuring pipets. Measuring pipets are straight-bore pipets marked at different volumes. They are less accurate than volumetric pipets. Measuring pipets are straight-bore pipets marked at different volumes. They are less accurate than volumetric pipets. ©Gary Christian, Analytical Chemistry, 6 th Ed. (Wiley)

15. These syringe pipets can reproducibly deliver a selected volume. They come in fixed and variable volumes. The plastic tips are disposable These syringe pipets can reproducibly deliver a selected volume. They come in fixed and variable volumes. The plastic tips are disposable ©Gary Christian, Analytical Chemistry, 6 th Ed. (Wiley)

16. Volumetric Analysis - Principles Standard Solutions “Primary Standard “ – ◦highly purified compound used as a reference material in titrimetry Properties: High purity Stable in air Independent of relative humidity Readily available Reasonable solubility Large formula weight

17. Volumetric Analysis - Principles Examples of Standard Materials Primary Potassium Acid Phthalate KHC 8 H 4 O 4 (FW 204.23) Benzoic Acid C 6 H 5 COOH (FW 122.12) Na 2 CO 3, KH(IO 3 ) 2 Arsenious Oxide (As 2 O 3 ) Sodium Oxalate (Na 2 C 2 O 4 ) KI, K 2 Cr 2 O 7, Fe(pure) Secondary NaOH, KOH, Ba(OH) 2 HCl, HNO 3, HClO 4 Sulfamic Acid (HSO 3 NH 2 ) KMnO 4, Na 2 S 2 O 3 Ce(HSO 4 ) 4 (FW 632.6)

18. Volumetric Analysis-Principles Standardization – involves establishing the concentration of a “standard solution” Direct method: dissolve caarefully weighed quantity of primary standard; dilute to known volume Indirect methods: Titrate weighed quantity of primary standard Titrate weighed quantity of secondary standard Titrate measured volume of other standard solution

20. Typical buret. A 50-mL buret is marked in 0.1 mL increments. You interpolate to 0.01 mL, good to about ±0.02 mL. Two readings are taken for every volume measurement. A 50-mL buret is marked in 0.1 mL increments. You interpolate to 0.01 mL, good to about ±0.02 mL. Two readings are taken for every volume measurement. ©Gary Christian, Analytical Chemistry, 6 th Ed. (Wiley)

21. Meniscus illuminator. Position the black field just below the meniscus. Avoid parallax error by reading at eye level Position the black field just below the meniscus. Avoid parallax error by reading at eye level ©Gary Christian, Analytical Chemistry, 6 th Ed. (Wiley)

22. Proper technique for titration. Place the flask on a white background. Place the buret tip in the neck of the flask while your swirl Place the flask on a white background. Place the buret tip in the neck of the flask while your swirl ©Gary Christian, Analytical Chemistry, 6 th Ed. (Wiley)

23. Preparation of standard solution Making a calibration curves Measurement of analyte of interest Quantification of analytes using selected quantification techniques

24. Spectroscopic Technique The commonly spectroscopic techniques used in Food analysis are: Spectroscopy UV for transparent solution. For example analysis of DNA at 260 and 280 nm Spectroscopy visible for measurement of colouric solution. For example determination of amino acid using Lowry method. The commonly spectroscopic techniques used in Food analysis are: Spectroscopy UV for transparent solution. For example analysis of DNA at 260 and 280 nm Spectroscopy visible for measurement of colouric solution. For example determination of amino acid using Lowry method. The technique for both spectroscopic is different

25. Operational time in Visible spectroscopy

26. Quantification Techniques External Standard Internal standard Internal Normalization Standard Addition

27. Standar Eksternal Konsentrasi baku etanol (ppm)Luas puncak (y) 0,5044 1,009 1,513 2,018 2,522 17994 36109 54121 71988 89984 y = bx + a y = respon detektor x = konsentrasi Kita bisa menghitung konsentrasi sampel dengan memasukkan luas puncak (y) sampel lalu memasukkannya ke persamaan regresi linier

28. Standar internal Kadar diazinon (ppm) (x) Luas puncak diazino n Luas puncak paration Luas puncak diazinon/ Luas puncak paration (y) 6,5341167289011,31129730 9,8011686892131,83093824 13,0682593094182,753275216 19,0683687787144,231929888 26,1365438490196,029886393

29. Normalisasi Internal

30. Ada dua hal yang harus diperhatikan : (i)kita harus yakin bahwa kita telah menghitung semua komponen (ii)kita harus mengasumsikan bahwa kita memperoleh respons detektor yang sama untuk setiap komponen. Untuk mengatasi kesulitan ini, maka diperlukan kalibrasi detektor.

31. Standard Addition

32. Method Validation Precision Accuracy Limit of detection Limit of quantification Spesificity System suitability Linearity Kisaran (range) Robutness METHOD VALIDATION

34. How to determine limit of detection (LOD) and limit of quantification (LOQ) for sensitivity????

37. YOG-08. HPLC37

38. YOG-08. HPLC38