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

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Page 1: Basic Analytical Techniques in Analytical Chemistry Laboratory Abdul Rohman Laboratory of Analytical Chemistry, Department of Pharmaceutical Chemistry,

Basic Analytical Techniques in Analytical Chemistry Laboratory

Abdul RohmanLaboratory of Analytical Chemistry, Department of Pharmaceutical Chemistry, Gadjah Mada University, Indonesia

Page 2: Basic Analytical Techniques in Analytical Chemistry Laboratory Abdul Rohman Laboratory of Analytical Chemistry, Department of Pharmaceutical Chemistry,

Contents

Weighing Procedures

Preparation of standard solution

Basic techniques in spectroscopic and Chromatographic

Method Validation

Page 3: Basic Analytical Techniques in Analytical Chemistry Laboratory Abdul Rohman Laboratory of Analytical Chemistry, Department of Pharmaceutical Chemistry,

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

Page 4: Basic Analytical Techniques in Analytical Chemistry Laboratory Abdul Rohman Laboratory of Analytical Chemistry, Department of Pharmaceutical Chemistry,

4

YOG-08. Gra

v.

TEKNIK MENIMBANGTEKNIK MENIMBANGWeighing by difference:

Wadah + zat = A gWadah + sisa = B gBerat zat = A - B g

Weighing by addition:Wadah ditimbang = C gWadah + zat = D gBerat zat = D - C g

Page 5: Basic Analytical Techniques in Analytical Chemistry Laboratory Abdul Rohman Laboratory of Analytical Chemistry, Department of Pharmaceutical Chemistry,

5

YOG-08. Gra

v.

N E R A C A

Jenis neraca Sensitivitas (mg)

Makro/analitik 0,1

Semimikro 0,01

Mikro 0,001

Page 6: Basic Analytical Techniques in Analytical Chemistry Laboratory Abdul Rohman Laboratory of Analytical Chemistry, Department of Pharmaceutical Chemistry,

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

Page 7: Basic Analytical Techniques in Analytical Chemistry Laboratory Abdul Rohman Laboratory of Analytical Chemistry, Department of Pharmaceutical Chemistry,

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??????

Page 8: Basic Analytical Techniques in Analytical Chemistry Laboratory Abdul Rohman Laboratory of Analytical Chemistry, Department of Pharmaceutical Chemistry,

Preparation of standard solutions

Molarity (M)= moles/liter or mmoles/mLNormality(N) = equivalence/liter or meq/mLFormality(F)= is identical to molarityMolality(m) = moles/1000g solvent

Page 9: Basic Analytical Techniques in Analytical Chemistry Laboratory Abdul Rohman Laboratory of Analytical Chemistry, Department of Pharmaceutical Chemistry,

Expression of Analytical ResultsExpression of Analytical ResultsSo Many WaysSo 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 %

Page 10: Basic Analytical Techniques in Analytical Chemistry Laboratory Abdul Rohman Laboratory of Analytical Chemistry, Department of Pharmaceutical Chemistry,

Density CalculationsDensity CalculationsHow do we convert to MolarityHow do we convert to Molarity

Density = mass solute /unit volumeSpecific Gravity = Dsolute/DH20

DH2O = 1.00000 g/mL @ 4oCDH2O = 0.99821 g/mL @ 20oCDHCl = 1.19 g/mLDH2SO4 = 1.84 g/mL

Page 11: Basic Analytical Techniques in Analytical Chemistry Laboratory Abdul Rohman Laboratory of Analytical Chemistry, Department of Pharmaceutical Chemistry,

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.

Page 12: Basic Analytical Techniques in Analytical Chemistry Laboratory Abdul Rohman Laboratory of Analytical Chemistry, Department of Pharmaceutical Chemistry,

. 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

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, 6th Ed. (Wiley)

Page 13: Basic Analytical Techniques in Analytical Chemistry Laboratory Abdul Rohman Laboratory of Analytical Chemistry, Department of Pharmaceutical Chemistry,

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, 6th Ed. (Wiley)

Page 14: Basic Analytical Techniques in Analytical Chemistry Laboratory Abdul Rohman Laboratory of Analytical Chemistry, Department of Pharmaceutical Chemistry,

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, 6th Ed. (Wiley)

Page 15: Basic Analytical Techniques in Analytical Chemistry Laboratory Abdul Rohman Laboratory of Analytical Chemistry, Department of Pharmaceutical Chemistry,

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, 6th Ed. (Wiley)

Page 16: Basic Analytical Techniques in Analytical Chemistry Laboratory Abdul Rohman Laboratory of Analytical Chemistry, Department of Pharmaceutical Chemistry,

Volumetric Analysis - Principles Standard Volumetric Analysis - Principles Standard Solutions Solutions

“Primary Standard “ – ◦highly purified compound used as a reference

material in titrimetryProperties:High purity Stable in airIndependent of relative humidityReadily availableReasonable solubilityLarge formula weight

Page 17: Basic Analytical Techniques in Analytical Chemistry Laboratory Abdul Rohman Laboratory of Analytical Chemistry, Department of Pharmaceutical Chemistry,

Volumetric Analysis - Volumetric Analysis - PrinciplesPrinciplesExamples of Standard MaterialsExamples of Standard Materials

Primary Potassium Acid Phthalate KHC8H4O4 (FW 204.23) Benzoic Acid C6H5COOH (FW 122.12) Na2CO3 , KH(IO3)2

Arsenious Oxide (As2O3) Sodium Oxalate (Na2C2O4) KI , K2Cr2O7 , Fe(pure)

Secondary NaOH , KOH , Ba(OH)2

HCl , HNO3 , HClO4

Sulfamic Acid (HSO3NH2) KMnO4 , Na2S2O3

Ce(HSO4)4 (FW 632.6)

Page 18: Basic Analytical Techniques in Analytical Chemistry Laboratory Abdul Rohman Laboratory of Analytical Chemistry, Department of Pharmaceutical Chemistry,

Volumetric Analysis-PrinciplesVolumetric Analysis-PrinciplesStandardization – involves establishing the

concentration of a “standard solution”Direct method: dissolve caarefully weighed quantity of primary

standard; dilute to known volumeIndirect methods:Titrate weighed quantity of primary standardTitrate weighed quantity of secondary standardTitrate measured volume of other standard

solution

Page 19: Basic Analytical Techniques in Analytical Chemistry Laboratory Abdul Rohman Laboratory of Analytical Chemistry, Department of Pharmaceutical Chemistry,
Page 20: Basic Analytical Techniques in Analytical Chemistry Laboratory Abdul Rohman Laboratory of Analytical Chemistry, Department of Pharmaceutical Chemistry,

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, 6th Ed. (Wiley)

Page 21: Basic Analytical Techniques in Analytical Chemistry Laboratory Abdul Rohman Laboratory of Analytical Chemistry, Department of Pharmaceutical Chemistry,

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, 6th Ed. (Wiley)

Page 22: Basic Analytical Techniques in Analytical Chemistry Laboratory Abdul Rohman Laboratory of Analytical Chemistry, Department of Pharmaceutical Chemistry,

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, 6th Ed. (Wiley)

Page 23: Basic Analytical Techniques in Analytical Chemistry Laboratory Abdul Rohman Laboratory of Analytical Chemistry, Department of Pharmaceutical Chemistry,

Preparation of standard solution

Making a calibration curves

Measurement of analyte of interest

Quantification of analytes using selected quantification techniques

Page 24: Basic Analytical Techniques in Analytical Chemistry Laboratory Abdul Rohman Laboratory of Analytical Chemistry, Department of Pharmaceutical Chemistry,

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

Page 25: Basic Analytical Techniques in Analytical Chemistry Laboratory Abdul Rohman Laboratory of Analytical Chemistry, Department of Pharmaceutical Chemistry,

Absorbansi

waktu pengukuran

waktu operasional

Operational time in Visible spectroscopy

Page 26: Basic Analytical Techniques in Analytical Chemistry Laboratory Abdul Rohman Laboratory of Analytical Chemistry, Department of Pharmaceutical Chemistry,

Quantification Techniques

External Standard

Internal standard

Internal Normalization

Standard Addition

Page 27: Basic Analytical Techniques in Analytical Chemistry Laboratory Abdul Rohman Laboratory of Analytical Chemistry, Department of Pharmaceutical Chemistry,

Standar Eksternal

x

x

x

x

x

Konsentrasi baku

5

10

15

20

25

1,0 2,0 3,0 4,0 5,0

respon sampel

konsentrasi sampel hasil interpolasi

respon detektor

0,0

Konsentrasi baku etanol (ppm) Luas puncak (y)

0,50441,0091,5132,0182,522

1799436109541217198889984

y = bx + a y = respon detektorx = konsentrasi

Kita bisa menghitung konsentrasi sampel dengan memasukkan luas puncak (y) sampel lalu memasukkannya ke persamaan regresi linier

Page 28: Basic Analytical Techniques in Analytical Chemistry Laboratory Abdul Rohman Laboratory of Analytical Chemistry, Department of Pharmaceutical Chemistry,

Standar internal

Kadar diazinon

(ppm) (x)

Luas puncak diazinon

Luas puncak paration

Luas puncak diazinon/ Luas puncak paration (y)

6,534 11672 8901 1,31129730

9,801 16868 9213 1,83093824

13,068 25930 9418 2,753275216

19,068 36877 8714 4,231929888

26,136 54384 9019 6,029886393

Page 29: Basic Analytical Techniques in Analytical Chemistry Laboratory Abdul Rohman Laboratory of Analytical Chemistry, Department of Pharmaceutical Chemistry,

Normalisasi Internal

Page 30: Basic Analytical Techniques in Analytical Chemistry Laboratory Abdul Rohman Laboratory of Analytical Chemistry, Department of Pharmaceutical Chemistry,

Normalisasi Internal 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.

Page 31: Basic Analytical Techniques in Analytical Chemistry Laboratory Abdul Rohman Laboratory of Analytical Chemistry, Department of Pharmaceutical Chemistry,

Standard Addition

Page 32: Basic Analytical Techniques in Analytical Chemistry Laboratory Abdul Rohman Laboratory of Analytical Chemistry, Department of Pharmaceutical Chemistry,

Method Validation

Precision

Accuracy

Limit of detection

Limit of quantification

Spesificity

System suitability

Linearity

Kisaran (range)

Robutness

METHOD VALIDATION

Page 33: Basic Analytical Techniques in Analytical Chemistry Laboratory Abdul Rohman Laboratory of Analytical Chemistry, Department of Pharmaceutical Chemistry,
Page 34: Basic Analytical Techniques in Analytical Chemistry Laboratory Abdul Rohman Laboratory of Analytical Chemistry, Department of Pharmaceutical Chemistry,

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

Page 35: Basic Analytical Techniques in Analytical Chemistry Laboratory Abdul Rohman Laboratory of Analytical Chemistry, Department of Pharmaceutical Chemistry,
Page 36: Basic Analytical Techniques in Analytical Chemistry Laboratory Abdul Rohman Laboratory of Analytical Chemistry, Department of Pharmaceutical Chemistry,
Page 37: Basic Analytical Techniques in Analytical Chemistry Laboratory Abdul Rohman Laboratory of Analytical Chemistry, Department of Pharmaceutical Chemistry,

YOG-08. HPLC 37

Page 38: Basic Analytical Techniques in Analytical Chemistry Laboratory Abdul Rohman Laboratory of Analytical Chemistry, Department of Pharmaceutical Chemistry,

YOG-08. HPLC 38

Page 39: Basic Analytical Techniques in Analytical Chemistry Laboratory Abdul Rohman Laboratory of Analytical Chemistry, Department of Pharmaceutical Chemistry,