Anfisko I/3Anfisko I/3

Basic Principles of Basic Principles of Analytical MethodsAnalytical Methods

QuizQuiz

Jelaskan hubungan antara pH dan ionisasi Jelaskan hubungan antara pH dan ionisasi molekul obat, pH dan koefisien partisimolekul obat, pH dan koefisien partisi

Untuk tujuan apa sajakah perlu diketahui Untuk tujuan apa sajakah perlu diketahui sifat sifat fisikakimia obat.sifat sifat fisikakimia obat.

I.I. Classification of Analytical MethodsClassification of Analytical Methods

•• Analytical chemistryAnalytical chemistry is the science of identifying the is the science of identifying the components in a sample (components in a sample (qualitative analysisqualitative analysis) and the ) and the relative amounts of each of the components (relative amounts of each of the components (quantitative quantitative analysisanalysis). Generally a separation step is required to ). Generally a separation step is required to isolate the components in a sample prior to analysis.isolate the components in a sample prior to analysis.

•• The methods used for analysis fall into two general classes: Classical Methods & Instrumental Methods.

I.I. Classification of Analytical MethodsClassification of Analytical Methods

A.A. Classical MethodsClassical Methods – Commonly referred to as “wet chemistry”. – Commonly referred to as “wet chemistry”.

1.1. Separation of AnalytesSeparation of Analytes - -common procedures include common procedures include extraction, distillation, extraction, distillation, precipitation, filtration, etc. precipitation, filtration, etc.

2.2. Quantitative AnalysisQuantitative Analysis - -titration and gravimetric analysis. titration and gravimetric analysis.

3.3. Qualitative AnalysisQualitative Analysis - - BP, BP, MP, color, odor, density, reactivity, MP, color, odor, density, reactivity, refractive index, etc. refractive index, etc.

CH 321CH 321

I.I. Classification of Analytical MethodsClassification of Analytical Methods B.B. Instrumental MethodsInstrumental Methods - - exploit exploit

the physical properties of an analyte to obtain the physical properties of an analyte to obtain qualitative and quantitative information. qualitative and quantitative information.

I.I. Classification of Analytical MethodsClassification of Analytical Methods B.B. Instrumental MethodsInstrumental Methods

1.1. Separation of AnalytesSeparation of Analytes - - can can be accomplishedbe accomplished (dicapai) (dicapai) in two in two ways. ways.

a.a. Physical separation of analytesPhysical separation of analytesi.i. Chromatography – gas or liquid (GC, LC)Chromatography – gas or liquid (GC, LC)ii.ii. Electrophoresis – gel or capillary gel (GE, CGE) Electrophoresis – gel or capillary gel (GE, CGE)

b.b. Spectroscopic separation of analytesSpectroscopic separation of analytes•• Isolate the signal arising from the analyte by Isolate the signal arising from the analyte by spectroscopy. spectroscopy.

I.I. Classification of Analytical MethodsClassification of Analytical Methods..........B.B. Instrumental Instrumental

MethodsMethods..........

2. 2. AnalysisAnalysis Quantitative Quantitative•• Ultraviolet-Visible spectrophotometry (UV-Vis) Ultraviolet-Visible spectrophotometry (UV-Vis) •• Atomic emission and absorption spectroscopy (AES, Atomic emission and absorption spectroscopy (AES, AAS) AAS) •• Conductivity (pH, ISE) Conductivity (pH, ISE)

3. 3. Qualitative AnalysisQualitative Analysis•• X-ray spectrometry X-ray spectrometry •• Infrared spectroscopy (IR) Infrared spectroscopy (IR) •• Mass Spectrometry (MS) Mass Spectrometry (MS) •• Nuclear magnetic resonance (NMR)Nuclear magnetic resonance (NMR)

II.II. Instrument ComponentsInstrument Components General Instrument ComponentsGeneral Instrument Components

III.III. Selecting an Analytical MethodSelecting an Analytical Method A. Defining the ProblemA. Defining the Problem

•• To determine the best method of analysis the analyst To determine the best method of analysis the analyst should ask the following questions.should ask the following questions.

1.1. What accuracy is required?What accuracy is required?

2.2. How much sample is available?How much sample is available?

3.3. What is the concentration range of the sample? What is the concentration range of the sample?

4.4. Are there components in the sample that will cause Are there components in the sample that will cause interferences? interferences?

6.6. How many samples are to be analyzed? How many samples are to be analyzed?

5.5. What are the physical and/or chemical properties of What are the physical and/or chemical properties of the sample matrix? the sample matrix?

III.III. Selecting an Analytical MethodSelecting an Analytical Method B. Performance Characteristics & Figures of MeritB. Performance Characteristics & Figures of Merit

•• Performance CharacteristicsPerformance Characteristics - - criteria used to compare criteria used to compare which of several instrumental methods would be the best for which of several instrumental methods would be the best for a particular analysis.a particular analysis.

•• Figures of MeritFigures of Merit - - quantitative (numerical) measures of quantitative (numerical) measures of performance characteristics.performance characteristics.

III.III. Selecting an Analytical MethodSelecting an Analytical Method B. Performance Characteristics & Figures of MeritB. Performance Characteristics & Figures of Merit

•• Performance CharacteristicsPerformance Characteristics - - criteria used to compare criteria used to compare which of several instrumental methods would be the best for which of several instrumental methods would be the best for a particular analysisa particular analysis ..

•• Other performance characteristics to consider when choosing a method:Other performance characteristics to consider when choosing a method:

1)1) Speed of analysisSpeed of analysis2)2) Ease & convenienceEase & convenience (Kenyamanan) (Kenyamanan)3)3) Operator skill levelOperator skill level4)4) Cost and availability of equipment (instrumentation)Cost and availability of equipment (instrumentation)5)5) Cost of analysis per sampleCost of analysis per sample

1.1. PrecisionPrecision -- measure of the reproducibility of a set measure of the reproducibility of a set of determinations. of determinations.

b) Coefficient of variation (CV)

100x

sCV

Figures of M eritFigures of M erit

a) a) Estimated standard deviation (Estimated standard deviation (ss))

1

1

22

2

or

N

Nxx

sN

xxs

ii

i

xxii = individual determination = individual determination

= mean value of determinations= mean value of determinationsNN = number of determinations = number of determinations  

xx

Example:Example: Four students determine the concentration of chloride Four students determine the concentration of chloride in a tap water sample and obtain the following results:in a tap water sample and obtain the following results:

Conc. of ClConc. of Cl-- (ppm) (ppm)153.6153.6149.2149.2158.5158.5161.1161.1

Calculate the average concentration of chloride in the Calculate the average concentration of chloride in the water sample, the estimated standard deviation in the water sample, the estimated standard deviation in the concentration and the coefficient of variation.concentration and the coefficient of variation.

Is the determined concentration more precise than 256.2 ± 7.8 ppm ClIs the determined concentration more precise than 256.2 ± 7.8 ppm Cl-- determined for another tap water sample using the same method?determined for another tap water sample using the same method?

0

20

40

60

80

100

0 0.2 0.4 0.6 0.8 1

Sign

al (S

)

Concentration (c)

m2

m1

Sbl

3.3. SensitivitySensitivity - - the ability to discriminatethe ability to discriminate (membedakan)(membedakan) between small differences in analyte between small differences in analyte concentration.concentration.

Sm1

C

Sm2

Figures of MeritFigures of MeritSS = signal or instrument response = signal or instrument responseSSblbl = signal from blank sample = signal from blank sample

cc = sample concentration = sample concentrationm m = = calibration sensitivitycalibration sensitivity(slope of calibration curve)(slope of calibration curve)

a)a) Calibration sensitivity (Calibration sensitivity (mm))**** blSmcS

** IUPAC Definition** IUPAC Definition

b)b) Analytical sensitivity (Analytical sensitivity ())

Ss

m

= analytical sensitivity= analytical sensitivitym = calibration sensitivitym = calibration sensitivityssSS = std. dev. in signal measurement = std. dev. in signal measurement

2.2. BiasBias -- measure of the systematicmeasure of the systematic (ukuran sistematik) (ukuran sistematik), , or determinate, error in an analytical analysis. Also or determinate, error in an analytical analysis. Also referred to as referred to as accuracyaccuracy..

Figures of MeritFigures of Merit

b)b) Percent bias, or error Percent bias, or error

100 Error %

x

a)a) Absolute bias, or error (EAbsolute bias, or error (Eaa) )

xEa

= = mean of a small (sample) set of replicate measurementsmean of a small (sample) set of replicate measurements = = true or accepted valuetrue or accepted value

xx

4.4. Detection LimitDetection Limit - - the minimum concentration or mass of the minimum concentration or mass of analyte that can be detected by an instrumental method at a known analyte that can be detected by an instrumental method at a known level of confidence (usually 95% confidence level). level of confidence (usually 95% confidence level).

Figures of MeritFigures of Merit

a)a) Minimum detectable signal (Minimum detectable signal (SSmm) )

blblavgm ksSS ,

SSmm = minimum detectable signal= minimum detectable signal

SSavg,blavg,bl = = average signal of the blankaverage signal of the blank

ssblbl = = standard deviation in the blank signal standard deviation in the blank signal

k k = multiple of variation in the blank signal= multiple of variation in the blank signal

• • The analytical signal must be larger than the blank signal (The analytical signal must be larger than the blank signal (SSavg,blavg,bl) by some factor () by some factor (kk) of ) of

the standard deviation in the blank (the standard deviation in the blank (ssblbl). ). kk is usually set to a value of three.is usually set to a value of three.

b)b) Minimum detectable concentration (Minimum detectable concentration (ccmm) ) • • Limit of Detection (Limit of Detection (LODLOD))

m

SSc blavgm

m,

ccmm = minimum detectable concentration= minimum detectable concentration

mm = slope of the calibration curve = slope of the calibration curve

• • Expressed in terms of Expressed in terms of ssblbl m

ksc bl

m

5.5. Dynamic RangeDynamic Range - - the range for an analytical method the range for an analytical method which extend form the lowest concentration at which a which extend form the lowest concentration at which a quantitative measure can be made (quantitative measure can be made (LOQLOQ) to the ) to the concentration at which the calibration curve departs from concentration at which the calibration curve departs from linearity (linearity (LOLLOL). ).

b)b) Limit of linearity (Limit of linearity (LOLLOL)) – – point where the calibration curve point where the calibration curve

departs from linearity. (somewhat arbitrary) departs from linearity. (somewhat arbitrary)

a)a) Limit of quantitation (Limit of quantitation (LOQLOQ) )

m

sLOQ bl10

ssblbl = standard deviation in the blank = standard deviation in the blank

signalsignalmm = slope of the calibration curve = slope of the calibration curve

Inst

rum

ent R

espo

nse

Concentration

Dynamic Range

cm

LOL

LOQ

Figures of MeritFigures of Merit

Kisaran suatu metode Kisaran suatu metode analitik yang mencakup analitik yang mencakup konsentrasi terkecil yang konsentrasi terkecil yang dpt diukur sec kuantitatif dpt diukur sec kuantitatif (LOQ) sampai (LOQ) sampai konsentrasi dimana kurva konsentrasi dimana kurva kalibrasi meiyimpang kalibrasi meiyimpang dari linieritasdari linieritas

6.6. SelectivitySelectivity - - the degree to which an analytical method is the degree to which an analytical method is free from interferences from other species free from interferences from other species contained in the sample matrix.contained in the sample matrix.

sejauh mana sebuah metode analitis bebas sejauh mana sebuah metode analitis bebas dari gangguan dari spesies lain yang dari gangguan dari spesies lain yang terkandung dalam matriks sampel.terkandung dalam matriks sampel.

  

Selectivity coefficients are not widely use to compare different methods of Selectivity coefficients are not widely use to compare different methods of analysis.analysis.

Koefisien selektivitas tidak secara luas digunakan untuk membandingkan Koefisien selektivitas tidak secara luas digunakan untuk membandingkan berbagai metode analisis.berbagai metode analisis.

Example:Example: A calibration curve is determined for lithium (Li) using A calibration curve is determined for lithium (Li) using flame atomic emission spectroscopy. The slope of the flame atomic emission spectroscopy. The slope of the calibration curve is 1221 emission units per calibration curve is 1221 emission units per concentration unit (concentration unit (g/mL, ppm). Five replicate blank g/mL, ppm). Five replicate blank analyses resulted in the following instrument responses analyses resulted in the following instrument responses for the blank: 54, 61, 57, 60, 57 emission units.for the blank: 54, 61, 57, 60, 57 emission units.

A) What is the calibration sensitivity of the method? A) What is the calibration sensitivity of the method?

B) What is the limit of detection for the method? B) What is the limit of detection for the method?

C) What is the limit of quantitation for the method?  C) What is the limit of quantitation for the method?  

IV.IV. Instrument Calibration Methods Instrument Calibration Methods

Calibration Calibration – – the process of relating the measured the process of relating the measured analytical signal (instrument response) to analytical signal (instrument response) to the concentration of the analyte.the concentration of the analyte.

Suatu Suatu proses yang menghubungkan sinyal proses yang menghubungkan sinyal analitik (instrumen respons) terhadap analitik (instrumen respons) terhadap konsentrasi analyte.konsentrasi analyte.  

Common Methods of Calibration:Common Methods of Calibration:

A.A. Preparation of a calibration curve.Preparation of a calibration curve.

B.B. Standard addition methodStandard addition method

C.C. Internal standard methodInternal standard method

A.A. Calibration CurvesCalibration Curves •• Several Several standardsstandards of known analyte concentrations are of known analyte concentrations are

prepared, introduced into the instrument, and instrument prepared, introduced into the instrument, and instrument

response (absorbance, emission, pH, etc.) is recorded.response (absorbance, emission, pH, etc.) is recorded.

Standard SolutionsStandard Solutions – – Solutions of known analyte concentrations Solutions of known analyte concentrations usually prepared by the experimenter. The usually prepared by the experimenter. The standards are prepared over a concentration that standards are prepared over a concentration that encompasses the expected concentration of the encompasses the expected concentration of the analyte, but not beyond the LOL for the analyte, but not beyond the LOL for the instrument. instrument.

Calibration CurveCalibration Curve – – A plot of standard concentration (x) vs. A plot of standard concentration (x) vs. instrument response (y). Preferably the instrument response (y). Preferably the relationship between standard concentration and relationship between standard concentration and instrument response is linear. instrument response is linear.

1.1. Preparation of a Calibration Curve Preparation of a Calibration Curve •• Measure instrument response for a set of standards Measure instrument response for a set of standards

•• Plot standard concentration vs. instrument response. Plot standard concentration vs. instrument response.

•• Use least squares method (linear regression) to Use least squares method (linear regression) to calculate equation for best fit line (y = mx + b).calculate equation for best fit line (y = mx + b).

intercept)-(Y (Slope)

yyxx

xy

xxxy

iiiiiixy

iiiyy

iiixx

SS

Sr

mx -ybSSm

NyxyxyyxxS

NyyyySN

xxxxS

2

222

22

2.2. Interpretation of Calibration CurvesInterpretation of Calibration Curves •• Use the equation for the line (y = mx + b) to calculate Use the equation for the line (y = mx + b) to calculate

the concentration of the standard.the concentration of the standard.

m = 0.0236 & b = -0.00881m = 0.0236 & b = -0.00881y = 0.0236x – 0.00881y = 0.0236x – 0.00881

Rearrange:Rearrange:x = (y – b)/mx = (y – b)/m

x = (y + 0.00881)/0.0236x = (y + 0.00881)/0.0236

And:And:x (sample concentration) = 37.4x (sample concentration) = 37.4

B.B. Method of Standard AdditionsMethod of Standard Additions•• Used for analytes in a complex matrix where interferences in Used for analytes in a complex matrix where interferences in

the IR for the analyte will occur.the IR for the analyte will occur.

i.e. blood, sediment, human serum, etc.i.e. blood, sediment, human serum, etc.

•• Often referred to as Often referred to as SpikingSpiking the sample. the sample.

•• Method:Method:

1)1) Prepare several identical aliquots, VPrepare several identical aliquots, Vxx, of the unknown sample., of the unknown sample.

2)2) Add a variable volume, VAdd a variable volume, Vss, of a standard solution of known , of a standard solution of known

concentration, cconcentration, css, to each unknown aliquot., to each unknown aliquot.3)3) Dilute each solution to an equal volume, VDilute each solution to an equal volume, Vtt..

4)4) Make instrumental measurements of each sample to get an Make instrumental measurements of each sample to get an instrument response, S.instrument response, S.

5)5) Calculate unknown concentration, cCalculate unknown concentration, cxx, from the following , from the following

equation.equation.

B.B. Method of Standard AdditionsMethod of Standard Additions•• Standard Additions EquationStandard Additions Equation

•• Where:Where:

S =S =signal or instrument responsesignal or instrument response

k =k =proportionality constantproportionality constant

VVss = =volume of standard addedvolume of standard added

ccss = =concentration of the standardconcentration of the standard

VVxx = =volume of the sample aliquotvolume of the sample aliquot

ccxx = =concentration of the sampleconcentration of the sample

VVtt = =total volume of diluted solutionstotal volume of diluted solutions

t

xx

t

ss

V

ckV

V

ckVS

B.B. Method of Standard AdditionsMethod of Standard Additions•• A plot of instrument response (S) vs. standard volume (VA plot of instrument response (S) vs. standard volume (Vss) )

yields a straight line of the form:yields a straight line of the form:

V s

Inst

rum

ent

Res

pon

se (

S)

m = y/ x

b = y-intercept

(V s ) 0

bmVS s

B.B. Method of Standard AdditionsMethod of Standard Additions

•• Combine:Combine:

bmVS s

•• Calculating concentration of the sample.Calculating concentration of the sample.

andandt

xx

t

ss

V

ckV

V

ckVS

x

sx mV

bcc

Example: Standard Additions MethodExample: Standard Additions Method

Arsenic in a biological sample is determined by the method of standard Arsenic in a biological sample is determined by the method of standard additions. 10-mL aliquots of the sample are pipetted into each of five additions. 10-mL aliquots of the sample are pipetted into each of five 100-mL volumetric flasks. Various volumes of a 22.1 ppm As 100-mL volumetric flasks. Various volumes of a 22.1 ppm As standard were added to four of the five flasks and each solution was standard were added to four of the five flasks and each solution was diluted to volume with deionized water. The absorbance of each diluted to volume with deionized water. The absorbance of each solution was determined.solution was determined.

Sample (mL)Sample (mL) Standard (mL)Standard (mL) AbsorbanceAbsorbance

10.010.0 0.000.00 0.1560.156

10.010.0 5.005.00 0.1950.195

10.010.0 10.0010.00 0.2390.239

10.010.0 15.0015.00 0.2760.276

10.010.0 20.0020.00 0.3200.320

Calculate the concentration of the sample and its standard deviation.

Y= 0,03701x + 0,1554Y= 0,03701x + 0,1554R = 0,999R = 0,999

Kadar sampelKadar sampelY=0 maka x = 0,1554/0,03701= 4,19 ppmY=0 maka x = 0,1554/0,03701= 4,19 ppm

Karena sample mengalami pengenceran 10x maka konsentrasi sampel Karena sample mengalami pengenceran 10x maka konsentrasi sampel = 4,19 x 10= 4,19 x 10= 41,9 ppm= 41,9 ppm

Volume Volume yang yang ditambahditambahknkn

KonsentrasiKonsentrasi

(ppm)(ppm)

AbsorbansiAbsorbansi

00 00 0,1560,156

55 1,1051,105 0,1950,195

1010 2,212,21 0,2390,239

1515

2020

3,3153,315

4,424,42

0,2760,276

0,3200,320

Arsenic Standard Addition

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0 5 10 15 20

Volume Standard (Vs)

Abs

orba

nce

m = 0.00818b = 0.1554

sm = 0.000119

sb = 0.001463

•• Using Standard Addition to estimate sample concentrationUsing Standard Addition to estimate sample concentration..

1)1) Make two solutions containing equal aliquots of sample and Make two solutions containing equal aliquots of sample and add standard to one of the solutions. Dilute solutions to add standard to one of the solutions. Dilute solutions to volume. volume.

2)2) Measure instrument response for both solutions.Measure instrument response for both solutions.

3)3) Calculate the concentration of the sample with the following Calculate the concentration of the sample with the following equation.equation.

x

ssx VSS

VcSc

12

1

Where:Where:

SS11 = instrument response sample = instrument response sample

SS22 = instrument response sample + spike = instrument response sample + spike

Example: Two-point Standard AdditionExample: Two-point Standard AdditionA 25.0-mL aliquot of an aqueous quinine solution was A 25.0-mL aliquot of an aqueous quinine solution was diluted to 50.0 mL and determined to have an absorbance of diluted to 50.0 mL and determined to have an absorbance of 0.416 at 348 nm when measured in a 1.00 cm cell. A second 0.416 at 348 nm when measured in a 1.00 cm cell. A second 25 mL aliquot was mixed with 10.0 mL of a solution 25 mL aliquot was mixed with 10.0 mL of a solution containing 23.4 ppm of quinine. After diluting the solution containing 23.4 ppm of quinine. After diluting the solution to 50.0 mL, this solution had an absorbance of 0.610 (1.00 to 50.0 mL, this solution had an absorbance of 0.610 (1.00 cm cell). Calculate the concentration (ppm) of quinine in the cm cell). Calculate the concentration (ppm) of quinine in the sample.sample.

C.C. Internal Standard MethodInternal Standard Method•• Commonly used in quantitative liquid and gas chromotography.Commonly used in quantitative liquid and gas chromotography.

•• A constant volume or mass of a standard compound is added to A constant volume or mass of a standard compound is added to each standard and sample solution.each standard and sample solution.

•• The ratio of analyte to the internal standard is used to plot the The ratio of analyte to the internal standard is used to plot the calibration curve and to determine the concentration of the calibration curve and to determine the concentration of the sample.sample.

C.C. Internal Standard MethodInternal Standard Method

Hexane Calibration Curve

y = 31641x + 142659

R2 = 0.8171

0.00E+00

5.00E+05

1.00E+06

1.50E+06

2.00E+06

2.50E+06

0 10 20 30 40 50 60

Conc. (ppt hexane)

Pea

k A

rea

C.C. Internal Standard MethodInternal Standard Method

Hexane/Octane Calibration Curve

y = 0.0359x + 0.0772

R2 = 0.9998

0

0.5

1

1.5

2

2.5

0 10 20 30 40 50 60

Conc. (ppt hexane)

Hea

xne/

Oct

ane

Pea

k A

rea