Analysis Pharmacy Method

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Anfisko I/3

Basic Principles ofAnalytical Methods


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Quiz

Jelaskan hubungan antara pH dan ionisasi

molekul obat, pH dan koefisien partisi

Untuk tujuan apa sajakah perlu diketahuisifat sifat fisikakimia obat.


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I. Classification of Analytical Methods

Analytical chemistryis the science of identifying the

components in a sample (qualitative analysis) and the

relative amounts of each of the components (quantitative

analysis). Generally a separation step is required to

isolate the components in a sample prior to analysis.

The methods used for analysis fall into two general

classes: Classical Methods& Instrumental Methods.


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A. Classical MethodsCommonly referred to as wet chemistry.

1. Separation of Analytes- common procedures include

extraction, distillation,precipitation, filtration, etc.

2. Quantitative Analysis- titration and gravimetric analysis.

3. Qualitative Analysis- BP, MP, color, odor, density,

reactivity, refractive index, etc.

CH 321


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B. Instrumental Methods- exploit the physical properties of ananalyte to obtain qualitative andquantitative information.


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I. Classification of Analytical MethodsB. Instrumental Methods

1. Separation of Analytes- can be accomplished (dicapai) in

two ways.

a. Physical separation of analytesi. Chromatographygas or liquid (GC, LC)

ii.Electrophoresisgel or capillary gel (GE, CGE)

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

spectroscopy.


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I. Classification of Analytical Methods.....

B. Instrumental Methods.....

2. Analysis Quantitative

Ultraviolet-Visible spectrophotometry (UV-Vis)

Atomic emission and absorption spectroscopy (AES, AAS)

Conductivity (pH, ISE)

3. Qualitative Analysis

X-ray spectrometry

Infrared spectroscopy (IR)

Mass Spectrometry (MS)

Nuclear magnetic resonance (NMR)


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Electrical or

Mechanical

Signal

Signal

Generator

Detector

or

Input Transducer

Signal

Processor

Analytical

SignalTransduced

Signal

Display Unit

Chart RecorderDigital ReadoutComputer

Digital Data Analog Data

Meter

II. Instrument Components

EElleeccttrriiccaalloorr

MMeecchhaanniiccaall

SSiiggnnaall

Signal

Generator

Detector

or

Input Transducer

Signal

Processor

AAnnaallyyttiiccaall

SSiiggnnaallTTrraannssdduucceedd

SSiiggnnaall

Display Unit

or

Output

Transducer

General Instrument Components


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III. Selecting an Analytical Method

A. Defining the Problem To determine the best method of analysis the analyst

should ask the following questions.

1.What accuracy is required?

2.How much sample is available?

3.What is the concentration range of the sample?

4.Are there components in the sample that will cause

interferences?

6.How many samples are to be analyzed?

5.What are the physical and/or chemical properties of

the sample matrix?


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III. Selecting an Analytical MethodB. Performance Characteristics & Figures of Merit

Performance Characteristics- criteria used to compare

which of several instrumental methods would be the best for

a particular analysis.

Figures of Merit- quantitative (numerical) measures of

performance characteristics.


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III. Selecting an Analytical MethodB. Performance Characteristics & Figures of Merit

Performance Characteristics- criteria used to compare

which of several instrumental methods would be the best for

a particular analysis .

Other performance characteristics to consider when choosing a method:

1) Speed of analysis

2) Ease & convenience (Kenyamanan)

3) Operator skill level4) Cost and availability of equipment (instrumentation)

5) Cost of analysis per sample


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1. Precision-measure of the reproducibility of a set ofdeterminations.

b) Coefficient of variation(CV)

100x

sCV

Figures of Merit

a) Estimated standard deviation (s)

1

1

22

2

or

NN

xx

sN

xxs

ii

i

xi= individual determination

= mean value of determinations

N= number of determinationsx


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Example:Four students determine the concentration of chloride

in a tap water sample and obtain the following results:

Conc. of Cl-(ppm)

153.6

149.2

158.5

161.1

Calculate the average concentration of chloride in the

water sample, the estimated standard deviation in the

concentration and the coefficient of variation.

Is the determined concentration more precise than 256.2 7.8 ppm Cl-

determined for another tap water sample using the same method?


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0

20

40

60

80

100

0 0.2 0.4 0.6 0.8 1

S

ignal(S)

Concentration (c)

m2

m1

Sbl

3. Sensitivity- the ability to discriminate (membedakan) betweensmall differences in analyte concentration.

DSm1

DC

DSm2

Figures of Merit

S= signal or instrument response

Sbl= signal from blank sample

c= sample concentration

m = calibration sensitivity

(slope of calibration curve)

a) Calibration sensitivity (m)** blSmcS

** IUPAC Definition

b) Analytical sensitivity ()Ss

mg

g= analytical sensitivity

m = calibration sensitivity

sS= std. dev. in signal measurement


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2. Bias-measure of the systematic (ukuran sistematik), ordeterminate, error in an analytical analysis. Alsoreferred to as accuracy.

Figures of Merit

b) Percent bias, or error

100Error% x

a) Absolute bias, or error (Ea)

xEa

= mean of a small (sample) set of replicate measurements

= true or accepted valuex


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4. Detection Limit- the minimum concentration or mass of analytethat can be detected by an instrumental method at a known level ofconfidence (usually 95% confidence level).

Figures of Merit

a) Minimum detectable signal (Sm

)

blblavgm ksSS ,

Sm= minimum detectable signal

Savg,bl

= average signal of the blank

sbl

=standard deviation in the blank signal

k = multiple of variation in the blank signal

The analytical signal must be larger than the blank signal (Savg,bl

) bysome factor (k) of the standard deviation in the blank (s

bl). kis

usually set to a value of three.

b) Minimum detectable concentration (cm

)

Limit of Detection (LOD)mSSc blavgmm

,

cm= minimum detectable concentration

m= slope of the calibration curve

Expressed in terms of sbl

m

ksc blm


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5. Dynamic Range-the range for an analytical method whichextend form the lowest concentration at which a quantitativemeasure can be made (LOQ) to the concentration at whichthe calibration curve departs from linearity (LOL).

b) Limit of linearity (LOL )point where the calibration curve departs

from linearity. (somewhat arbitrary)

a) Limit of quantitation (LOQ)

m

sLOQ bl

10

sbl= standard deviation in the blank

signal

m= slope of the calibration curve

InstrumentResponse

Concentration

Dynamic Range

cm

LOL

LOQ

Figures of Merit

Kisaran suatu metodeanalitik yang mencakupkonsentrasi terkecil yangdpt diukur sec kuantitatif

(LOQ) sampaikonsentrasi dimana kurvakalibrasi meiyimpangdari linieritas


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6. Selectivity- the degree to which an analytical method isfree from interferences from other species

contained in the sample matrix.

sejauh mana sebuah metode analitis bebasdari gangguan dari spesies lain yangterkandung dalam matriks sampel.

Selectivity coefficients are not widely use to compare different methods of

analysis.

Koefisien selektivitas tidak secara luas digunakan untuk membandingkan

berbagai metode analisis.


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Example: A calibration curve is determined for lithium (Li) usingflame atomic emission spectroscopy. The slope of the

calibration curve is 1221 emission units perconcentration unit (g/mL, ppm). Five replicate blankanalyses resulted in the following instrument responsesfor the blank: 54, 61, 57, 60, 57 emission units.

A) What is the calibration sensitivity of the method?

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

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


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IV. Instrument Calibration Methods

Calibration the process of relating the measured

analytical signal (instrument response) to

the concentration of the analyte.

Suatuproses yang menghubungkan sinyal

analitik (instrumen respons) terhadapkonsentrasi analyte.

Common Methods of Calibration:

A. Preparation of a calibration curve.

B. Standard addition method

C. Internal standard method


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A. Calibration Curves

Several standards of known analyte concentrations are

prepared, introduced into the instrument, and instrumentresponse (absorbance, emission, pH, etc.) is recorded.

Standard Solutions Solutions of known analyte concentrations

usually prepared by the experimenter. The

standards are prepared over a concentration thatencompasses the expected concentration of the

analyte, but not beyond the LOL for the

instrument.

Calibration Curve A plot of standard concentration (x) vs.instrument response (y). Preferably the

relationship between standard concentration and

instrument response is linear.


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0.0

0.5

1.0

1.5

2.0

2.5

0 20 40 60 80 100

Concentration

CorrectedIR

1. Preparation of a Calibration Curve

Measure instrument response for a set of standards

Plot standard concentration vs. instrument response.

0.0

0.5

1.0

1.5

2.0

2.5

0 20 40 60 80 100

Concentration

CorrectedIR

0.0

0.5

1.0

1.5

2.0

2.5

0 20 40 60 80 100

Concentration

CorrectedIR

Concentration InstrumentResponse (IR)

Corrected IR

0.000 0.013 0.000

6.010 0.101 0.088

31.800 0.811 0.798

63.500 1.498 1.485

88.900 2.094 2.081

Blank 0.049

Sample 0.924 0.875


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Use least squares method (linear regression) to

calculate equation for best fit line (y = mx + b).

xi yi xi

2

yi

2

xiyiConcentration IR Corr. IR

0.000 0.013 0.000 0.000000 0.000000 0.000000

6.010 0.101 0.088 36.120100 0.007744 0.528880

31.800 0.811 0.798 1011.240000 0.636804 25.376400

63.500 1.498 1.485 4032.250000 2.205225 94.297500

88.900 2.094 2.081 7903.210000 4.330561 185.000900

190.210 4.452 12982.820100 7.180334 305.203680Mean 38.042 0.890

intercept)-(Y(Slope)

yyxx

xy

xxxy

iiiiiixy

iiiyy

iiixx

SS

Sr

mx-ybSSm

N

yx

yxyyxxS

Ny

yyySN

xxxxS

222

222


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

the concentration of the standard.

m = 0.0236 & b = -0.00881

y = 0.0236x0.00881

Rearrange:

x = (yb)/m

x = (y + 0.00881)/0.0236

And:x (sample concentration) = 37.4


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B. Method of Standard Additions

Used for analytes in a complex matrix where interferences in

the IR for the analyte will occur.i.e. blood, sediment, human serum, etc.

Often referred to as Spikingthe sample.

Method:

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

2) Add a variable volume, Vs, of a standard solution of known

concentration, cs, to each unknown aliquot.

3) Dilute each solution to an equal volume, Vt.

4) Make instrumental measurements of each sample to get an

instrument response, S.

5) Calculate unknown concentration, cx, from the following

equation.


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Standard Additions Equation

Where:

S = signal or instrument response

k = proportionality constant

Vs= volume of standard added

cs= concentration of the standardVx= volume of the sample aliquot

cx= concentration of the sample

Vt= total volume of diluted solutions

t

xx

t

ss

V

ckV

V

ckVS


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A plot of instrument response (S) vs. standard volume (Vs)

yields a straight line of the form:

V s

In

strumentRespons

e(S)

m = Dy/Dx

b = y-intercept

(Vs) 0

bmVS s


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Combine:

bmVS s

Calculating concentration of the sample.

andt

xx

t

ss

V

ckV

V

ckVS

x

sx

mV

bcc


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Example: Standard Additions Method

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

100-mL volumetric flasks. Various volumes of a 22.1 ppm As standardwere added to four of the five flasks and each solution was diluted to

volume with deionized water. The absorbance of each solution was

determined.

Sample (mL) Standard (mL) Absorbance

10.0 0.00 0.156

10.0 5.00 0.195

10.0 10.00 0.239

10.0 15.00 0.27610.0 20.00 0.320

Calculate the concentration of the sample and its standard deviation.

Volume Konsentrasi Absorbansi


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Y= 0,03701x + 0,1554

R = 0,999

Kadar sampel

Y=0 maka x = 0,1554/0,03701= 4,19 ppm

Karena sample mengalami pengenceran 10x maka konsentrasi sampel

= 4,19 x 10

= 41,9 ppm

Volume

yang

ditambah

kn

Konsentrasi

(ppm)

Absorbansi

0 0 0,156

5 1,105 0,195

10 2,21 0,23915

20

3,315

4,42

0,276

0,320


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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)

Absorba

nce

m = 0.00818

b = 0.1554

sm = 0.000119

sb = 0.001463


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Using Standard Addition to estimate sample concentration.

1) Make two solutions containing equal aliquots of sample and

add standard to one of the solutions. Dilute solutions to

volume.

2) Measure instrument response for both solutions.

3) Calculate the concentration of the sample with the followingequation.

xss

xVSS

VcSc

12

1

Where:

S1= instrument response sample

S2= instrument response sample + spike


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Example: Two-point Standard Addition

A 25.0-mL aliquot of an aqueous quinine solution wasdiluted 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

25 mL aliquot was mixed with 10.0 mL of a solution

containing 23.4 ppm of quinine. After diluting the solution

to 50.0 mL, this solution had an absorbance of 0.610 (1.00cm cell). Calculate the concentration (ppm) of quinine in the

sample.


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C. Internal Standard Method

Commonly used in quantitative liquid and gas chromotography.

A constant volume or mass of a standard compound is added to

each standard and sample solution.

The ratio of analyte to the internal standard is used to plot the

calibration curve and to determine the concentration of thesample.


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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)

PeakArea


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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)

Heaxne/OctanePeak

Area

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