Anfisko I/3

Basic Principles of
Analytical Methods
Quiz
Jelaskan hubungan antara pH dan ionisasi
molekul obat, pH dan koefisien partisi
Untuk tujuan apa sajakah perlu diketahui
sifat sifat fisikakimia obat.
I. Classification of Analytical Methods
Analytical chemistry is 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.
I. Classification of Analytical Methods
A. Classical Methods Commonly 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

I. Classification of Analytical Methods
B. Instrumental Methods - exploit the physical properties of an
analyte to obtain qualitative and
quantitative information.
I. Classification of Analytical Methods
B. Instrumental Methods
1. Separation of Analytes - can be accomplished (dicapai) in
two ways.
a. Physical separation of analytes
i. Chromatography gas or liquid (GC, LC)
ii. Electrophoresis gel or capillary gel (GE, CGE)
b. Spectroscopic separation of analytes
Isolate the signal arising from the analyte by
spectroscopy.
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)

Electrical or
Mechanical
Signal
Signal
Generator
Detector
or
Input Transducer
Signal
Processor
Analytical
Signal
Transduced
Signal
Display Unit
Chart Recorder Digital Readout Computer
Digital Data Analog Data
Meter
II. Instrument Components

E El le ec ct tr ri ic ca al l o or r
M Me ec ch ha an ni ic ca al l
S Si ig gn na al l
Signal
Generator
Detector
or
Input Transducer
Signal
Processor
A An na al ly yt ti ic ca al l
S Si ig gn na al l
T Tr ra an ns sd du uc ce ed d
S Si ig gn na al l
Display Unit
or
Output
Transducer
General Instrument Components
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?
III. Selecting an Analytical Method
B. 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.
III. Selecting an Analytical Method
B. 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 level
4) Cost and availability of equipment (instrumentation)
5) Cost of analysis per sample
1. Precision - measure of the reproducibility of a set of
determinations.
b) Coefficient of variation (CV)
100 =
x
s
CV
Figures of M erit
a) Estimated standard deviation (s)
( )
( )
1

1
2
2
2
or

|
|
.
|

\
|

N
N
x
x
s
N
x x
s
i
i
i
x
i
= individual determination
= mean value of determinations
N = number of determinations


x
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?
0
20
40
60
80
100
0 0.2 0.4 0.6 0.8 1
S
i
g
n
a
l

(
S
)
Concentration (c)
m2
m1
S
bl
3. Sensitivity - the ability to discriminate (membedakan) between
small differences in analyte concentration.
AS
m1

AC
AS
m2

Figures of Merit
S = signal or instrument response
S
bl
= signal from blank sample
c = sample concentration
m = calibration sensitivity
(slope of calibration curve)
a) Calibration sensitivity (m)
**

bl
S mc S + =
** IUPAC Definition
b) Analytical sensitivity ()
S
s
m
=
= analytical sensitivity
m = calibration sensitivity
s
S
= std. dev. in signal measurement

2. Bias - measure of the systematic (ukuran sistematik), or
determinate, error in an analytical analysis. Also
referred to as accuracy.
Figures of Merit
b) Percent bias, or error
100 Error %

x
a) Absolute bias, or error (E
a
)
= x E
a
= mean of a small (sample) set of replicate measurements
= true or accepted value
x
4. Detection Limit - the minimum concentration or mass of analyte
that can be detected by an instrumental method at a known level of
confidence (usually 95% confidence level).
Figures of Merit
a) Minimum detectable signal (S
m
)
bl bl avg m
ks S S + =
,
S
m
= minimum detectable signal
S
avg,bl
= average signal of the blank
s
bl
= standard deviation in the blank signal
k = multiple of variation in the blank signal
The analytical signal must be larger than the blank signal (S
avg,bl
) by some factor (k) of
the standard deviation in the blank (s
bl
). k is usually set to a value of three.
b) Minimum detectable concentration (c
m
)
Limit of Detection (LOD)
m
S S
c
bl avg m
m
,

=
c
m
= minimum detectable concentration
m = slope of the calibration curve
Expressed in terms of s
bl

m
ks
c
bl
m
=
5. Dynamic Range - the range for an analytical method which
extend form the lowest concentration at which a quantitative
measure can be made (LOQ) to the concentration at which
the 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
s
LOQ
bl
10
=
s
bl
= standard deviation in the blank
signal
m = slope of the calibration curve
I
n
s
t
r
u
m
e
n
t

R
e
s
p
o
n
s
e
Concentration
Dynamic Range
c
m
LOL
LOQ
Figures of Merit
Kisaran suatu metode
analitik yang mencakup
konsentrasi terkecil yang
dpt diukur sec kuantitatif
(LOQ) sampai
konsentrasi dimana kurva
kalibrasi meiyimpang
dari linieritas
6. Selectivity - the degree to which an analytical method is
free from interferences from other species
contained in the sample matrix.

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

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

IV. Instrument Calibration Methods
Calibration the process of relating the measured
analytical signal (instrument response) to
the concentration of the analyte.
Suatu proses yang menghubungkan sinyal
analitik (instrumen respons) terhadap
konsentrasi analyte.




Common Methods of Calibration:
A. Preparation of a calibration curve.
B. Standard addition method
C. Internal standard method
A. Calibration Curves
Several standards of known analyte concentrations are
prepared, introduced into the instrument, and instrument
response (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 that
encompasses 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.
Calibration Curve
0.0
0.5
1.0
1.5
2.0
2.5
0 20 40 60 80 100
Concentration
C
o
r
r
e
c
t
e
d

I
R
1. Preparation of a Calibration Curve
Measure instrument response for a set of standards
Plot standard concentration vs. instrument response.
Calibration Curve
0.0
0.5
1.0
1.5
2.0
2.5
0 20 40 60 80 100
Concentration
C
o
r
r
e
c
t
e
d

I
R
Calibration Curve
0.0
0.5
1.0
1.5
2.0
2.5
0 20 40 60 80 100
Concentration
C
o
r
r
e
c
t
e
d

I
R
Concentration Instrument
Response (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

Concentration Instrument
Response (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

Concentration Instrument
Response (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

Use least squares method (linear regression) to
calculate equation for best fit line (y = mx + b).
x
i
y
i
x
i
2
y
i
2
x
i
y
i

Concentration 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
E =
190.210 4.452 12982.820100 7.180334 305.203680
Mean 38.042 0.890


( )
( )
( )
( )
( )( )

intercept) - (Y (Slope)

yy xx
xy
xx xy
i i
i i i i xy
i
i i yy
i
i i xx
S S
S
r
m x - y b S S m
N
y x
y x y y x x S
N
y
y y y S
N
x
x x x S

=
= =

= =

= =

= =
2
2 2
2
2 2
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 = 0.0236x 0.00881

Rearrange:
x = (y b)/m
x = (y + 0.00881)/0.0236

And:
x (sample concentration) = 37.4

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 Spiking the sample.
Method:
1) Prepare several identical aliquots, V
x
, of the unknown sample.
2) Add a variable volume, V
s
, of a standard solution of known
concentration, c
s
, to each unknown aliquot.
3) Dilute each solution to an equal volume, V
t
.
4) Make instrumental measurements of each sample to get an
instrument response, S.
5) Calculate unknown concentration, c
x
, from the following
equation.
B. Method of Standard Additions
Standard Additions Equation
Where:
S = signal or instrument response
k = proportionality constant
V
s
= volume of standard added
c
s
= concentration of the standard
V
x
= volume of the sample aliquot
c
x
= concentration of the sample
V
t
= total volume of diluted solutions
t
x x
t
s s
V
c kV
V
c kV
S + =
B. Method of Standard Additions
A plot of instrument response (S) vs. standard volume (V
s
)
yields a straight line of the form:
V
s
I
n
s
t
r
u
m
e
n
t

R
e
s
p
o
n
s
e

(
S
)
m = Ay/ Ax
b = y-intercept
(V
s
)
0
b mV S
s
+ =
B. Method of Standard Additions
Combine:
b mV S
s
+ =
Calculating concentration of the sample.
and
t
x x
t
s s
V
c kV
V
c kV
S + =
x
s
x
mV
bc
c =
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 standard
were 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 0.276
10.0 20.00 0.320
Calculate the concentration of the sample and its standard deviation.
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 0,239
15
20
3,315
4,42
0,276
0,320
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 (V
s
)
A
b
s
o
r
b
a
n
c
e
m = 0.00818
b = 0.1554
s
m
= 0.000119
s
b
= 0.001463
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 following
equation.
( )
x
s s
x
V S S
V c S
c
1 2
1

=
Where:
S
1
= instrument response sample
S
2
= instrument response sample + spike

Example: Two-point Standard Addition
A 25.0-mL aliquot of an aqueous quinine solution was
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
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 (1.00
cm cell). Calculate the concentration (ppm) of quinine in the
sample.

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 the
sample.
C. Internal Standard Method
Hexane Calibration Curve
y = 31641x + 142659
R
2
= 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)
P
e
a
k

A
r
e
a
C. Internal Standard Method
Hexane/Octane Calibration Curve
y = 0.0359x + 0.0772
R
2
= 0.9998
0
0.5
1
1.5
2
2.5
0 10 20 30 40 50 60
Conc. (ppt hexane)
H
e
a
x
n
e
/
O
c
t
a
n
e

P
e
a
k

A
r
e
a