anfisko i-3 dasar dasar metode analisis
TRANSCRIPT
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