Langkah Analisis Toksikologi Forensik

Penyiapan Sampel
Analisis:

Uji Penapisan (Screening test)

Uji Pemastian (Confirmation test)

Penetapan kadar (determinasi)

Data Analisis
Interpretasi
Penulisan Laporan
(Bukti Surat / Surat Keterangan / Keterangan Ahli)

PENETAPAN KADAR
Penetapan Kadar = KUANTITATIF
SPEKTROFOTOMETER
KROMATOGRAFI LAPIS TIPISSPEKTROFOTODENSITOMETER
KROMATOGRAFI CAIR KINERJA TINGGI (HPLC)
KROMATOGRAFI GAS
AAS

CALIBRATION METHODS

CALIBRATION METHODS
1. Calibration Curve
Method (External
Standard)
2. Standard Additions
Method
3. Internal Standard
Method

CALIBRATION CURVE
METHOD
1.

2.
3.

Most convenient when a large

number of similar samples are to be
analyzed.
Most common technique.
Facilitates calculation of Figures of
Merit.

CALIBRATION CURVE
PROCEDURE
1.

Prepare a series of standard solutions

(analyte solutions with known
concentrations).

2.

Plot [analyte] vs. Analytical Signal.

3.

Use signal for unknown to find [analyte].

For many analytical techniques, we need to evaluate the response of

the unknown sample against a set of standards (known quantities).

This involves a calibration!

1) Determine the instrumental responses for the

standards.

2) Find the response of the unknown sample.

3) Compare the response of the unknown sample to that from the
standards to determine the concentration of the unknown.

Example 1
I prepared 6 solutions with a known concentration of Cr 6+ and added the
necessary colouring agents. I then used a UV-vis spectrophotometer and
measured the absorbance for each solution at a particular wavelength. The results
are in the table below.
Concentration
/ mg.l-1

Absorbance

Corrected
absorbance

0.002

0.000

0.078

0.076

0.163

0.161

0.297

0.295

0.464

0.462

0.600

0.598

Corrected absorbance = (sample absorbance) (blank absorbance)

Fit best straight line:

0.7
0.6

Abs

0.5
0.4
0.3
0.2

y = 0.075x + 0.003

0.1
0
0

Conc / mg/l

I then measured my sample to have an absorbance of 0.418 and the

blank, 0.003. I can calculate the concentration using my calibration
curve.
y = 0.0750x + 0.0029
Abs = (0.0750 x Conc) + 0.0029
Conc = (Abs 0.0029)/0.0750

For my unknown:
Corrected absorbance = 0.418 0.003 = 0.415
Conc = (0.415 0.0029)/0.0750
Conc = 5.49 mg.l-1

Check on your calibration curve!!

Absorbance = 0.415
Conc = 5.49 mg.l-1
0.7
0.6

Abs

0.5
0.4
0.3
0.2

y = 0.075x + 0.003

0.1
0
0

Conc / mg/l

Not Linear??
2.50

y = 0.0865 x + 0.853

log(Signal)

2.00

1.50

1.00

0.50

0.00
-1.00

-0.50

0.00

0.50

log(Pb concentration)

1.00

1.50

Remember

S = mC + b
log(S) = log (mC + b)
b must be ZERO!!
log(S) = log(m) + log(C)
The original curve did not pass through the origin. We
must subtract the blank signal from each point.

Corrected Data
[Pb]
(ppb)
0.20
0.50
1.50
2.50
3.50
4.50
5.50
18.50

Signal
(mAbs)
1.07
2.83
8.23
14.10
19.49
24.40
30.94
97.59

log[Pb]

log(S)

-0.70
-0.30
0.18
0.40
0.54
0.65
0.74
1.27

0.03
0.45
0.92
1.15
1.29
1.39
1.49
1.99

Linear!
2.50

log(signal)

2.00

y = 0.9965x + 0.7419

1.50

1.00

0.50

0.00
-1.00

-0.50

0.00

0.50

log(Pb concentration)

1.00

1.50

Standard Addition Method

1. Most convenient when a small
number of samples are to be
analyzed.
2. Useful when the analyte is
present in a complicated matrix
and no ideal blank is available.

Standard Addition
Procedure
1. Add one or more increments of a
standard solution to sample aliquots of
the same size. Each mixture is then
diluted to the same volume.
2. Prepare a plot of Analytical Signal
versus:
a) volume of standard solution added, or
b) concentration of analyte added.

Standard Addition
Procedure
3. The x-intercept of the standard addition
plot corresponds to the amount of
analyte that must have been present in
the sample (after accounting for
dilution).
4. The standard addition method assumes:
a) the curve is linear over the concentration
range
b) the y-intercept of a calibration curve would
be 0

Example: Fe in Drinking
Water
Sample
Volume
(mL)
10
10
10
10
10

Standard
Volume
(mL)
Signal (V)
0
5
10
15
20

0.215
0.424
0.685
0.826
0.967

The concentration of
the Fe standard
solution is 11.1 ppm
All solutions are
diluted to a final
volume of 50 mL

Contoh

[Fe] = ?

x-intercept = -6.08 mL
Therefore, 10 mL of sample diluted to 50 mL would give a
signal equivalent to 6.08 mL of standard diluted to 50 mL.
Vsam x [Fe]sam = Vstd x [Fe]std
10.0 mL x [Fe] = 6.08 mL x 11.1 ppm
[Fe] = 6.75 ppm

Internal Standard
Method

A second compound, not the analyte,

added at an appropriate stage in the
assay to correct for systematic errors in
the analysis.
WHY?
1.Most convenient when variations in
analytical sample size, position, or
matrix limit the precision of a
technique.
2.May correct for certain types of

Internal Standard
An internal standard must:
Be completely resolved from the known and unknown
substances in the chromatogram
Elute near to (preferably just after the last) peak(s) of
interest
Have a similar detector response (peak height or
area) to the analyte(s)
Have similar chemical and physical properties to the
analyte(s)
Undergo any derivatization reaction in the same way
as the analyte(s)
Be chemically and physically stable on storage in
solution and during the analysis
Be easily available with adequate purity

Internal Standard
Method
Internal standard method is often used to reduce

the impact of systematic errors such as variations

in injection volume or evaporation of extraction
solvent during the analysis.
A known amount of the internal standard that
behaves similarly to the analyte during the
analysis, but elutes at a different place on the
chromatogram or is otherwise detected
independently of the analyte is added at an
appropriate stage in the analysis.
Subsequently, the detector response of the
analyte relative to the response of the internal
standard is plotted against analyte concentration
when constructing a calibration graph.

Internal Standard
Procedure
1. Prepare a set of standard solutions
for analyte (A) as with the
calibration curve method, but add
a constant amount of a second
species (B) to each solution.
2. Prepare a plot of SA/SB versus [A].

Example: Pb by ICP
Emission
Each Pb solution contains 100
ppm Cu.

Signal
[Pb]
(ppm)

Pb

Cu

20
40
60
80
100

112
243
326
355
558

1347
1527
1383
1135
1440

Pb/Cu
0.083
0.159
0.236
0.313
0.388

No Internal Standard Correction

600

Pb Emission Signal

500

400

300

200

100

0
0

20

40

60

[Pb] (ppm)

80

100

120

Internal Standard Correction

0.450
0.400

Pb Emission Signal

0.350
0.300
0.250
0.200
0.150
0.100
0.050
0.000
0

20

40

60

[Pb] (ppm)

80

100

120

Results for an unknown sample after

adding 100 ppm Cu
Run

Pb

Signal
Cu

1
2
3
4
5

346
297
328
331
324

1426
1229
1366
1371
1356

mean

S/N

325
17.8
18.2

1350
72.7
18.6

Pb/Cu
0.243
0.242
0.240
0.241
0.239
0.241
0.00144
167

TERIMA KASIH