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d3 Poltekes Pertemuan 2

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  • IDENTIFIKASI SENYAWA PENYEBAB KERACUNANPande M. Nova Armita S., S.Farm., M.Si., Apt.

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  • Langkah Analisis Toksikologi Forensik

  • PENETAPAN KADARPenetapan Kadar = KUANTITATIF

    SPEKTROFOTOMETER

    KROMATOGRAFI LAPIS TIPIS-SPEKTROFOTODENSITOMETER

    KROMATOGRAFI CAIR KINERJA TINGGI (HPLC)

    KROMATOGRAFI GAS

    AAS

  • CALIBRATION METHODS

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  • CALIBRATION METHODS1. Calibration Curve Method (External Standard)2. Standard Additions Method3. Internal Standard Method

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  • CALIBRATION CURVE METHODMost convenient when a large number of similar samples are to be analyzed.Most common technique.Facilitates calculation of Figures of Merit.

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  • CALIBRATION CURVE PROCEDUREPrepare a series of standard solutions (analyte solutions with known concentrations).

    Plot [analyte] vs. Analytical Signal.

    Use signal for unknown to find [analyte].

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  • For many analytical techniques, we need to evaluate the response of the unknown sample against a set of standards (known quantities).Determine the instrumental responses for the standards.Find the response of the unknown sample.Compare the response of the unknown sample to that from the standards to determine the concentration of the unknown.This involves a calibration!

  • Corrected absorbance = (sample absorbance) (blank absorbance)Example 1I prepared 6 solutions with a known concentration of Cr6+ 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 Correctedabsorbance00.0020.00010.0780.07620.1630.16140.2970.29560.4640.46280.6000.598

  • Fit best straight line:

    Chart1

    0

    0.076

    0.161

    0.295

    0.462

    0.598

    Conc / mg/l

    Abs

    y = 0.075x + 0.003

    Graph

    Conc/ppmAbs

    00

    10.076

    20.161

    40.295

    60.462

    80.598

    Graph

    0

    0

    0

    0

    0

    0

    Conc / mg/l

    Abs

    Calc

    0

    0

    0

    0

    0

    0

    Conc / mg/l

    Abs

    Sheet3

    xiyixi.yixi2didi2sysm2sc2

    0000-0.00298.41E-06

    10.0760.0761-0.00193.61E-06

    20.1610.32240.00816.56E-05

    40.2951.1816-0.00796.24E-05

    60.4622.772360.00918.28E-05

    80.5984.78464-0.00492.40E-05

    211.5929.1341212.47E-040.00785588959.257E-050.0018668788

    0.0096210.04321

    m=0.07500.075smsc

    c=0.00290.0029

    m=0.0748+-0.009

    c=0.003+-0.04??

    xiyixi.yixi2yi2

    00000.00

    10.0760.07610.00578

    20.1610.32240.0259

    40.2951.18160.0870

    60.4622.772360.213

    80.5984.784640.358

    211.5929.1341210.690

    r=1.000

  • 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 + 0.0029Abs = (0.0750 x Conc) + 0.0029Conc = (Abs 0.0029)/0.0750For my unknown: Corrected absorbance = 0.418 0.003 = 0.415Conc = (0.415 0.0029)/0.0750 Conc = 5.49 mg.l-1Check on your calibration curve!!

  • Absorbance = 0.415Conc = 5.49 mg.l-1

    Chart1

    0

    0.076

    0.161

    0.295

    0.462

    0.598

    Conc / mg/l

    Abs

    y = 0.075x + 0.003

    Graph

    Conc/ppmAbs

    00

    10.076

    20.161

    40.295

    60.462

    80.598

    Graph

    0

    0

    0

    0

    0

    0

    Conc / mg/l

    Abs

    Calc

    0

    0

    0

    0

    0

    0

    Conc / mg/l

    Abs

    Sheet3

    xiyixi.yixi2didi2sysm2sc2

    0000-0.00298.41E-06

    10.0760.0761-0.00193.61E-06

    20.1610.32240.00816.56E-05

    40.2951.1816-0.00796.24E-05

    60.4622.772360.00918.28E-05

    80.5984.78464-0.00492.40E-05

    211.5929.1341212.47E-040.00785588959.257E-050.0018668788

    0.0096210.04321

    m=0.07500.075smsc

    c=0.00290.0029

    m=0.0748+-0.009

    c=0.003+-0.04??

    xiyixi.yixi2yi2

    00000.00

    10.0760.07610.00578

    20.1610.32240.0259

    40.2951.18160.0870

    60.4622.772360.213

    80.5984.784640.358

    211.5929.1341210.690

    r=1.000

  • Not Linear??

  • 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

  • Linear!

  • Standard Addition MethodMost convenient when a small number of samples are to be analyzed.

    Useful when the analyte is present in a complicated matrix and no ideal blank is available.

  • Standard Addition ProcedureAdd one or more increments of a standard solution to sample aliquots of the same size. Each mixture is then diluted to the same volume.

    Prepare a plot of Analytical Signal versus:volume of standard solution added, orconcentration of analyte added.

  • Standard Addition ProcedureThe 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).

    The standard addition method assumes:the curve is linear over the concentration rangethe y-intercept of a calibration curve would be 0

  • Example: Fe in Drinking WaterThe 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 MethodA second compound, not the analyte, added at an appropriate stage in the assay to correct for systematic errors in the analysis.

    WHY?Most convenient when variations in analytical sample size, position, or matrix limit the precision of a technique.May correct for certain types of noise.

  • Internal StandardAn 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 MethodInternal 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 ProcedurePrepare 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.

    Prepare a plot of SA/SB versus [A].

  • Example: Pb by ICP EmissionEach Pb solution contains 100 ppm Cu.

  • No Internal Standard Correction

  • Internal Standard Correction

  • Results for an unknown sample after adding 100 ppm Cu

  • TERIMA KASIH

    **

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