Analytical Issues

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Analytical Issues. The measurement of any physical quantity (e.g., mass, concentration, length) is subject to some uncertainty - i.e. the error in the measurement. In general, the average of several measurements will be considerably more reliable than a single measurement.

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Analytical Issues
• The measurement of any physical quantity (e.g., mass, concentration, length) is subject to some uncertainty - i.e. the error in the measurement.
• In general, the average of several measurements will be considerably more reliable than a single measurement.
• However, the fact that the measured values agree well among themselves is no guarantee of their accuracy.
Analytical Issues (Glossary)

accuracy- a measure of the departure of the measurement from the true value

bias (related to systematic error) - in error by a constant amount (or direction)

Analytical Issues (Glossary)

precision (related to indeterminate errors) - a measure of the reproducibility of a measurement

absolute error - error in the measurement (in the same units as the measurement)

PRECISE BUT

INACCURATE

ACCURATE BUT

IMPRECISE

Precision vs Accuracy
Analytical Issues (Glossary)

relative error - error expressed as a fractional part of the value measured

significant figures - for reporting; use relative error (e.g., for accuracy of 1 in 1000, use four significant figures); however, for computing purposes, more significant figures may be carried to avoid round-off errors.

Measures of Deviation
• range - interval between the greatest and least measurements
• average deviation - sum of deviations (without regard to sign) divided by number of measurements
• standard deviation - square root of the sum of the square of deviations divided by the number of measurements

X  1 = 68.3% X  2 = 95.5%

X  3 = 99.7%

• coefficient of variation:
• std. dev./mean x100 = %
Analytical Issues (Glossary)
• quantitation limit - lowest concentration reliably measured
• detection limit - lowest concentration at which presence of analyte can be reliably confirmed
• recovery rate - amount measured relative to amount added, or known to be present, usually as a percentage
• known addition - used to test “recovery” on an actual sample
Quality Assurance/Control (QA/QC)
• protocol set up to insure accuracy of results within some set limit.
• Document all handling and processes
• Monitor all storage conditions
• Account for the buoyancy of air in all weightings
Weight Correction
• Wapp = apparent weight, uncorrected for buoyancy of the air
• Wvac = weight in a vacuum
• dair = density of air
• dsub = density of the substance
• dwt = density of the measuring weights
Analytical Techniques
• Chromatographic Methods of Analysis
• Analysis of Pesticides in ppb range
• Gas Chromatography (GC)
• High Pressure Liquid Chromatography (HPLC)
• Colorimetric Methodsof Analysis
• Analysis of Nutrients (eg nitrate) in ppm range
• Spectrophotometry
• Flow Injection Analysis (FIA)
• Ion specific electrodes, Immunoassay

Chromatographic Methods of AnalysisHistoryTsweet - 1910 First coined the term chromatography which means color mapping from his separation of plant pigments on magnesium silicate columns Planar chromatography -1930’s to 1950’sMartin and Synge - 1959 Development of gas chromatography Modern liquid chromatography - developed in the early 1970’s with the introduction of efficient columns, high pressure metering pumps and flow through detectors

What is a Chromatograph ???

Pump Injector Column Detector Recorder

Schematic of a Gas Chromatograph

Sawyer and McCarty, 1967

Fig.10.22, p 278

Chromatograph Detectors
• FID: Flame Ionization Detector
• ECD: Electron Capture Detector
• NPD: Nitrogen-Phosphorus Detector
• MS: Mass Spectrometer
• UV: Ultra Violet Light Detector
• Fluorescence Detector
Chromatographic Subsystems
• Pump
• GCHPLC
• Gas Cylinder He, H2 High Pressure Pump
• Column
• Open Capillary Packed Column
• 30 m x 0.25mm 2 m x 4.6 mm
• Detectors
• FID, ECD, NPD, MS UV, Fluoresence, MS
Chromatographic Columns
• “Better Chromatography through Silicon Chemistry”
• Developments pushed by analytical needs
• Environmental Analysis
• Hundreds to thousands of compounds
• Method 524 for Drinking Water 84 compounds
• Method 525 for Drinking Water Pesticides
• 53 Nitrogen containing herbicides
• 36 Organochlorine herbicides and insecticides
• 26 organophosphate insecticides
• Difficult analysis required by federal regulations pushed development of better columns
Column Improvements
• Gas chromatography
• Fused Silica Capillaries
• Consistent, inert, and relatively unbreakable
• Bonded Stationary Phases
• low bleed, solvent resistant, long lasting, rinseable
• HPLC
• Bonded Phases C18, C8, glycol
• Inert uniform silica particles
Nitrogen Containing Herbicides

8-TBA

9-Acetochlor

10-Alachlor

11-Metribuzin

12-Metolachlor

13-Chlorpyrifos

14-Cyanizine

13

1-Propachlor

2-Trifluralin

3-DEA

4-DIA

5-Propazine

6-Atrazine

7-Simazine

5

8

6

1

11

7

10

4

9

12

14

3

2

10

20

Time, minutes

Solid lines represent third order best fit equations

area

peak

Peak area

Peak height

Nanograms of alachlor

Pesticides (herbicides and insecticides)

GLC (gas-liquid chromatography)

• Extract sample (water, solid, biological material) with an organic solvent (e.g., 1500 mL of water with 100 mL of CH2Cl2)
• Possibly concentrate, make derivative, and/or “clean-up,” e.g., concentrate organic solvent from 100 mL to 2 mL)
• Inject into a GLC (0.5 to 10 µL)
• Compare peak height (or area) and retention time with standard(s)

5. Compute concentration

from graph p.h. of 1.5 = 0.30 ng

(i.e. 3 x 10-9g)

amount in extracted sample =

0.30 ng x 2 mL/2.5 µL =

0.24 µg

conc. = 0.24 µg/1500 mL

= 0.16 µg/L

= 0.16 ppb

Legend

Pesticide Conjugated with Enzyme

Pesticide

Chromogen/Substrate

Colored Product

Pesticide Immunoassay

Magnetic Particle with Antibody Attached

Pesticide Immunoassay

1).Immunological Reaction

The pesticide-containing sample is incubated with enzyme conjugate

(same pesticide conjugated to enzyme horseradish peroxidase)

and magnetic particles with attached antibody that is specific for

this pesticide. Both pesticide and pesticide conjugate compete for the same antibody sites on magnetic particles.

magnet

Pesticide Immunoassay

2). Separation

The magnetic field is applied to hold the magnetic particles (with pesticide and pesticide conjugate attached in proportion to their original concentration) in the tube and to allow excess reagents to be decanted.

Pesticide Immunoassay

3). Color Development

The presence of labeled pesticide is detected by adding enzyme substrate, hydrogen peroxide, and chromogen, 3.3’, 5.5’- Tetra Methylbenzidine, and measuring the colored product at 450 nm. Since labeled pesticide (enzyme conjugate) was in competition with unlabeled pesticide (sample), the color developed is inversely proportional to the concentration of pesticide in the sample.

Pesticide Immunoassay
• Only works with certain organic compounds including some pesticides
• Not very accurate; used more for screening than for quantitative analysis.
• Only works over a narrow range of concentrations.
• Reacts with families of compounds rather than a specific compound
Photoelectric Colorimeter

Sawyer and McCarty, 1967

Fig.4.8, p 77

Colorimeter Schematic

Sawyer and McCarty, 1967

Fig.4.7, p 76

COLORIMETRIC METHODS Nutrients (nitrogen and phosphorus)

Soil or biological samples

1. Extract (or digest) with water or solutions

2. Measure absorbance

3. Compute concentration from calibration constant or curve

COLORIMETRIC METHODS Nutrients (nitrogen and phosphorus)

Beer’s law: A = a b c

where

A = absorbance

= log (incoming light/outgoing light),

a = extinction coefficient (cm2/µg),

A unique value for each compound

b = path length (cm),

c = concentration (mg/1000 cm3)

COLORIMETRIC METHODS Nutrients (nitrogen and phosphorus)

Example:

A = 0.301

a = 0.462 cm2/mg

b = 1cm

A = abc = 0.301 = 0.462 x 1 x c

c = 0.301/0.462 = 0.65 ppm

• Relatively easy to use
• Results will be influenced by impurities in the solution
Ion-Selective ElectrodesNernst Equation
• E = Electrode Potential (volts)
• n = valence (equivalents/mole)
• R = the gas constant
• T = The absolute Temperature (K)
• [A] = activity of the selected ion
Ion-Selective ElectrodesExample Analytes
• pH (most common use)
• Dissolved Oxygen
• Ammonia
• Fluoride
• Nitrate
• Potassium
• Chloride
• Relatively inexpensive per sample analyzed
• Relatively simple to use
• Not influenced by impurities in the solution
• Relatively easy to make in-situ field measurements
• Need a separate probe for each analyte
Stable Isotope Nitrogen Tracing

Atmospheric N2

14N 99.6337 atom %;

15N 0.3663 atom %;

13N 0 atom %; (Th= 603sec)

Typical Material Costs:

15N depleted – 99.99% 14N

0.01% 15N NH4NO3 1 kg (365 g N) \$230

15N enriched – 95.0% 14N

5.0% 15N NH4NO3 1 kg (365 g N) \$2,500

200 kg 14N equivalent/ha ~ \$1,400,000/ha

Labeled N-tracingAnalytical Procedure
• Conversion of sample N to NH4-N by means of acid digestion
• Oxidation of NH3 to N2

2NH3+3NaOBr = 3NaBr + 3H2O+N2

• Determination of isotope composition

(+-0.001atom% of15N)

mass spectroscopy 14N2+,14N15N+,15N2+

(automated total N, 15N analysis less than \$10/sample)

Labeled N-tracingCalculations
• As=15N atom% in the sample
• Am=15N atom% in applied material
• ref = atom% non-labeled N in system
Labeled N-tracingExample Calculations
• Given: 200 kg/ha applied nitrogen fertilizer containing 5 atom% 15N
• 0.367 atom% non-labeled N in system
• 5 cm runoff depth drain sample; Cw = 24.0 mg/L N; As = 3.64 atom% 15N
• Find: Percent of fertilizer lost in this runoff event.
Labeled N-tracingExample Calculations

Loss from fertilizer

Drainage water loss

Percent of applied fertilizer lost