ANALYTICAL METHODS

Prepared by: ALVIN A. ALDEA, RMT, MSc©

NOTE: This handout is for EXCLUSIVE USE of University of Saint Louis’ BS Medical Technology II
Students AY 2019-2020. Unauthorized reproduction is prohibited.

Electromagnetic Energy
´ Radiant energy from short wavelength gamma rays to long wavelength radio waves. They
are photon of energy traveling in a wavelike manner:
Wavelength = Electromagnetic energy
´ Energy is transmitted via electromagnetic waves and is characterized by its frequency and
wavelength
´ The relationship between Energy and Wavelength is described by PLANCK’S FORMULA:
E=hv
h: a constant 6.62 x 10-27 erg sec
v: frequency
Wavelength
´ Distance between 2 successive peaks and it is expressed in terms of nanometer

Frequency
´ The number of vibration of wave motion per second
´ The lower the wave frequency the longer the wavelength
1
𝑊∝
𝐹 𝑎𝑛𝑑 𝐸

Light Spectra
1. Visible: 400 to 700 nm
2. Invisible:
A. UV region: <400nm
B. IR region: >700nm
B. IR region: >700 nm

Colorimetry
´ Two considerations:
1. Quality of the color
2. Intensity of the color
´ Kinds of Colorimetry

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 ANALYTICAL METHODS
Prepared by: ALVIN A. ALDEA, RMT, MSc©

NOTE: This handout is for EXCLUSIVE USE of University of Saint Louis’ BS Medical Technology II
Students AY 2019-2020. Unauthorized reproduction is prohibited.

1. Visual Colorimetry
-uses the eye in determining end point
2. Photoelectric Colorimetry
A. Photometric/Filter Photometry
-measurement of light intensity WITHOUT consideration of wavelength
B. Spectrophotometric Measurement
E.g. Spectrophotometer, AAS, FEP

Spectrophotometry
´ Involves measurement of the light transmitted by a solution to determine the
concentration of light absorbing substances in the solution
´ Parts:
1. Light Source:
-provides radiant energy
A. Continuum source
-emits radiation that changes in intensity
B. Line Source
-emits radiation (limited) and wavelength
-an intense beam of light is directed though monochromator and the
sample
-factors for choosing line source: Range, spectral distribution, source,
stability, and temperature
-Types of LIght Source:
A. Tungsten Iodine Lamp
-provides WL from 340-700 nm
-in visible (15%) and near IR (85%)
-used in moderately diluted sample
B. Deuterium Discharge Lamp
-UV down to 165 nm
C. Merst Glower
-an electrically heated rod of rare earth
-IR region
D. Globar
-uses Silicon carbide
-IR region
E. Mercury Vapor Lamp/Arc
-UV and Visible region
F. Hallow Cathode Lamp
-Light source of AAS
2. Monochromator
-isolates specific or individual wavelength
-Monochromatic light: Light radiation of single WL
-Types:
A. Prisms
-wedge shaped pieces of glass, quartz or NaCl
-a narrow light focused on a prism is refracted as it enters the
more dense glass

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 ANALYTICAL METHODS
Prepared by: ALVIN A. ALDEA, RMT, MSc©

NOTE: This handout is for EXCLUSIVE USE of University of Saint Louis’ BS Medical Technology II
Students AY 2019-2020. Unauthorized reproduction is prohibited.

-can be rotated allowing only the desired WL to pass through an

exit slit
B. Diffraction Gratings
-most commonly used; better resolution than prism
-based on the principle that WL are bent as they pass a sharp
corner
-made by cutting grooves or slits into a surface of flat piece of
crown glass
C. Filters
-simple, least expensive but useful
-made by placing semi-transparent silver films on both sides of
dielectric such as Magnesium chloride
-produce monochromatic light based on principle of
CONSTRUCTIVE INTERFERENCE of waves enter one side of
the filter and are reflected at the second surface
-Advantage of gratings over prisms
1. Production of linear spectrum: constant band pass which is
simple
2. Used in the region of spectrum where light energy is
absorbed by glass prisms
-Bandpass: 10-20 nm
3. Entrance Slit
-minimizes unwanted stray light and prevents the entrance of scattered
light into the monochromator
***Stray Light: refers to any WL outside the band transmitted by the
monochromator
-causes absorbance errors
-deleted by cut-off filters
-most common cause of stray light: scratches on optical
surface and dust particles
4. Exit Slit
-controls the width of light beam (band pass)
-allows only narrow fraction of the spectrum to reach the sample cuvet
***Band pass: the range of WL between points at which transmittance is
half peak transmittance
5. Cuvet/Analytical Cell/Sample Cell
-holds the solution whose concentration is to be determined
-Types:
A. Borocate Glass Cuvet
-for solution that do not etch glass
-for strong alkaline solution
B. Quartz/Plastic
-does not absorb UV radiation at WL below 320 nm
-used for measurement of solution requiring UV spectum
C. Alumina Silica Glass
-good for 340 nm and above (Visible)
-most commonly used

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 ANALYTICAL METHODS
Prepared by: ALVIN A. ALDEA, RMT, MSc©

NOTE: This handout is for EXCLUSIVE USE of University of Saint Louis’ BS Medical Technology II
Students AY 2019-2020. Unauthorized reproduction is prohibited.

D. Soft Glass
-used for strong acidic solution
-cuvets with scratches scatter light so it must be discarded
6. Detector/Photodetector
-detects and converts transmitted light into photoelectric energy
-detects amount of light that passes through the sample in the cuvet
-Types:
A. Barrier Layer Cell/Photocell/Photovoltaic cell
-simplest detector, least expensive, temperature sensitive
-composition: Selenium on a plate of iron covered with
transparent layer of silver
-requires no external voltage source but utilizes internal electron
transfer for current production—low internal resistance
B. Phototube/Photoemissive tube
-contains cathode and anode enclosed in a glass case
-it has a photosensitive material that gives off electron when light
energy strikes it
-requires external voltage
C. Photomultiplier tube
-most common type
-measures visible and UV regions
-excellent sensitivity and rapid response
-detects low level of light and amplifies radiant energy

4. Photodiode
-not as sensitive as PM
-excellent linearity

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 ANALYTICAL METHODS
Prepared by: ALVIN A. ALDEA, RMT, MSc©

NOTE: This handout is for EXCLUSIVE USE of University of Saint Louis’ BS Medical Technology II
Students AY 2019-2020. Unauthorized reproduction is prohibited.

-measures light at a multitude of WL

7. Meter or Read-out device

-galvanometer or ammeter
-displays output of the detection system
´ Principle: Beer’s Law/Beer-Lambert/s Law
-states that
𝐶 ∝ 𝐴
or
,
𝐶 ∝ -./ 0
-Mathematically establishes the relationship between concentration and
absorbance
-%T: ratio of the radiant energy transmitted (I) divided by radiant energy
incident on the sample (Io)
4
%𝑇 = 𝑥 100
45
-In commercial spectrophotometer, it is measured as:
𝑠𝑎𝑚𝑝𝑙𝑒 𝑏𝑒𝑎𝑚 𝑠𝑖𝑔𝑛𝑎𝑙
%𝑇 = 𝑥 100
𝑏𝑙𝑎𝑛𝑘 𝑏𝑒𝑎𝑚 𝑠𝑖𝑔𝑛𝑎𝑙
-The amount of light absorbed at a particular wavelength depends on:
Molecular type
Ion type
pH
Temperature
-Absorbance is mathematically derived from %T:
4
1. 𝐴 = − log E45F
2. 𝐴 = log(100%) − log %𝑇
3. 𝐴 = 2 − log %𝑇
-Absorbance is also known as OPTICAL DENSITY
-Formula in Beer’s Law:
𝐴 = 𝑎𝑏𝑐
a: molecular absorptivity

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 ANALYTICAL METHODS
Prepared by: ALVIN A. ALDEA, RMT, MSc©

NOTE: This handout is for EXCLUSIVE USE of University of Saint Louis’ BS Medical Technology II
Students AY 2019-2020. Unauthorized reproduction is prohibited.

b: length of light through the solution

c: concentration of absorbing molecules/solution
´ Double Beam Spectrophotometry
-Instrument that splits the monochromator light into two components
-One passes through the sample and other on the blank
-Additional beam corrects for variation in light source intensity
-Types:
1. Double-beam in space
-uses 2 photodetectors; one for sample and one for reference beam

2. Double-beam in time
-uses one photodetector and alternately passes monochromator light
through the sample and the reference cuvet

´ Quality Control of Spectrophotometer

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 ANALYTICAL METHODS
Prepared by: ALVIN A. ALDEA, RMT, MSc©

NOTE: This handout is for EXCLUSIVE USE of University of Saint Louis’ BS Medical Technology II
Students AY 2019-2020. Unauthorized reproduction is prohibited.

• Wavelength Accuracy: the WL indicated in the dial is the actual WL of light passed
by the monochromator
• WL Accuracy checkers:
Didynium (600nm)
Holmium oxide filter (360nm)

Flame Emission Photometry

´ Measures the light emitted by a single atom burned in flame
´ Principle: Excitation of lower to higher energy state
´ Parts:
A. Light source: Flame (also serve as cuvet)
-the purpose of flame in FEP:
1. Breaks the chemical bonds to produce atoms
2. Source of energy by the atoms to enter an excited state
B. Gases: mixture of H and O gas (acetylene propane, or natural gas)
C. Atomizer or Burner: breaks up the solution into finer droplets so that atom will
absorb heat and get excited
D.. Monochromator: Interference filters
Na filter- Yellow @ 589nm
K filter- Purple @ 767nm
Li filter- Red @ 761nm
E. Detector: Photocell

´ Method: Indirect Internal Standard Method

´ Use: measurement of excited ions (Na and K)
´ Internal standard: Lithium
-Purpose: to achieve stability
-Li also acts as radiation buffer
-Lithium is preferred because it is similar to Na and K
-if Li is the analyte, CESIUM is used as a standard

Page 7 of 15
 ANALYTICAL METHODS
Prepared by: ALVIN A. ALDEA, RMT, MSc©

NOTE: This handout is for EXCLUSIVE USE of University of Saint Louis’ BS Medical Technology II
Students AY 2019-2020. Unauthorized reproduction is prohibited.

Atomic Absorption Spectrophotometry

´ Absorption of EM radiation by atoms than molecules
´ Measures the light absorbed by atoms dissociated by heat
´ Principle: Element is not excited by merely dissociated form its chemical bond and placed
in an unionized, unexcited, ground state
´ Most/more sensitive, very specific, accurate compared to FEP
´ Used to measure unexcited trace metals Ca2+ and Mg2+
´ Lanthanum or Strontium chloride is added to samples to form stable complexes with
phosphate to avoid Calcium interference.
´ Parts:
A. Light Source:
Hallow Cathode Lamp
Electrodeless Lamp
B. Mechanical Rotating Chopper
-modulates the beam coming from the Hallow Cathode Lamp
C. Burner
-uses flame to dissociate chemical bonds and form free, unexcited atoms
-flame is the sample cells
-alternative to burner: Electric furnace (uses Deuterium lamp as secondary LS)
D. Monochromator
E. Photodetector: PM tubes
F. Read-out Device/Meter
´ Interferences:
1. Chemical
-flame cannot dissociate the sample into neutral atoms
-lowers the value
2. Ionization
-atoms in the flame becomes excited and emit energy rather than staying in
ground state
3. Matrix
-sample droplets due to enhancement of light absorption by organic solvents
-may be overcome by pretreatment of sample by extraction
´ Recently, Inductive Coupled Plasma (ICP) is used to increase the sensitivity for atomic
emission.

Volumetry/Titimetry
´ Unknown sample is made to react with a known solution in the presence of an indicator
´ Example:
Cl- determination: Schales-Schales
Ca2+ determination: EDTA Titration Method

Gravimetry
´ Isolation of pure form of the sample and its derivatives and the determination of dry weight
´ Example: Total Lipid Determination
´ Steps:
Preparation Washing
Digestion Drying

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 ANALYTICAL METHODS
Prepared by: ALVIN A. ALDEA, RMT, MSc©

NOTE: This handout is for EXCLUSIVE USE of University of Saint Louis’ BS Medical Technology II
Students AY 2019-2020. Unauthorized reproduction is prohibited.

Precipitation Weighing

Turbidimetry
´ Turbidimetry
´ Principle: Measurement of the light BLOCKED by a suspension of particulate matter as
light passes through the cuvet
´ Measurement of abundant large particle (e.g. Protein)
´ Factors affecting turbidimetry:
1. Size and number of particles
2. Depth of the tube
3. Cross-sectional area of each particle
´ Disadvantage: Variable absorption due to aggregation of particles which tend to settle out
of the bottom

Nephelometry
´ Measures the amount of antigen-antibody complexes
´ Principle: Determines the amount of SCATTERED LIGHT by a particulate matter
suspended in a turbid solution
´ Light scattering depends on:
1. Wavelength
2. Particulate
´ More specific than turbidimetry
´ The higher the light scatter, the higher the concentration
´ For the size close to or larger than the WL of the incident light, sensitivity is increased by
measuring forward light scatter

LASER Application
´ LASER: Light Amplification by Stimulated Emission of Radiation (LASER)
´ Based on the interaction of radiant energy and suitable excited atoms or molecules
´ Can serve as LS of Incident Energy in a spectrophotometer or nephelometer
´ LASER spectrophotometry can be used for the determination of structure and ID of sample
as well as for diagnosis
´ Clinical Application: Coulter counter

Electrophoresis
´ Migration of charged particles in an electric field
´ Separates protein on the basis of their electrical charge densities
´ Acidic and Basic amino acids determines the net charge on a protein; hence, its
electrophoretic mobility
´ On electrophoresis, proteins are NEGATIVELY charged therefore moves towards the
anode
´ Electrophoresis
´ Terminologies:
1. Iontophoresis
-migration of small charged ions
2. Zone electrophoresis
-migration of charged macromolecules

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 ANALYTICAL METHODS
Prepared by: ALVIN A. ALDEA, RMT, MSc©

NOTE: This handout is for EXCLUSIVE USE of University of Saint Louis’ BS Medical Technology II
Students AY 2019-2020. Unauthorized reproduction is prohibited.

-electrophoresis on a support media

3. Amphoteric
-molecule whose net charge can either be positive or negative
-e.g., Protein: Acidic(+); Alkaline (-)
4. Electroendosmosis/Endosmosis
-movement of buffer ion and solvent relative to the fixed support
´ Factors Affecting the Rate of Migration
1. Net electric charge
-the higher the charge, the faster the electrophoretic separation
2. Shape and Size of the Molecule
-Generally, the greater the size, the slower the separation
3. Electric Fluid Strength
-the higher the ionic strength, the slower the movement
-mobility also depends greatly on the buffer type and concentration
-e.g., Barbital (Veronal) buffer: pH 8.6
Tris-Boric EDTA buffer: pH 8.7
´ Supporting Media:
1. Paper electrophoresis
-earliest support media
-Disadvantage: fragile and easily damaged and staining of protein is variable
2. Starch Gel
-separates by surface charge and molecular size
-larger samples could be employed
-disadvantage: fragile and unable to store results permanently
3. Cellulose acetate
-separates by molecular size
-requires 2-5 mL
-sample is diffused for 5 minutes
-Advantage:
Serial and structure of particles can be carefully controlled
Greater resolution
Faster separation
Permanent record can be maintained
4. Agarose Gel
-neutral: separates by electrical charge; it does not bind protein
5. Polyacrilamide Gel Electrophoresis
-neutral: separates by electrical charge and molecular size
-separates CHON to fractions
-used in the study of isoenzymes
´ Components:
1. Electrical power
2. Support medium
3. Buffer
4. Sample
5. Detecting system
´ New approaches to Electrophoresis
1. High Resolution CHON Electrophoresis

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 ANALYTICAL METHODS
Prepared by: ALVIN A. ALDEA, RMT, MSc©

NOTE: This handout is for EXCLUSIVE USE of University of Saint Louis’ BS Medical Technology II
Students AY 2019-2020. Unauthorized reproduction is prohibited.

-Permits the detection of 12-15 CHON fractions

-Modification: addition of calcium chloride and cooling system
2. Isoelectric focusing
-Ideal for separating CHON pf identical sizes but with different net charges
-Advantages:
(1) Resolve the mixture of protein;
(2) detects the isoenzyme of CK and ALP in the serum;
(3) Identify genetic variants of protein such as AAT;
(4) detects CSF oligoclonal banding
3. 2-Dimentional Protein Fractionation
-Combination of IEF and PAGE
-Identification of most unique protein that can’t be measured by single
techniques
4. Immunoelectrophoresis (IEP)
-Separation of protein fractions based on Ag-Ab reactions
5. Capillary Electrophoresis
-Sample molecules are separated by electro-osmotic flow
- (+) charged ions move early at the capillary outlet; (-) charged ions move at
the same direction but on a slower rate
6. Western blotting (Immunoblotting)
-Method used to separate, detect and ID one or more proteins in a complex
mixture
´ Temperature of operation
1. More voltage, more movement of protein
2. Limitations: the higher the current, the higher the tempt.
3. Higher tempt: (1) proteins are denatured (2) evaporation of solvent
´ Protein Detection after Separation
1. Colored dyes
Amido Black (Naphtol Blue Black): Dark blue band
S Ponceau in 5% TCA: Red bands
Coomasie blue: For CSF
Sudan Black
Fat Red 7B
Oil Red O
Gold/Silver
2. Enzyme Reaction
-Detection of specific enzymes
3. Immunofixation
-For specific protein which are not enzymes
4. Silver staining
-Useful for urine and CSF for detection of extremely low levels of protein
´ Clearing agents
-Used to dry up the supporting medium thereby preventing the diffusion of the
separated fractions to other fractions
-E.g., Methanol, Acetic acid, Cyclohexanol, Dioxane
´ Quantitation of Separated Protein Fractions
1. Elution Technique followed by Spectrophotometry

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 ANALYTICAL METHODS
Prepared by: ALVIN A. ALDEA, RMT, MSc©

NOTE: This handout is for EXCLUSIVE USE of University of Saint Louis’ BS Medical Technology II
Students AY 2019-2020. Unauthorized reproduction is prohibited.

-Done be eluting each fraction into solution for further chemical analysis
-Time consuming
2. Densitometry/Absorptiometry
-Method of choice
-Measurement of the density of light passing through the fraction
´ Specimen: CSF, Serum, Urine
´ Clinical Application
1. For analysis of protein that can provide quick and useful information regarding the
presence or absence of disease entities

Chromatography
´ Involves the separation of soluble component in a solution by specific differences in
physical and chemical characteristics of the different components on a supporting medium
called adsorbent or sorbent
´ Forms
1. Planar
1.1. Paper Chromatography
- Sorbent: Special grades of Filters (e.g., Whatman phase separating
paper)
-Clinical use: Fractionation of sugars, AA, Barbiturates
1.2. Thin Layer Chromatography
-Used for drug screening (semiquantitative test)
-Sample components are identified by comparison with standards
on the same plate
-Sorbent: Thin plastic plates impregnanted with a layer of silica gel or
alumina, PAGE or starch sgel
-Retention factor (Rf) value:
KLMNOPQR SROKLPT RKTR 5U Q5VW5PRPN V5XRM
Rf=
N5NOS KLMNOPQR M5SXRPN UY5PN V5XRM
2. Column
2.1. Gas Chromatography
-Useful for compounds that are naturally volatile or can be easily converted
into a volatile form
-Kinds:
2.1.1. Gas Solid Chromatography
-Sorbent is a solid of large surface
-Differences in absorption at the solid phase surfaces
2.1.2. Gas Liquid Chromatography
-Separation occurs by differences in solute partitioning
between gaseous mobile phase and liquid stationary
phase
-Sorbent: Non volatile liquid
-Clinical Use:
Drug Screening,
Fractionation of steroids, lipids, barbiturates, blood alcohol and other
toxicologic substances
**Mass Spectrophotometry

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 ANALYTICAL METHODS
Prepared by: ALVIN A. ALDEA, RMT, MSc©

NOTE: This handout is for EXCLUSIVE USE of University of Saint Louis’ BS Medical Technology II
Students AY 2019-2020. Unauthorized reproduction is prohibited.

-Used to measure compounds separated by GC

2.2. Liquid Chromatography
-Based on the distribution between a liquid mobile phase and a stationary
phase
-HPLC is most widely used LC
**HPLC (High Performance Liquid Chromatography)
-Principle: It follows the concept of selective adsorption. It applies 4000
to 10000 lbs/sq inch pressure for rapid separation of HMW
components and many labile biologic compounds such as
peptides, drugs, hormones, barbiturates, lipids, steroids, and antibiotics
-Separation Mechanism
2.2.1. Gel/Gel Permeation/Gel Filtration/Size Exclusion/Molecular
Sieve Chromatography/Molecular Exclusion
-Separates molecules based on differences in their size and
-Types:
[Link]. Gel Filtration/Hydrophilic gel
-Used in the separation of solute soluble in
aqueous medium such as enzymes,
antibodies, and other protein
[Link]. Gel Permeation/Hydrophobic gel
-Used in the separation of soluble only in
organic solvents such as triglyceride
and fatty acids
2.2.2. Ion Exchange Chromatography
-Sorbent: Anion or cation resin with functional groups
-Clinical use:
(1) separation of unwanted subs present in a cool
mixture;
(2) concentration of solute of interest suspended in
highly diluted sample can be determined
-Example: Natural purification of water as it perforates through
soil
2.2.3. Partition (Liquid-Liquid) Chromatography
-Based on relative solubility in an organic solvent and aqueous
solvent
-“Like dissolves like”
-Clinical use: (1) fractionation of therapeutic drugs; (2) Lipid
studies
2.2.4. Affinity Chromatography
-Used to separate and prepare larger quantities of LPP, CHO,
and HBA1c
1.25. Adsorption Chromatography (Liquid-Solid)
-Based on the difference between adsorption and desorption of
solutes at the surface of a solid particle
-Most soluble remain in the mobile phase

Fluorometry

Page 13 of 15
 ANALYTICAL METHODS
Prepared by: ALVIN A. ALDEA, RMT, MSc©

NOTE: This handout is for EXCLUSIVE USE of University of Saint Louis’ BS Medical Technology II
Students AY 2019-2020. Unauthorized reproduction is prohibited.

´ Determines the amount of light emitted by a molecule after excitation by EM radiation

´ LS: Mercury Arc, Xenon lamp
´ Light detector: PM tubes
´ Uses 2 Monochromator
-The WL that is best absorbed by the solution to be measured is selected by the primary
filter
-Secondary filter prevents incident light from striking the solution
´ Measures the amount of light intensity present over zero background
´ Affected by quenching

Electrochemistry
´ Measurement of current or voltage generated by the activity of specific ion
´ Types
1. Potentiometry
-Measurement of the differences of voltage (potential) at a constant current
-Based on NERST EQUATION
-Reference electrode with constant voltage
Saturated calomel: External
Silver-silver chloride: Internal
**Ion Selective Electrode (ISE)- very sensitive and selective to the ion it measures; ionic
selectivity depends on the membrane used
2. Polarography
-Measurement of differences in current at a constant voltage
-Based on ILKOVIC EQUATION
3. Coulometry
-Measurement of the amount of electricity at fixed potential

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 ANALYTICAL METHODS
Prepared by: ALVIN A. ALDEA, RMT, MSc©

NOTE: This handout is for EXCLUSIVE USE of University of Saint Louis’ BS Medical Technology II
Students AY 2019-2020. Unauthorized reproduction is prohibited.

-It is an electromechanical titration in which the titrant is electrochemically generated

and the endpoint is detected by AMPEROMETRY
-Based on FARADAY’S LAW
4. Amperometry
-Measurement of the current flow produces by Redox reaction
5. Voltametry
-Measurement of current after the potential is applied to an electromechanical cell
-It allows chemical sample to be preconcentrated thus utilizing minimal dye
-Anodic stripping voltametry (used for lead testing)

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