CLINICAL CHEMISTRY Pau’s Notes

1ST SEMESTER – MIDTERMS

CHAPTER 2 – ANALYTICAL METHODS
ANALYTICAL METHODS
- How the machines work
RODRIGUEZ BISHOP
I. COLORIMETRY I. SPECTROMETRY V.
a. Spectrophotometry a. Spectrophotometry
b. Flame Emission b. AAS
Photometry (FEP) c. Mass Spectrometry
c. Atomic Absorption II. LUMINESCENCE
Spectrophotometry a. Fluorescence
(AAS) b. Chemiluminescence
II. VOLUMETRIC III. ELECTROANALYTIC
(TITRIMETRIC) METHODS
III. TURBIDIMETRY a. Electrophoresis
IV. NEPHELOMETRY b. Potentiometry
V. ELECTROPHORESIS
c. Amperometry COLORIMETRY
VI. CHROMATOGRAPHY
a. Planar
IV. CHROMATOGRAPHY • Photoelectric Colorimetry – the primary analytical
a. Gas
i. Paper Chromatography utility of spectrophotometry/filter photometry is the
b. Liquid
ii. Thin Layer isolation of discreet portions of the spectrum for
Chromatography c. Thin Layer
b. Column purposes of measurement
i. Gas Chromatography • 2 measurements:
ii. Liquid Chromatography
VII. FLUOROMETRY/
o Photometric measurement: measurement of light
MOLECULAR intensity
LUMINESCENCE o Spectrophotometric measurement: measurement
SPECTROMETRY
VIII. CHEMILUMINESCENCE of light intensity in a narrower wavelength
IX. OSMOMETRY SPECTROPHOTOMETER
X. ELECTROCHEMISTRY
TECHNIQUES
a. Potentiometry
b. Coulometry
c. Amperometry
d. Voltammetry
Light energy, wavelength, & radiant energy spectrum:
‒ Continuous flowing motion
‒ Analytes prefer specific wavelengths ‒ Single Beam Spectrophotometer – simplest type of
The relationship b/w wavelength & energy (E) is described absorption spectrophotometer
by Planck’s formula: E = hv ‒ One measurement at a time in a single wavelength
Where: ‒ The max absorption of the analyte must be known
E – energy of a photon in Joules or eV in advance when using this spectrophotometer
h – constant (6.626 × 10−34 𝑒𝑟𝑔 sec) ‒ Double Beam Spectrophotometer – an instrument that
v – frequency splits the monochromatic light into 2 components; 1
• Energy (E) – transmitted via electromagnetic waves beam thru the sample & 1 thru the reference sol’n
that are characterized by their frequency & (standard/control)
wavelength ‒ The additional beam corrects for variation in light
• Wavelength (λ) – distance b/w 2 successive peaks & is source intensity
expressed in terms of nanometer (nm) ‒ In this type of spectrophotometer, the absorbance
• Frequency (v) – # of vibrations of wave motion per of the sample can be recorded directly as the
second electrical output of the sample beam
o The lower the wave frequency, the longer the ‒ 2 types of Double Beam Spectrophotometer:
wavelength ▪ DOUBLE BEAM IN SPACE – w/ 2 photodetectors
o The wavelength is inversely related to frequency & (for sample beam & reference beam)
energy; the shorter the wavelength, the higher the
frequency & energy and vice versa…

▪ DOUBLE BEAM IN TIME – w/ 1 photodetector &

➢ 103 = Radio Waves alternately passes the monochromatic light thru
➢ 10−2= Microwaves – excites the water molecules the sample cuvette & the reference cuvette
➢ 10−5= Infrared – there are analytes that prefer this; using a chopper/rotating sector mirror
invisible waves
➢ 0.5 × 0−6= Visible – colors; RGB
➢ 10−8= Ultraviolet – if body receives ↑UV, it mutates the
cell → cancer; there are cells/analytes/proteins that
need UV to be seen
➢ 10−10= X-Ray – may be dangerous (3x a year);
radiology lab should not be beside clinical lab (should
be separated w/ lead wall if is inevitable) 6 BASIC COMPONENTS OF A SPECTROPHOTOMETER
➢ 10−12= Gamma Ray – if we are exposed from it, we will 1. RADIANT ENERGY (Light Source)
die; causes TOO MUCH MUTATION ‒ Provides polychromatic light & must generate
Infrared  ROYGBIV → Ultraviolet sufficient radiant energy/power to measure the
>> weak >>strong analyte of interest
No light source can do the 3 regions (INFRA, VISIBLE, ULTRA) ‒ Factors for choosing a light source:
so one should choose carefully depending on what you ▪ Range
need most ▪ Spectral distribution w/in the range
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CLINICAL CHEMISTRY Pau’s Notes
1ST SEMESTER – MIDTERMS
▪ Source of radiant prod 3. SAMPLE HOLDER (CUVET)
▪ Stability of the radiant energy ‒ Also called:
▪ Temp = ↑ temps shuts off machine; ↑ temp, ↑ results ▪ Absorption cell
‒ Continuum Source – emits radiation that changes ▪ Analytical cell
in intensity ▪ Sample cell
▪ Widely used in the lab ‒ It holds the sol’n whose concentration is to be
▪ Egs: measured
→ Tungsten light bulb: commonly used in the ▪ May be:
visible & near infrared region → Blank – set 0; default
→ Deuterium Lamps: routinely used to provide → Standard – pure sol’n; known con of reagent
UV rad in analytical spectrometers → Control – mimics human sample; also known con
→ Unknown – human sample
→ Xenon Discharge Lamps: covers both UV &
‒ Should not intervene to measurement (NO
visible range
microscratches/blur in cuvette = wash by water blanking)
‒ Line Source – emits limited rad & wavelength
‒The path length must be 1 cm/less (automated
▪ Emits a few discrete lights & are widely used in
analyzers)
atomic absorption, molecular, & fluorescent
‒ Kinds of Cuvets:
spectrophotometry
▪ Alumina Silica Glass – most commonly used (350-
▪ Egs:
2000 nm)
→ Mercury & Sodium Vapor Lamps: UV & visible
▪ Quartz/Plastic – used for measurement of sol’n
regions
requiring visible & UV spectra
→ Hollow Cathode Lamps: AAS
▪ Borosilicate Glass
→ *Light Amplification by Stimulated Emission of
▪ Soft Glass
Radiation (LASER) – also used as a light source
4. PHOTODETECTOR
for spectrophotometry
‒ Detects & converts transmitted light of analyte into
‒ Alternative for Tungsten Bulb:
photoelectric energy
Alternative Light Source UV Visible Infrared
Mercury Arc / / ‒ Detects the amt of light that passes thru the sample
Deuterium Lamp (165 nm) / in the cuvet
Hydrogen Lamp / ‒ Kinds of Photodetectors:
Xenon Lamp / ▪ Barrier Layer Cell/Photocell/Photovoltaic Cell –
Nernst Glower Lamp / the simplest detector; least expensive; temp-
Globar (Silicone Carbide) / sensitive
‒ ENTRANCE SLIT: allows polychromatic light to reach → Used in filter photometers w/ a wide bandpass
monochromator and minimizes unwanted/stray → A basic photo transducer that is used for
light & prevents the entrance of scattered light into detecting & measuring rad in the visible
the monochromator system; minor part region
▪ Stray Light – any wavelength outside the band ▪ Phototube – contains cathode & anode enclosed
transmitted by the monochromator in a glass case
→ Does not originate from the polychromatic → Has a photosensitive mat that gives off
light source & causes absorbance error electrons when light energy strikes it
→ Limits the max absorbance that a ▪ Photomultiplier Tube (PMT) – most commonly
spectrophotometer can achieve used detector (visible & UV regions)
→ The most common cause of loss of linearity at → Has excellent sensitivity & has a rapid response
high analyte concentration (detects very low level of light)
2. MONOCHROMATOR ▪ Photodiode – not as sensitive as PMT but w/
‒ Isolates specific/indiv wavelength of light excellent linearity
‒ Should be linear to exit slit → Most useful as simultaneous multichannel
‒ Kinds of Monochromators: detector
▪ Prisms – wedge-shaped pieces of glass, quartz/ ‒ Transmitted light release electron (dynode chain)
NaCl when they hit photocathodes/anodes =
→ Can be rotated, allowing only the desired photoenergy (does not quantify)
wavelength to pass thru the exit slit 5. SIGNAL PROCESSOR
▪ Diffraction Gratings (Spectrometers) – most ‒ Photoenergy converts into electrons where they
commonly used bcoz it provides better resolution are quantified – measurement of transmitted light
than prism (not yet the final results of absorbed light)
▪ Filters (Photometers) – simple, least expensive, not ‒ Beer’s law/Beer-Lambert’s law is used to quantify
precise but useful 6. READOUT DEVICE (METER)
▪ Holographic Gratings – exhibit less scattered light ‒ Displays the output of the detection system
than diffraction gratings due to their sinusoidal ‒ Egs:
groove profile ▪ Galvanometer
→ Efficiency is usually considerably less than a ▪ Ammeter
comparable rule diffraction grating ▪ Light emitting Diode (LED) Display
‒ EXIT SLIT: controls the width of the light beam, that BEER’S LAW (Beer-Lambert’s Law)
we call incident light (monochromatic light), w/c - The law was discovered by Pierre Bouguer before
allows only a narrow fraction of the spectrum to 1729
reach the sample cuvette - Johann Heinrich Lambert cited Bouguer in his book
▪ Bandpass – the total range of wavelength Photometria (1760)
transmitter - Formulated by German mathematician & chemist
→ The degree of wavelength isolation is a August Beer in 1852
function of the type of device used & the - It states that the concentration of the unknown
width of the entrance & exit slits substance is directly proportional to the absorbed
→ Spectral purity of the spectrophotometer is light (absorbance/optical density) & inversely
reflected by bandpass (narrower bandpass = proportional to the amt of light transmitter
greater resolution) (%Transmittance)

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CLINICAL CHEMISTRY Pau’s Notes
1ST SEMESTER – MIDTERMS
- It mathematically establishes the relationship b/w FLAME EMISSION PHOTOMETRY
concentration & absorbance
- Absorbance (A) – the amt of light absorbed
→ Proportional to the inverse log of transmittance
→ Cannot be measured directly by a
spectrophotometer but rather is mathematically
𝐼
derived from %T as follows: %𝑇 = × 100
𝐼𝑜
𝐴 = 𝑎𝑏𝑐 = 2 − 𝑙𝑜𝑔%𝑇
→ Where:
▪ A = absorbance
▪ a = molar absorptivity
▪ b = length of light thru the sol’n
▪ c = concentration of absorbing molecules/sol’n
- %Transmittance – the ratio of the radiant energy ‒ Principle: Excitation of electrons from lower to higher
Transmitted (T) divided by the radiant energy incident energy state
on the sample (I) ‒ Light Source: Flames (also serves as the cuvet)
→ The %T measured by commercial ‒ Method: Indirect Internal Standard Method
spectrophotometers is the ratio of the sample ‒ Internal Standard: Lithium/Cesium (corrects variations
transmitted beam divided by the blank transmitted in flame & atomizer characteristics)
beam ‒ Purpose: measurement of excited ions (electrolytes
→ In actual practice, the light transmitted by a blank such as Na, K, & Li)
substitutes for Io ‒ Sample sol’n in liquid form undergo nebulization & will
become vapor going inside the mixing chamber, air is
incorporated while gas inlet provide fuel until they
result in flame
‒ Any unwanted/unnecessary component will go to
waste if partial burner
‒ If total burner, there is no waste
‒ Flame produces is a scattered light, so the biconcave
mirror collects the light & bounces back to the
biconvex mirror w/c collects all the extra light & goes
to monochromator (filter)
‒ Diff ions emit diff light color upon excitation:
Li – crimson red Mg – blue
Na – yellow/yellow-orange Ca – orange to red
𝐼 K – lilac/violet/purple Cu – green/blue
%𝑇 = × 100
𝐼𝑜 ATOMIC ABSORPTION SPECTROPHOTOMETRY
→ Where:
▪ It = transmitted light thru the sample
▪ Io = intensity of light striking the sample (incident
light)
𝐴𝑢
𝑈𝑛𝑘𝑛𝑜𝑤𝑛 𝑆𝑜𝑙′𝑛 = × 𝐶𝑠
𝐴𝑠
We must be reminded that manual computation is a part
of quality control!!
BLANKING TECHNIQUE
1. Water Blanking = flush out previous sample; correct/
calibrate machine; no absorbed light [0]
2. Reagent Blanking = not all reagent are colorless; this ‒ Principle: Element is not excited by merely dissociated
method lets the machine know that the reagent from its chemical bond & place in an unionized,
already has a color (introduced); don’t replace w/ unexcited, ground state
water if it’s just bcoz it’s colorless ‒ Light Source: Hollow-Cathode Lamp
3. Sample Blanking = hemolysis, lipemia, ictericia; dilute ‒ Interferences: Chemical, Matrix (difference in
the sol’n viscosity), & Ionization
Light blocked → no light transmitted → negative [-] ‒ Reference method for measurement of unexcited ions
- To correct for artifactual absorbance readings, ‒ More sensitive than flame photometry bcoz it has a
“blanking” procedures/dual wavelength method PMT
may be used ‒ It is accurate, precise, & very specific
- Lipids interfere by mainly increasing light scatter ‒ It follows the Beer-Lambert’s Law
(turbidity) ‒ Sample sol’n in liquid form also undergo nebulization &
- A blanking process may not be effective in some will become vapor going inside the mixing baffles,
causes of turbidity, & ultracentrifugation may be incorporated w/ oxygen & fuel until they result in flame
necessary to clear the serum/plasma of chylomicrons ‒ Any unwanted/unnecessary component will go to
Reagent Blank Sample Blank
waste if partial burner
- corrects the - corrects the
absorbance caused by measurement for optical ‒ If total burner, there is no waste
color of the reagents interference (Hgb, ‒ Atomizer – allow the ions to go to higher energy state
- The absorbance of the bilirubin, & lipids) & wait till it goes back to ground state – can’t emit their
reagents is absorbing the own light so they only absorb & transmit
automatically wavelength of
‒ Flame (atomization; chemically dissociate ions) is
subtracted from each of measurement
unknown reading - Measures absorbance 1st produced to make ions excited w/c makes
of the sample & reagent them have higher energy state (removing bonds =
in the absence of the
end product

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CLINICAL CHEMISTRY Pau’s Notes
1ST SEMESTER – MIDTERMS
freedom of ions) & emit their own light (light is ELECTROPHORESIS
continuous) • Terminologies:
▪ Not all ions can be excited!! o Amphoteric – has a net charge that can be either
‒ Light source – series of activity; light from inert gases; (+)/(-) depending on pH conditions
hollow cathode lamp ▪ Amphoterism – proteins → amino acid sequence;
‒ Inert gases emit light that is the same wavelength slight changes = flexibility of charges of amino acids
w/ ions so it is compatible to be absorbed o Electroendosmosis/Endosmosis – the movement of
‒ Chopper – cuts continuous light from light source by buffer ions & solvent relative to the fixed support
rotation so broken light hits the sample o Iontophoresis – the migration of small charged ions
‒ The sample (unexcited ions) absorbs broken light, so it o Zone electrophoresis – the migration of charged
also transmits broken light macromolecules
‒ Monochromator – isolate the wavelength necessary
for detection
‒ Photodetector – differentiate light:
‒ Continuous light = from excited ions
‒ Broken light = from unexcited ions
VOLUMETRIC

• It is the migration of charged particles in an electric

field
• It separates proteins on the basis of their electric
charge densities, shape, size, & pH
• Principle: The unknown sample is made to react w/ a IOTHOPHORESIS
known sol’n in the presence of an indicator • opposite attracts
o Change of color = presence of analyte (quali) o Anode [+] – will go towards (-) region
o Can be placed in spectrometry o Cathode [-] – will go towards (+) region
• Examples: Schales & Schales method (Chloride test) • SERUM PROTEIN ELECTROPHORESIS
o EDTA Titration method (Calcium test) ▪ Band 1 – fastest; only 1 = Albumin (referred to as
TURBIDIMETRY jeepney)
▪ Band 2 – α1 region
▪ Band 3 – α2 region
▪ Band 4 – β region = sometimes presents 2 peaks
▪ Band 5 – γ region = Ig
o Bands 1-4 are from the liver, while band 5 is from the
plasma
• Principle: It determines the amt of light blocked ▪ So when band 5 has high results, there pt has
(reduction of light) by a particulate matter in a turbid cancer of plasma/multiple myeloma
sol’n
• Use: Protein measurements (CSF & urine)
o Detection of bacterial growth in broth cultures
▪ The machine alarms when there is growth of
bacteria, w/c means the light is totally blocked in
less than 24 hrs
▪ The machine also alarms when there is no growth
of bacteria w/in 48 hrs (no blocked light)
o Antimicrobial test (broth method)
o Detect clot formation
▪ Optical turbidity (Electronic impedance in Hema)
▪ Coagulation studies
NEPHELOMETRY

o Rocket Electrophoresis – looks like a rocket that had

• Principle: It determines the amt of scattered light by a taken off
particulate matter suspended in a turbid sol’n ▪ ↑ graph, ↑ concentration
• Light scattering depends on wavelength & particle ▪ Multiple myeloma = γ spike (like rockstar hand sign)
size ▪ Fibrinogen = migrates b/w β & γ = β-γ bridging
• Light scattered by particles is measured at an angle, According to SIZE
typically 15-90º to the beam incident on the cuvet • LIPOPROTEINS (LP) = HDL, LDL, VLDL, & Chylomicrons
o Each angle gives us diff info: back = size, 90º= (doesn’t show, but accumulate; bigger so causes ↑ friction)
granules, forward = nucleus, transmitted light o Band 1 – none
• The detector (PM tube) output is proportional to o b/w Band 2 & 3 – HDL region
concentration o b/w Band 3 & 4 – VLDL
• For antigen/antibody reaxn & for proteins o Band 4 – LDL
o Band 5 – none
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CLINICAL CHEMISTRY Pau’s Notes
1ST SEMESTER – MIDTERMS
• GLOBIN CHAINS IN HGB - Isoelectric Focusing – separates molecules by
migration thru a pH gradient
→ Ideal for separating proteins of identical sizes but
w/ diff net charges
→ pH gradient is created by adding acid to the
anodic area of the electrolyte cell & adding base
to the cathode area
→ Advantages:
▪ The ability to resolve mixture of proteins
▪ To detect isoenzymes of ACP, CK, & ALP in serum
▪ To identify genetic variants of proteins such as
alpha-1-antitrypsin
▪ To detect CSF oligoclonal banding
CAPILLARY ELECTROPHORESIS

According to pH gradient
• ISOELECTRIC FOCUSING = ALP (Alkaline phosphatase)
o Bone = if ↑ (Flip pattern, Pre-hepatic), Paget’s
disease; total ALP = ↑
o Liver
o Intestinal
o Placental

- ONE-WAY!
→ Fastest migrators = cations (by EOF & attracted to
cathod region)
→ 2nd fastest = neutrals (by EOF)
→ Slowest/don’t move = anions
- Sample molecules are separated by electro-osmotic
flow (EOF)
- It utilizes nanoliter quantities of spxms
- Uses:
→ Separation
→ Quantitation & determination of molecular weights
Components of electrophoresis: of proteins & peptides
o Electrical power → Analysis of PCR products
o Support medium → Analysis of organic & inorganic substances & drugs
o Buffer: Barbital (pH 8.6) - Less sensitive than the electrophoresis
o Sample → To improve, add luminescence
o Detector (Chemiluminescence)
Factors Affecting Rate of Migration: CHROMATOGRAPHY
o Net electric charge of the molecule • History:
o Size & shape of the molecule o Mikhail Tswett, a Russian botanist used to
o Electric field strength chromatography to separate plant pigments (1906)
o Nature of the supporting medium o He called the new technique chromatography bcoz
o Temp of operation the result of the analysis was ‘written in color’ along
Supporting Media: the length of the adsorbent column
o Cellulose acetate – separates by molecular size o Chroma means “color” & graphein means to “write”
o Agarose gel - separates by electrical charge; does • It refers to the group of test/techniques used to
not bind protein separate complex mixtures on the basis of diff physical
o Polyacrylamide gel - separates on the basis of interactions b/w the indiv compounds & the stationary
charge & molecular size; separates proteins into 20 phase of the system
fractions; used to study isoenzymes • It involves separation of soluble components in a sol’n
TWO-DIMENSION ELECTROPHORESIS by specific differences in physical-chemical
- Same pts, 2 diff sample (eg. blood & tissue) characteristics of the diff constituents
- Certain proteins might be present in blood but present Basic Chromatography & Hyphenated Techniques
in tissue • According to type of Chromatography:
DENSITOMETRY Planar Column
Paper Chroma Gas
Thin Layer Chroma Gas-Solid
Gas-Liquid
Liquid
HPLC
LC-MS
• According to Phase:
Stationary Mobile
- Responsible for scanning & quantifying Paper Chroma Gas
- Measures the absorbance of stain – concentration of Thin Layer Chroma Liquid
the dye & protein fraction Column
- It scans & quantitates electrophoretic pattern • According to Force of Separation:
→ Looks for pattern & intensity of stain/light o Adsorption Chromatography
→ ↑ intensity, ↑ concentration o Partition Chromatography
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CLINICAL CHEMISTRY Pau’s Notes
1ST SEMESTER – MIDTERMS
o Ion Exchange Chromatography COLUMN CHROMATOGRAPHY
o Gel Filtration Chromatography - Stationary phase is held in a narrow tube thru w/c the
o Affinity Chromatography mobile phase is forced under pressure/under the
• Basic Components: effect of gravity
o Mobile Phase (Gas/Liquid) – carries the sample Classification according to Force of Separation:
o Stationary Phase (Solid/Liquid) 1. Gel/Gel Permeation/Gel Filtration/Size
o Column Holding the Stationary Phase Exclusion/ Molecular Sieve/Molecular
o Separated Components (Eluate) Exclusion Chromatography
PLANAR CHROMATOGRAPHY ‒ Mobile phase: Water/dilute alcohol
PAPER CHROMATOGRAPHY ‒ Separation mechanism:
o Based on difference b/w the solutes
molecular weights
o Molecules will distribute themselves
outside & inside the pores according
to their size
‒ Albˉ
‒ Medium sized proteins will slow down;
large will be stuck
2. Ion Exchange Chromatography
‒ The mechanism in this type of chromatography is
the exchange of sample ions & mobile-phase ions
w/ the charged group of the stationary phase
‒ For separation of amino acids, proteins, & nucleic
acids
‒ Ion Exchange Resin = Drug resin
- Used for fractionation of sugar & amino acid ‒ Opposite attracts
- Sorbent (stationary phase) – Whatman paper
- Despite the tissue being white, it consists of diff colors
- Eg.: β-HCG (Pregnancy Test), Dengue Duo, & Drug
Testing
→ Line is a line regardless of thickness = [+]
→ Cross reactivity = if too much Ab, can react w/ Ags
even tho it is not for them
→ Hook effect = sa sobrang [+], nag-[-] sa test; Ag
excess
→ in drug testing, if there is line, [-] test
THIN LAYER CHROMATOGRAPHY (TLC)
3. Partition Chromatography (Liquid-Liquid
Chromatography)
‒ Separation of compounds based on their partition
b/w a liquid mobile phase & a liquid stationary
phase coated on a solid support
‒ Is for separation of therapeutic drugs & their
metabolites
4. Affinity Chromatography
‒ Uses immobilized
- plate; Thin layer coated plate biochemical ligands as the
- A method for identifying substances & testing the stationary phase to separate
purity of compounds a few solutes from other
- Useful technique bcoz it is relatively quick & requires unretained solutes
small quantities of material ‒ This type of separation uses
- Separations in TLC involve distributing a mixture of the so-called lock-&-key
2/more substance b/w a stationary phase & a mobile binding that is widely present
phase in biologic systems
→ Stationary phase: thin layer of adsorbent (usually o Ag – lock
silica gel/alumina) coated on a plate o Ab – key; since it is only
→ Mobile phase: developing liquid w/c travels up the produced when there is
stationary phase, carrying the samples w/ it Ags & so it is specific w/ 1
- Retention factor (Rf) value = relative distance of Ag (IgM – master key)
migration from the point application ‒ Ligand – inert matrix
→ Movement bcoz of mobile phase ‒ Use: (separation)
→ Every drug has its own rf value o Lipoproteins
𝐷𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑙𝑒𝑎𝑑𝑖𝑛𝑔 𝑜𝑓 𝑐𝑜𝑚𝑝𝑜𝑛𝑒𝑛𝑡 𝑚𝑜𝑣𝑒𝑠 o Carbs & glycated Hgb
𝑅𝑓 =
𝑇𝑜𝑡𝑎𝑙 𝑑𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑠𝑜𝑙𝑣𝑒𝑛𝑡 𝑓𝑟𝑜𝑛𝑡 𝑚𝑜𝑣𝑒𝑠 o Antibodies
- TLC is a semi quantitative drug screening test 5. Adsorption Chromatography (Liquid-Solid
- Sample components are identified by comparison w/ Chromatography)
standards on the same plate ‒ Separation is based on the differences
- Biological samples such as blood, urine & gastric fluid (competition) b/w the adsorption & desorption of
can be used for the test solutes at the surface of a solid particle
- Sorbent: thin plastic plates impregnated w/ a layer of ‒ The compounds are adsorbed to a solid support
silica gel/alumina such as silica/alumina
- Each drug has a characteristic Rf value & it must ‒ Adsorb = binds (not combine) w/ molecule
match the Rf value of the drug standard ‒ Adsorptive stationary phase complementary to
molecule properties
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CLINICAL CHEMISTRY Pau’s Notes
1ST SEMESTER – MIDTERMS
MASS SPECTROSCOPY

- Based on the fragmentation & ionization of molecules

using a suitable source of energy
- It can also detect structural info & determination of
molecular weight
- Doesn’t work on its own
→ Before a compound can be detected & quantified
by MS, it must be separated by GC
- Ion acceleration = gives change to ions of energy
- Magnet = moves fragment of ions toward the
detector
GAS CHROMATOGRAPHY-MASS SPECTROSCOPY (GC-MS)

Factors Effect
Particle size of solid Decrease of size improves separation
stationary phase (or of (but very small particles need high
support) pressure)
Column dimensions Efficiency increases as ration
length/width increases
Uniformity of packing Non uniform packing results in irregular
movement of solutes thru column & less
uniform zone formation, (i.e. band
broadening/tailing)
Column temp Increase in column temp results in - Gold standard for drug testing
speed of elution but does not improve
- Also used for xenobiotics, anabolic steroids, &
separation (tailing)
Eluting solvent Solvents should be of low viscosity (to pesticides
give efficient resolution) & high volatility - In this method, quanti measurement of drug can be
(to get rapid recovery of the
substances)
performed by selective ion monitoring
Solvent flow rate Uniform & low flow rate gives better - It uses an electron beam to split the drug emerging
resolution from the column into its component ions – drugs are
Continuity of flow Discontinuous flow disturbs resolution
Condition of adsorbent Deactivation of adsorbent decreases
detected by means of the presence of
separation decomposition fragments w/c arise after
Concentration of solutes Substances of high concentration degradation of the analytes
move slowly
- Tandem mass spectroscopy (MS/MS) – can detect 20
GAS CHROMATOGRAPHY
inborn errors of metabolism from a single blood spot
- There is pattern recognizable based on library
- Gas-solid – most common
- Hyphenated = more sophisticated
- There might still be errors, but more trials ↓ errors
LIQUID CHROMATOGRAPHY
- Based on the distribution of solutes b/w a liquid mobile
phase & a stationary phase
- HPLC is the most widely used liquid chromatography
= Rapid HbA1C
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)
- Used for separation of steroids, barbiturates, blood,
alcohol, & lipids
- It is useful for compounds that are naturally volatile/
can be easily converted into a volatile form
- Elution order of volatiles is based on their boiling point
- tR – the retention time of the solute in GC/HPLC
- Mobile phase: N, He, H, Ar (inert gases)
- Used as confirmatory test:
→ Looks for fingerprint (each drug has fingerprint)
→ Any interferences (unnecessary analytes) are
denaturized
- Gas Solid Chromatography (GSC) – separation occurs
based on differences in absorption at the solid phase - Uses pressure for fast separations, controlled temp, in-
surfaces line detectors & gradient elution technique
- Gas Liquid Chromatography (GLC) – separation - In reverse phase HPLC, the mobile phase is more polar
occurs by differences in solute partitioning b/w the than stationary phase
gaseous mobile phase & the liquid stationary phase - Uses:
→ Fractionation of drugs, hormones, lipids, carbs, &
proteins

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CLINICAL CHEMISTRY Pau’s Notes
1ST SEMESTER – MIDTERMS
→ Separation & quantitation of various Hgb assoc w/ • PHOTODETECTORS: Photomultiplier tube (Luminometer)
specific diseases (e.g. thalassemia) o Luminol – reagent
→ Rapid HbA1c test (w/in 5 mins) o Bioluminescence – life
LIQUID CHROMATOGRAPHY-MASS SPECTROSCOPY o Thermoluminescence – heat
(LC-MS) o Radioluminescence
▪ Nuclear medicine: combi of radiology & clinical
▪ Tumor markers
o Electroluminescence – light bulb
OSMOMETRY

- For detecting nonvolatile substances in body fluids

- Utilized to confirm (+) results from screening of elicited
drugs – it is a complementary method to GC-MS
- It is also used in therapeutic drug monitoring,
toxicology & studies of drug metabolites
• The measurement of the osmolality of an aqueous
- It requires interface methods to convert nonvolatile to
sol’n such as serum, plasma, or urine
volatile compounds
o (1) Boiling Point
FLUOROMETRY/MOLECULAR LUMINESCENCE
o (2) Freezing Point
SPECTROPHOTOMETRY
o (3) Vapor Pressure
o (4) Osmotic Pressure
• ↑ Osmolal blood sample
o ↑ Vapor pressure & Boiling point
o ↓ Osmotic pressure & Freezing point (depresses)
• If balance is disrupted, the body will react.
o Blood will thicken, making it hard for the kidneys to
filter → UTI
o Dehydrated = viscous blood
• Principle: It is based on measuring changes in the
• Principle: It determines the amt of light emitted by a
colligative properties of sol’ns that occur owing to
molecule after excitation by electromagnetic
variations in particle concentration
radiation
• Osmotic particles: Glucose, Urea, N, & Na
• LIGHT SOURCE: Mercury arc or Xenon lamp
• REFRIGERANT: to make temp drop/make sample cold
o Can excite molecule whether it is absorbed/not;
• At a certain point, the temp will become steady
the only goal is to excite molecules
making the thermistor know that the sol’n sample has
• LENS: collects light & focuses on the filter
reached its freezing point so that the machine will stop
• EXCITATION FILTER: isolates light to excite molecules
& results are displayed (the temp of freezing point)
• TRANSMITTED/WIDESPREAD LIGHT: excited molecule
ELECTROCHEMISTRY TECHNIQUES
o 1 line/emitted light
• The measurement of the current/voltage generated
• EMISSION FILTER: 2nd monochromator; isolates
by the activity of a specific ion
emitted light from the analyte of interest, increase
• Replaced FEP & AAS
sensitivity of fluorescence
• LIGHT DETECTOR: Photomultiplier tube/Phototube
• Use: Porphyrins, Mg, Ca, & catecholamines
• It is affected by quenching – pH & temp changes,
chemical contaminants, UV light changes
CHEMILUMINESCENCE

• It differs from fluorescence & phosphorescence in that

the emission of light is created from a chemical/ - Internal sol’n is the reason why ion gives electrons
electrochemical reaxn & not from absorption of → It is sensed by ion-sensing electrode
electromagnetic energy - Control electrode doesn’t have ion-selective
• It is more sensitive than fluorescence membrane that is why anything can enter
o Doesn’t require light source → This ensures that the machine is measuring the
• Principle: The chemical reaxn yields an electronically electrons
excited compound that emits light as it returns to its - Ion-selective membrane expires/thin out, especially
ground state, or that transfer its energy to another when used too much, making it possible for other ions
compound, w/c then produces emission to enter
• Use: Immunoassays → More Na buildup
• Ions become excited & goes back to ground state as → Expired calibration = control problem
it emits light
8|Pa ge
CLINICAL CHEMISTRY Pau’s Notes
1ST SEMESTER – MIDTERMS
- Ion-selective electrode = inside the electrode is - An electrochemical titration in w/c the titrant is
sensitive but not specific electrochemically generated & the endpoint is
→ Direct ISE (w/o sample dilution) = serum detected by amperometry
→ Indirect ISE (w/ sample dilution) = urine → has - Use: Chloride test (CSF, serum, & sweat)
bacteria & ↑ acidity - Interference: Bromide, Cyanide, & Cysteine
POTENTIOMETRY AMPEROMETRY

- It is the measurement of the current flow produced by

- It is the measurement of electrical potential due to the
an oxidation-reduction
activity of free ions – change in voltage indicates
- Use: pO2, glucose, chloride & peroxidase
activity of each analyte
determinations
- It is also the measurement of differences in voltage
- Polarography = the measurement of differences in
(potential) at a constant current
current at a constant voltage
- It follows the Nernst equation
VOLTAMETRY
- Reference electrodes: Calomel & Silver-Silver Chloride
- Use: pH & pCO2 tests
ION SELECTIVE ELECTRODE (ISE)

- It is the measurement of the current after w/c a

- It is an electrochemical transducer capable of potential is applied to an electrochemical cell
responding to 1 given ion - It allows sample to be preconcentrated, thus utilizing
- It is very sensitive & selective for the ion it measures – it minimal analyte
measures the activity of 1 ion much more than other - Anodic stripping voltammetry – for lead & iron testing
ions present in the sample
- It is a sensitive method but not specific – it does not
discriminate b/w ions in causing voltage differences
b/w the measuring electrode & the standard
electrode
- 2 Types of ISE:
→ 1. Direct ISE (w/o sample dilution)
→ 2. Indirect ISE (w/ sample dilution)
- ISE Membrane:
→ Glass aluminum silicate (Na)
→ Valinomycin gel (K)
→ Organic liquid membrane ion exchangers (Ca & Li)
→ Gas & enzyme electrodes
- Causes of Malfunctions of ISE:
→ Defective ISE membrane
→ Buildup of counter voltages from liquid junction
potentials at the salt bridge
→ Buildup of proteins at the electrodes
COULOMETRY

- It is the measurement of the amt of electricity (in

coulombs) at a fixed potential

9|Pa ge