Review of Analytical Methods

Part 1: Spectrophotometry

Roger L. Bertholf, Ph.D.

Associate Professor of Pathology
Chief of Clinical Chemistry & Toxicology
University of Florida Health Science
Center/Jacksonville
 Analytical methods used in
clinical chemistry
• Spectrophotometry
• Electrochemistry
• Immunochemistry
• Other
– Osmometry
– Chromatography
– Electrophoresis
 Introduction to spectrophotometry
• Involves interaction of electromagnetic
radiation with matter
• For laboratory application, typically
involves radiation in the ultraviolet and
visible regions of the spectrum.
• Absorbance of electromagnetic radiation is
quantitative.
Electromagnetic radiation

Velocity = c
A

Wavelength
(λ)
Wavelength, frequency, and
energy

E = energy
hc h = Plank’s constant
E = hν = ν = frequency
λ c = speed of light
λ = wavelength
 The Electromagnetic Spectrum

Wavelength ( λ, cm)
10-11 10-9 10-6 10-5 10-4 10-2 102

γ x-ray UV visible IR µ Rf

1021 1019 1016 1015 1014 1012 108

Frequency (ν, Hz)
Inner shell Outer shell Molecular Molecular Nuclear
Nuclear
electrons electrons vibrations rotation Spin
 Visible spectrum

Wavelength (nm)
390 450 520 590 620 780

←UV ←Increasing Energy IR →

Increasing Wavelength→

“Red-Orange-Yellow-Green-Blue”
 Absorption of EM radiation

I0 (radiant intensity) I (transmitted intensity)

I N
dI dI I
dn
= −kI 0 ; ∫I I = −k ∫0 dn ; ln I 0 = −kN ; log T = −k ′bc ; A = abc
0
 Manipulation of Beer’s Law

1 100
A = abc = − log T = log = log
T %T
100
log = log(100) − log(%T ) = 2 − log(%T )
%T
∴ A = 2 − log(%T )
and , %T = 10 ( 2− A)

Hence, 50% transmittance results in an absorbance of 0.301, and

an absorbance of 2.0 corresponds to 1% transmittance
 Beer’s Law error in
Error (dA/A) → measurement

0.0 0.434 2.0

Absorbance →
 Design of spectrometric
methods
• The analyte absorbs at a unique
wavelength (not very common)
• The analyte reacts with a reagent to
produce an adduct that absorbs at a
unique wavelength (a chromophore)
• The analyte is involved in a reaction
that produces a chromophore
 Measuring total protein
• All proteins are composed of 20 (or so) amino
acids.
• There are several analytical methods for
measuring proteins:
– Kjeldahl’s method (reference)
– Direct photometry
– Folin-Ciocalteu (Lowery) method
– Dye-binding methods (Amido black; Coomassie
Brilliant Blue; Silver)
– Precipitation with sulfosalicylic acid or
trichloracetic acid (TCA)
– Biuret method
Kjeldahl’s method

Specimen
Hot H2SO4 digestion
Correction for non-protein nitrogen

NH4+
Titration or Nessler’s
reagent (KI/HgCl2/KOH)

Protein nitrogen
Multiply by 6.25 (100%/16%)

Total protein
 Direct photometry
H2 H
C COOH H2 H
C C COOH
C
NH2
OH CH NH2
HN

Tyrosine Tryptophan

λmax= 280 nm

• Absorption at 200–225 nm can also be

used (λmax for peptide bonds)
• Free Tyr and Trp, uric acid, and bilirubin
interfere at 280 nm
Folin-Ciocalteu (Lowry) method

Protein
Phosphotungstic/phosphomolybdic acid Reduced form (blue)
(Tyr, Trp)

• Sometimes used in combination with

biuret method
• 100 times more sensitive than biuret
alone
• Typically requires some purification,
due to interferences
 Biuret method
H
H2N N NH2

Cu++
O O
or . . . O Blue adduct (λ = 540 nm)
-
OH
H H
N C C
C N
H

• Sodium potassium tartrate is added to

complex and stabilize the Cu++ (cupric)
ions
• Iodide is added as an antioxidant
 Measuring albumin
• Albumin is the most abundant protein in serum (40-
60% of total protein)
• Albumin is an anionic protein (pI=4.0-5.8)
– Enriched in Asp, Glu
∀ ∴ Albumin reacts with anionic dyes
– BCG (λmax= 628 nm), BCP (λmax= 603 nm)
• Binding of BCG and BCP is not specific, since
other proteins have Asp and Glu residues
– Reading absorbance within 30 s improves specificity
Specificity of bromocresol dyes

BCG (pH 4.2)

Albumin green or purple adduct
BCP (pH 5.2)
Absorbance →

30 s
Time →
 Measuring glucose
Glucose
Glucose + O2 oxidase Gluconic acid + H2O2
Peroxidase
o-Dianiside
Oxidized o-dianiside
λmax= 400–540 (pH-dependant)

• Glucose is highly specific for β-D-Glucose

• The peroxidase step is subject to interferences
from several endogeneous substances
– Uric acid in urine can produce falsely low results
– Potassium ferrocyanide reduces bilirubin
interference
• About a fourth of clinical laboratories use the
glucose oxidase method
 Glucose isomers
CH2OH CH2OH

H O H H O OH

OH H OH H

OH OH OH H

H OH H OH

α-D-glucose (36%) β-D-glucose (64%)

• The interconversion of the α and β isomers of

glucose is spontaneous, but slow
• Mutorotase is added to glucose oxidase
reagent systems to accelerate the
interconversion
 Measuring creatinine
H
N
O- -
O NH
NH
O2 N NO2 O2N
H3C OH-
NH + CH3
-
O NO2

O
NO2

Creatinine Picric acid O2N

Janovski complex
λmax= 485 nm

• The reaction of creatinine and alkaline picrate

was described in 1886 by Max Eduard Jaffe
• Many other compounds also react with picrate
 Modifications of the Jaffe
method

• Fuller’s Earth (aluminum silicate, Lloyd’s

reagent)
– adsorbs creatinine to eliminate protein interference
• Acid blanking
– after color development; dissociates Janovsky
complex
• Pre-oxidation
– addition of ferricyanide oxidizes bilirubin
• Kinetic methods
 Absorbance (λ = 520 nm)

0
Fast-reacting
(pyruvate, glucose,
ascorbate)

20
∆t
∆A

∆t
= rate

Time (sec) →
creatinine (and α-keto acids)
∆A

80
Kinetic Jaffe method

Slow-reacting
(protein)
 Enzymatic creatinine method
CH3 CH3
H COOH
Creatinine N-Methylhydantoin
N N N
iminohydrolase amidohydrolase
H
NH O N
C
N N O CH3
O H O H
Creatinine N-Methylhydantoin N-Carbamoylsarcosine

H COOH N-Carbamoylsarcosine Sarcosine

N amidohydrolase H oxidase H2N COOH
H N COOH C + CH2O
C N H3C H2
O CH3 H2O H2O2
NH3 + CO2
N-Carbamoylsarcosine Sarcosine Glycine

• H2O2 is measured by conventional

peroxidase/dye methods
 Enzymatic creatinine method
CH3 CH3
Creatinine Creatine
N H
amidohydrolase N NH amidohydrolase
N COOH
NH H3C
N COOH NH2
O H
Urea
Creatinine Creatine Sarcosine

Sarcosine
H H2N COOH
N oxidase C + CH2O
COOH
H3C H2
Sarcosine O2 H2O2 Glycine

• H2O2 is measured by conventional

peroxidase/dye methods
 Measuring urea (direct
method)
O
O O
O
CH3
+
H CH3 H+, ∆
+ N N
H3C H3C
H2N NH2
NOH O
H3C CH3
Diacetyl monoxime Diacetyl Urea Diazone
λmax= 540 nm

• Direct methods measure a chromagen

produced directly from urea
• Indirect methods measure ammonia,
produced from urea
 Measuring urea (indirect
method)
OH

H2N NH2 N
-
Urease OH
2 NH4+ +

O -
O O
Urea Phenol Indophenol
λmax = 560 nm

• The second step is called the Berthelot reaction

• In the U.S., urea is customarily reported as “Blood
Urea Nitrogen” (BUN), even though . . .
– It is not measured in blood (it is measured in serum)
– Urea is measured, not nitrogen
 Conversion of urea/BUN

28 mg N / mmol urea
BUN ( mg / dL ) = urea (mg / L) × × 0.1 dL / L
60 mg urea / mmol
60 mg urea / mmol
Urea ( mg / L ) = BUN (mg / dL) × × 10 L / dL
28 mg N / mmol urea
 Measuring uric acid
O
Phosphotungstic acid Tungsten blue O H
N N
HN H2N
O- O

O N N O2 H2O2 N
H O N H
H H
Uric Acid Allantoin

• Tungsten blue absorbs at λ = 650-700 nm

• Uricase enzyme catalyzes the same reaction,
and is more specific
– Absorbance of uric acid at λ ∼ 585 nm is monitored
• Methods based on measurement of H2O2 are
common
 Measuring total calcium
O CH3 CH3 O
AsO3H2 H2O3As HO OH
OH OH -
O O
-

N N N N
N N
O
- -
O 3S SO3 -
O O O O-
O

Arsenazo III o-Cresolphthalein complexone

λmax= 650 nm λmax= 570 - 580 nm

• More than 90% of laboratories use one or the

other of these methods.
• Specimens are acidified to release Ca++ from
protein, but the CPC-Ca++ complex forms at
alkaline pH
 Measuring phosphate

H+ Red. Molybdenum
H3PO4 + (NH4)6Mo7O24 (NH4)3[PO4(MoO3)12]
blue
λmax= 340 nm λmax= 600-700 nm

• Phosphate in serum occurs in two

forms:
– H2PO4- and HPO4-2
• Only inorganic phosphate is measured
by this method. Organic phosphate is
primarily intracellular.
 Measuring magnesium
H3C CH3 H3C CH3
O O
OH
HO O
-
O O-
N
N N
-
N SO3

H3C CH3 CH3

- - -
O O SO3 O O
HO

Calmagite Methylthymol blue

λmax= 530 - 550 nm λmax= 600 nm

• Formazan dye and Xylidyl blue (Magnon) are also

used to complex magnesium
• 27Mg neutron activation is the definitive method,
but atomic absorption is used as a reference
 Measuring iron
Bathophenanthroline Ferrozine SO3Na

SO3H

N N N N

Fe++ Fe++

λ max= 534 nm λ max= 562 nm

• The specimen is acidified to release iron

from transferrin and reduce Fe3+ to Fe2+
(ferrous ion)
 Measuring bilirubin
OH

O
Azobilirubin (Isomer II)
O OH
HO3S N N
HO N N
O HO3S N N+Cl- H H
O
Diazotized sulfanilic acid
O N N N N HO
H H H H Azobilirubin (Isomer I)
Bilirubin (unconjugated)
O N N SO3H
N N
H H

• Diazo reaction with bilirubin was first described by

Erlich in 1883
• Azobilirubin isomers absorb at 600 nm
 Evolution of the diazo method

• 1916: van den Bergh and Muller discover that

alcohol accelerates the reaction, and coined the
terms “direct” and “indirect” bilirubin
• 1938: Jendrassik and Grof add caffeine and
sodium benzoate as accelerators
– Presumably, the caffeine and benzoate displace un-
conjugated bilirubin from albumin
• The Jendrassik/Grof method was later modified by
Doumas, and is in common use today
 Bilirubin sub-forms

• HPLC analysis has demonstrated at least 4

distinct forms of bilirubin in serum:
α-bilirubin is the un-conjugated form (27% of total
bilirubin)
β-bilirubin is mono-conjugated with glucuronic acid
(24%)
γ-bilirubin is di-conjugated with glucuronic acid (13%)
δ-bilirubin is irreversibly bound to protein (37%)
• Only the β, γ, and δ fractions are soluble in water, and
therefore correspond to the direct fraction
∀ α-bilirubin is solubilized by alcohols, and is present, along
with all of the other sub-forms, in the indirect fraction
 Measuring cholesterol by L-B

L-B reagent
H2SO4/HOAc
HO HOO2S

Cholesterol Cholestahexaene sulfonic acid

λmax = 620 nm

• The Liebermann-Burchard method is used by

the CDC to establish reference materials
• Cholesterol esters are hydrolyzed and
extracted into hexane prior to the L-B reaction
 Enzymatic cholesterol
methods
Cholesterol esters
Cholesteryl
ester
hydroxylase Cholesterol
Cholesterol
oxidase
Choles-4-en-3-one + H2O2
Phenol
4-aminoantipyrine
Quinoneimine dye (λmax∼500 nm) Peroxidase

• Enzymatic methods are most commonly

adapted to automated chemistry analyzers
• The reaction is not entirely specific for
cholesterol, but interferences in serum are
minimal
 Measuring HDL cholesterol
• Ultracentrifugation is the most accurate method
– HDL has density 1.063 – 1.21 g/mL
• Routine methods precipitate Apo-B-100
lipoprotein with a polyanion/divalent cation
– Includes VLDL, IDL, Lp(a), LDL, and chylomicrons

Dextran sulfate
HDL, IDL, LDL, VLDL HDL + (IDL, LDL, VLDL)↓
Mg ++

• Newer automated methods use a modified form of

cholesterol esterase, which selectively reacts with
HDL cholesterol
 Measuring triglycerides
Triglycerides
Lipase
Glycerol + FFAs
Glycerokinase
ATP Glycerophosphate + ADP
Glycerophasphate
oxidase
Dihydroxyacetone + H2O2
Peroxidase
Quinoneimine dye (λmax ∼500 nm)

• LDL is often estimated based on

triglyceride concentration, using the
Friedwald Equation:
[LDL chol] = [Total chol] – [HDL chol] – [Triglyceride]/5
 Spectrophotometric methods
involving enzymes

• Often, enzymes are used to facilitate a

direct measurement (cholesterol,
triglycerides)
• Enzymes may be used to indirectly
measure the concentration of a substrate
(glucose, uric acid, creatinine)
• Some analytical methods are designed to
measure clinically important enzymes
 Enzyme kinetics

k1 k2
E+S ES E+P
k-1

The Km (Michaelis constant)

for an enzyme reaction is a
Km =
[ E ][ S ]
measure of the affinity of
substrate for the enzyme. [ ES ]
Km is a thermodynamic
quantity, and has nothing to
[ E ] = [ Etotal ] − [ ES ]
do with the rate of the
( [ Etot ] − [ ES ] )[ S ] k −1
enzyme-catalyzed reaction.
∴ Km = =
[ ES ] k1
 Enzyme kinetics

k1 k2
E+S ES E+P
k-1

v = k 2 × [ ES ]

substituting for [ ES ] , v = k 2 ×
[ Etot ] × [ S ]
Km + [S]
when enzyme is saturated , [ Etot ] = [ ES ] , and v = Vmax
Vmax × [ S ]
so, v =
Km + [S]
 The Michaelis-Menton
equation
Vmax × [ S ]
rearranging v =
Km + [S]
Vmax − v K m
we get = ( Michaelis − Menton)
v [S]
Vmax × [ S ]
or , taking the reciprocal of v =
Km + [S]

1  Km 1  1
we get =  ×  + ( Lineweaver − Burk )
v  Vmax [ S ]  Vmax

The Lineweaver-Burk equation is of the form y = ax + b, so a plot

of 1/v versus 1/[S] gives a straight line, from which Km and Vmax can
be derived.
 The Michaelis-Menton curve

Vmax

Vmax × [ S ]
v=
Km + [S]
v→

½Vmax
Vmax
when v = , Km = [S]
2

Km [S] →
 The Lineweaver-Burk plot

1  Km 1  1
=  ×  +
v  Vmax [ S ]  Vmax

1/v →
1/Vmax

-1/Km 1/[S] →
 Enzyme inhibition

• Competitive inhibitors compete with the

substrate for the enzyme active site (Km)
• Non-competitive inhibitors alter the ability of
the enzyme to convert substrate to product
(Vmax)
• Un-competitive inhibitors affect both the
enzyme substrate complex and conversion of
substrate to product (both Km and Vmax)
 M-M analysis of an enzyme
inhibitor
Vmax
Vmax(i)
v→

Non-competitive

Km [S] →
 Competitive

Km(i)
L-B analysis of an enzyme
inhibitor
Non-competitive
Competitive

1/v →

1/Vmax

-1/Km 1/[S] →
Measuring enzyme-catalyzed
reactions
Enzyme
Substrate Product
Cofactor Cofactor*

• The progress of an enzyme-catalyzed

reaction can be followed by measuring:
– The disappearance of substrate
– The appearance of product
– The conversion of a cofactor
 Measuring enzyme-catalyzed
reactions
Enzyme
Substrate Product
Cofactor Cofactor*

• When the substrate is in excess, the rate of the

reaction depends on the enzyme activity
• When the enzyme is in excess, the rate of the
reaction depends on the substrate concentration
 Enzyme cofactors
O

NH2
O
-
O O
P CH2 N+
NH2 O
O H H
-
O O H
N H
P OH OH
N
O
N N H2C

OH OH
O
H
H

Nicotinamide adenine dinucleotide (NAD+, oxidized form)

 NAD UV absorption spectra

NAD+
NADH
Absorbance →

λmax= 340 nm

250 300 350 400

λ (nm)
 Enzyme reaction profile

Linear phase

Substrate depletion
Product →

Lag phase

∆A
∝ [ ES ]
∆t

Time →
Mix
 Measuring glucose by
hexokinase
Glucose-6-phosphate
Hexokinase dehydrogenase
Glucose Glucose-6-phosphate 6-Phosphogluconate

ATP ADP NAD+ NADH

• The hexokinase method is used in about half

of all clinical laboratories
• Some hexokinase methods use NADP,
depending on the source of G-6-PD enzyme
• A reference method has been developed for
glucose based on the hexokinase reaction
 Measuring bicarbonate
O-
-
P O
PEP O COO- Malate HO COO-
O O carboxylase dehydrogenase
C CH
C
+ O
- H 2C H2C
HO O -
C COO + COO-
NADH NAD
Bicarbonate H2C COO- Oxaloacetate Malate

Phosphoenolpyruvate

• The specimen is alkalinized to convert all forms

of CO2 to HCO3-, so the method actually
measures total CO2
• Enzymatic methods for total CO2 are most
common, followed by electrode methods
 Measuring lactate
dehydrogenase
O Lactate OH
dehydrogenase
O- O-
H 3C H3C

O NADH NAD+ O
Pyruvate Lactate

• Both P→L and L→P methods are available

– At physiological pH, P→L reaction if favored
– L→P reaction requires pH of 8.8-9.8
• LD (sometimes designated LDH) activity will
vary, depending on which method is used
 Measuring creatine kinase
(CK)
O
H
HN NH2 HN N P O-
Creatine kinase
O
N N
H3C CH2 H3C CH2
ATP ADP
COO- COO-
Creatine Phosphocreatine

• Both creatine and phosphocreatine spontaneously

hydrolyze to creatinine
• The reverse (PCr→Cr) reaction is favorable,
although the reagents are more expensive
• All methods involve measurement of ATP or ADP
 Measuring creatine kinase
CK
pH 6.7
Creatine phosphate Creatine

ADP ATP ADP

G-6-PDH
Glucose Glucose-6-phosphate 6-Phosphogluconate
HK
NADP+ NADPH

• Potential sources of interferences include:

– Glutathione (Glutathione reductase also uses
NADPH as a cofactor)
– Adenosine kinase phosphorylates ADP to ATP
(fluoride ion inhibits AK activity
– Calcium ion may inhibit CK activity, since the
enzyme is Mg++-dependent.
 Measuring creatine kinase
CK
pH 9.0
Creatine Creatine phosphate

ATP ADP ATP

LD
Phosphoenolpyruvate Pyruvate Lactate
PK
NADH NAD+

• Since the forward (Cr →PCr) reaction is

slower, the method is not sensitive
• Luminescent methods have been
developed, linking ATP to luciferin
activation
Measuring alkaline phosphatase
O O-
N+ p-Nitrophenoxide

O- -
O O O-
N+ N+

Alkaline phosphatase
pH 10.3, Mg++

O P O H2O PI
O- O
-
O
Benzoid Quinonoid
p-Nitrophenol (colorless) (λmax= 404 nm)
phosphate

• The natural substrate for ALKP is not

known
 Measuring transaminase enzymes
O COO-

H2N CH C OH C O

CH3 CH2
-
COO COO-
L-Aspartate COO-
C O Aspartate HC NH2
O transaminase Oxaloacetate
+ CH2 + CH2
H2N CH C OH Alanine -
COO
CH2 transaminase CH2
CH2
C O
COO- COO-
C O
CH3 L-Glutamate
2-Oxyglutarate
OH Pyruvate
L-Alanine

• Pyridoxyl-5-phosphate is a required cofactor

• Oxaloacetate and pyruvate are measured with their
corresponding dehydrogenase enzymes, MD and LD
 Measuring gamma glutamyl transferase

H COOH
O
O N NO2
C HN CH2
C CH2 γ-Glutamyl
transferase CH2 C O
CH2 NH pH 8.2
NO2 + + CH2 NH
CH2 C O
HC NH2 CH2
HC NH2 CH2
NH2
COOH COOH
COOH NH2

γ-glutamyl-p-nitroanalide Glycylglycine p-Nitroanaline γ-Glutamylglycylglycine

λmax= 405 nm

• Method described by Szasz in 1969,

and modified by Rosalki and Tarlow
 Measuring amylase
CH2OH CH2OH

O O
α-Amylase
OH OH Glucose, Maltose
Ca++
O
α(1→4)
OH OH
α-Amylose

• Hydrolysis of both α(1→4) and α(1 →6)

linkages occur, but at different rates.
• Hence, the amylase activity measured will
depend on the selected substrate
• There are more approaches to measuring
amylase than virtually any other common
clinical analyte
 Amyloclastic amylase method
Amylase
Starch + I2 Blue complex Red complex

• The rate of disappearance of the blue

complex is proportional to amylase activity
• Starch also can be measured
turbidimetrically
• Starch-based methods for amylase
measurement are not very common any
more
 Saccharogenic amylase
method
Amylase
Starch Glucose + Maltose Reduced substrate

• Several methods can be used to quantify the

reducing sugars liberated from starch
• Somogyi described a saccharogenic amylase
method, and defined the units of activity in
terms of “reducing equivalents of glucose”
• Alternatively, glucose or maltose can be
measured by conventional enzymatic methods
Chromogenic amylase method
Amylase
Dye-labeled starch Small dye-labeled fragments
Separation
step
Photometric measurement of dye

• J&J Vitros application allows small dye-

labeled fragments to diffuse through a
filter layer
• Abbott FP method uses fluorescein-
labeled starch
 Defined-substrate amylase
method
Amylase
4-NP-(Glucose)7 4-NP-(Glucose)4,3,2
α-Glucosidase

4-NP-(Glucose)4 + Glucose + NP
λmax= 405 nm

∀ α-Glucosidase does not react with

oligosaccharides containing more than 4
glucose residues
• A modification of this approach uses β-2-chloro-
4-NP, which has a higher molar absorptivity
than 4-NP
 Measuring lipase (direct)
H2C OFA H2C OH H2C OH H2C OH
Lipase Lipase Lipase
HC OFA HC OFA HC OH HC OH

H2C OFA H2C OFA H2C OFA H2C OH

FA FA FA
Triglyceride α,β-Diglyceride α-Monoglyceride Glycerol

• The Cherry/Crandall procedure involves lipase

degradation of olive oil and measurement of liberated
fatty acids by titration
• Alternatively, the decrease in turbidity of a triglyceride
emulsion can be monitored
• For full activity and specificity, addition of the coenzyme
colipase is required
 Measuring lipase (indirect)

• Indirect methods for lipase

measurement focus on:
– Enzymatic phosphorylation (Glycerol
kinase) and oxidation (L-α-
Glycerophosphate oxidase) of glycerol, and
measurement of liberated H2O2
– Dye-labeled diglyceride that releases a
chromophore when hydrolyzed by lipase
• Several non-triglyceride substrates
have been proposed, as well
 Post-test
Identify the methods proposed by the following:

• Folin-Wu Glucose
• Jendrassik-Grof Bilirubin
• Somogyi-Nelson Glucose/Amylase
• Kjeldahl Total protein
• Lieberman-Bourchard Cholesterol
• Rosalki-Tarlow GGT
• Jaffe Creatinine
• Bertholet Urea
• Fisk-Subbarrow Phosphate