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
E
A

Velocity = c

Wavelength ()

Wavelength, frequency, and energy

hc
E h

E = energy
h = Planks constant
= frequency
c = speed of light
= wavelength

The Electromagnetic Spectrum

Wavelength (, cm)
10-11

10-9

10-6 10-5 10-4

10-2

102

x-ray

UV visible IR

Rf

1021

1019

1016 1015 1014

1012

108

Frequency (, Hz)
Nuclear

Inner shell
electrons

Outer shell
electrons

Molecular Molecular
vibrations rotation

Nuclear
Spin

Visible spectrum
390

UV

450

Wavelength (nm)
520
590

620

Increasing Energy
Increasing Wavelength

Red-Orange-Yellow-Green-Blue

780

IR

Molecular orbital energies

* or *
molecular
orbital

Energy

s or p
atomic
orbital

n*
n *

non-bonding
orbital
or
molecular
orbital

n
n

*
*

Molecular electronic energy

transitions
Singlet
E4
E3
E2

Triplet

VR

E1

IC

10-6-10-9 sec

P
E0

10-4-10 sec

Absorption of EM radiation

I0 (radiant intensity)
dI
kI 0 ;
dn

I (transmitted intensity)

dI
I
I I k 0 dn ; ln I 0 kN ; log T k bc ; A abc
0

Manipulation of Beers 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

Error (dA/A)

Beers Law error in measurement

0.0

0.434
Absorbance

2.0

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:

Kjeldahls 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

Kjeldahls method
Specimen
Hot H2SO4 digestion
Correction for non-protein nitrogen

NH4+
Titration or Nesslers
reagent (KI/HgCl2/KOH)

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

Total protein

Direct photometry
H2
C

H2
C

COOH
C

COOH
C

NH2

OH

HN

Tyrosine

CH

NH2

Tryptophan

max= 280 nm

Absorption at 200225 nm can also be used

(max for peptide bonds)
Free Tyr and Trp, uric acid, and bilirubin
interfere at 280 nm

Folin-Ciocalteu (Lowry) method

Phosphotungstic/phosphomolybdic acid

Protein
(Tyr, Trp)

Reduced form (blue)

Sometimes used in combination with biuret

method
100 times more sensitive than biuret alone
Typically requires some purification, due to
interferences

Biuret method
H
N

H2N
O

NH2
O

or . . .

H
N

OH

H
C
C

Cu++
-

Blue adduct ( = 540 nm)

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 (max= 628 nm), BCP (max= 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

BCP (pH 5.2)

green or purple adduct

Absorbance

Albumin

BCG (pH 4.2)

30 s

Time

Measuring glucose
Glucose + O2

Glucose
oxidase

Gluconic acid + H2O2

Oxidized o-dianiside
max= 400540 (pH-dependant)

Peroxidase
o-Dianiside

Glucose is highly specific for -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
O

H
OH

OH
OH

OH
H

OH

-D-glucose (36%)

OH

H
H

OH
H

OH

-D-glucose (64%)

The interconversion of the and isomers of

glucose is spontaneous, but slow
Mutorotase is added to glucose oxidase reagent
systems to accelerate the interconversion

Measuring creatinine
O-

NH
O2 N
H3C

NH

NO2

Creatinine

NH

O2N

OH-

CH3
-

H
N

NO2

NO2

Picric acid

O2N

Janovski complex
max= 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
Fullers Earth (aluminum silicate, Lloyds reagent)
adsorbs creatinine to eliminate protein interference

Acid blanking
after color development; dissociates Janovsky complex

Pre-oxidation
addition of ferricyanide oxidizes bilirubin

Kinetic methods

Fast-reacting
(pyruvate, glucose,
ascorbate)

Absorbance ( = 520 nm)

0
A

creatinine (and -keto acids)

20
Time (sec)
80

Slow-reacting
(protein)

Kinetic Jaffe method

A
rate
t

Enzymatic creatinine method

CH3

CH3
Creatinine
iminohydrolase

N
NH

COOH
N
C

H
N

H
N

Sarcosine
oxidase

H2O

CH3
NH3 + CO2

N-Carbamoylsarcosine

COOH

H3C

N-Carbamoylsarcosine

CH3

N-Methylhydantoin
N-Carbamoylsarcosine
amidohydrolase

H
N

N
H

Creatinine
H

N
H

COOH

N-Methylhydantoin
amidohydrolase

H2O2

Sarcosine

H2O2 is measured by conventional

peroxidase/dye methods

H2N

COOH
C
H2

Glycine

+ CH2O

Enzymatic creatinine method

CH3

CH3

Creatinine
amidohydrolase

Creatine
NH amidohydrolase

NH
O

H3C

N
H

COOH

COOH NH2

Creatinine

H3C

H
N

Urea

Creatine
H
N

COOH

Sarcosine

Sarcosine
oxidase
O2

H2N

H2O2

C
H2

COOH

Sarcosine

+ CH2O

Glycine

H2O2 is measured by conventional

peroxidase/dye methods

Measuring urea (direct method)

O
O

H3C

CH3
NOH

Diacetyl monoxime

CH3

H3C

+
H2N

NH2

Diacetyl

H+,

H3C

Urea

CH3

Diazone
max= 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

Urease

OH

2 NH4+ +

O
Urea

Phenol

O
Indophenol

max = 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
BUN mg / dL urea (mg / L)

28 mg N / mmol urea
0.1 dL / L
60 mg urea / mmol

Urea mg / L BUN (mg / dL)

60 mg urea / mmol
10 L / dL
28 mg N / mmol urea

Measuring uric acid

O
N

HN
O

Phosphotungstic acid Tungsten blue

H2N

ON
H

N
H

Uric Acid

O2

H2O2

H
N
O

N
H

N
H

Allantoin

Tungsten blue absorbs at = 650-700 nm

Uricase enzyme catalyzes the same reaction, and is
more specific
Absorbance of uric acid at 585 nm is monitored

Methods based on measurement of H2O2 are common

Measuring total calcium

O
AsO3H2

OH

OH

H2O3As

CH3
HO

CH3

O
OH

N
O

O 3S

SO3

O
O

Arsenazo III
max= 650 nm

o-Cresolphthalein complexone
max= 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
H3PO4 + (NH4)6Mo7O24

H+

(NH4)3[PO4(MoO3)12]

max= 340 nm

Red. Molybdenum

blue

max= 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

OH
HO

O
N

SO3

H3C
HO

Calmagite
max= 530 - 550 nm

CH3

CH3
SO3-

Methylthymol blue
max= 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 method

Measuring iron
Bathophenanthroline

Ferrozine

SO3Na

SO3H
N

Fe++

Fe++

max= 534 nm

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

HO

HO3S

N+Cl-

HO3S

N
H

N
H

O
O

N
H

N
H

N
H

Bilirubin (unconjugated)

N
H

Diazotized sulfanilic acid

HO

N
H

Azobilirubin (Isomer I)
N
H

SO3H

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:
-bilirubin is the un-conjugated form (27% of total bilirubin)
-bilirubin is mono-conjugated with glucuronic acid (24%)
-bilirubin is di-conjugated with glucuronic acid (13%)
-bilirubin is irreversibly bound to protein (37%)

Only the , , and fractions are soluble in water, and

therefore correspond to the direct fraction
-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

max = 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

Quinoneimine dye (max500 nm)

Phenol
4-aminoantipyrine
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
HDL, IDL, LDL, VLDL

Dextran sulfate
Mg

++

HDL + (IDL, LDL, VLDL)

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

Quinoneimine dye (max 500 nm)

Peroxidase

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
E+S
The Km (Michaelis constant)
for an enzyme reaction is a
measure of the affinity of
substrate for the enzyme.
Km is a thermodynamic
quantity, and has nothing to
do with the rate of the
enzyme-catalyzed reaction.

k1
k-1

ES

k2

E+P

E S
Km
ES
E Etotal ES
Etot ES S k 1
Km

ES
k1

Enzyme kinetics
E+S

k1
k-1

ES

k2

E+P

v k 2 ES

Etot S
substituting for ES , v k 2
Km S
when enzyme is saturated , Etot ES , and v Vmax
Vmax S
so, v
Km S

The Michaelis-Menton equation

rearrangin g v
Vmax v K m
we get

v
S

Vmax S
Km S

Michaelis Menton

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

Vmax

Vmax
when v
, Km S
2

Km

[S]

1 Km
1
1


v Vmax S Vmax

1/v

The Lineweaver-Burk plot

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)

Competitive
Non-competitive
Km Km(i)

[S]

L-B analysis of an enzyme

inhibitor
1/v

Non-competitive
Competitive

1/Vmax

-1/Km

1/[S]

Measuring enzyme-catalyzed
reactions
Substrate

Enzyme

Cofactor

Product
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
Substrate

Enzyme

Cofactor

Product
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
P

NH2
-

O
O
O

O
P

CH2
H
H
OH

N+
H
H
OH

O
N

H2C

N
OH
H

OH
H

Nicotinamide adenine dinucleotide (NAD+, oxidized form)

Enzyme cofactors
H

NH2
-

O
P

NH2
-

O
O
O

O
P

CH2
H

H
OH

N
H
H
OH

O
N

H2C

N
OH
H

OH
H

Phosphate attachment
(NADP+ and NADPH)

NADH (reduced form)

NAD UV absorption spectra

NAD+
Absorbance

NADH
max= 340 nm

250

300

(nm)

350

400

Enzyme reaction profile

A
ES
t
Mix

Time

Substrate depletion

Lag phase

Product

Linear phase

Measuring glucose by
hexokinase
Glucose
ATP

Hexokinase

Glucose-6-phosphate
ADP

Glucose-6-phosphate
dehydrogenase

NAD+

6-Phosphogluconate

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-

O
HO

PEP
carboxylase

Bicarbonate

H2C

H2C
COO-

COO-

COO

Oxaloacetate

Malate
dehydrogenase

HO

CH

H2C
NADH

NAD

COO-

COO-

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
H3C
O
Pyruvate

O-

OH

Lactate
dehydrogenase

O-

H3C
NADH

NAD+

O
Lactate

Both PL and LP methods are available

At physiological pH, PL reaction if favored
LP 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
HN

NH2

H
N

HN
Creatine kinase

O-

N
H3C

N
CH2
COO-

Creatine

ATP

ADP

H3C

CH2
COO-

Phosphocreatine

Both creatine and phosphocreatine spontaneously

hydrolyze to creatinine
The reverse (PCrCr) 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
ADP

Creatine
ATP

ADP

Glucose-6-phosphate

Glucose
HK

G-6-PDH

NADP+

6-Phosphogluconate

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
ATP

Creatine phosphate
ADP

ATP

Pyruvate

Phosphoenolpyruvate
PK

NADH

LD

Lactate
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

N+

O-

p-Nitrophenoxide
O

N+

O-

N+

O-

Alkaline phosphatase
pH 10.3, Mg++
O
O

P
O

O
-

p-Nitrophenol
phosphate

H2O

PI
O-

Benzoid
(colorless)

Quinonoid
(max= 404 nm)

The natural substrate for ALKP is not known

Measuring transaminase enzymes

COO-

O
H2N

CH

OH

CH3

L-Aspartate
O
H2N

CH

OH

CH2

COO-

COOAspartate
transaminase
Alanine
transaminase

COO-

2-Oxyglutarate

HC

Oxaloacetate
+

CH2
CH2

CH2
C

OH

COO

COO

NH2

CH2
CH2

CH3

COO-

L-Glutamate

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
COOH

H
N

O
C

CH2

CH2

NH
NO2 +

CH2
HC

NO2

NH2

COOH

-glutamyl-p-nitroanalide

-Glutamyl
transferase
pH 8.2

CH2

NH2

p-Nitroanaline
max= 405 nm

HN

CH2

CH2

CH2

NH

HC

NH2

Glycylglycine

NH2

COOH

CH2
COOH

-Glutamylglycylglycine

Method described by Szasz in 1969, and

modified by Rosalki and Tarlow

Measuring amylase
CH2OH

CH2OH

-Amylase

OH

OH

Ca++

OH

(14)

Glucose, Maltose

OH

-Amylose

Hydrolysis of both (14) and (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

Starch + I2

Blue complex

Amylase

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

Starch

Amylase

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

Dye-labeled starch

Amylase

Small dye-labeled fragments

Photometric measurement of dye

Separation
step

J&J Vitros application allows small dyelabeled fragments to diffuse through a filter
layer
Abbott FP method uses fluorescein-labeled
starch

Defined-substrate amylase method

4-NP-(Glucose)7

Amylase

4-NP-(Glucose)4,3,2
-Glucosidase

4-NP-(Glucose)4 + Glucose + NP

max= 405 nm

-Glucosidase does not react with oligosaccharides

containing more than 4 glucose residues
A modification of this approach uses -2-chloro-4NP, which has a higher molar absorptivity than 4-NP

Measuring lipase (direct)

H2C

OFA

HC

OFA

H2C

OFA

Lipase

Triglyceride

FA

H2C

OH
Lipase

HC

OFA

H2C

OFA

,-Diglyceride

FA

H2C

OH

HC

OH

H2C

Lipase

OFA

-Monoglyceride

FA

H2C

OH

HC

OH

H2C

OH

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--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
Jendrassik-Grof
Somogyi-Nelson
Kjeldahl
Lieberman-Bourchard
Rosalki-Tarlow
Jaffe
Bertholet
Fisk-Subbarrow

Glucose
Bilirubin
Glucose/Amylase
Total protein
Cholesterol
GGT
Creatinine
Urea
Phosphate