Basic Techniques In Clinical

Chemistry Laboratory

临床生物化学基本技术
Computerization and the use of automated methods of
analysis allow a high degree of productivity and improve the
quality of services.

Understanding the basic principles of techniques and the

theory of instrumental analysis will provide a working
knowledge of instruments, applications to patients testing in
the clinical chemistry laboratory.
A variety of techniques have been used in clinical chemistry
laboratory for sample testing
Most Fundamental Methods Include:
Spectrophotometry 分光光度技术
Nephelometry 散射比浊法
Turbidimetry 比浊法
Fluorometry 荧光测定法
Electrophoresis 电泳
Chromatography 层析法
Mass spectrometry 质谱法
Biochip(Protein and DNA Chip/Array) 生物芯片
Biosensor 生物传感技术
 Photometry

Photometry 光 度 测 定 is defined as the

measurement of the luminous intensity of
light or the amount of luminous light falling
on a surface from such a source.
 Photometry

Many determinations made in the clinical

laborotory are based on radiant energy emitted,
transmitted, absorbed, or reflected under
controlled conditions. The principles invovled in
such measurements are considered in the
following spectrophotometry, nephelometry,
turbidimetry, fluorometry.
 SPECTROPHOTOMETRY
分光光度技术

The absorbance吸光率of a solution is the amount

of light absorbed by that solution. According to
Beer’s Law the absorbance(A) varies directly with the
concentration of the solution(c) in question. It is
equals to the concentration of a substance in solution
multiplied by the length of the path(b) that the light
must pass through the solution, multiplied by the
molar absorptivity摩尔吸收率(a)of the substance of
interest. A=abc(formula)
Diagram of internal parts of a
spectrophotometer
Schematic diagram of
Spectrophotometer

In practice, a beam of
light is passed through a
monochromator 单色光仪
that provides selection of
the desired region of the
spectrum to be used for
measurements.
The light next passes through
an absorbance cell, where a
portion of the radiant energy
is absorbed, depending on
the nature and the
concentration of the solution.
Any light not absorbed is
transmitted to a photo-
detector which converts light
energy to electrical energy
that can be registered on a
galvanometer.
 In operation, an opaque block is substituted for
the cuvette, so that no light reaches the photocell,
and the meter is adjusted to read
0%T(transmittance). Next, a cuvette containing a
reagent blank 空 白 is inserted and the meter is
adjusted to read 100%T (i.e., zero absorbance).
Calibrating solutions containing various known
concentrations of the substance are inserted,
and readings are recorded. Finally a reading is
made of the unknown solution, and its
concentration is determined by comparison with
the readings obtained on the calibrator.
Determination of the concentration of
the unknown using the standard curve
 TURBIDIMETRY

Turbidity causes the decrease of the

intensity of the incident beam of light入射光
束as it passes through a solution of particles.
The measurement of this decrease in
intensity of the incident light beam that is
caused by scattering, reflectance反射, and
absorption of the light is called turbidimetry
 NEPHELOMETRY

Nephelometry is defined as the detection of light

energy scattered or reflected toward a detector
that is not in the direct path of the transmitted
light.
Common nephelometers measure scattered light
at right angles to the incident light.
 Spectrophotometry has been successfully
applied to the analysis of different kinds of
enzymes in serum, such as ALT, AST, ALP,GGT,
and quantitative analysis of total protein and
albumin in serum.
 Turbidimetry can be used to determine the
concentration of apolipoprotein，
immunoglobulin，alexin and rheumatoid
factors.
 FLUOROMETRY荧光测定法

The interaction of radiant energy with molecules

or particles in solution can result in either
fluorescence荧光 or light scattering散射光 .
Fluorescence occurs when a molecule absorbs light at
one wavelength波长 and remits light at a longer
wavelength.
Light scattering occurs when radiant energy passing
through a solution meets a molecule in an elastic
collision, which results in the light being dispersed分散
in all directions.
Several automated fluorometric analyzers have been
developed for special applications in clinical
laboratory, because of their sensitivity, speed,
simplicity,and reliability.These include the flow
cytometer, hematofluorometer, fluorescence
microscopy.
The relationship of concentration to intensity of
fluorescence emission may be derived from the
Beer- Lambert law: the fluorescence intensity is
directly proportional to the concentration of the
fluorophore荧光基团and the excitation intensity.
 Electrophoresis

Electrophoresis refers to the migration(移动) of

charged solutes or particles in a liquid or a porous
supporting medium, such as cellulose acetate乙酸纤维
素sheets or agarose gel film琼脂糖凝胶 , under the
influence of an electrical field电场.
 Theory of electrophoresis

Definitions
1) anode阳极: the positively charged electrode电极
in electrophoresis system.
2) cathode 阴极: negative electrode.
3) isoelectric point (pI)等电点of a molecule:
is the pH at which it has no net charge净电荷and
will not move in an electrical field.
4) ampholyte or zwitterion两性离子 : is a molecule that can be
either positively or negatively charged; example: proteins,
amino acids.
Chemical species carrying an electrical charge
move either to the cathode or the anode in an
electrophoresis system, depending on the kind of
charge they carry.
In a solution more acid than the isoelectric point of
the solute, an ampholyte takes on a positive charge
and migrates toward the cathode. In the reverse
situation, it migrates toward the anode.
The rate of migration 迁移率is dependent on factors
such as:
1) the net electrical charge净电荷of the molecule,
2) the size 大小 and shape 形状 of the molecule,
3) the electric field strength电场强度 ,
4) the characteristics of the supporting medium支持物,
5) and the operation temperature.
Description of Electrophoresis

A schematic diagram of an
electrophoresis system shows:
 two buffer boxes缓冲液箱(l)
with baffle plates隔板 contain
the buffer used in the process.
 In each buffer box is an
electrode电极(2) of either
platinum铂 or carbon, the
polarity极性 of which is fixed
by the mode of connection to
the power supply.
 The electrophoresis support支持物
(3) on which separation take place
is in contact with buffer by means
of wicks (strips) 条子 (4).
 The entire device is covered 覆盖
(5) to minimize evaporation蒸发
and protect the system.
 The direct current power supply
may be either constant current 恒
流 or constant voltage or both.
 Automated Electrophoresis Systems

Although electrophoresis was traditionally a manual

technique, it has been improved by the introduction of
prepackaged gels预先包装的凝胶 and electronic
systems电子系统 that incorporated all the necessary
components and reagents to perform the procedure
easily.
Example of rapid electrophoresis (REP) analyzer, (Helena Laboratories).
Different types of electrophoresis
Starch Gel Electrophoresis淀粉凝胶电泳

Starch gel electrophoresis separates macromolecular

ions on the basis of both surface charge表面电荷 and
molecular size.
Because proper preparation of gels is relatively
difficult and requires considerable skill, this technique
is now rarely used in the clinical laboratory.
 Agarose Gel Electrophoresis 琼脂糖凝胶电泳
It is a convenient method of electrophoresis that uses
a purified, essentially neutral fraction of agar called
agarose as a medium.
It has been successfully applied to the analysis of
serum proteins, hemoglobin variants, isoenzymes,
lipoproteins fractions and other substances.
The advantages of agarose gel include its lower affinity
低亲合力 for proteins and its native clarity after drying,
which permits excellent densitometric examination光密
度测量 (扫描).
 Cellulose Acetate Electrophoresis
(CAE)乙(醋）酸纤维（素）电泳

Cellulose acetate is a thermoplastic resin热塑树脂 of

cellulose that is made by treating cellulose with acetic
anhydride乙酸酐to acetylate the hydroxyl groups to form
the raw material for membranes contain about 80% air
space in the form of pockets.
An advantage of CAE is the speed of separation (20
min-1h) and the ability to store the transparent
membranes for long periods.
Cellulose Acetate Electrophoresis
 Disc Electrophoresis 圆盘电泳

Serum protein zones determined by ordinary

electrophoretic techniques contain several proteins
with the same electrophoretic mobility 电 泳 迁 移 率
and they tend to be large because proteins diffuse
during electrophoresis. Disc electrophoresis was
introduced in 1964 to overcome these deficiencies.
 Polyacrylamide Gel Electrophoresis
(PAGE) 聚丙烯酰胺凝胶电泳

In this technique, individual gels are prepared in situ原位

in glass tubes by polymerizing a gel monomer凝胶单体
and a cross-linking agent交联物 with the aid of an
appropriate catalyst. The first gel to be poured into the
tubular-shaped electrophoresis cell is the small-pore
separation gel小孔分离胶. After 30 min, during
which gelation takes place, a large pore gel, the spacer
gel浓缩胶 is thrown on top of the separation gel.
Then a large-pore monomer solution containing a
small amount of sample (serum) is polymerized聚合
above the spacer gel so that the finished product is
composed of three different layers of gel.

When electrophoresis begins, all protein ions

migrate through the large-pore gel and amass on
the separation gel in a very fine zone.
This process improves the resolution 清 晰 度 and
concentrate protein components at the border zone,
so that pre-concentration of specimens with low
protein content may not be necessary. Separation
then takes place in the bottom separation gel with
the retardation of some proteins due to the
molecular sieve分子筛 phenomenon.
Polyacrylamide Gel Electrophoresis
Polyacrylamide Gel Electrophoresis
 Isoelectric Focusing (IEF) Electrophoresis
等电聚焦电泳

+ –
pH 3 pH 7.5 pH 10 Amphoteric compounds两性化合物,
such as proteins, can be separated
by virtue of migration in a medium
+ –
possessing a stable pH gradient
pH 3 pH 7.5 pH 10
using isoelectric focusing
electrophoresis(IEF) 等 电 聚 焦 . The
– protein moves to a zone in the
+
pH 3 pH 7.5 pH 10
medium where the pH is equal to its
isoelectric point (pI). At this pH, the
+ – charge becomes zero and migration
pH 3 pH 7.5 pH 10 ceases.
 Two-Dimensional (2D) Electrophoresis
双向电泳

In the first dimension, it uses charge-dependent电

荷依赖的 IEF electrophoresis in a large-pore medium
such as agarose or large-pore polyacrylamide gel;
and in the second dimension, molecular weight-
dependent 分 子 量 依 赖 的 electrophoresis in
polyacrylamide.
第一向：等电聚焦

+ –
pH 3 pH 7.5 pH 10

+ –
pH 3 pH 7.5 pH 10

+ –
pH 3 pH 7.5 pH 10

+ –
pH 3 pH 7.5 pH 10
 第二向：SDS-PAGE电泳

+ –
pH 3 pH 7.5 pH10
Proteins migrate
through the gel at
a rate proportional
to their size.

Smallest proteins
travel the furthest size
distance

charge
 Capillary Electrophoresis (CE)
毛细管电泳

In CE, the classic techniques of electrophoresis are

carried out in a small-hollow小洞(10 - 100μm of
diameter) fused silica熔融石英 capillary tube of
20 to 200 cm in length. This capillary tube is
connected to a detector at its terminal end, via
buffer reservoirs to a high-voltage power supply高
压电.
 Two distinct advantages of the capillary format
include the ease of automation and the efficient
heat dissipation （ 热 分 散 ） that permits the
application of much high voltages (25 to 30 kV)
than in traditional electrophoresis. This high
voltage enhances separation efficiency and
reduces separation time in some cases to less
than 1 min.
 CHROMATOGRAPHY
层析法，色谱法

Chromatography is a physical method of

separation in which the components to be separated
are distributed between two phases两相, one of which
is stationary (stationary phase固定相) while the other
(mobile phase流动相) moves in the definite direction.
 Basic concepts and definitions

The primary goal of the chromatographic process

is to separate a mixture混合物into its individual
components 成 分 , which are called solutes or
analytes.
A chromatographic separation requires a sample
to be introduced into a flowing stream流动的 of
gas or liquid (mobile phase) that passes through
a bed, layer, or column containing the stationary
phase.
If the mobile phase is a gas, the technique is
known as gas chromatography (GC)气相层析,
if a liquid, it is called liquid chromatography (LC)
液相层析.
The stationary phase may be particles of a solid or
gel or a liquid.
As the mobile phase carries the sample through
the stationary phase, the solutes with lesser
affinity亲合力for the stationary phase remain in
the mobile phase and travel faster and separate
from those that have a greater affinity for it.
 Separation mechanisms

Adsorption, affinity, ion exchange, partition,

and steric exclusion chromatography describe
the predominant chemical or physical
mechanisms used to separate solutes.
 Gel-Filtration Chromatography
凝胶过滤层析

It is also known as steric exclusion chromatography空

间排阻层析,gel-permeation, size exclusion, molecular
exclusion, molecular sieve chromatography分子排阻层
析 and separate solutes on the basis of their molecular
size.
A variety of materials have been used as
stationary phases including cross link dextran交联葡聚
糖 (Sephadex), polyacrylamide 聚 丙 烯 酰 胺 凝 胶 (Bio-
Gel), agarose (Sepharose), etc.
Molecular size chromatography

Molecules too large to

enter the pores remain
exclusively in the mobile
phase and rapidly elude
洗脱 from the column.
Molecules that are
intermediates in size (and
small molecules) have
access to various fractions
of the pore volume and
elude slowly.
 In addition to preparative applications (制备应用),
gel-filtration chromatography has been used in the
clinical laboratory to ：
1. to determine molecular weights of
macromolecules，
2. to remove low-molecular-weight salts or buffer
ions from protein solutions.
 Adsorption chromatography 吸附层析

Adsorption chromatography exploits the polarity极性,

or the related tendency for hydrogen binding氢键 of
molecules in order to partition between a polar
sorbent and a less polar solvent, or vice-versa, as the
mobile phase moves through the stationary sorbent.
 Affinity chromatography 亲和层析

The term affinity chromatography describes a

number of separation mechanisms with interactions
that occur between biochemical species (enzyme-
substrate, hormone-receptor, or antigen-antibody
complexes).
The stationary phase in affinity chromatography is
prepared by immobilizing a ligand配体 on particles of
a support either directly or via a spacer.
Adjustments of pH and ionic strength are required
to achieve optimum binding of the analyte to the
ligand. If this interaction is specific, the analyte
may be removed in a single step by addition of a
substrate or an inhibitor, or by a pH change, an
ionic strength change, or addition of a hydrogen
bond-breaking agent, such as urea.
In the clinical laboratory, it has been used to
separate proteins and antibiotics.
 Ion-exchange chromatography
离子交换层析

In ion-exchange chromatography 离 子 交 换 层 析 ,
solutes in a sample are separated by their
differences in sign and magnitude of ionic charge.
In practice, ionic analytes are selectively eluted
from ion-exchange resins by varying the pH
and/or ionic strength of the mobile phase.
 Cation-exchange resins阳离子交换树脂 contain
covalently bound, negatively charged functional
groups, such as sulfonate ions, carboxylate ions or
carboxy-methyl (CM) groups.
This technique is most useful for separation of
organic and inorganic ions, amino acids,
nucleotides, and proteins.
Anion-exchange resins 阴 离 子 交 换 树 脂 are
characterized by the presence of strongly basic
quaternary amines (triethylamino-ethyl groups) or
weakly basic groups (aminoethyl, diethylaminoethyl)
which can bear a positive charge.
Ion-exchange chromatography is widely used to
separate and remove inorganic ion from aqueous
mixtures.
 Partition chromatography
分配层析
In partition chromatography (also called thin-layer
chromatography)分配层析, a thin film 薄膜of liquid is
adsorbed onto the surfaces of support particles.
Separation is based on differences in the relative
solubility溶解度 of solute molecules in this film and the
mobile phase.
FIG： Two-dimensional thin layer or paper chromatography
 Gas chromatography (GC)

GC is a process by which a mixture is separated

into its constituent components by forcing a gaseous
mixture of it and mobile phase (carrier gas运载气体)
through a column containing the stationary phase.
Separation of the solutes in the mixture is based on
the relative differences in their vapor pressures蒸汽
压 and their interaction with the stationary phase.
A compound with a high vapor pressure will be eluted
more rapidly than compounds with lower vapor
pressures.
 The effluent 洗 脱 液 from the column carries the
separated sample constituents to the detector
which produces a signal that is displayed as a
series of peaks高峰
The volume and time at which an unknown solute
elutes 洗脱from a column are used to identify the
unknown compound. These values are compared
and matched with those obtained from reference
compounds.
Peak size (area or height) is proportional to the
amount of the compound detected and can be used
to quantify the amount of analyte in the sample.

Depending on the nature of the stationary phase,

GC techniques are divided into two categories:
gas-solid chromatography (GSC) and gas-liquid
chromatography (GLC).
 High-Performance Liquid
Chromatography (HPLC)

In LC液相层析, separation is based on the distribution of

the solutes between a liquid mobile phase and a
stationary phase. When an efficient column is used in a
liquid chromatograph, the technique is HPLC高效液相层
析. Because column efficiency is inversely related to the
particle size of the column packing, relatively high
pressure is required to pump liquid through an efficient
column.
Different components of HPLC
A basic liquid chromatograph consists of a solvent
reservoir 储溶剂器, a pump泵 to force the liquid
mobile phase through the system; an injector注射器
for introducing an aliquot of sample into the column;
a chromatographic column to separate the analytes
being measured; an on-line detector to detect the
separated analytes as they elute from the column;
and a computer to control the system and process
data.
Different components of HPLC
glycosylated hemoglobin
 MASS SPECTROMETRY (MS)
A mass spectrometry 质 谱 法 is an analytical
technique that first ionizes a target molecule and
then separates and measures the mass of a
molecule or its fragments. Mass analysis is the
process by which a mixture of ionic species is
identified according to the mass-to-charge (m/z)
ratios (ions).
The analysis is qualitative, quantitative, and
extremely useful for determining the elemental
composition and structure of both inorganic and
organic compounds.
Mass spectrometry when coupled with either gas or
liquid chromatography, the resultant technique is a
particularly powerful analytical technique that has
found extensive use for clinical applications.
 Coupled Techniques

Gas Chromatography/Mass Spectrometry

is a powerful analytical technique that combines
gas chromatograph resolving power with the
Excellent specificity and sensitivity of the mass
spectrometer.
 High Performance Liquid
Chromatography/ Mass
Spectrometry

Compared to gas chromatographs, it has been

more difficult to interface liquid chromatographs
with mass spectrometers because the analytes are
dissolved in a liquid, rather than a gas phase.
 SUMMARY

METHODS
APPLICATIONS