Faculty of science and natural resources

department of biotechnology

2018

Instrumentation

OSAMA S ALRAWAB (BVM&S;BSc,

MSc)
 1- Centrifugation

Definition: Biological centrifugation is a process that uses

centrifugal force to separate and Purify mixtures of biological
particles in a liquid medium according to their size, shape,
density, viscosity of the medium and rotor speed. This process is
used to separate two immiscible liquids. More dense components
of the mixture migrate away from the axis of the centrifuge,
while less dense components of the mixture migrate towards the
axis.

BASIC PRINCIPLES OF SEDIMENTATION

The effect of sedimentation due to the influence of the Earth’s

gravitational field (g=981 cm s-2) versus the increased rate of
sedimentation in a centrifugal field (g>981 cm s-2) is apparent.
To give a simple but illustrative example, crude sand particles

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added to a bucket of water travel slowly to the bottom of the
bucket by gravitation, but sediment much faster when the bucket
is swung around in a circle. Similarly, biological structures exhibit
a drastic increase in sedimentation when they undergo
acceleration in a centrifugal field. When designing a
centrifugation protocol, it is important to keep in mind that:

 Dense structure  sediment faster.

 Massive particle  move faster.

 Greater frictional coefficient  particle move slower.

 Greater centrifugal force  faster particle sediment.

 The sedimentation rate of a given particle will be zero when the density
of the particle and the surrounding medium are equal.

Types of centrifuges

A- According to Rotor Type :

They divided into three categories:

1- swinging-bucket rotors:

o Particles travel longer distance before pelleting  allow better

separation.

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 o Excellent for gradient centrifugation.

o Easier to withdraw supernatant without disturbing pellet.

o Preferred for rate-zonal separations.

2- Fixed angle rotors:

o Particles travel only short distance before pelleting.

o Excellent for fractionation purposes.

o The most widely use rotor type.

3- Vertical tube rotors.

o Vertical rotors are highly specialized. They are typically used to

band DNA in cesium chloride.

o Useful if the particle must only move a short distance until it

pellets .

o Run time on vertical rotors is short.

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B- According to Attainable Speed :

1- Low speed :

o Max ~ 20 X 103 rpm.

o Most laboratories have a standard

low-speed centrifuge used for

routine sedimentation of heavy

particles. Low speed centrifuge

o Called microfuge , clinical or table top

o Operate at room temperatures with no means of

temperature control.

o Two types of rotors are used in it, fixed angle and

swinging bucket.

o It is used for rapid separation of red blood cells and

DNA from proteins.

2- High speed centrifuges:

o Max ~ 80 X 103 rpm.

o Three types of rotors are

available for high speed

High speed centrifuge

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 centrifugation-fixed angle, swinging bucket, vertical

rotors.

o Used in sophisticated biochemical applications such as

separation of macromolecules (proteins or nucleic acids)

during purification or preparative work.

o Can be used to estimate sedimentation coefficient and

molecular weight.

3- Ultracentrifuge :

o Max ~ 10 5 rpm.

o It is the most
Ultracentrifuge
sophisticated instrument.

o Intense heat is generated due to high speed thus the

spinning chambers must be refrigerated and kept at

high vacuum.

o Used for both preparative work and analytical work.

o Uses small sample size < 1 ml.

o Uses pure sample.

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 Types of Centrifugation Techniques

Preparative Centrifugation Analytical Centrifugation

Differential centrifugation Density gradient centrifugation

Isopycnic centrifugation
Rate zonal centrifugation

A- Preparative
Centrifugation

1-Differential centrifugation:

Proper separation of many subcellular structures is absolutely

dependent on preparative ultracentrifugation. Cellular and

subcellular fractionation techniques are indispensable methods

used in biological research. The isolation of large cellular

structures, the nuclear fraction, mitochondria, chloroplasts or

large protein precipitates can be achieved by conventional high-

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speed centrifugation. Differential centrifugation is based upon

the differences in the sedimentation rate of biological particles of

different size and density. Crude tissue homogenates containing

Organelles, membrane vesicles and other structural fragments

are divided into different fractions by the stepwise increase of

the applied centrifugal field.

2-Density-gradient centrifugation:

Used to separate biological particles of similar size but

differing density. And subdivided into two techniques:

i- Rate zonal centrifugation.

ii- Isopycnic centrifugation.

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Rate zonal centrifugation

 In Rate zonal centrifugation the solution have a density

gradient.

 The sample has a density greater than all the layers in the

solution.

 The particles will begin sedimenting in separate zones

according to their size shape and density.

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Isopycnic centrifugation

 This type of centrifugation is slightly different than zonal

centrifugation but still the basic idea remains the same.

 We need to perform isopycnic centrifugation a uniform

mixture of sample and a gradient-forming substance.

 The difference between ispycnic and rate-zonal is in case of

rate-zonal the gradient forming substance form the

gradient texture before the reaction start.

 As gradient forming substance for isopycnic cesium chloride

is a choice.

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 Safety parameters:

1- Always use correct size tube to prevent tube damage.

2- Always use by mass no a volume counterbalance.

3- Put tubes opposite to each other.

4- Close lid.

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 2-Chromatography
 Chromatography is a physical method of separation in

which the components to be separated are distributed

between two phases:

 One of which is stationary (stationary phase) while the

other (the mobile phase) moves through it in a definite

direction.

 The chromatographic process occurs due to differences in

the distribution or partition coefficient Kd of the individual

sample components:

Kd =

 The Term Chromatography (chroma = a colour; graphein =

to write) is the collective term for a set of laboratory

techniques for the separation of mixtures.

 Chromatography involves a sample (or sample extract)

being dissolved in a mobile phase (which may be a gas or a
liquid).

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 Classification of Chromatography

A- On the basis of
B- On the basis of C- On the basis of
interaction of
chromatographic physical state of mobile
solute to the
bead Shape. phase
stationary phase

1- Two Dimensional:
1- Adsorption chromatography 1-Liquid Chromatography
i- Paper Chromatography.
2- Ion Exchange Chromatography 2-Super Critical Fluid
ii-Thin Layer Chromatography(TLC)
3-Partition Chromatography. Chromatography

2-Three Dimensional:
4-Size Exclusion Chromatography
3-Gas Chromatography

i-Column Chromatography

: ‫الجىسي ابدا اى طزيقة مه طزق الفصل الكزوماجوجزافي جحكون مه قسميه‬

Stationary phase ‫طور ساكه او ثابث‬

Mobile Phase ‫طور مححزك‬

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1- ION-EXCHANGE CHROMATOGRAPHY

 This form of chromatography relies on the attraction

between oppositely charged stationary phase, known as an

ion exchanger, and mobile phase solvent (‫ )المذيب‬+ analyte

(‫)المادة المراد تحليلها او فصلها‬.

 It is frequently chosen for the separation and purification of

proteins, peptides and nucleic acids.

 Ion exchange is used for the removal of toxic heavy metals

such as lead, mercury, and chromium from a variety of

industrial processes.

 There are two types of ion exchanger, namely 1-cation and

2-anion exchangers.

‫تسمى‬ +ve ‫اذا كانت شحنة الجزيئ المراد فصله موجب‬

cation ion .exchange ‫الطريقة‬

‫تسمى‬ –ve ‫ و بالحالة‬chromatography

anion ion exchange

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 We use beads(‫ )خرزات‬of polymeric particles sucha as Cellulose

as stationary phase.

 In case of Anion exchange DEAE Cellulaose is used as

stationary phase .

N.B DEAE is abbreviation for Diethylaminoethyl.

 The charge of DEAE-C is positive (+ve).

 In case of Cation exchange carboxy methyl cellulose is used

as stationary phase .

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  The charge of carboxy methyl cellulose is negative (-ve).

Principle of Ion Exchange Chromatography

Separation in Ion exchange chromatography depends upon the

reversible adsorption of charged solute molecule to immobilized

ion exchange group of opposite charge.

Ion exchange performed in four main steps :

1- Equilibration.

2- Sample application and wash.

3- Elution.

4- Regeneration.

 The first step in ion exchange is Equilibration of

stationary phase.

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 When equilibrium is reached all stationary phase

charged group are associated with exchange counter-

ion such as chloride or sodium.

 The goal of this step is to bind the target molecules

and wash out all unbounded materials.

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 The sample buffer should have the same PH and ionic

strength as the starting buffer in order to bind all

appropriately charged proteins.

 The third step is elution (‫)الشطف‬,biomolecules are released

from the ion exchanger by a change in the buffer

composition.

 A common way is to increase the ionic strength with

sodium chloride or another simple salt.

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 In order to desorb the bound proteins.

 Proteins are desorbed relative to the number of

charged group on their surface.

The final step in exchange process, regeneration means

remove all molecules still bound on stationary phase

surface .and ensure that the full capacity of the

stationary phase surface is available for next run.

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2-Gas chromatography (GC)

 It exploits the differences in partition coefficients between a

stationary liquid phase and a mobile gas phase.

 Stationary phase is an

inert solid material

impregnated with a non-

volatile liquid.

 In gas chromatography, a

sample is rapidly heated

and vaporized at the

injection port (‫)منفذ‬.

 The sample is transported through the column by a mobile

phase consisting of an inert gas (‫ )غاز خامل‬such as Helium.

 Sample components are separated based on their boiling

points and relative affinity for the stationary phase, which

is most often a viscous liquid (wax) within the column.

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 It is well suited for use in the petrochemical, environmental

monitoring and industrial chemical fields.

 GC is Sensitive, rapid and reliable .

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3-HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC )

 Liquid chromatography (LC) is a separation technique in

which the mobile phase is a liquid.

 Chromatographic techniques are slow & time consuming,

hence the separation can be greatly improved by using high

pressure in the range of 5000-10000 psi(pounds per

square inch),hence this technique is also referred to as high

pressure liquid chromatography.

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 In HPLC the sample is forced by a liquid at high pressure

(the mobile phase) through a column ) stationary phase(

composed of irregularly or spherically shaped particles.

 The interaction between the mobile and the stationary

Phase leads to the separation of the mixture.

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Types of HPLC According to Mode of Separation

1. Normal phase chromatography (NPLC):

 Stationary phase is polar (hydrophilic)  Silicon dioxide

SiO2 and Aluminium oxide Al2O3.

 mobile face is non-polar (hydrophobic) heptane and

hexane

Mechanism:

- Polar compounds travels slower and eluted slowly due to

higher affinity to stationary phase

- Non-polar compounds travels faster and eluted first due to

lower affinity to stationary phase.

NPLC technique is not widely used in pharmaceutical separations.

2. Reverse phase chromatography (RPLC):

 Stationary face is non-polar (hydrophobic)  ethyl and

phenyl diol, hydrophobic polymers.

 mobile face is Polar (hydrophilic)  methanol

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Mechanism:

 Polar compounds travels faster and eluted first due to lesser

affinity to stationary phase.

 Non-Polar compounds travels slower and eluted slowly due

to higher affinity to stationary phase.

Applications of HPLC( ‫)استخدامات‬:

- Shelf life ‫عمر الرف تعنى صالحية‬determinations ‫تحديد‬of pharmaceutical

products ‫ المنتجات الصيدالنية‬.

- Identification of counterfeit ‫ تزييف‬drug products ‫ المنتجات الدوائية‬.

- Pharmaceutical quality control.

ADVANTAGES OF HPLC (‫)مميزات‬:

1- Separations fast and efficient.

2- Accurate quantitative measurements.

3- Repetitive and reproducible analysis using the same

column.

4- Don’t use heat safer for biological samples.

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 Spectroscopic techniques ‫الحقىيات الطيفية‬

The term Spectroscopic techniques

‫مصطلح التقنيات الطيفية يشير الى‬
.
refers to techniques which employ ‫التقنيات التى توظف الضؤ وتفاعله مع‬

light to interact with matter and thus ‫المادة وبالتالى التحقق من مميزات‬
‫محددة للعينة وذلك لدراسة قوامها‬
probe certain features of a sample to
‫اوتركيبها‬
learn about its consistency or

Properties of electromagnetic radiation

 Electromagnetic radiation consist of discrete packages ‫حزم‬

‫ منفصلة‬of energy ‫ الطاقة‬which are called as photons.

 A photon consists of an oscillating ‫ متذبذب‬electric field(E) & an

oscillating magnetic field (M) which are perpendicular ‫ متعامد‬to

each other.

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 Frequency ‫( التردد‬ν):

- It is defined as the number of times electrical field

radiation oscillates in one second.

- The unit for frequency is Hertz (Hz).

1 Hz = 1 cycle per second

 Wavelength ‫الطول الموجى‬ (λ):

- It is the distance between two nearest parts of the wave

in the same phase i.e. distance between two nearest

crest ‫ قمة‬or troughs ‫قاع او منخفض‬.

 The Electromagnetic Spectrum ‫الطيف االلكترومغناطيسى‬:

- The electromagnetic (EM) spectrum is the range of all

types of EM radiation.

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Types of Spectroscopy

1. Absorption Spectroscopy ‫مطيافية االمتصاص‬:

• An analytical technique which concerns with the measurement

of absorption of electromagnetic radiation. • e.g. UV (185 - 400

nm) / Visible (400 - 800 nm) Spectroscopy.

2. Emission Spectroscopy ‫مطيافية االنبعاث‬:

• An analytical technique in which emission (of a particle or

radiation) is dispersed ‫ تتشتت‬according to some property of the

emission & the amount of dispersion is measured. • e.g. Mass

Spectroscopy.

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 1- UV–VIS SPECTROSCOPY

o UV-Visible spectroscopy is a technique capable of both

quantitative and qualitative analysis of liquid, solid and

gaseous samples.

o The UV radiation region extends from 10 nm to 400 nm

and the visible radiation region extends from 400 nm to

800 nm.

o Near UV Region: 200 nm to 400 nm & Far UV Region:

below 200 nm.

o Far UV spectroscopy is studied under vacuum condition.

o The common solvent used for preparing sample to be

analyzed is either ethyl alcohol or hexane.

Terms used in UV / Visible Spectroscopy

 Chromophore: The part of a molecule responsible for

imparting ‫ وقل‬color  Acetone, carbonyl group and

Ethylene group .

 Auxochrome: The functional groups attached to a

chromophore which modifies the ability of the chromophore
to absorb light  Benzene, Phenol

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Instrumention

A spectrophotometer is an instrument for measuring the

Transmittance or absorbance of a sample as a function ‫ دالة‬of the

Wavelength of electromagnetic radiation there are two types of

uv/vis spec devices:

1- Single beam instrument ‫جهاز مفرد الطيف‬

 The absorbance of a sample is determined by measuring the

intensity of light reaching the detector without the sample

(the blank) and comparing it with the intensity of light

reaching the detector after passing through the sample.

 The blank and the sample are measured consecutively.

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 2-Double beam instrument ‫جهاز مزدوج الطيف‬

 Compensate for these changes in lamp intensity between

measurements on blank and sample cuvettes.

 The chopper switches the light path between a reference

optical path and a sample optical path to the detector.

 instrument components ‫مكونات الجهاز‬

1. Light Source ‫مصدر ضوئي‬

2. Sample Chamber ‫غزفة العيىة‬

3. Optical System ‫الىظام البصزى‬

4. Detector ‫الكشاف‬

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1. Light Source

Characteristics of good light source:

1. High intensity but small surface area.

2. Wide spectral range.

3. Stable output.

4. Long life at reasonable cost.

Types of Light Sources :

A- UV source:

o Both deuterium and hydrogen lamps emit

radiation in the range 160 - 375 nm.

o Quartz Cuvettes must be used in these lamps

because glass absorbs radiation of wavelengths

less than 350 nm.

B- VIS. Source
o The tungsten filament lamp is

commonly employed as a source of

visible light. Lamps emit radiation in

the range 380 - 780 nm.

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C- Xenon Source :

o The wavelength range of 190-1100 nm.

o The xenon gas inside the glass bulb is

under extremely high pressure, the xenon

Lamp can explode.

2. Sample Chamber (Cuvettes)

o Cuvettes: The containers for the

sample and reference solution.

o They must be transparent to the

radiation which will pass through

them.

o For Uv we use Quartz cuvettes .

o For visible we use glass cuvettes .

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3. Optical System (monochromator ‫)موحد االطوال الموجية‬:

Accepts polychromatic input light from a lamp and outputs

monochromatic light.

Characteristics desired of a monochromator

1. Minimum absorption of light as it passes through the system

2. High degree of accuracy in wavelength selection

3. High spectral purity over a broad spectral range

Components :

1-Entrance slit.

2-Dispersion device.

3- Exit slit.

Dispersion device:

They are of two kinds namely:

1-Prisms :Simple glass prisms are used for visible range. For

UV region silica, fused silica or quartz prism is used.

Fluorite is used in vaccum UV range.

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2-Gratings ‫محزوز الحيود‬: are often used in the monochromators of

spectrophotometers operating in UV, visible and infra- red

Regions. Their resolving power is far superior to that of

Prisms & they yield a linear resolution of spectrum.

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‫الكشاف ‪4. Detector‬‬

‫‪Detectors to measure the intensity of radiation and it should‬‬

‫‪give:-‬‬

‫‪ Low noise.‬‬ ‫ضجيج او معدل شوشرة منخفض‬

‫حساسية عالية ‪ High sensitivity.‬‬

‫‪Types of detectors :‬‬

‫‪1-The photomultiplier tube (PTM):‬‬

‫اكثو نوع استخداما ‪Is a commonly used detector in UV-Vis spectroscopy.‬‬

‫هذا النوع من الكشفات عبارة عن خلية ضؤية التى درستها بالمرحلة الثانوية وتقوم بقياس شدة الضوء الساقط عليها وتحولها الى طاقة او‬
‫اشارة يمكن قياسها ‪.‬‬

‫‪2- Silicon detectors‬‬

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‫استخدامات او تطبيقات ‪APPLICATIONS OF U.V. SPECTROSCOPY‬‬

‫‪1- Detection of Impurities‬‬ ‫الكشف عن الشوائب‬

‫‪2- Structure elucidation of organic compounds‬‬ ‫توضيح الصيغ التركيبية للمركبات العضوية‬

‫التحليل الكمى ‪3- QUANTITATIVE ANALYSIS‬‬

‫التحليل النوعى ‪4- QUALITATIVE ANALYSIS‬‬
‫تحديد المجموعات الوظيفية ‪5- DETECTION OF FUNCTIONAL GROUPS‬‬
‫تحديد الوزن الجزيئى ‪6- MOLECULAR WEIGHT DETERMINATION‬‬
‫يستخدم ككشاف الجهزة الكروماتوجرافيا السائلة عالية االداء ‪7- AS HPLC DETECTOR‬‬

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 Mass spectrometer (MS)

‫مطياف الكتلة‬

 It is a technique used for measuring the molecular weight of

organic compounds.

 In a mass spectrometer, a molecule is vaporized and ionized

by bombardment ‫قصف‬with a beam of high-energy electrons.

 The energy of the electrons is ~1600 kcal (or 70 ev).

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)‫كلية العلوم والموارد الطبيعية قسم التقنيات الحيوية – جامعة الجفارة (االجهزة‬
  Since it takes ~ 100 Kcal of energy to cleave a typical S

bond, 1600 kcal is an enormous amount of energy to come

into contact with a molecule.

What does a mass spectrometer do?

1-It measures better than any other technique.

2-It can give information about chemical structure.

What are mass measurements good for?

To identify, verify and quantitate: metabolites, proteins

isolated from natural sources ,oligonucleotides ,drugs and
organic chemicals .

Application of mass spectroscopy

1- Biomolecule characterization :

-proteins and peptide

-oligonucleotides

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)‫كلية العلوم والموارد الطبيعية قسم التقنيات الحيوية – جامعة الجفارة (االجهزة‬
 2- Environmental analysis:

- Pesticides on food

- Soil and groundwater contamination

3- Pharmaceutical analysis :

- Bioavailability studies.

- Drug metabolism studies

- Drug degradation studies

4-Forensic and clinical analysis.

How Does Mass Spec Work‫كيف يعمل مطياف الكتلة‬

1- Vaporization ‫ التبخير‬:

Molecules in a sample are vaporized (converted to

the gas phase by heating by using a coil ‫)ملف‬.

2- Ionization ‫ التايين‬:

 Electron beam bombards the vapors, which

converts the vapors to ions. Because mass

spectroscopy measures the mass of charged

particles, only ions will be detected, and

neutral molecules will not be seen.

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)‫كلية العلوم والموارد الطبيعية قسم التقنيات الحيوية – جامعة الجفارة (االجهزة‬
 Electron beam  ionize the molecule the species formed

called radical cation and symbolized as M +*

 Radical cation M+* is called molecular ion or parent ion

 The mass of M +* represent the molecular weight of the

molecule .

 Because M+* is unstable it decomposed to form fragments of

radicals and cations that have lower molecular weight of

M +*

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)‫كلية العلوم والموارد الطبيعية قسم التقنيات الحيوية – جامعة الجفارة (االجهزة‬
 3- Acceleration ‫التسريع او التعجيل‬

Acceleration is simple attraction. The positive ions created

in the ionization stage accelerate towards negative plates at

a speed dependent on their mass. In other words, lighter

molecules move quicker than heavier ones.

4- Deflection ‫االنعطاف‬

The ions are deflected by a magnetic field, and the deflection is

again dependent on mass. So, ions of different mass travel
through the spectrometer at different speeds  amount of
deflection depends on m/z ratio (where m is a mass and z is the
charge).

5- Detection ‫الكشف‬

Ions of increasing mass reach the detector one after another, and

then it’s over to the computer to provide a spectrum. A mass

spectrum is a plot ‫رسم بيانى‬ of each the amount of each cation

(relative abundance) versus ‫ضذ‬ its mass-to-charge ratio m/z.

Since z is almost always +1 then m/z is actually measure the

mass (m) of the individual ion .

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)‫كلية العلوم والموارد الطبيعية قسم التقنيات الحيوية – جامعة الجفارة (االجهزة‬
Ionization methods

1- Electron Ionization (EI):

Most common ionization technique , limited to relatively low MW

compounds .

2-chemical ionization (CI):

Ionization with very little fragmentation ,still for low MW

compounds .

3-Desorption Ionization (DI):

For higher MW or very labile compounds .

5-Spray Ionization (SI)

For LC-MS, GS-MS and biomolecules .

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)‫كلية العلوم والموارد الطبيعية قسم التقنيات الحيوية – جامعة الجفارة (االجهزة‬