CONTENTS

1. Introduction

2. Western blotting

3. Agarose gel electrophoresis

4. PCR

5. Lymphocyte proliferation assay(MTT assay)

6. HPLC

7. ELISA

8. Isolation of PBMC

9. Cell culture techniques

o Spleenocyte culture

o NO estimation

o Inverted microscope

o Cell counting and cell viability (haemocytometer)

[Link]

1
 INTRODUCTION

As a part of [Link] Biotechnology degree award from AMITY University, Noida U.P. I have studied some basic
instrumentation techniques from DIPAS (DRDO), Lucknow road, New Delhi. A brief introduction of the institute
and work that I have done has been given as followed-

In DIPAS I have basically done the instrumentation techniques in different labs. In biochemistry lab, I have studied
about CHR(cold hypoxia restrain), it is an instrument which provides hypoxia conditions to the animal that we want
for further experiment (estimations),etc. In endocrinology lab, I have studied about bone densitometer, through
which the density of bone can be calculated. I have also been given the overview of flow cytometer, HPLC, BOD
incubator hypoxia chamber, etc. In hematology lab, I have studied western blotting, pH meter and its calibration,
pipette calibration, etc. In immuno modulation lab, I have studied the cell culture techniques, PCR, RT-PCR,
agarose gel electrophoresis, inverted microscope, etc. In cell culture techniques I have studied about spleenocyte
culture, MTT assay, ELISA, NO estimation, isolation of PBMC, spleen from mouse, cell counting, etc.

The main of DIPAS is to test different drugs on mouse or any other organism under hypoxia conditions and the
following result is used for thewelfare of soldiers who are at high altitude. As at high altitude it’s really difficult to
live there in less air, so in DIPAS we basically feed the mouse with the drug like sea buck thorn, olive oil, picric
acid, statin, etc. and they are tested for their gasping time, etc that either it is decreasing or increasing when given a
particular drug under hypoxia and normal conditions and depending on the observations different estimations are
done like GSH and many more and then the result is obtained and we get to conclusion that particular drug is
effective for a soldier or not.

2
 WESTERN BLOTTING

The Western blot alternatively also known as “Protein Immunoblot” is an analytical technique used to detect

specific proteins in a given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native or
denatured proteins by the length of the polypeptide or by the 3-D structure of the protein. The proteins are then
transferred to a membrane typically nitrocellulose or PVDF, where they are probed using antibodies specific to the
target protein.

Steps in a western blot

Tissue preparation
Samples may be taken from whole tissue or from cell culture. In most cases, solid tissues are first broken down
mechanically by a Blender (larger sample volumes), a homogenizer (smaller volumes) or by sonication. However,
Western blotting is not restricted to cellular studies only.

Assorted detergents, salts, and buffers may be employed to encourage Lysis of cells and to solubilize

proteins. Protease and phosphatase inhibitors are often added to prevent the digestion of the sample by its own
enzymes. Tissue preparation is often done at cold temperatures to avoid protein denaturing.

The proteins of the sample are separated using gel electrophoresis. Separation of proteins may be by isoelectric
point, molecular weight, electric charge, or a combination of these factors. The nature of the separation depends on
the treatment of the sample and the nature of the gel. This is the most useful way for protein determination.

By far the most common type of gel electrophoresis employs polyacrylamide gels and buffers loaded with sodium
dodecyl sulfate (SDS). SDS-PAGE (SDS polyacrylamide gel electrophoresis) maintains polypeptides in a denatured
state once they have been treated with strong reducing agents to remove secondary and tertiary structure (e.g.
disulfide bonds [S-S] to sulfhydryl groups [SH and SH]) and thus allows separation of proteins by their molecular
weight. Sampled proteins become covered in the negatively charged SDS and move to the positively charged
electrode through the acrylamide mesh of the gel. Smaller proteins migrate faster through this mesh and the proteins
are thus separated according to size (usually measured in kilo Daltons, kDa). The concentration of acrylamide
determines the resolution of the gel - the greater the acrylamide concentration the better the resolution of lower
molecular weight proteins. The lower the acrylamide concentration the better the resolution of higher molecular
weight proteins.

3
Samples are loaded into wells in the gel. One lane is usually reserved for a marker or ladder, a commercially
available mixture of proteins having defined molecular weights, typically stained so as to form visible, coloured
bands. When voltage is applied along the gel, proteins migrate into it at different speeds. These different rates of
advancement (different electrophoretic mobilities) separate into bands within each lane.

It is also possible to use a two-dimensional (2-D) gel which spreads the proteins from a single sample out in two
dimensions. Proteins are separated according to isoelectric point (pH at which they have neutral net charge) in the
first dimension, and according to their molecular weight in the second dimension.

Transfer

In order to make the proteins accessible to antibody detection, they are moved from within the gel onto a membrane
made of nitrocellulose or polyvinylidene difluoride (PVDF). The membrane is placed on top of the gel, and a stack
of filter papers placed on top of that. The entire stack is placed in a buffer solution which moves up the paper by
capillary action, bringing the proteins with it. Another method for transferring the proteins is called electro
blotting and uses an electric current to pull proteins from the gel into the PVDF or nitrocellulose membrane. The
proteins move from within the gel onto the membrane while maintaining the organization they had within the gel.
As a result of this "blotting" process, the proteins are exposed on a thin surface layer for detection (see below). Both
varieties of membrane are chosen for their non-specific protein binding properties (i.e. binds all proteins equally
well). Protein binding is based upon hydrophobic interactions, as well as charged interactions between the
membrane and protein. Nitrocellulose membranes are cheaper than PVDF, but are far more fragile and do not stand
up well to repeated probings.

4
The uniformity and overall effectiveness of transfer of protein from the gel to the membrane can be checked by
staining the membrane with Coomassie Brilliant Blue or Ponceau S dyes. Ponceau S is the more common of the two,
due to Ponceau S's higher sensitivity and its water solubility makes it easier to subsequently destain and probe the
membrane as described below.

Blocking
Since the membrane has been chosen for its ability to bind protein and as both antibodies and the target are proteins,
steps must be taken to prevent interactions between the membrane and the antibody used for detection of the target
protein. Blocking of non-specific binding is achieved by placing the membrane in a dilute solution of protein -
typically 3-5% Bovine serum albumin (BSA) or non-fat dry milk (both are inexpensive) in Tris-Buffered
Saline (TBS), with a minute percentage of detergent such as Tween 20 or Triton X-100. The protein in the dilute
solution attaches to the membrane in all places where the target proteins have not attached. Thus, when the antibody
is added, there is no room on the membrane for it to attach other than on the binding sites of the specific target
protein. This reduces "noise" in the final product of the Western blot, leading to clearer results, and eliminates false
positives.

Detection
During the detection process the membrane is "probed" for the protein of interest with a modified antibody which is
linked to a reporter enzyme, which when exposed to an appropriate substrate drives a colourimetric reaction and
produces a colour. For a variety of reasons, this traditionally takes place in a two-step process, although there are
now one-step detection methods available for certain applications.

5
Two steps

 Primary antibody

Antibodies are generated when a host species or immune cell culture is exposed to the protein of interest (or a part
thereof). Normally, this is part of the immune response, whereas here they are harvested and used as sensitive and
specific detection tools that bind the protein directly.

After blocking, a dilute solution of primary antibody (generally between 0.5 and 5 micrograms/mL) is incubated
with the membrane under gentle agitation. Typically, the solution is composed of buffered saline solution with a
small percentage of detergent, and sometimes with powdered milk or BSA. The antibody solution and the membrane
can be sealed and incubated together for anywhere from 30 minutes to overnight. It can also be incubated at
different temperatures, with warmer temperatures being associated with more binding, both specific (to the target
protein, the "signal") and non-specific ("noise").

 Secondary antibody

After rinsing the membrane to remove unbound primary antibody, the membrane is exposed to another antibody,
directed at a species-specific portion of the primary antibody. Antibodies come from animal sources (or animal
sourced hybridoma cultures); an anti-mouse secondary will bind to almost any mouse-sourced primary antibody,
which allows some cost savings by allowing an entire lab to share a single source of mass-produced antibody, and
provides far more consistent results. This is known as a secondary antibody, and due to its targeting properties, tends
to be referred to as "anti-mouse," "anti-goat," etc. The secondary antibody is usually linked to biotin or to a
reporter enzyme such as alkaline phosphatase orhorseradish peroxidase. This means that several secondary
antibodies will bind to one primary antibody and enhance the signal.

Most commonly, a horseradish peroxidase-linked secondary is used to cleave a chemiluminescent agent, and the
reaction product produces luminescence in proportion to the amount of protein. A sensitive sheet of photographic
film is placed against the membrane, and exposure to the light from the reaction creates an image of the antibodies
bound to the blot. A cheaper but less sensitive approach utilizes a 4-chloronaphthol stain with 1% hydrogen
peroxide; reaction of peroxide radicals with 4-chloronaphthol produces a dark brown stain that can be
photographed without using specialized photographic film.

6
 .

As with the ELISPOT and ELISA procedures, the enzyme can be provided with a substrate molecule that will be
converted by the enzyme to a colored reaction product that will be visible on the membrane (see the figure below
with blue bands).

Another method of secondary antibody detection utilizes a near-infrared (NIR) fluorophore-linked antibody. Light
produced from the excitation of a fluorescent dye is static, making fluorescent detection a more precise and accurate
measure of the difference in signal produced by labeled antibodies bound to proteins on a Western blot. Proteins can
be accurately quantified because the signal generated by the different amounts of proteins on the membranes is
measured in a static state, as compared to chemiluminescence, in which light is measured in a dynamic state. [7]

A third alternative is to use a radioactive label rather than an enzyme coupled to the secondary antibody, such as
labeling an antibody-binding protein like Staphylococcus Protein A or Streptavidin with a radioactive isotope of
iodine. Since other methods are safer, quicker, and cheaper, this method is now rarely used; however, an advantage
of this approach is the sensitivity of auto-radiography based imaging, which enables highly accurate protein
quantification when combined with optical software (e.g. Optiquant).

One step
Historically, the probing process was performed in two steps because of the relative ease of producing primary and
secondary antibodies in separate processes. This gives researchers and corporations huge advantages in terms of
flexibility, and adds an amplification step to the detection process. Given the advent of high-throughput protein
analysis and lower limits of detection, however, there has been interest in developing one-step probing systems that
would allow the process to occur faster and with less consumables. This requires a probe antibody which both
recognizes the protein of interest and contains a detectable label, probes which are often available for known protein
tags. The primary probe is incubated with the membrane in a manner similar to that for the primary antibody in a
two-step process, and then is ready for direct detection after a series of wash steps.

7
Western blot using radioactive detection system

Analysis
After the unbound probes are washed away, the Western blot is ready for detection of the probes that are labeled and
bound to the protein of interest. In practical terms, not all Westerns reveal protein only at one band in a membrane.
Size approximations are taken by comparing the stained bands to that of the marker or ladder loaded during
electrophoresis. The process is repeated for a structural protein, such as actin or tubulin, that should not change
between samples. The amount of target protein is indexed to the structural protein to control between groups. This
practice ensures correction for the amount of total protein on the membrane in case of errors or incomplete transfers.

Colorimetric detection

The colorimetric detection method depends on incubation of the Western blot with a substrate that reacts with the
reporter enzyme (such as peroxidase) that is bound to the secondary antibody. This converts the soluble dye into an
insoluble form of a different color that precipitates next to the enzyme and thereby stains the membrane.
Development of the blot is then stopped by washing away the soluble dye. Protein levels are evaluated
through densitometry (how intense the stain is) or spectrophotometry.

Chemiluminescent detection

Chemiluminescent detection methods depend on incubation of the Western blot with a substrate that will luminesce
when exposed to the reporter on the secondary antibody. The light is then detected by photographic film, and more
recently by CCD cameras which capture a digital image of the Western blot. The image is analysed by densitometry,
which evaluates the relative amount of protein staining and quantifies the results in terms of optical density. Newer

8
 software allows further data analysis such as molecular weight analysis if appropriate standards are used.

Radioactive detection

Radioactive labels do not require enzyme substrates, but rather allow the placement of medical X-ray film directly
against the Western blot which develops as it is exposed to the label and creates dark regions which correspond to
the protein bands of interest (see image to the right). The importance of radioactive detections methods is declinin,
because it is very expensive, health and safety risks are high and ECL provides a useful alternative.

Fluorescent detection

The fluorescently labeled probe is excited by light and the emission of the excitation is then detected by a
photosensor such as CCD camera equipped with appropriate emission filters which captures a digital image of the
Western blot and allows further data analysis such as molecular weight analysis and a quantitative Western blot
analysis. Fluorescence is considered to be among the most sensitive detection methods for blotting analysis.

Secondary probing
One major difference between nitrocellulose and PVDF membranes relates to the ability of each to support
"stripping" antibodies off and reusing the membrane for subsequent antibody probes. While there are well-
established protocols available for stripping nitrocellulose membranes, the sturdier PVDF allows for easier stripping,
and for more reuse before background noise limits experiments. Another difference is that, unlike nitrocellulose,

9
PVDF must be soaked in 95% ethanol, isopropanol or methanol before use. PVDF membranes also tend to be
thicker and more resistant to damage during use.

Materials

1. Acryl amide +bis acryl

29g acryl amide

1g bis amide

Make up volume 100ml and filter

2. Tris (1.5M,pH8.8)

18.171g Tris in 60ml distilled water

Adjust pH to 8.8 with HCl

Make up volume to 100ml

3. Tris (15M,pH6.8)

18.171g Tris in 60ml distilled water

Adjust pH to 6.8 with HCl

Make up volume to 100ml

4. Sample Buffer

For 100ml

SDS 9.2g

B-mercaptaethanol 20ml

Glycerol 40ml

Tris HCl 25ml

pH 6.8

0.1% BPB dye 4ml

10
 Make up volume to 100ml with distilled water

1X Running buffer

5. Electrophoretic buffer(pH8.3,1lt)

(maintains the charge/ion conc.)

Glycine 14.3g

Tris 3.023g

SDS 1g

Distilled water 1lt

Adjust pH to 8.3

6. 1 X Transfer buffer(500ml)

Methanol 100ml

Tris 1.51g

Glycine 7.2g

Make up volume upto 500ml with distilled water

10% Running gel 4% stacking gel

Acryl + bis acryl 3.32ml 1.3ml

Distilled water 4.08ml 6.1ml

Tris(pH 8.8) 2.4ml 2.5ml

10% SDS 0.1ml 100ul

10% APS 100ul 100ul

TEMED 10ul 10ul

11
 Lysis buffer for tissue homogenization for western blot

DTT 0.1mM

Tris 0.01M

NaCl 0.1M

EDTA 0.001M

NaN3 0.1%

Protein Separation by SDS-PAGE

Solutions:

 Running Buffer: Add 50 ml 20X concentrate to 850ml dH2O

 Loading Samples: Add 8μl of sample (at concentration of 1mg/ml), 1μl Reducing Agent (10X), and 2.5μl LDS
Sample Buffer. MAINTAIN THIS RATIO if loading a different amount

Procedure:

1. Place gel into the core and put into tank. Wells should face the inside for loading.

2. Close apparatus and use running buffer to make sure that there are no leaks.

3. Add Buffer to the tank and inside the core

4. Add 500μl Antioxidant to the middle of the tank

5. Fill wells with loading samples. Leave space for the molecular weight markers. If you are running a
western on the SDS-PAGE, leave a space in between sample groups

6. Run @ 100 volts (30 mA) until dye line runs off the gel.

 
Protein Transfer to PVDF

Solutions:

Dilute transfer buffer to 1x and add 100ml MeOH - store @ 4°C. For NuPAGE Novex Bis-Tris or Tris-Acetate
Gels: 50ml NuPage Transfer Buffer (20x), 100ml methanol, 850ml dH2O

12
 Proceedure:

1. Prepare a dish of MeOH and of transfer buffer for PVDF

2. Soak pads in cold transfer buffer

3. Dip both sides of the membrane in MeOH, water, and soak in cold transfer buffer

4. Load transfer apparatus

5. Dip filter paper in TB before putting on apparatus

a. Put 2 pads/side.

b. Remove gel from its case. Add filter paper and gel to top of pads

c. Add PVDF making sure there are no air bubbles and another piece of filter paper. Use a small
glass test tube to gently roll out air bubbles. Complete the set up.

6. Place inside transfer apparatus and fill with cold transfer buffer - make sure it does not leak and do not fill
all the way to the top. Add COLD H2O or TB to outside

7. Set up transfer apparatus on ice (or in cold room). For Bis-Tris gels, run @ 30 V volts for 1 hr. Expected
current starts at 170mA and ends at 110mA.

8. Remove membrane paper from transfer buffer apparatus. Wet in MeOH and dry on filter paper overnight

 
Detection

Solutions:

 Wash Buffer: 1x TBS-T - Add 5ml of 10% Tween-20 per liter of TBS (final concentration ~0.05%)

 Blocking buffer: Combine 100 ml TBS, 10 ml Tween-20, 10 g BSA fraction V, and 890 ml dH2O. AVOID
using milk when using Avidin/biotin systems.

 Primary Antibody: For SuperSignal West Dura Extended Duration Substrate, the concentration of primary
antibody needs to be between 20ng/ml and 1μg/ml. Dilute in blocking buffer (1:1000 = 10 μl per 10 ml) - for a
1mg/ml antibody solution, dilutions should be in the range of 1:1,000 to 1:50,000.

 Secondary antibody: For SuperSignal West Dura Extended Duration Substrate, the concentration of
secondary antibody needs to be between 4-20ng/ml. Dilute in blocking buffer (1:50,000 = 1 μl per 50ml) - for a
1mg/ml antibody solution, dilutions should be in the range of 1:50,000 to 1:250,000.

 Detection regent: 10 ml enhancer solution + 10 ml peroxide (DO NOT MIX UNTIL READY; 1:1 ratio)

13
Procedure:

1. Reactivate in MeOH

2. Block: place membrane in blocking roughly 50 ml buffer for 1.5 hours at room temperature or overnight @
4°C. Incubate on a rocker platform.

3. Dilute primary antibody in blocking buffer and incubate according to manufacturer's instructions. Incubate
on a rocker platform. Incubate 1st @ 4°C overnight

4. Wash blot 4-6 X 5 minutes in Wash Buffer on a rocker platform

5. Prepare secondary antibody in blocking buffer (12ml for one blot). Incubate on a rocker platform.

a. Place membrane in secondary antibody solution @ RT for 1 hr.

b. Additional blocking agents may be added if background staining is an issue.

6. Wash 4-6 X 5 min in Wash Buffer on a rocker platform

7. Mix together detection reagent and soak membrane for 5 minutes

a. Rock manually or on rocking platform and observe membrane while color development takes
place.

b. Optional: Stop color development when bands are easily visualized or when background color
development begins to be excessive, by decanting the development solution into a waste container, and
adding approximately 50 ml of deionized water to the incubation tray.

c. Optional: Return the tray to the rocking platform and check the color of the water after 5-10
minutes. Decant and replace with fresh water if there is a significant purple tinge to the water, or if band
intensity has continued to increase.

8. Place blot on sheet protector and make sure the outside is clean and the inside is bubble free. Remove
excess liquid/Keep in dark

9. Visualize blot using x-ray film or chemiluminescent gel imager

14
Result

After performing the experiment bands were obtained.

[ Western blot analysis of proteins separated by SDS-PAGE.]

15
 AGAROSE GEL ELECTROPHORESIS

Agarose gel electrophoresis is a method used in biochemistry and molecular biology to separate a mixed

population of DNA and RNAfragments by length, or to separate proteins by [Link] acid molecules are
separated by applying an electric field to move the negatively charged molecules through an agarose matrix. Shorter
molecules move faster and migrate farther than longer ones because shorter molecules migrate more easily through
the pores of the [Link] phenomenon is called [Link] are separated by charge in agarose because the pores
of the gel are too large to sieve proteins.

Factors affecting migration of nucleic acids

The most important factor is the length of the DNA molecule, smaller molecules travel farther. But conformation of
the DNA molecule is also a factor. To avoid this problem linear molecules are usually separated, usually DNA
fragments from a restriction digest, linear DNA PCR products, or RNAs.

Increasing the agarose concentration of a gel reduces the migration speed and enables separation of smaller DNA
molecules. The higher the voltage, the faster the DNA moves. But voltage is limited by the fact that it heats and
ultimately causes the gel to melt. High voltages also decrease the resolution (above about 5 to 8 V/cm).

Conformations of a DNA plasmid that has not been cut with a restriction enzyme will move with different speeds
(slowest to fastest: nicked or open circular, linearised, or supercoiled plasmid).

Visualisation: ethidium bromide(EtBr)

The most common dye used to make DNA or RNA bands visible for agarose gel electrophoresis is ethidium
bromide, usually abbreviated as EtBr. It fluoresces under UV light when intercalated into DNA (or RNA). By
running DNA through an EtBr-treated gel and visualizing it with UV light, any band containing more than ~20 ng
DNA becomes distinctly visible. EtBr is a known mutagen, however, safer alternatives are available.

Percent agarose and resolution limits

16
Agarose gel electrophoresis can be used for the separation of DNA fragments ranging from 50 base pair to several
megabases (millions of bases) using specialized apparatus. The distance between DNA bands of a given length is
determined by the percent agarose in the gel. In general lower concentrations of agarose are better for larger
molecules because they result in greater separation between bands that are close in size. The disadvantage of higher
concentrations is the long run times (sometimes days). Instead high percentage agarose gels should be run with
a pulsed field electrophoresis (PFE), or field inversion electrophoresis.

Most agarose gels are made with between 0.7% (good separation or resolution of large 5–10kb DNA fragments) and
2% (good resolution for small 0.2–1kb fragments) agarose dissolved in electrophoresis buffer. Up to 3% can be used
for separating very tiny fragments but a vertical polyacrylamide gel is more appropriate in this case. Low percentage
gels are very weak and may break when you try to lift them. High percentage gels are often brittle and do not set
evenly. 1% gels are common for many applications.

Buffers

There are a number of buffers used for agarose electrophoresis. The most common
being: Tris/Acetate/EDTA (TAE), Tris/Borate/EDTA (TBE) and lithium borate (LB). TAE has the lowest buffering
capacity but provides the best resolution for larger DNA. This means a lower voltage and more time, but a better
product. LB is relatively new and is ineffective in resolving fragments larger than 5 kbp; However, with its low
conductivity, a much higher voltage could be used (up to 35 V/cm), which means a shorter analysis time for routine
electrophoresis. As low as one base pair size difference could be resolved in 3 % agarose gel with an extremely low
conductivity medium (1 mM Lithium borate).

Analysis

After electrophoresis the gel is illuminated with an ultraviolet lamp (usually by placing it on a light box, while using
protective gear to limit exposure to ultraviolet radiation) to view the DNA bands. The ethidium
bromide fluoresces reddish-orange in the presence of DNA. The DNA band can also be cut out of the gel, and can
then be dissolved to retrieve the purified DNA. The gel can then be photographed usually with a digital or polaroid
camera. Although the stained nucleic acid fluoresces reddish-orange, images are usually shown in black and white.

17
Gel electrophoresis research often takes advantage of software-based image analysis tools, such as ImageJ.

1 2 3

A 1% agarose 'slab' gel prior to UV

The gel with UV illumination, theethidium
illumination, behind a perspex UV shield. Digital photo of the gel. Lane 1. Commercial
bromide stained DNA glows orange
Only the marker dyes can be see DNA Markers (1kbplus), Lane 2. empty,
Lane 3. a PCR product of just over 500
bases, Lane 4. Restrictiondigest showing a
similar fragment cut from a 4.5
kb plasmid vector

18
Typical methods

Materials

Typically 10-30 μl/sample of the DNA fragments to separate are obtained, as well as a mixture of DNA fragments
(usually 10-20) of known size (after processing with DNA size markers either from a commercial source or prepared
manually).

 Buffer solution, usually TBE buffer or TAE 1.0x, pH 8.0

 Agarose
 An ultraviolet-fluorescent dye, ethidium bromide, (5.25 mg/ml in H2O). The stock solution be careful
handling this. Alternative dyes may be used, such as SYBR Green.
 Nitrile rubber gloves. Latex gloves do not protect well from ethidium bromide
 A color marker dye containing a low molecular weight dye such as "bromophenol blue" (to enable tracking
the progress of the electrophoresis) and glycerol (to make the DNA solution denser so it will sink into the wells
of the gel).
 A gel rack
 A "comb"
 Power Supply
 UV lamp or UV lightbox or other method to visualize DNA in the gel

Reagents/Solutions

 50 x TAE : 242g Tris base, 57.1 ml of glacial Acetic acid, 100ml of 0.5M EDTA (pH 8.0), Make up to 1 L
with water

19
  5 x TBE: 54g Tris base: 27.5g of boric acid: 20ml of 0.5M EDTA (pH 8.0), Make up to 1 L with water

 20 x Sodium Boric Acid: 40.2g Borax Anhydrous, Make up to 1 L with water

 20X Lithium Boric Acid: Low-conductance DNA lithium borate electrophoretic media.

Preparation
There are several methods for preparing gels. A common example is shown here. Other methods might differ in the
buffering system used, the sample size to be loaded, the total volume of the gel (typically thickness is kept to a
constant amount while length and breadth are varied as needed). Most agarose gels used in
modern biochemistry and molecular biology are prepared and run horizontally.

1. Make a 1% agarose solution in 100ml TAE, for typical DNA fragments (see figures). A solution of up to 2-
4% can be used if you analyze small DNA molecules, and for large molecules, a solution as low as 0.7%
can be used.
2. Carefully bring the solution just to the boil to dissolve the agarose, preferably in a microwave oven.
3. Let the solution cool down to about 60 °C at room temperature, or water bath. Stir or swirl the solution
while cooling.

Wear gloves from here on, ethidium bromide is a mutagen, for more information on safety see ethidium bromide

1. Add 5 µl ethidium bromide stock (10 mg/ml) per 100 ml gel solution for a final concentration of 0.5 ug/ml.
Be very careful when handling the concentrated stock. Some researchers prefer not to add ethidium
bromide to the gel itself, instead soaking the gel in an ethidium bromide solution after running.
2. Stir the solution to disperse the ethidium bromide, then pour it into the gel rack.
3. Insert the comb at one side of the gel, about 5-10 mm from the end of the gel.
4. When the gel has cooled down and become solid, carefully remove the comb. The holes that remain in the
gel are the wells or slots.
5. Put the gel, together with the rack, into a tank with TAE. Ethidium bromide at the same concentration can
be added to the buffer. The gel must be completely covered with TAE, with the slots at the end electrode
that will have the negative current.

Procedure
After the gel has been prepared, use a micropipette to inject about 2.5 µl of stained DNA (a DNA ladder is also
highly recommended). Close the lid of the electrophoresis chamber and apply current (typically 100 V for 30

20
minutes with 15 ml of gel). The colored dye in the DNA ladder and DNA samples acts as a "front wave" that runs
faster than the DNA itself. When the "front wave" approaches the end of the gel, the current is stopped. The DNA is
stained with ethidium bromide, and is then visible under ultraviolet light.

1. The agarose gel with three slots/wells (S).

2. Injection of DNA ladder (molecular weight markers) into the first slot.
3. DNA ladder injected. Injection of samples into the second and third slot.
4. A current is applied. The DNA moves toward the positive anode due to the negative charges on
its phosphate backbone.
5. Small DNA strands move fast, large DNA strands move slowly through the gel. The DNA is not normally
visible during this process, so the marker dye is added to the DNA to avoid the DNA being run entirely off
the gel. The marker dye has a low molecular weight, and migrates faster than the DNA, so as long as the
marker has not run past the end of the gel, the DNA will still be in the gel.
6. Add the color marker dye to the DNA ladder.

A pattern of DNA-bands under UV light

21
Figure 1: Schematic drawing of the electrophoresis process, see text for description of steps

Applications

 Estimation of the size of DNA molecules following restriction enzyme digestion, e.g. in restriction
mapping of cloned DNA.
 Analysis of PCR products, e.g. in molecular genetic diagnosis or genetic fingerprinting
 Separation of restricted genomic DNA prior to Southern transfer, or of RNA prior to Northern transfer.

Agarose gels are easily cast and handled compared to other matrices and nucleic acids are not chemically altered
during electrophoresis. Samples are also easily recovered. After the experiment is finished, the resulting gel can be
stored in a plastic bag in a refrigerator.

There are limits to electrophoretic techniques. Since passing current through a gel causes heating, gels may melt
during electrophoresis. Electrophoresis is performed in buffer solutions to reduce pH changes due to the electric
field, which is important because the charge of DNA and RNA depends on pH, but running for too long can exhaust

22
the buffering capacity of the solution. Further, different preparations of genetic material may not migrate
consistently with each other, for morphological or other reasons.

Result
After performing the experiment bands were observed.

23
Digital image of 3 plasmid restriction digests run on a 1% w/v agarose gel, 3 Volts/cm, stained with ethidium bromide. The DNA size
marker is a commercial 1 kbp ladder. The position of the wells and direction of DNA migration is noted.

POLYMERASE CHAIN REACTION(PCR)

24
The polymerase chain reaction (PCR) is a technique in molecular biology to amplify a single or few copies of a
piece of DNAacross several orders of magnitude, generating thousands to millions of copies of a particular DNA
sequence. The method relies on thermal cycling, consisting of cycles of repeated heating and cooling of the reaction
for DNA melting and enzymaticreplication of the DNA. Primers (short DNA fragments) containing sequences
complementary to the target region along with aDNA polymerase (after which the method is named) are key
components to enable selective and repeated amplification. As PCR progresses, the DNA generated is itself used as
a template for replication, setting in motion a chain reaction in which the DNA template is exponentially amplified.
PCR can be extensively modified to perform a wide array of genetic manipulations.

Almost all PCR applications employ a heat-stable DNA polymerase, such as Taq polymerase, an enzyme originally
isolated from the bacterium Thermus aquaticus. This DNA polymerase enzymatically assembles a new DNA strand
from DNA building blocks, the nucleotides, by using single-stranded DNA as a template and DNA
oligonucleotides (also called DNA primers), which are required for initiation of DNA synthesis. The vast majority
of PCR methods use thermal cycling, i.e., alternately heating and cooling the PCR sample to a defined series of
temperature steps. These thermal cycling steps are necessary first to physically separate the two strands in a DNA
double helix at a high temperature in a process called DNA melting. At a lower temperature, each strand is then used
as the template in DNA synthesis by the DNA polymerase to selectively amplify the target DNA. The selectivity of
PCR results from the use of primers that are complementary to the DNA region targeted for amplification under
specific thermal cycling conditions.

Developed in 1983 by Kary Mullis,PCR is now a common and often indispensable technique used in medical and
biological research labs for a variety of [Link] include DNA cloning for sequencing, DNA-
based phylogeny, or functional analysis of genes; the diagnosis of hereditary diseases; the identification of genetic
fingerprints (used in forensic sciences and paternity testing); and the detection and diagnosis of infectious diseases.
In 1993, Mullis was awarded the Nobel Prize in Chemistry for his work on PCR.

25
A strip of eight PCR tubes, each containing a 100 μL reaction mixture

PCR Principles and Procedures

PCR is used to amplify a specific region of a DNA strand (the DNA target). Most PCR methods typically amplify
DNA fragments of up to ~10 kilo base pairs (kb), although some techniques allow for amplification of fragments up
to 40 kb in size.

A basic PCR set up requires several components and [Link] components include:

 DNA template that contains the DNA region (target) to be amplified.( it’s not usually necessary to be
incredibly fastidious about how much template we add to a [Link] can get product with incredibly
small amounts of starting DNA. I usually do a 3mL plasmid prep and use 1/6 of a microliter per PCR
[Link] can use more or less -- it doesn’t seem to matter that much.)
 Two primers that are complementary to the 3' (three prime) ends of each of the sense and anti-sense strand
of the DNA target.( a good place to start with primer concentration is 50pmol of each primer per reaction. If we
don’t get our desired product, we can increase to 75pmol or 100pmol. This usually does the trick. I wouldn’t go
past 200pmol of primers unless it’s a special protocol that recommends using more.)
 Taq polymerase or another DNA polymerase with a temperature optimum at around 70 °C.
 Deoxynucleoside triphosphates (dNTPs; also very commonly and erroneously called deoxynucleotide
triphosphates), the building blocks from which the DNA polymerases synthesizes a new DNA strand. (we want
the final concentration to be 200μM, so a 2mM stock is essentially 10X -- use 2.5μL per reaction.)
 Buffer solution, providing a suitable chemical environment for optimum activity and stability of the DNA
polymerase.(with concentration 1X, usually comes as 10X stock. For 25μL reactions, this means 2.5μL.)
 Divalent cations, magnesium or manganese ions; generally Mg2+ is used, but Mn2+ can be utilized for PCR-
mediated DNA mutagenesis, as higher Mn2+ concentration increases the error rate during DNA synthesis.( this
is the greatest variable in PCR. The success of a PCR is very dependent on how much magnesium is present in
the reaction./. For this reason, it is usually advisable to do a magnesium optimization when performing new
PCRs. I do my reactions in sets of six, keeping all variables constant except for magnesium. I usually go from
1mM to 6mM MgCl2. Since the stock is 25mM, usually, this means that 1μL of stock equals 1mM MgCl2 in a
25μL reaction -- it’s convenient.)
 Monovalent cation potassium ions.

The PCR is commonly carried out in a reaction volume of 10–200 μl in small reaction tubes (0.2–0.5 ml volumes) in
a thermal cycler. The thermal cycler heats and cools the reaction tubes to achieve the temperatures required at each
step of the reaction (see below). Many modern thermal cyclers make use of the Peltier effect which permits both

26
heating and cooling of the block holding the PCR tubes simply by reversing the electric current. Thin-walled
reaction tubes permit favorable thermal conductivity to allow for rapid thermal equilibration. Most thermal cyclers
have heated lids to prevent condensation at the top of the reaction tube. Older thermocyclers lacking a heated lid
require a layer of oil on top of the reaction mixture or a ball of wax inside the tube.

Figure 1a: A thermal cycler for PCR

Procedure

27
Figure 2: Schematic drawing of the PCR cycle. (1) Denaturing at 94–96 °C. (2) Annealing at ~65 °C (3) Elongation at 72 °C. Four
cycles are shown here. The blue lines represent the DNA template to which primers (red arrows) anneal that are extended by the DNA
polymerase (light green circles), to give shorter DNA products (green lines), which themselves are used as templates as PCR progresses.

Typically, PCR consists of a series of 20-40 repeated temperature changes, called cycles, with each cycle commonly
consisting of 2-3 discrete temperature steps, usually three (Fig. 2). The cycling is often preceded by a single

28
temperature step (called hold) at a high temperature (>90°C), and followed by one hold at the end for final product
extension or brief storage. The temperatures used and the length of time they are applied in each cycle depend on a
variety of parameters. These include the enzyme used for DNA synthesis, the concentration of divalent ions and
dNTPs in the reaction, and the melting temperature (Tm) of the primers.

 Initialization step: This step consists of heating the reaction to a temperature of 94–96 °C (or 98 °C if
extremely thermostable polymerases are used), which is held for 1–9 minutes. It is only required for DNA
polymerases that require heat activation by hot-start PCR.

 Denaturation step: This step is the first regular cycling event and consists of heating the reaction to 94–
98 °C for 20–30 seconds. It causes DNA melting of the DNA template by disrupting the hydrogen bonds
between complementary bases, yielding single-stranded DNA molecules.

 Annealing step: The reaction temperature is lowered to 50–65 °C for 20–40 seconds allowing annealing of
the primers to the single-stranded DNA template. Typically the annealing temperature is about 3-5 degrees
Celsius below the Tm of the primers used. Stable DNA-DNA hydrogen bonds are only formed when the primer
sequence very closely matches the template sequence. The polymerase binds to the primer-template hybrid and
begins DNA synthesis.

 Extension/elongation step: The temperature at this step depends on the DNA polymerase used; Taq
polymerase has its optimum activity temperature at 75–80 °C,[10][11] and commonly a temperature of 72 °C is
used with this enzyme. At this step the DNA polymerase synthesizes a new DNA strand complementary to the
DNA template strand by adding dNTPs that are complementary to the template in 5' to 3' direction, condensing
the 5'-phosphate group of the dNTPs with the 3'-hydroxyl group at the end of the nascent (extending) DNA
strand. The extension time depends both on the DNA polymerase used and on the length of the DNA fragment
to be amplified. As a rule-of-thumb, at its optimum temperature, the DNA polymerase will polymerize a
thousand bases per minute. Under optimum conditions, i.e., if there are no limitations due to limiting substrates
or reagents, at each extension step, the amount of DNA target is doubled, leading to exponential (geometric)
amplification of the specific DNA fragment.

 Final elongation: This single step is occasionally performed at a temperature of 70–74 °C for 5–15 minutes
after the last PCR cycle to ensure that any remaining single-stranded DNA is fully extended.

 Final hold: This step at 4–15 °C for an indefinite time may be employed for short-term storage of the
reaction.

29
Figure 3: Ethidium bromide-stained PCR products after gel electrophoresis. Two sets of primers were used to amplify a target sequence
from three different tissue samples. No amplification is present in sample #1; DNA bands in sample #2 and #3 indicate successful
amplification of the target sequence. The gel also shows a positive control, and a DNA ladder containing DNA fragments of defined
length for sizing the bands in the experimental PCRs.

To check whether the PCR generated the anticipated DNA fragment (also sometimes referred to as the amplimer
or amplicon),agarose gel electrophoresis is employed for size separation of the PCR products. The size(s) of PCR
products is determined by comparison with a DNA ladder (a molecular weight marker), which contains DNA
fragments of known size, run on the gel alongside the PCR products.

PCR Stages
The PCR process can be divided into three stages:

1. Exponential amplification: At every cycle, the amount of product is doubled (assuming 100%
reaction efficiency). The reaction is very sensitive: only minute quantities of DNA need to be
present.

2. Levelling off stage: The reaction slows as the DNA polymerase loses activity and as consumption
of reagents such as dNTPs and primers causes them to become limiting.

3. Plateau: No more product accumulates due to exhaustion of reagents and enzyme.

30
PCR optimization
In practice, PCR can fail for various reasons, in part due to its sensitivity to contamination causing amplification of
spurious DNA products. Because of this, a number of techniques and procedures have been developed for
optimizing PCR [Link] with extraneous DNA is addressed with lab protocols and procedures that
separate pre-PCR mixtures from potential DNA [Link] usually involves spatial separation of PCR-setup
areas from areas for analysis or purification of PCR products, use of disposable plasticware, and thoroughly cleaning
the work surface between reaction setups. Primer-design techniques are important in improving PCR product yield
and in avoiding the formation of spurious products, and the usage of alternate buffer components or polymerase
enzymes can help with amplification of long or otherwise problematic regions of DNA.

Applications of PCR

Selective DNA isolation

PCR allows isolation of DNA fragments from genomic DNA by selective amplification of a specific region of DNA.
This use of PCR augments many methods, such as generating hybridization
probes for Southern or northern hybridization and DNA cloning, which require larger amounts of DNA, representing
a specific DNA region. PCR supplies these techniques with high amounts of pure DNA, enabling analysis of DNA
samples even from very small amounts of starting material.

Other applications of PCR include DNA sequencing to determine unknown PCR-amplified sequences in which one
of the amplification primers may be used in Sanger sequencing, isolation of a DNA sequence to
expedite recombinant DNAtechnologies involving the insertion of a DNA sequence into a plasmid or the genetic
material of another organism. Bacterial colonies ([Link]) can be rapidly screened by PCR for correct
DNA vector [Link] may also be used for genetic fingerprinting; a forensic technique used to identify a
person or organism by comparing experimental DNAs through different PCR-based methods.

Some PCR 'fingerprints' methods have high discriminative power and can be used to identify genetic relationships
between individuals, such as parent-child or between siblings, and are used in paternity testing (Fig. 4). This
technique may also be used to determine evolutionary relationships among organisms.

31
Figure 4: Electrophoresis of PCR-amplified DNA fragments. (1) Father. (2) Child. (3) Mother. The child has inherited some, but not all
of the fingerprint of each of its parents, giving it a new, unique fingerprint.

Amplification and quantification of DNA

Because PCR amplifies the regions of DNA that it targets, PCR can be used to analyze extremely small amounts of
sample. This is often critical for forensic analysis, when only a trace amount of DNA is available as evidence. PCR
may also be used in the analysis of ancient DNA that is tens of thousands of years old. These PCR-based techniques
have been successfully used on animals, such as a forty-thousand-year-old mammoth, and also on human DNA, in
applications ranging from the analysis of Egyptian mummies to the identification of aRussian tsar.

Quantitative PCR methods allow the estimation of the amount of a given sequence present in a sample—a technique
often applied to quantitatively determine levels of gene expression. Real-time PCR is an established tool for DNA
quantification that measures the accumulation of DNA product after each round of PCR amplification.

 In Use of DNA in forensic entomology

32
PCR in diagnosis of diseases
PCR permits early diagnosis of malignant diseases such as leukemia and lymphomas, which is currently the highest
developed in cancer research and is already being used [Link] assays can be performed directly on genomic
DNA samples to detect translocation-specific malignant cells at a sensitivity which is at least 10,000 fold higher
than other methods.

PCR also permits identification of non-cultivatable or slow-growing microorganisms such

as mycobacteria, anaerobic bacteria, or viruses fromtissue culture assays and animal models. The basis for PCR
diagnostic applications in microbiology is the detection of infectious agents and the discrimination of non-
pathogenic from pathogenic strains by virtue of specific genes.

Viral DNA can likewise be detected by PCR. The primers used need to be specific to the targeted sequences in the
DNA of a virus, and the PCR can be used for diagnostic analyses or DNA sequencing of the viral genome. The high
sensitivity of PCR permits virus detection soon after infection and even before the onset of disease. Such early
detection may give physicians a significant lead in treatment. The amount of virus ("viral load") in a patient can also
be quantified by PCR-based DNA quantitation techniques.

33
 LYMPHOCYTE PROLIFERATION ASSAY(MTT Assay)

The lymphocyte proliferation assay is used as an in vitro surrogate, similar to the in vivo delayed-type
hypersensitivity assay, to assess the overall quality and character of the cellular arm of the immune response. The
lymphocyte proliferation assay is used to assess congenital immune deficiencies, transient immune compromised
states, and more recently, the progressive immune deficiency of HIV infection.

The MTT assay and the MTS assay are colorimetric assays for measuring the activity of enzymes that reduce MTT
or close dyes (XTT, MTS, WSTs) to formazan dyes, giving a purple color. A main application allows to assess the
viability (cell counting) and the proliferation of cells (cell culture assays). It can also be used to
determine cytotoxicity of potential medicinal agents and toxic materials, since those agents would stimulate or
inhibit cell viability and growth.

  Lymphocyte proliferation assay is used to determine lymphocyte activation and the cell-mediated
immune responses.  When B cells encounter their specific antigens, with the help of T cells, B cells are
stimulated to undergo proliferation.  When T cells are activated by antigen-presenting cells and cytokines,
T cells undergo proliferation.   The proliferation of B and T cells leads to clonal expansion and the
initiation of the specific immune responses.

            Cells undergoing proliferation increase their rate of protein and DNA synthesis.  The increase in
DNA synthesis can be measured by adding [3H]thymidine, a radioisotope-labeled DNA precursor, to the
cell culture medium.  The amount of tritium taken up by the dividing cells is correlated to the level of
cellular proliferation.  Cells undergoing proliferation are also metabolically active and increase their
cellular level of dehydrogenase and their product NADH and NADPH.  The levels of NADH and
NADPH can be measured by their ability to reduce yellow colored MTT (3-(4, 5-dimethylthiazolyl-2)-2,
5-diphenyltetrazolium bromide) to intracellular purple formazan.  The resulting purple products can be
solubilized and quantified by spectrophotometric means.  MTT and [3H]thymidine incorporation are two
common methods used to measure cell proliferation.

            Cytokines, such as IL-2, can stimulate T cells to proliferate.  T cells proliferate in an IL-2
concentration-dependent manner.  The level of T cell proliferation can be used as a measurement of IL-2
concentration.  Using IL-2 with a known concentration as a standard, the concentration of IL-2 in an
unknown sample can be measured by its ability to stimulate T cell proliferation

34
 96 well plate showing MTT assay

MTT

MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a yellow tetrazole), is reduced

to purple formazan in living cells.A solubilization solution (usually either dimethyl sulfoxide, an acidified ethanol
solution, or a solution of the detergent sodium dodecyl sulfate in diluted hydrochloric acid) is added to dissolve the
insoluble purple formazan product into a colored solution. The absorbance of this colored solution can be quantified
by measuring at a certain wavelength (usually between 500 and 600 nm) by a spectrophotometer. The absorption
maximum is dependent on the solvent employed.

Measurement of Cell Viability: MTT Assay

1. Plate 1.0 ml of cells (50,000-100,000 cells/ml) into each well of 24-well culture plate.
2. Incubate the cells for 24 h in CO2 incubator.
3. After treatment of cells for 24-72 h, experimental media are removed, and cells are washed with PBS.
4. The cells are incubated with basal medium containing 0.5 mg/ml MTT in CO2 incubator at 37 C for 4 h.
5. The medium is aspirated, and the formazan product is solubilized with dimethyl sulfoxide (DMSO).
6. Absorbance at 570 nm is measured for each well using a microplate reader.

35
Applications

The MTT assay is an alternative to the thymidine incorporation test which measures cell proliferation by
determining the amounts of incorporated tritiated thymidine into freshly synthesized DNA. It can be used also to
determine proliferation of mycoplasm-infected cells, which would interfere with thymidine incorporation
measurements. The proliferative profiles of cells as determined by the colorimetric assay, which essentially
measures energy metabolism, or radioisotope assay usually do not show large differences.

The MTT assay has been adapted also to quantitate cytotoxic activities and growth inhibitory activities of cytokines.

36
 High Performance Liquid Chromatography

High performance liquid chromatography(or high pressure liquid chromatography, HPLC is a form of column
chromatography used frequently in biochemistry and analytical chemistry . HPLC is used to separate components of
a mixture by using a variety of chemical interactions between the substance being analysed (analyte) and the
chromatography column. The sample to be analysed is introduced in small volume to the stream of mobile phase and
is retarded by specific chemical or physical interactions with the stationary phase as it traverses the length of the
column. The amount of retardation depends upon the nature of the analyte, stationary phase and mobile phase
configuration. The time at which a specific analyte elutes from the end of the column is called the retention time
and is considered a rather unique identifying characteristic of a given analyte. The use of pressure increases the
linear velocity (speed) giving the components less time to diffuse within the column leading to improved resolution
in the resulting chromatogram. Common solvents used include any miscible combinations of water or various
organic liquids (the most common are methanol and acetonitrile). Water may contain buffers or salts to assist in the
separation of the analyte components, or compounds such as trifluoroacetic acid which acts as an ion pairing agent.

A further refinement to HPLC has been to vary the mobile phase composition during the analysis; this is known as
gradient elution. A normal gradient for reversed phase chromatography might start at 5% methanol and progress
linearly to 50% methanol over 25 minutes, depending on how hydrophobic the analyte is. The gradient separates the
analyte mixtures as a function of the affinity of the analyte for the current mobile phase composition relative to the
stationary phase. This partioning process is similar to that which occurs during a liquid-liquid interaction but is
continuous but not stepwise. In this example using a water-methanol gradient, the more hydrophobic
components will elute under the conditions of relatively high methanol whereas the more hydrophilic compounds
will elute out under condition of relatively low methanol. The choice of solvents, additives and gradient depend on
the nature of the stationary phase and the analyte. Often a series of tests are performed on the analyte and a number
of generic runs may be processed in order to find the optimum HPLC method for the analyte- the method which
gives the best separation of peaks.

Types of HPLC:

Normal phase chromatography

Also known as Normal phase HPLC (NP-HPLC) was the first kind of HPLC chemistry used, and separates analytes
based on polarity. This method uses a polar stationary phase and a non polar mobile phase, and is used when the
analyte of interest is fairly polar in nature. The polar analyte associates with and is retained with the polar stationary
phase. Adsorption strengths increase with the increase in analyte polarity, and the interaction between the polar
analyte and the polar stationary phase (relative to the mobile phase) increases the elution time. The interaction
strength not only depends on the functional groups in the analyte molecule but also on steric factors and structural
isomers are often resolved from one another. Use of more polar solvents in the mobile phase will decrease the
retention time of the analytes while more hydrophobic solvents tend to increase retention times. Particularly polar

37
solvents in a mixture tend to deactivate the column by occupying the stationary phase surface. This is somewhat
particular to normal phase because it is most purely an adsorptive mechanism (the interactions are with a hard
surface rather than a soft layer on a surface).

NP-HPLC had fallen out of favor in the 1970s with the development of reversed phase HPLC because of a lack of
reproducibility of retention times as water or protic organic solvents changed the hydration state of the silica or
alumina chromatographic media. Recently it has become useful again with the development of HILIC bonded
phases which utilize a partition mechanism which provides reproducibility.

Reversed phase chromatography

Reversed phase HPLC (RP-HPLC or RPC) consists of a non polar stationary phase and an aqueous, moderately
polar mobile phase. One common stationary phase is silica which has been treated with RMe2SiCl, where R is a
straight chain alkyl group such as C18H37 or C8H17. The retention time is therefore longer for molecules which are
more non polar in nature, allowing polar molecules to elute more readily. Retention Time (RT) is increased by the
addition of polar solvent to the mobile phase and decreased by the addition of more hydrophobic solvent. Reversed
phase chromatography is so commonly used that it is not uncommon for it to be incorrectly referred to as “HPLC”
without further specification. The pharmaceutical industry regularly employs RPC to qualify drugs before their
release.

RPC operates on the principle of hydrophobic interactions, which result from repulsive forces between a polar
eluent, the relatively non polar analyte and the non polar stationary phase. The binding of the analyte to the
stationary phase is proportional to the contact surface area around the non polar segment of the analyte molecule
upon association with the ligand in the aqueous eluent. This solvophobic effect is dominated by the force of water
for “cavity reduction” around the analyte and the C18 chain versus the complex of both. The energy
released in this process is proportional to the surface tension of the eluent (water: 73 erg/cm2, methanol:22
erg/cm2) and to the hydrophobic surface of the analyte and the ligand respectively. The retention can be decreased
by adding less-polar solvent (MeOH, ACN) in to the mobile phase to reduce the surface tension of water.
Gradient elution uses this effect by automatically changing the polarity of the mobile phase during the course of the
analysis.

Structural properties of the analyte molecule play an important role in its retention characteristics. In general, an
analyte with a larger hydrophobic surface area (C-H,C-C, and generally non polar atomic bonds such as S-S and
others) results in a longer retention time because it increases the molecule’s non polar surface area which is non
interacting with the water structure. On the other hand polar groups such as –OH,-NH2,-COO- or NH3+ reduce
retention as they are well integrated into water. Very large molecules, however can result in an incomplete
interaction between the large analyte surface and the ligands alkyl chains and can have problems entering the pores
of the stationary phase.

38
RT increases with hydrophobic-non polar-surface area. Branched chain compounds elute more rapidly than their
corresponding linear isomers because the overall surface area is decreased. Similarly organic compounds with single
C-C bonds elute later than the ones with a C=C or C-C triple bond, as the double or triple bond is shorter than a
single C-C bond.

Aside from mobile phase surface tension (organizational strength in eluent structure), other mobile phase modifiers
can affect analyte retention. For example, the addition of inorganic salts causes a moderate linear increase in the
surface tension of aqueous solutions (ca. 1.5 erg/cm3 pro Mol for (NH4)2SO4), and because the entropy of the
analyte-solvent interface is controlled by surface tension, the addition of salts tend to increase the retention time.
This technique is used for mild separation and recovery of proteins and protection of their biological activity in
protein analysis (hydrophobic interaction chromatography, HIC).

Another important component is the influence of the pH since this can change the hydrophobicity of the analyte. For
this reason most methods use a buffering agent, such as sodium phosphate, to control the pH. A volatile organic acid
such as formic acid or more commonly trifluoroacetic acid is is often added to the mobile phase, if mass
spectrometry is applied to the eluent fractions. The buffers serve multiple purposes: they control pH, neutralize the
charge on any residual exposed silica on the stationary phase and act as ion pairing agents to neutralize charge on
the analyte. The effect varies depending upon the use but generally improves the chromatography.

Reversed phase columns are quite difficult to damage compared with normal silica columns, however many
reversed phase columns consist of alkyl derivatized silica particles and should never be used with aqueous bases as
these will destroy the underlying silica particle. They can be used with aqueous acid , but the column should not be
exposed to the acid for long , as it can corrode the metal parts of the HPLC equipment. The metal content of HPLC
columns should be kept low if the best possible ability to separate substances is to be retained. A good test for the
metal content of the column is to inject a sample which is a mixture of 2,2’- and 4,4’-bipyridine. Because the 2,2’-
bipy can chelate the metal, the shape of the peak for the 2,2’-bipy will be distorted(tailed) when metal ions are
present on the surface of the silica.

39
PARAMETERS

Internal Diameter

The internal diameter (ID) of an HPLC column is a critical aspect that determines quantity of analyte that can be
loaded onto the column and also influences sensitivity. Larger columns are usually seen in industrial applications
such as the purification of a drug product for later use. Low ID columns have improved sensitivity and lower solvent
consumption at the expense of loading capacity. Larger ID columns (over 10mm) are used to purify usable amounts
of materials because of their large loading capacity.

Analytical scale columns (4.6mm) have been the most common type of columns though smaller columns are rapidly
gaining in popularity. They are used in traditional quantitative analysis of samples and often use a UV-V as
absorbance detector.

Narrow bore columns (1-2 mm) are used for applications when more sensitivity is desired either with special UV-
visible detectors, fluorescence detection or with other detection methods like liquid chromatography–mass
spectrometry.

Capillary columns (under 0.3mm) which are used almost exclusively with alternate detection means such as mass
spectrometry. They are usually made from fused silica capillaries, rather than the stainless steel tubing that larger
columns employ.

Particle Properties:

Irregular Shape

40
The first available HPLC columns were packed using irregularly shaped silica particles. Because of this, many
standard analytical methods are still based on these materials. Irregular particles are also used in large- scale
preparative applications because of their high surface area, capacity and low cost.

Spherical Shape

The majority of new HPLC methods are performed on spherical shaped or spheroidal (almost spherical) particles.
Spherical particles provide higher efficiency, better column stability and lower back-pressures compared to
irregularly shaped particles.

Particle Size

Particle size for HPLC column packings refers to the average diameter of the packing particles. Most HPLC
packings contain a narrow range of particle diameters. Particle size affects the back-pressure of the column and the
separation efficiency. Column back-pressure and column efficiency are inversely proportional to the square of the
particle diameter. This means that as the particle size decreases, the column back-pressure and efficiency increase. A
well packed column with 3 μm packings produces almost twice the separation efficiency of a comparable 5 μm
column. However, the 3 μm column will have about a three-fold higher back-pressure compared to the 5 μm column
when operated with the same mobile phase and at the same flow rate. Highly efficient, small-particle (3 μm and 4
μm) columns are ideal for complex mixtures with similar components. Fast, high-resolution separations can be
achieved with small particles packed in short (10-50 mm length) columns. Grace Vydac offers SHORTFAST™ and
LIGHTNING™ HPLC columns specifically for these fast-HPLC applications.

Larger particle (5 μm and 7 μm) columns are typically used for routine analyses where analytes have greater
structural differences. Large 10 μm packings have only moderate column efficiencies. Columns packed with 10 μm
packings are generally used as scout columns for future preparative separations, semi- preparative applications, or
routine QA/QC methods where high chromatographic efficiencies are not required. Large particles (15- 20 μm) are
used for preparative-scale separations.

Most traditional HPLC is performed with the stationary phase attached to the outside of small spherical silica
particles (very small beads). These particles come in a variety of sizes with 5µm beads being the most common.
Smaller particles generally provide more surface area and better separations, but the pressure required for optimum
linear velocity increases by the inverse of the particle diameter squared.

41
This means that changing to particles that are half as big, keeping the size of the column the same will double the
performance, but increase the required pressure by a factor of four. Larger particles are more often used in non-
HPLC applications such as solid phase extraction.

Pore Size

The pore size of a packing material represents the average size of the pores within each particle. The size of the
analyte should be considered when choosing the appropriate pore size for the packing material. The molecular
weight of an analyte can be used to estimate the size of the molecule. As a general rule, a pore size of 100 Å or less
should be used for analytes below 3,000 MW. A pore size of 100 Å -130 Å is recommended for samples in the range
of 3,000 MW - 10,000 MW. For samples above10, 000MW, including peptides and proteins, a 300 Å materials
provides the best efficiency and peak shape.

Many stationary phases are porous to provide greater surface area. Small pores provide greater surface area while
larger pore size has better kinetics especially for larger analytes. For example a protein which is only slightly smaller
than a pore might enter the pore but not easily leave once inside.

Pore Volume

Pore volume is a measurement of the empty space within a particle. Pore volume is a good indicator of the
mechanical strength of a packing. Particles with large pore volumes are typically weaker than particles with small
pore volumes. Pore volumes of 1.0 mL/g or less are recommended for most HPLC separations. Pore volumes of
greater than 1.0mL/g are preferred for size-exclusion chromatography and useful for low-pressure methods.

Surface Area

The physical structure of the particle substrate determines the surface area of the packing material. Surface area is
determined by pore size. Pore size and surface area are inversely related. A packing material with a small pore size
will have a large surface area, and vice versa. High surface area materials offer greater capacity and longer analyte
retention times. Low surface area packings offer faster equilibration time and are often used for large molecular
weight molecules.

Carbon Load

The carbon load is a measure of the amount of bonded phase bound to the surface of the packing. High carbon loads
provide greater column capacities and resolution. Low carbon loads produce less retentive packings and faster
analysis times.

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Surface Coverage

Surface coverage is calculated from the carbon load and surface area of a packing material. Surface coverage affects
the retention, selectivity and stability of bonded phases.

End-Capping

A reversed-phase HPLC column that is end-capped has gone through a secondary bonding step to cover unreacted
silanols on the silica surface. End-capped packing materials eliminate unpredictable secondary interactions. Basic
analytes tend to produce asymmetric tailed peaks on non end-capped columns, requiring the addition of modifiers to
the mobile phase. Non end-capped materials exhibit different selectivity than end-capped columns. This selectivity
difference can enhance separations of polar analytes by controlling the secondary silanol interactions.

Pump Pressure

Pumps vary in pressure capacity, but their performance is measured on their ability to yield a consistent and
reproducible flow rate. Modern HPLC systems have been improved to work at much higher pressures, and therefore
be able to use much smaller particle sizes in the columns (<2 micrometers). These “Ultra High Performance Liquid
Chromatography” or UHPLCs can work at 15000 lbf/in2. Note that the term “UPLC”, sometimes found instead is a
trademark of Waters Corporation and not the name for the technique in general.

Parts of HPLC Instrument

The Column

HPLC analysis is based on separation of different molecules or chemicals by interaction with beads in a column.
The column is the heart of the HPLC system and consists of a metal housing in a tube shape that is filled with tiny
beads with characteristics that have an affinity for the chemicals that are being analyzed. The chemicals to interact
with the beads as they flow through the column in a solvent. Tiny molecular differences in similar compounds
causes them to migrate through the column at different velocities, and separates them according to migration time
through the column. The column has a frit, a screen-like filter that allows solvent to flow through but keeps the
beads from leaving the column.

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Substrate Materials:

1Silica

Porous silica particles are the most common substrate material used for HPLC column packing. Silica-based
columns can withstand high pressures, are compatible with most organic and aqueous mobile-phase solvents, and
come in a wide range of bonded phases. Silica-based columns are often used for separations of low molecular
weight analytes using mobile phase solvents and samples with a pH range of 2 to 7.5. Some new-generation silica
materials, such as EVEREST™, DENALI™, and GENESIS™, have extended pH ranges.

Polymeric

Highly cross-linked styrene-divinylbenzene based packings are compatible with most mobile phase solvents and
samples with a pH of 1 to 14. Polymer-based columns tend to have lower efficiencies for small molecules compared
to silica-based columns due to their smaller average surface area. In addition, polymer-based columns typically have
lower mechanical strength and therefore cannot withstand the highest system backpressures. A polymer-based
packing is often a good alternative if the sample requires a mobile phase pH outside the normal operating range of
standard silica-based columns. Polymeric packings are often used for ion-exchange separations, and are also useful
in non-aqueous GPC size-exclusing analyses and ion exclusion analyses of organic acids and carbohydrates.

Column Length, Diameter and Volume

Once a packing material has been selected, the physical dimensions of the HPLC column hardware should also be
optimized for the desired separation. It is important to understand the relationship between column length, diameter
and volume. The column length (L) and internal diameter (d) determine the bed volume (V) of an HPLC column by
the following equation: V = L x πd2/4 This equation does not account for the reduction in liquid volume due to the
packing material. It is an upper limit on the column volume – the minimum volume of mobile phase required to
elute an unretained analyte from the column. Small column diameters provide higher sensitivity than larger column
diameters for the same injected mass because the concentration of the analyte in the mobile phase is greater. Smaller
diameter columns also use less mobile phase per analysis because a slower flow rate is required to achieve the same
linear velocity through the column. HPLC instrumentation may need to be modified for columns with very small
internal diameters to eliminate band broadening due to extra-column effects, i.e., mixing volumes outside the
column. Longer columns often provide increased resolution. Larger-diameter columns provide greater sample
loading and lower back-pressure. Column back-pressure for a given flow rate increases as the column length
increases and as internal diameter decreases.

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Column Flow Rate

When the column dimensions are changed to optimize a separation, or to scale a separation to a preparative or
narrow-bore application, the mobile phase flow rate should be adjusted proportionally to cross-sectional area of the
column to maintain consistent linear velocity and retention times. The following equation will assist in adjusting the
flow rate (F) for columns of different inside diameters (d). F2 = F1 x (d2/d1)2

Pumps and Injectors

Because of the small particles used in modern HPLC, modern LC pumps need to operate reliably and precisely at
pressures of 10,000 p.s.i. or at least 6,000 p.s.i. To operate at these pressures and remain sensibly inert to the wide
variety of solvents used HPLC pumps usually have sapphire pistons, stainless steel cylinders and return valves fitted
with sapphire balls and stainless steel seats. For analytical purposes HPLC pumps should have flow rates that range
from 0 to 10 ml/min., but for preparative HPLC, flow rates in excess of 100 ml/min may be required. It is extremely
difficult to provide a very constant flow rate at very low flow rates. If 0.1% is considered acceptable then for
100uls/min a flow variation of less than 0.1ul/min is required. This level of constancy is required because most
HPLC detectors are flow sensitive and errors in quantization will result from changes in flow rate. Solvent is flowed
through the column under high pressure.

The pumps used for HPLC are carefully machined for exact flow rates and high pressure performance. The flow
rate, in addition to how a molecule reacts with the beads in the column, determines how fast a particular chemical
will migrate through the column. The migration time is used to identify individual chemicals. The injector is used to
place a sample into the solvent stream flowing through the column. It is designed to place a liquid sample into the
solvent stream with as little perturbation as possible.

There are a number of different types of pumps that can provide the necessary pressures and flow-rates required by
the modern liquid chromatograph. In the early years of the LC renaissance, there were two types of pump in
common use; they were the pneumatic pump, where the necessary high pressures were achieved by pneumatic
amplification, and the syringe pump, which was simply a large, strongly constructed syringe with a plunger that was
driven by a motor. Today the majority of modern chromatographs are fitted with reciprocating pumps fitted with
either pistons or diaphragms.

The pneumatic pump has a much larger flow capacity than the piston type pumps but, nowadays, is largely used for
column packing and not for general analysis. The pneumatic pump can provide extremely high pressures and is
relatively inexpensive, but the high pressure models are a little cumbersome and, at high flow rates, can consume
considerable quantities of compressed air.

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Detector

Various types of detectors are used to determine when the chemical of interest exits the column. Optical detectors
use a beam of light to determine when a particular chemical passes out of the column. As the solvent stream exits
the column, it passed through the detector's light path. Ultraviolet and visible light detectors operate by recording the
absorption of light caused by the chemical of interest. The change in absorption caused by the chemical indicates the
presence of the chemical. Other detectors commonly found on HPLC instruments include fluorescence and
electrochemical detectors.

LC detectors have been extensively discussed in Liquid Chromatography Detectors and HPLC detectors use the
same detection principals with extra care being given to the small solute elution volumes that result from the
combination of high column efficiencies with small volumes. In order to give an accurate chromatographic profile
the detector sampling (cell) volume must be a small fraction of the solute elution volume. If the detector volume
were larger than the elution volume then you would have peaks that appeared with flat tops as the whole peak would
be resident in the detector at the same time. This means that as column volumes decrease and system efficiencies
increase the volume of the detector cell volume must also decrease. This is of course at odds for the requirement for
detector to maintain high sensitivity as this is usually dependant on having a larger cell volume. Again, this requires
the very careful design of modern detectors.

HPLC Data Acquisition

Data acquisition was discussed in Liquid Chromatography Detectors and the only extra consideration required for
HPLC is the higher sampling rate needed for the rapidly eluting narrow peaks of the HPLC chromatogram.
Although the theoretical number of samples needed for good quantization are actually quite small, for real systems a
hundred samples or more per peak is recommended; thus, for a 4 sec wide peak, a rate of 25 samples per second
may be required. The same data analysis and reporting software can be used as in ordinary LC.

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MATERIALS AND METHODS

1) HPLC Instruments

Make Detector Pump Software

HITACHI UV-L-2400 L-2130 EZ Chrome Elite
Agilent DAD G1316A G1311A EZ Chrome Elite
Jasco UV-2075 Plus PU 2089 Plus Jasco Chrom Pass
Waters Water TM 966 515 HPLC Pump Empower Version 5.0

2) Disintegration Apparatus(Electrolab Disintegration Tester USP-ED2L)

3) Dissolution Apparatus

Electrolab Tablet Dissolution Tester USP (XXIII) TDT-06T (Manual)

Electrolab Tablet Tester USP Tester USP (XXIII) TDT-08T (Auto Sampler)

4) pH Meter (LabIndia, PN 05130711)

5) Karl Fischer Auto Titrator(Mettlet-Toledo DL55)

6) Polarimeter(Jasco, P2000)

7) UV-Visible Spectrophotometer (SHIMADZU, UV-1601)

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