GASSCHROMATOGRAPHY[GC], ADVANCED STUDY OF THE FOLLOWING AND THEIR APPLICATIONS, INTRODUCTION, THEORY, COLUMN OPERATION,INSTRUMENTATION AND DETECTION,APPLICATIONS AND ADVANTAGES OF GC,PRINCIPLE OF SEPARATION IN GC, HOW GC MECHINE WORKS? COLUMN, DETECTORS. BY P.RAVISANKAR, VIGNAN PHARMACY COLLEGE, VADLAMUDI, GUNTUR, A.P, INDIA.

1. HPLC
Prof. Ravisankar
Vignan Pharmacy college
Valdlamudi
Guntur Dist.
Andhra Pradesh
India.
banuman35@gmail.com
00919059994000

2. Brief History and Definition
Liquid chromatography was defined in the early 1900s by the work of the Russian
botanist, Mikhail S. Tswett. His studies focused on separating compounds [leaf
pigments], extracted from plants using a solvent, in a column packed with particles.
Tswett filled an open glass column with particles. Two specific materials that he found
useful were powdered chalk [calcium carbonate] and alumina. He poured his sample
[solvent extract of homogenized plant leaves into the column and allowed it to pass into the
particle bed.. The compounds that were more strongly attracted to the particles slowed
down, while other compounds more strongly attracted to the solvent moved faster. This
process can be described as follows: the compounds contained in the sample distribute, or
partition differently between the moving solvent, called the mobile phase, and the
particles, called the stationary phase. This causes each compound to move at a different
speed, thus creating a separation of the compounds.
Tswett coined the name chromatography
[from the Greek words chroma, meaning color,
and graph, meaning writing—literally, color writing]
to describe his colorful experiment.
Curiously, the Russian name Tswett means color. Tswett's Experiment
The concept of the first HPLC was put forth by Kirkland
and Huber
b/n 1967 and 1969.

3. History of HPLC:
Prior to the 1970's, few reliable chromatographic methods were commercially
available to the laboratory scientist were carried out using a variety of techniques
including open-column chromatography, paper chromatography, and thin-layer
chromatography.
However, these chromatographic techniques were inadequate for quantification of
compounds and did not achieve sufficiently high resolution to distinguish between similar
compounds.
During this time, pressure liquid chromatography began to be used to decrease flow through
time, thus reducing purification times of compounds being isolated by column
chromatography. However, flow rates were inconsistent, and the question of whether it was
better to have constant flow rate or constant pressure was debated.
High pressure liquid chromatography was developed in the mid-1970's and quickly improved
with the development of column packing materials and the additional convenience of on-line
detectors. In the late 1970's, new methods including reverse phase liquid chromatography
allowed for improved separation between very similar compounds.

4. • By the 1980's HPLC was commonly used for the separation of chemical
compounds. New techniques improved
separation, identification, purification and quantification far above those
obtained using previous techniques. Computers and automation added
to the convenience of HPLC. Additional column types giving better
reproducibility were introduced and such terms as micro-column, affinity
columns, and Fast HPLC began to immerge.
• The past decade has seen a vast undertaking in the development of
micro-columns, and other specialized columns. The usual diameter of
micro-columns, or capillary columns, ranges from 3 µm to 200 µm. Fast
HPLC utilizes a column that is shorter than the typical column. A Fast
HPLC column is about 3 mm long and is packed with smaller particles.
• Although HPLC is widely considered to be a technique mainly for
biotechnological, biomedical, and biochemical research as well as for the
pharmaceutical industry,in actual fact these fields currently comprise
only about 50% of HPLC users. Currently HPLC is used in a variety of
fields and industries including the cosmetics, energy, food, and
environmental industries.

5. Introduction: HLC is basically a high improved form of column chromatography.
Instead of solvent being allowed to drip through a column under gravity, it is
Forced through under high pressure up to 400 atmospheres.
That makes it much faster.
In HPLC it is also allows you to use a very much smaller particle size (3-5µm)for column
packing. Material which gives a much greater surface area for interactions b/n
the stationary phase And the mobile phase it. This allows the better separation
of the components of the complex mixture.
This in turn Increase the elution by over 100 folds.
The other major improvement over column chromatography concerns the
Detection methods which can be used.
These methods are highly automated and extremely sensitive.
What Is UltraPerformance Liquid Chromatography (UPLC Technology)?
In 2004, further advances in instrumentation and column technology were
made to achieve very significant increases
in resolution, speed, and sensitivity in liquid chromatography. Columns with
smaller particles [1.7 micron]and instrumentation with specialized capabilities
designed to deliver mobile phase at 15,000 psi [1,000 bar]
were needed to achieve a new level of performance. A new system had to be holistically
created to perform ultra-performance liquid chromatography, now known as
UPLC technology.

11. H
P
igh
ressure
L iquid
C hromatography
Since high pressure is used when compared to classical chromatography
Separation of the components from a mixture is achieved by pumping
mobile phase at High pressure using appropriate displacement pumps
or gas pressure. (Due to the small particle size (3-5 um).

12. H
P
igh
erformance
L iquid
C hromatography
More ever it is improved performance when compared to other
conventional column Chromatographic techniques.

13. (advantages)

15. Determination
Peak area (or height) is proportional to the concentration
(or amount) of the component.
The concentration of component A(caffeine) is determined by
comparing the peak area with that of the standard caffeine
peak.
What is the concentration of component A?
A
B
C
Caffeine (1mg/ml)
5ul injection (5ug)

19. A
B
C
Results obtained by HPLC
Chromatogram containing three peaks
Qualitative analysis (identification) and
Quantitative analysis (determination)
Can be performed using the information contained in the
chromatogram
Chromatography : Method
Chromatogram : Results
Chromatograph : Instrument

20. Separation Mechanism
A
B
C
time
.
CBA
Column
Packing
material
↓ ↓ ↓ ↓
Mobile phase (solvent)
C > B > A

22. CLASSIFICATION OF HPLC:
BASED ON MODE OF RETENTION
BASED ON OPERATING METHOD
BASED ON MODE OF SEPARATION

23. BASED ON MODE OF RETENTION
ADSORPTION PARTITION
SIZE EXCLUSION
AFFINITY
ION
EXCHANGE
Liquid mobile
phase and solid
adsorbent
Liquid
mobile
phase and
ionic
stationary
phase
Liquid mobile
phase and
liquid stationary
phase
Molecular
modelling
Lock & key

24. (A) uses charge, (B) uses
pores, and (C) uses
covalent bonds to create
the differential affinities
among the mixture
components for the
stationary phase.

27. BASED ON OPERATING METHOD
ELUTION DISPLACEMENT
ISOCRATIC GRADIENT
FRONTAL

28. Based on elution Technique
There are 2 types of elution :
1- Isocratic elution : fixed ratio of mobile phase
components during the analysis.
2- Gradient elution (mobile phase programming) :the
components of mobile phase ratio is changed
through out the analysis for separation of components very
close in polarity.

30. BASED ON MODE OF SEPARATION;
MODE OF
SEPARATION
STATIONARY
PHASE
MOBILE
PHASE
NORMAL PHASE POLAR NON POLAR
REVERSE PHASE NON POLAR POLAR

31. 1.Based on Modes of chromatography:
There are 2modes (Normal phase mode and Reverse phase mode)
depending on the polarity of the stationary and mobile phase.
Polar- polar --- interaction (or) affinity is more.
Nonpolar- Nonpolar– interaction (or) affinity is more.
Polar- Nonpolar --- Interaction is less.
Remember, "Like attracts like” in polarity
Those with dissimilar polarity exhibit much
weaker attraction, if any, and may even repel one another.

33. Normal Phase Mode HPLC or NP- HPLC or Normal phase HPLC:
This is essentially just the same as you will already have read about in TLC or
Column chromatography.
Although it is described as “Normal” it is not most commonly used form of HPLC.
In This separations of plant extracts, Tswett was successful using a polar stationary
phase [chalk in a glass column; see Figure A] with a much less polar [non-polar]
mobile phase. This classical mode of chromatography became known as normal phase.
normal-phase chromatographic separation of our three-dye test mixture.,The stationary
phase is polar and retains the polar yellow dye most strongly. The relatively non-polar
blue dye is won in the retention competition by the mobile phase, a non-polar solvent,
and elutes quickly. Since the blue dye is most like the mobile phase [both are non-polar],
it moves faster. It is typical for normal-phase chromatography on silica that the mobile
phase is 100% organic; no water is used.
The commonly used columns for NP-HPLC include Cyano, amino and diol bonded phases
As well as base silica or its derivatives.
Stationary phase
(Polar)
Mobile phase (Non-
Polar)
Non polar material
elutes first.

34. Normal Phase Chromatography
Interaction : Adsorption
Packing materials : Polar ex. Silica gel
Silica-NH2
Silica-CN
Silica-OH
Mobile phase : Non-polar ex. n-Hex/CH2CL2
iso-Oct/IPA
iso-Oct/AcOEt
Sample : Fat-soluble
Different polarity

35. Stationary phases for Normal-Phase
chromatography:
Common porous adsorbents such as silica & Alumina,
that have polar hydroxyl groups on surface.
In addition to porous adsorbents, a variety of polar bonded phases
exist in some functional groups, such as
cyano [-(CH2)3CΞN],
diol [-(CH2)3OCH2CH(OH)CH2OH]
Amino groups [-(CH2)n NH2, where n is 3 or 4],

36. Solvent Characteristics
n-Hexane Mobile phase
n-Heptane Mobile phase
Isooctane Mobile phase
1-chlorobutane Large dipole
Chloroform Proton donor
Methylene chloride Large dipole
Ethylene acetate Proton donor
Tetrahydrofuron Proton acceptor
Propylamine Proton acceptor
Acetonitrile Dipole
Mobile phases for Normal-phase chromatography:

37. Applications of Normal phase
chromatography:
Analysis of samples that are soluble in
non polar solvents.
Separation of isomers.
Fat and water soluble vitamins
,Hydrocarbons and Pesticides have been
separated using Hexane as mobile phase.
Separation of saccharides in food and
biological products.

39. CH3 CH2COOCH3
CH3 CH2COOCH3
Stationary phase (Non polar)
Mobile phase (Polar)
P0lar material elutes first.

40. 3. Separation mode
Reversed Phase Chromatography
Si
Si
O－Si－CH2(CH2)16CH3
CH3
CH3
O－Si－CH2(CH2)16CH3
CH3
CH3
O－Si－CH2(CH2)16CH3
CH3
CH3
CH3
CH3
O－Si－CH3
Silica-C18 Packing materials
Commonly used packing materials are
hydrocarbons having 18 carbon atoms
(called the Octadecyl radical) which are
chemically bonded to silica gel (Silica-
ODS).Since the surface of the Silica-
ODS is covered with hydrocarbon, the
polarity of the packing material itself is
very low.
Today, because it is more reproducible and has
broad applicability, reversed-phase
chromatography is used for approximately 75% of
all HPLC methods
In this technique the silica is modified to make it non-polar by attaching long hydrocarbon
Chain to its surface typically with either 8 or 18 carbon atoms in them.

41. Stationary phases for Reverse phase chromatography:
 Functional group chemically attached to a
silica support- Bonded phase.
 Popular bonded phases;
•Alkyl-C4H9,C8H17,C18H37
•Phenyl-C6H5

42. solvent Solvent polarity(P’)
Water
Dimethyl sulfoxide
Ethylene glycol
Acetonitrile
Methanol
Acetone
Dioxane
Ethanol
Tetrahydrofuran
2-Propanol
10.2
7.2
6.9
5.8
5.1
5.1
4.8
4.3
4.0
3.9
Mobile phases for Reverse-phase
chromatography:

43. Applications of Reverse phase chromatography:
Pharmaceutical industry; steroids,vitamins,beta blockers
Clinical laboratories; catecholamines
Chemical industry; polymer additives
Environmental pesticides and herbicides
Food and beverage industy; sweetners and additives

44. of
C
Stationary phases Mobile phases Applications
al phase
C
Bonded phase silica gel with functional groups
such as
Cyano [-(CH2)3 C=N]
Diol [-(CH2)3 OCH2-CH-OH CH2OH
Amino [-(CH2)2NH2] (n=3 or 4)
n-Hexane, n-heptane,
isooctane,
1-chlorobutane, CHCL3,
CH2CL2, Ethyl acetate, THF,
propylamine, acetonitrile and
MeOH
- Fat & oil soluble
vitamins
- Hydrocarbons
- Pesticides
- 8,9-oxide of avermectin
B1
sed
HPLC
Bonded phase silica gel with functional groups
(-C4 H8) – butyl
(-C8 H17) – octyl
(-C6 H5) – phenyl
C18 – octadecyl
Water, dimethyl sulfoxide,
ethylene glycol, ACN,
MeOH, acetone, dioxane,
EtOH, THF &
2-propanol.
- Most popular mode for the
separation of low molecular weight
neutral species that are soluble in
H2O
- Steroids
- B Vitamins
- β-blockers
- Catecholamines
- Polymer additives
- Pesticides & herbicides
- Carbohydrates
- Sweeteners
- Food additives
- Aminoacids, peptides,
nuclesedes,
oligosaccharides
xchange
C
Organic ion-exange resins containing
exchange sites. The resin is composed of
polystyrene- divinyl benzene
Functional groups present in different ion
exchange resins:
Acids, Alkali, Buffers,
Acetate buffer, Borate buffer
Analyses of amino acids,
nucleotides, (carbohydrates at PH
>12) & proteins
Inorganic anions, organic acids,
inorganic cations etc.
Fractionation of cations inorganic
lanthanides peptides, amino acids,
B.vitamins
Strong Cation Exchange Resin:
Sulfonic acids (-SO-
3H+
)
(sulfonated polystyrene)
Weak Cation Exchange Resins:
(polystyrene with functional groups)
+
Fractionation of cations inorganic
lanthanides peptides, amino acids,
B.vitamins
Fractionation of cations
Biochemical separations organic

45. GPC : Gel Permeation Chromatography
GFC : Gel Filtration Chromatography
Separation modes and features
Mode Stationary phase Mobile phase Interaction Feature
Normal phase Silica gel Organic solvent Adsorption Fat-soluble
chromatography (n-Hexane/IPE)
Reversed phase Silica-ODS MeOH/Water Hydrophobic Most widely used
chromatography (Silica-C18)
Size exclusion Chromatography
Non-aqueous (GPC) Porous Polymer Organic solvent (THF) Gel permeation Molecular weight distribution
Aqueous (GFC) Aqueous porous Polymer Buffer solution Gel permeation Protein Separation
Ion exchange Ion exchange gel Buffer solution Ion exchange Separation of
Chromatography ionic substances
Affinity Packing with ligand Buffer solution Affinity Purification of
Chromatography enzymes and proteins

46. High performance (or high pressure) liquid chromatography (HPLC) is a separation
technique in which a sample mixture is introduced into a stream of solvent (the
mobile phase) which then flows over a surface (the stationary phase) consisting of
either spherical particles or a film coated onto such particles. Separation results
from differences between sample components in their relative affinity for the
stationary and mobile phases

47. o
Solvent reservoirs (mobile
o
Phase reservoirs)
o
Pump
o
Injector
o
Column
o
Detector
o
Recorder
o
Pulse Damper
o
Degasser
o
Column Heater
Typical HPLC system consists of followings

48. A Modern HPLC instrument contains Glass
or stainless steel reservoirs of capacity of
500mL,1000ml,2000ml,5000ml reservoir kit.
for storing the mobile phase.
Provisions are included to remove dissolved
gases and dust from the liquid.
Mobile phase reservoirs and Solvent
pretreatment systems:
1000 ml HPLC Solvent
Reservoir Kit. Comes
with 1L solvent
reservoir, cap and
teflon insert for 1/8"
OD tubing, 1 meter
1/8" OD PTFE tubing
and 10 um 316
stainless steel frit.
$60.00

49. •The reservoir and its attachment to the pump should be
made of materials that will not contaminate the
mobile phase: Teflon, glass, or stainless steel.
•The vessel should have some sort of cap to prevent particulate
matter from contaminating the mobile phase.
•If you are using a solvent bottle as a reservoir, the top of the bottle
can be wrapped in aluminium foil to keep dust out or the bottle
cap can be drilled to allow inserting the inlet line through the cap.
•Don't close the bottle too tightly or removal of mobile phase by
the pump will create a vacuum. This prevents mobile phase from
flowing the pump, creating a "vapour lock" within the pump.
•The material chosen for constructing mobile phase reservoirs
should not be reactive towards the mobile phase.
Examples:
Acidic or alkaline solvents can not be stored in metallic reservoirs.
Non-polar solvents (chloroform) cannot be stored in polymeric reservoirs.

50. It is important that particulate matter be kept out of the mobile phase since
particulates can damage the pump and injector as well as plug the column. Mobile
phases are often filtered before adding them to the reservoir. In addition, a 10-
micron frit or inlet filter should be connected to the end of the inlet line that dips into
the reservoir. This inlet frit serves more than one purpose. Besides providing extra
protection against particulates entering the pump, the inlet filter serves to hold the
inlet line at the bottom of the reservoir. The stiffness of the inlet line tends to allow
this Teflon tubing to creep out of the reservoir, preventing use of all the mobile
phase unless an inlet filter is used to weigh down the end of the tubing. For
this reason, inlet filters are often called "sinkers".
Sinker" frits help keep the
inlet line submerged and
provide a final line of defense
against particulate
contamination.
The sinker frit is not a substitute for filtering
the mobile phase. The typical pore size
used in a sinker frit is on the order of 5 - 10
microns frits of smaller pore size are too
likely to plug. In general, the mobile
phase should be filtered through a 0.3
to 0.5-micron frit after the solvents
and buffers are mixed in order to
remove particulate matter. The sinker frit
protects against the occasional larger dust
particle encountered after the mobile
phase has been filtered.
frit or inlet filter

51. Filter systems:
Micro filters are (1-5µm) are placed before the solvent reservoirs in modern
HPLC Instruments for removing the particulate matter as well as
dust under vacuum.
purity of the solvents is utmost important because the particulate matter
Even in trace amounts interferes with the detector performance.

52. SOLVENT DEGASSING IN HPLC:
Solvent De-gassing
External vacuum
Degassing
Helium
Degassing
On-line
Degassing
Source:
Phyllis R. Brown Kingston, Rhode island Pg. No:78-79

53. Sparging:
It is important to remove any dissolved gas from the mobile phase before it is
pumped into the system.
H PLC analysis may contain gases such as oxygen that are non-visible to our eyes.
When gas is present in the eluent, this is detected as a noise and causes unstable
baseline.
This prevents bubble formation that might damage the column and degrade system
performance.
Most systems provide a means to pass helium through the solvent reservoirs in a
process called “sparging”.
This strips out any dissolved gas, but helium’s low solubility and inert nature prevent
it from being a problem itself. The image above shows this procedure being performed
on all four reservoirs.

54. Generally used method includes sparging (bubbling of inert gas), use of
aspirator, distillation system, and/or heating and stirring.
However, the method is not convenient and also when the solvent is left for a certain time
period (e.g., during the long analysis), gas will dissolve back gradually. Degasser uses
special polymer membrane tubing to remove gases. The numerous very small pores on
the surface of the polymer tube allow the air to go through while preventing any liquid to
go through the pore.
By placing this tubing under low pressure container, it created pressure differences inside
and outside the tubing (higher inside the tubing). This difference let the dissolved gas to
move through the pores and remove the gas.
Compared to classical batch type degassing, the degasser can be used on-line, it is more
convenient and efficient. Many of new HPLC unit system contain a degasser.

55. Solvents used :
The mobile phase is used to transport the sample components through the column
over the surface of the stationary phase.
The exact choice of mobile phase depends on the nature of both the sample
components to be separated and the column stationary phase.
organic compounds are commonly separated using water-methanol or water-
acetonitrile mixtures.
For Normal phase HPLC ( or) NP_HPLC
(or) Straight -phase HPLC.
There are 4 commonly used solvents.
In HPLC pure analytical grade
Organic solvents are used
(known as HPLC solvents)
It has been duly established
That “the stronger the solvent
Or solvent mixture, the more
Quickly it will elute an analyte
(i.e., an organic compound) from
A specific HPLC column)

56. Solvent choice:
The mobile phase is used to transport the sample components through the
column over the surface of the stationary phase
. In normal phasehplc, it would typically be a non-polar solvent or mixture of
solvents, such as hexane, cyclohexane, toluene, or dichloromethane.
In reverse phase hplc, it is typically either one of, or a mixture of two or more of,
the following: water, an aqueous buffer, methanol, acetonitrile, or
tetrahydrofuran (THF).
The exact choice of mobile phase depends on the nature of both the sample
components to be separated and the column stationary phase. Ion
chromatography, for example, often uses an aqueous buffer, while organic
compounds are commonly separated using water-methanol or water-acetonitrile
mixtures.

57. HPLC Pump Classification based on the Flow Rate
Microbore Standard bore Preparative
(1-250µL/min) (10µL/min—100 µL/min) (>10 mL/min)
SOLVENT DELIVERY SYSTEM (PUMPS):

58. Classification According to mechanism of Eluent
Displacement
Solvent delivery system
Syringe pumps Reciprocating-piston pumps
Single head Multi head
SOLVENT DELIVERY SYSTEMS(PUMPS)

59. HPLC pumps can be single, binary, or
ternary, meaning that the can make use of
one, two, or more different solvent
reservoirs. (Most ternary pumps actually
have four solvent reservoirs.) The image on
the right shows a fairly typical ternary
pump, which can be controlled viaeither the
front panel or computer control. The key
parts of such a pump system are the
proportionating valve, the mixing
chamber, the purge valve, and the actual
pump mechanism.

60. Solvent mixing:
The proportionating valve (lower-front of image) allows the desired volume ratio of
the different solvents to be drawn from the reservoirs. For example, if reservoir
A contained water and B methanol, the following pump settings would give a 50:50
water:methanol mixture – A=50%, B=50%, C=0%, D=0%. To ensure a uniform
composition, a mixing chamber (middle-right of image) is used. This particular
pump uses a reciprocating piston (upper-centre of image) to actually deliver the
mobile phase mixture at the required flow rate.

61. Pump assembly
This view shows the pump dis assembled. The
glass rod in the centre of the image is the piston;
the pump chamber has been removed. The
solenoid valve to the upper-left of the piston is
one of the proportioning valves. Routine
maintenance requires that the seals preventing
leaks around the piston be replaced periodically
This view shows the pump body removed from the
piston assembly. The piston assembly connects to the
large collar on the left of the unit. The mobile phase
enters through the top-right connector and leaves
through the bottom-left connector. The nuts adjacent
to these on the upper and lower sides actually retain
the check valves, which are essential to the working of
the pump; they basically determine the direction
through the pump taken by the mobile phase.
Check valves:

62. Reciprocating piston
pumps do not work if there
is air in the lines, which can
be the case after changing a
solvent reservoir, or
leaving the pump off.
Opening the purge valve
allows the contents of the
lines to be flushed
out without the contents
entering the injector or
column. It’s important to
remember to close the
purge valve before trying to
perform an experiment!
Purge valve:

63. Classification According To Materials Of Construction
Solvent Delivery system
Metallic Non-metallic
Steel Titanium
PEEK Teflon Ceramic
SOLVENT DELIVERY SYSTEMS (PUMPS)

64. Pumps
In the earlier state of HPLC development, the pump was the most important part of
the system.
The development of HPLC can be said that it was a development of pump system.
However, nowadays, the high pressure generation is a “standard” requirement and
what is more concerned nowadays is to be able to provide a consistent pressure at
any condition,
Most pumps used in current HPLC systems generate the flow by back-and-forth
motion of a motor-driven piston (reciprocating pumps).
The pump is a heart of the instrument. It can deliver the solvent through the
chromatograph with a pressure not less than 3000 Psig and 6000 Psig.

65. SYRINGE PUMPS:
Syringe Pumps: These are also called as positive displacement pumps. These are popular
as pulse less solvent delivery systems in HPLC. The syringe pump contains a large barrel
syringe with the plunger connected to a motor. As the plunger moves forward, it drives the
eluent through the chromatograph with a pulse less flow; the main advantage of syringe
pumps is that it generates stable flow.

66. WORKING OF A RECIPROCATING PUMP:

69. Isocratic pumps versus Gradient pumping
systems:
Isocratic Pumping Systems Gradient Pumping Systems:
An Elution with single
solvent or mixture of solvent
of constant composition is
termed as Isocratic elution.
Two or more solvents are
used for the elution of
analytes is termed as Gradient
Elution.

70. High pressure mixing:
In high pressure mixing the eluents are blended on the injector side, or high pressure side of
the pump. For this purpose two isocratic pumps are required. High pressure mixing is widely
used in HPLC-MS and capillary-LC.
Low pressure mixing:
Low pressure mixing is a common design for modern gradient HPLC systems. In this the
solvents are blended at atmospheric pressure. By means of a small Teflon block with 3 or 4
proportioning values, a mixture of any combination of the four solvents is delivered to the
low pressure intake side of the pump.

71. Based on the elution technique there are 2 types of HPLC techniques:
There are 2 types of elution :
1- Isocratic elution : fixed ratio of mobile phase components during the analysis.
2- Gradient elution (mobile phase programming) :the components of mobile phase
ratio is changed through out the analysis for separation of components very close in polarity.
1. Isocratic elution(separation) technique:
Isocratic elution is where the composition of the mobile phase
is unchanged during the entire elution process.
a. In this elution technique in which the components of mobile phase are maintained
constant throughout the process of separation is known as isocratic elution.
b. In this technique the mobile phase may contains single or two or more solvents of
similar polarity to elute the sample through the column.
Examples of mobile phases for isocratic elution are Chloroform,Benzene:petether(1:1)
In this technique changing the length/internal diameter of the column, the elution
order of the peaks remains same i.e., selectivity does not change.
In gradient elution any change in the dimensions of the column immediately affects
the elution order.
An isocratic elution cannot separate a complex mixture with adequate resolution and
good detectability.
Hence it is used only in the following cases…
1. When sample mixture contains less than 10 weakly retained components.
2. When gradient base line negatively effects trace analysis.

72. Advantages of isocratic elution technique:
1. Components of mobile phase remains constant through the process. Hence easy
to handle and does not require continuous monitoring.
2. Any change in column dimensions does not change the elution order (peaks
are of same order.
Disadvantages:
1. Late eluting peaks are flat and broad.
2. Takes longer time than gradient elution for complex mixtures.

73. HPLC analysis is a prominent analytical tool in pharmaceutical laboratories.
Once a method for the separation is developed, students can conduct both qualitative
and quantitative analysis of an analgesic mixture. These experiments indicated that
the analgesic compounds could be divided into two groups of polar and non-polar
molecules.
a) Isocratic elution of polar analgesics:he group of polar molecules included Aspirin,
Acetaminophen and caffeine which could be eluted on an octylsiloxane, C-8, column.
Furthermore, the mobile phase was composed of 67% H 2O, 3% 5 mM KH 2PO 4 (pH
6.1) and 30% methanol (volume by volume). Note that the presence of the
potassium phosphate buffer was necessary in order to obtain reproducible results,
particularly with respect to each component’s retention time. The optimized
experimental conditions were as follows:

74. A multicomponent mixture of six analgesic drugs commonly found in over-the-counter
pain-relieving formulations: Aspirin, Acetaminophen, caffeine, Naproxen, Ibuprofen and
salicylamide.

75. Once a method for the separation is developed, students can conduct both qualitative
and quantitative analysis of an analgesic mixture In order to develop the gradient elution
analysis, it was first necessary to conduct many preliminary trial-and-error
runs. These experiments indicated that the analgesic compounds could be divided into
two groups of polar and non-polar molecules..

76. a) Isocratic elution of polar analgesics
The group of polar molecules included Aspirin, Acetaminophen and caffeine which
could be eluted on an octylsiloxane, C-8, column. Furthermore, the mobile phase was
composed of 67% H 2O, 3% 5 mM KH 2PO 4 (pH 6.1) and 30% methanol (volume
by volume). Note that the presence of the potassium phosphate buffer was necessary
in order to obtain reproducible results, particularly with respect to each component’s
retention time. The optimized experimental conditions were as follows:

77. The following HPLC chromatogram was obtained by elution of a mixture of the three
polar analgesics according to the conditions specified above

78. b) Isocratic elution of non-polar analgesics
The remaining compounds, namely, Naproxen, salicylamide, and Ibuprofen are
relatively non-polar and therefore could be eluted from a C-8 column using a mobile
phase (solvent) that was predominantly made up of methanol. The optimized
conditions for the separation of the non-polar analgesic group were as follow

79. The following HPLC chromatogram was obtained by elution of a mixture of non-
polar analgesics
Note that under these conditions, it was possible to resolve Naproxen and
Ibuprofen only. When a mixture of all three components was
chromatographed, considerable overlap between Ibuprofen and salicylamide took place.
The latter was probably due to the relatively polar nature of the octylsiloxane, C-

80. Gradient elution of polar and non-polar analgesics
According to the preceding isocratic elution experiments, the polar analgesic
compounds Aspirin, Acetaminophen and caffeine required a relatively polar solvent.
The mobile phase was made up of 67% H 2O, 3% 5 mM KH 2PO 4 (pH 6.1) and 30%
methanol (volume by volume). By contrast, the non-polar analgesics Naproxen and
Ibuprofen required a non-polar solvent. The mobile phase was made up of 7% H 2O,
3% 5 mM KH 2PO 4 (pH 6.1) and 90% methanol (volume by volume).
Because of the singular discontinuity in the polarity, the solvent ramp to resolve the
analgesic mixture involved two time-dependent steps of decreasing polarity: in step 1
the mobile phase contained 67% H 2O, 3% 5 mM KH 2PO 4 (pH 6.1) and 30%
methanol(volume by volume), for a period of two minutes. In step 2 the mobile phase
was made up of 100% methyl alcohol.
Thus, in the first transient, the mobile phase is predominantly aqueous and therefore
highly polar -- it elutes the polar analgesics. Conversely, in the second transient, the
solvent is predominantly methanol which is relatively non-polar -- it elutes the non-polar
analgesics.

81. The gradient method operated as follows. When the sample was first loaded onto the
column, the mobile phase was predominantly water. Consequently, the more polar
Aspirin, Acetaminophen and caffeine traversed the stationary phase. The relatively non-
polar Naproxen and Ibuprofen remained tightly adsorbed at the column’s gate.
After two minutes, as shown by the vertical white marker, switching to absolute
methanol decreased the polarity of the mobile phase. As a result, the non-polar
analgesics, Naproxen and Ibuprofen, were successfully dislodged from the non-polar
matrix and eluted by the solvent.
A mixture containing Aspirin, Acetaminophen, caffeine, Naproxen and Ibuprofen was
then analysed under gradient elution conditions. As the chromatogram below shows, all
five analgesics were successfully resolved.

82. Isocratic elution for simple mixtures.
Gradient elution for complex mixture.
Isocratic and Gradient(techniques) HPLC System Operation
Two basic elution Techniques (modes) are used in HPLC. The first is
called isocratic elution. In this technique (mode), the mobile phase,
either a pure solvent or a mixture, remains the same throughout
the run.
Isocratic HPLC System

83. Gradient elution Technique:
Gradient elution is where the solvent strength of the moblie phase in
increased during the elution starting with a solvent of relatively low
solvent strength.
Steady changes of the mobile phase composition during the chromatographic run is
called gradient elution. It may be considered as an analogy to the temperature
programming in gas chromatography.
The main purpose of gradient elution is to move strongly retained components of the
mixture faster, but having the least retained component well resolved.
Gradient HPLC also known as solvent programming is a technique employed in HPLC.
Starting with the low content of the organic component in the eluent we allow the least
retained components to be separated. Strongly retained components will sit on the
adsorbent surface on the top of the column, or will move very slowly.
When we start to increase an amount of organic component in the eluent (acetonitrile)
then strongly retained components will move faster and faster, because of the steady
increase of the competition for the adsorption sites.
For example in a HPLC technique mobile phase as a mixture of A and B components.
The initial concentration of the component A(organic solvent) is say 10%,then after
A time period of about 20 min. the concentration is steadily increased to about 90%.
The other component B, is water which is regarded as the weak solvent that allows the
Elution of solutes very slowly, while the organic solvent is considered as the strong
solvent that rapidly elutes analytes from the column.
The commonly employed organic solvents include
methonol,acetonitrile,isopropanol,THF etc.

84. Performance of the gradient elution is strongly dependent on the instrumentation.
Two main points the chromatographer needs to know about his instrument:
How large the volume between the component mixing point and column inlet is. For
the low-pressure gradient systems this volume usually correspond to the pump
volume, and about 2 - 3 ml.
How well does the system mix eluent components. If the system does not mix
components well then it will supply for the certain time one component then another
and so on. Chromatographic performance of such system will be very low
especially for the least retained components.
Advantages:
-The technique increases the efficiency of columns
-Complex sample mixtures with wide retention range can be separated.
-Samples which contain a unknown composition can be screened.
-Retention of late eluting components is decreased. Hence they elute faster and
give narrower peaks.
Disadvantages:
-Requires continuous monitoring.
-Maintenance cost is more.
-The additional mixing of liquids causes a delay in the formation of gradient.
-Any change in column dimension changes the elution order (peaks are of different
order)

85. The second type is called gradient elution technique, wherein, as its name
implies, the mobile phase composition changes during the separation. This mode
is useful for samples that contain compounds that span a wide range of
chromatographic polarity. As the separation proceeds, the elution strength of the
mobile phase is increased to elute the more strongly retained sample components.
High-Pressure-Gradient System

86. Column heater
The LC separation is often largely influenced by the column temperature
. In order to obtain repeatable results, it is important to keep the consistent temperature
conditions.
Also for some analysis, such as sugar and organic acid, better resolutions can be
obtained at elevated temperature (50~80 °C). Thus columns are generally kept inside
the column oven (column heater).

87. The function of injector in HPLC is to introduce the sample in the
flowing solvent so that it may be carried to the column.
Injector may be operated either manually or automatically
Manual has two different modes for injecting samples
Sample Injector Device:
1.Septum mode injection
2.Valve injection

88. 1. Septum injection:
• This allows injecting sample into pressurized solvent
stream using a self-elastic septum with a micro syringe
• It is the simplest form of injector in the septum device
(similar to one used in GC).
Greatest Drawback:
Limited to max operating pressure of 1500
psi. Therefore, not widely used in HPLC anymore.

89. Widely used in HPLC, Allows reproducible introduction
of sample into the pressurized M.P without interruption of
flow even at high temperature.
Six-port Valve Type Injector
 One of the simplest and most common valve injector in
HPLC is the “six-port valco”(or Rheodyne model) injector.
Inject valve has two position:
(a) Load
(b) Inject
2. Valve-Type Injection in HPLC:

94. COLUMNS FOR HPLC;
Empty Stainless steel HPLC column
hardware come with the threaded
tube, two 2um frits, and two
stainless steel end fitting nuts.
2.1, 3.0, 4.0, 4.6, 7.8, 10, 22,
and 30mmI.D.
and 50mm,100mm.150mm,
250mm and 300mm length

95. . For normal phase, SIL or DIOL is usually the best choice while C18, C8, C4 or
Phenyl is the best choice for reversed phase applications

97. Stationary phases in column:
Silica gel
Alumina
Zirconium
 Carbon
 Hydroxyapatite.
Polymeric adsorbents such as polystyrene-divinyl benzene.
Packed with 3-10µm particles.
Resin based packings such as Polystyrene-divinylbenzene
& Acrylic based polymers are used in HPLC columns.
:

98. COLUMNS FOR HPLC:
A column is a heart of the chromatograph for separating a mixture
into components.
Column
Packing
material
container
Nature of
particle
Particle
size
Length
Diameter
Material

99. Pore size:
Porous microparticles are most common particles used in
HPLC.
Particles with small pores exhibit a high surface area & greater
retention.
Pore size
Porous
particle
Pellicular
particle
Porous
microparticle
Stationary phases in column:

100. Particle size:
Small particle sizes result in high efficiencies with increased
back- pressure, results decreased column permeability.
Porous particle ranges from ~20-40µm.
Porous micro particles are chosen for high efficiency, ranges
from 3-10µm
Internal diameter of column:
Internal diameter will effect the sample load, peak dilution, flow
rate.
Larger the diameter, greater the sampling load, higher the flow
rate.
Internal diameter for analytical columns ranges from 2-5mm
Stationary phases in column :

101. Column length:
Effects both efficiency & speed of separation.
Column efficiency tends to increase with column length.
Longer columns tends to longer time analysis.
Ranges from 30-300mm in length.
Construction materials of column:
316stainless steel for rigidity & mechanical strength at high
pressures.
Glass, Teflon, & PEEK Columns are used for aggressive
mobile phases, such as hydrochloric acid, or with solutes such
as proteins may adsorb to stainless steel.
Polymeric columns for ion-exchange packings, glass for
protein separation

102. One common stationary phase is a
silica which has been treated with
RMe2SiCl, where R is a straight chain
alkyl group such as C18H37 or C8H17.
With these stationary
phases, retention time is longer for
molecules which are more non-
polar, while polar molecules elute
more readily.

103. SiliaChrom AQ C18 columns use a new generation
adsorbent based on high purity spherical silica and unique
bonding technology. SiliaChrom AQ C18 is a slightly polar
C18 column, and is the most versatile reversed phase
column offered by SiliCycle for highly aqueous mobile
phases. SiliaChrom AQ C18 is considered as the most
universal HPLC column for any type of analytes (acidic,
basic and neutral). Furthermore, SiliaChromAQ C18 is
applicable to a wide range of sample types: plasma, urine,
drug formulation, food extraction, forensic, clinical and
bioanalytical.
Main Characteristics
•Exceptional stability at pH 1,5 to 9,0
•Inertness for acidic and basic analytes
•Compatible from 100% aqueous mobile
phase to 100% organic
•Rapid equilibration
•Reduced need for mobile phase modifiers
•Partially endcapped

106. Reversed-Phase Column
1.1 ODS column
The packing material used for reversed-phase column is often made of silica gel
modified with functional grouphe most often used functional group is octadecyl.
Octadecyl functional group is a straight carbon chain of 18. Its molecular structure is
shown below.
.
Ideally, entire surface of ODS gel is modified with C18 functional group; however
there will be remaining spaces that are not modified. Those part are called “residual
silanol” and the presence of residual silanols can influence the separation. Often “end
capping” is applied to the gels to immobilize the residual silanol. Almost all ODS
columns nowadays are end-capped, however depending on the type of
analyte, presence of silanol may provide better separation results.

107. Other Silica-Based Columns
ODS is most popularly used RP column, however since C18 is a long chain, it may
retain compounds too much and consequently results in long analysis time. Referring to
our story, this maybe a case that promotion was “too effective” making the customer stay
at the bar forever, which is not a good management for the bar. So in those cases, it is
better to use functional group with shorter chain, such as C8, C4, and C3.
C8：Octyl functional group -CH2CH2CH2CH2CH2CH2CH2CH3
C4：Butyl functional group -CH2CH2CH2CH3
C3：Trimethyl functional group -CH2CH2CH3
Also there are silica gels modified with phenyl and cyanopropyl functional groups.
The most popular Shodex TM polymer column is ODP series. As you may guess, the
name of ODP came from OctaDecyl Polymer. We believe it is easy to remember, as S
(silica) in ODS was simply replaced with P (polymer)
Polymer-Based Columns
Long column life
In general, polymer gel is chemically more
stable than silica gel and deteriorates slow

108. •Despite that the column life of silica column is about 3 months whereas it is not
surprising to see a polymer column provides stable analysis over 1 year.
•Good repeatability
•Usability under alkaline conditions
Silica column cannot be used under alkaline conditions where polymer column can.
Some basic samples (often pharmaceutical compounds) require alkaline mobile phase
to obtain good separations, thus polymer columns are suitable for such analysis.
Resolution and price
General consensus is that polymer columns compared to silica columns are more
expensive and provide less resolution. Therefore even though polymer columns have
several advantages over silica columns, former is less often used. The price of the
polymer column is becoming lower as the technology has been improved. Also
considering the longer column life, from a long term point of view, polymer column is not
extremely more expensive than silica columns. The biggest concern will be the
resolution. However, again the performance of newer polymer column has been
improved and is becoming very comparable to silica columns
The most popular Shodex TM polymer column is ODP series. As you may guess, the
name of ODP came from OctaDecyl Polymer. We believe it is easy to remember, as S
(silica) in ODS was simply replaced with P (polymer).
The next popular polymer-based RP column is DE-413. The packing gel is made of
polymethacrylate.

109. Normal-Phase Column
Silica gel without modification has large polarity, but when C18 functional group is
modified, the polarity becomes small. The larger polarity of silica gel is due to these
oxygen and hydrogen; the composition of silica is SiO2, but it has hydroxide group
(oxygen + hydrogen) on its surface. RP mode uses gel with small polarity (e.g., ODS)
and mobile phase with large polarity (e.g., water, acetonitrile). Normal-phase mode
uses gel with large polarity (e.g. silica) and mobile phase with small polarity (e.g.
hexane, chloroform). Instead of using the word small or large polarity, it is also common
to use words, hydrophilic or hydrophobic. Something easy to dissolve in water
(i.e., large polarity) is called hydrophilic and something easy to dissolve in oil (i.e., smal
polarity) is called hydrophobic.
At the very early stage of HPLC development, silica gel without any functional group
was only used. Thus, historically normal-phase mode was developed first and so
named “Normal”. Then the separation mode which uses opposite separation theory to
normal phase was developed and named “Reversed”. RP mode is much more
popularly used than normal phase nowadays, but we cannot change the historical
background, and thus they are still called normal and reversed-phase modes.

110. The matrix of the primary silica gel particle consists of a core of silicon atoms joined
together with oxygen atoms by siloxane bonds (silicon-oxygen-silicon bonds). On the
surface of each primary particle some residual, uncondensed hydroxyl groups from the
original polymeric silicic acid remain. These residual hydroxyl groups confer upon silica gel
its polar properties. These hydroxyl groups react with the silane reagents to form bonded
phases
There are three types of hydroxyl group.
The first is a single hydroxyl group attached to a silicon atom which has three siloxane
bonds joining it to the gel matrix.
The second is one of two hydroxyl groups attached to the same silicon atom which, in
turn, is joined to the matrix by only twosiloxane bonds. These twin hydroxyl groups are
called Geminal hydroxyl groups.
The third is one of three hydroxyl groups attached to a silicon atom which is now only
joined to the silica matrix by only a single siloxane bond. An example of each type of
hydroxyl bond is ….
adsorption could also take place the surface of siloxane bonds as well.

111. Silica gel is manufactured by releasing silicic acid from a strong solution of sodium silicate
by hydrochloric acid. (Sodium silicate is prepared by heating sand at a high temperature in
contact with caustic soda or sodium carbonate).
Initially, silicic acid is released,
Na2SiO3 +H2O + 2HCl = Si(OH)4 + 2NaCl
and then the free acid quickly starts to condense with itself with the elimination of
water to form dimers, trimers and eventually polymeric silicic acid.
The polymer grows, initially forming polymer aggregates and then polymer spheres, a
few Angstrom in diameter. Thesepolymeric spheres are called primary silica particles.
These primary particles continue to grow until, at a particular size, the surface silanol
groups on adjacent primary polymer particles, condense with the elimination of water.
This condensation causes the primary particles to adhere to one another and at this
stage the solution begins to gel. During this process, the primary particles of silica gel
will have diameters ranging from a few Angstrom to many thousands of Angstrom
depending on the conditions of formation.

112. his unique ultra-solvent-resistant computer-
printable label range for HPLC columns is
designed to permanently adhere to all
surfaces and withstand aggressive HPLC
cleaning agents. when exposed to
methanol, ethanol, acetone, IPA, and
THF, providing lab managers with the
perfect HPLC identification solution.
variable data (batch
numbers, barcodes, etc

113. PRP Polymeric Reversed Phase Poly(styrene-divinylbenzene) HPLC Columns
are used in quality assurance as well as research and development laboratories
because they last significantly longer than silica-based reversed phase columns. This is
particularly true for difficult analyses like: high pH samples (pH 8-13); labile or
reactive samples (irreversible adsorption); high aqueous purifications (80-100%
water); and separations with ion pairing reagents. HxSil silica-based columns are ideal
for applications where a higher efficiency reversed phase C8 or C18 column is needed.
Hamilton PRP-1 reversed phase columns
Polymeric columns for general
separations.
•pH Stable from 1 to 13
•Better sample recovery than silica
based columns.
•Excellent durability (stable to any
concentration of water or organic
solvent).
Product Features
•Four particle sizes: 5, 7, 10 and 12-
20 µm.
•Ten column internal diameters: 1.0
to 101.6 mm.
•Two column materials: 316 stainless
steel and PEEK

114. APPLICATIONS of PRP columns (polymeric Reverse phase columns
Peptide Standards
Resveratrol
Folic Acid
Pregnenolone
Biotin, Folic Acid, Vitamin B12
Coenzyme Q10
Ginkgo Biloba
Soy Isoflavins
Fat Soluble Vitamins
Phenobarbital, Scopolamine,
Hyoscyamine, Ergotamine
Pregnenolone, progesterone
Hormones
Erythromycins
Pyridinoline
Diphenylguanidine
separations up to 9 times faster if required

115. Special fitting style guard column for
protect main column!
For 4.6mmI.D. HPLC column.
Features
・Special fitting style guard column
・Easy hand tight connection
Guard Column 3mm (Cartridge type)

117. 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. A
column is said to be endcapped when a small silylating agent, such as
trimethylchlorosilane, is used to bond residual silanol groups on a packing
surface. It is most often used with reversed-phase packings and may cut down on
undesirable adsorption of basic or ionic compounds.

118. what is the specific advantage in using endcapped columns against non
endcapped one?
Because of steric hindrance and other factors, it is not possible to react all
of the silanols with C8 or C18 alkyl groups. Because of this, after the initial
bonding, additional bondings are sometimes performed, attaching smaller
alkyl groups, quite often propyl or isopropyl groups, to the residual
silanols. There is no endcapping process that is complete. There are
always unreacted silanols.
The resulting stationary phase, having fewer residual silanols, will display
fewer secondary interactions characteristic of silanols than a non-
endcapped stationary phase.
The result is less peak broadening and reduced tailing -- especially with
organic acids and bases.
The common endcapping group is a trimethylsilyl group

119. The silica is first dried at 250oC to remove the majority of the strongly
bound water from the surfacehe silica is suspended in toluene that is
contained in a flask fitted with a reflux condenser and then treated with an
excess of dimethylchlorsilane (all the adsorbed water on the silica gel
surface my not have been completely removed and the excess reagent will
scavenge any residual water) The mixture is refluxed for about three hours
and finally any remaining reagent or hydrochloric acid is The surface is
now covered with a layer of dimethylsilane groups. Squalene is then added
to the suspension, accompanied by the catalyst chlorplatinic acid, and the
mixture is next refluxed for a further 6 hours. Squalene contains six double
bonds along its chain, each double bond being separated by two methylene
groups and thus, in the presence of the catalyst, any double bond coming
in contact with the silane hydrogen on the silica surface will open and
attach the squalene chain to that silyl group.
reacted with trimethylchlorsilane to end
cap the final
methyloctylmonohydroxysilane groups that
were generated on the surface

120. An end-capped
C8 sorbent that
offers moderate
hydrophobic
retention with
negligible
secondary polar
interactions from
active silanol
groups.
Description:
Octadecyl (non end-capped) functionalized
silica, manufactured using trifunctional silane.
Average Particle Size: 50 µm (irregular shaped
particles)
Nominal Porosity: 60 Å
Applications: Matrix – Aqueous
Analytes – Wide range polarity
Retention mechanism – Non-polar [polar, cation exchange]
Comments: Strong non-polar (hydrophobic) phase.
Non end-capped to provide additional silanol interactions.