NAME: PRERANA CHAKRABORTY

STREAM: MSC. MICROBIOLOGY

SUBJECT: LABORATORY TECHNIQUES(MSMC 102)
SEMESTER: 1
ROLL NO: 1

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

(HPLC)

# INTRODUCTION

High Performance Liquid Chromatography also known as High Pressure Liquid

Chromatography is a technique in analytical chemistry used to separate, identify, and
quantify each component in a mixture. It relies on pumps to pass a pressurized liquid
solvent containing the sample mixture through a column filled with a solid adsorbent
material. Each component in the sample interacts slightly differently with the adsorbent
material, causing different flow rates for the different components and leading to the
separation of the components as they flow out of the column.

HPLC has been used for manufacturing (e.g., during the production process of
pharmaceutical and biological products), legal (e.g., detecting performance
enhancement drugs in urine), research (e.g., separating the components of a complex
biological sample, or of similar synthetic chemicals from each other), and medical
(e.g., detecting vitamin D levels in blood serum) purposes. HPLC is distinguished from
traditional ("low pressure") liquid chromatography because operational pressures are
significantly higher (50–350 bar), while ordinary liquid chromatography typically relies
on the force of gravity to pass the mobile phase through the column. Due to the small
sample amount separated in analytical
HPLC, typical column dimensions are
2.1–4.6 mm diameter, and 30–250 mm
length. Also HPLC columns are made
with smaller adsorbent particles (2–50
μm in average particle size). This gives
HPLC superior resolving power (the
ability to distinguish between
compounds) when separating mixtures,
which makes it a popular
chromatographic technique.

# CONSTRUCTION

HPLC FLOW DIAGRAM

# 7 MAIN COMPONENTS OF HPLC
1. Infusion system: The infusion pump is one of the
most important components of the HPLC system.
Infusion pumps are classified into constant pressure
pumps and constant flow pumps according to the
factors of constant output liquid. The performance
of the pump directly affects the quality of the entire
system and the reliability of the analysis results.

2. Degassing device: Bubbles are often seen in the

mobile phase solution due to dissolved oxygen
or air mixed in. Bubbles entering the detector can
result in sharp noise peaks on the
chromatogram. Small bubbles slowly
accumulate and become large bubbles. When
large bubbles enter the flow path or the
chromatographic column, the flow rate of the
mobile phase will slow down or the flow rate will
become unstable, causing the baseline to
fluctuate. It takes time to expel these bubbles
once they enter the column. At present, the most
commonly used mobile phase degassing devices in liquid chromatography are
offline ultrasonic vibration degassing, online inert gas bubbling purge
degassing, and online vacuum degassing.

3. Gradient elution device: HPLC has two elution methods, isocratic and gradient.
Isocratic elution means that the composition of the mobile phase remains
constant during the same analysis cycle, which is suitable for samples with a
small number of
components and little
difference in properties.
Gradient elution is a
program to control the
composition of the mobile
phase within an analysis
cycle, such as the polarity of
the solvent, ionic strength,
and pH value. It is used to
analyze complex samples
with a large number of
components and large differences in properties. The use of gradient elution can
shorten the analysis time, increase the resolution, improve the peak shape, and
increase the detection sensitivity, but it often causes baseline drift and reduces
reproducibility.
4. Sampling system: The six-port injection valve or
autosampler is frequently used at present. This
sampling device is required to have good
tightness, small dead volume, and good
repeatability to ensure central sampling, and that
the pressure and flow rate of the chromatographic
system during sampling are small. It is generally
used during sample analysis. There are two
sampling methods for six-port valve, partial filling
method and complete filling method.

5. Separation system: The separation system includes a

chromatographic column, a guard column, and a column
oven.

6. Detector: The detector is a device that is used

to continuously monitor the composition and
content changes of the effluent separated by
the chromatographic column. It is one of the
three key components in an HPLC system.
The detector requires high sensitivity, low
noise (that is, insensitive to external changes
such as temperature and flow), wide linear
range, good repeatability, and wide application
range.
 7. Data
Processing
System: The
system can
collect, store,
display, print,
and process
test data, so
that sample
separation,
preparation, or
identification
can be carried
out correctly.

# PRINCIPLE OF HPLC

a. The separation principle of HPLC is based on the distribution of the analyte

(sample) between a mobile phase (eluent) and a stationary phase (packing
material of the column). Depending on the chemical structure of the analyte,
the molecules are retarded while passing the stationary phase. The specific
intermolecular interactions between the molecules of a sample and the packing
material define their time “on-column”. Hence, different constituents of a sample
are eluted at different times. Thereby, the separation of the sample ingredients
is achieved.

b. The principle of separation in normal phase and reverse phase mode is

adsorption.

c. When a mixture of components is introduced into a HPLC column, they travel

according to their relative affinities towards the stationary phase.

d. The component which has lower affinity towards the adsorbent travels slower.
 e. The component which has lower affinity towards stationary phase travels faster.
Since no two components have the same affinity towards the stationary phase,
the components are separated.

# OPERATION OF HPLC

1. Follow the rules and regulations and wear apron

 Clean the HPLC.

 Switch it on and wait for it to get started.
 Prepare the instrument for analysis.
 Keep the solvent/solvents in the mobile phase in solvent reservoir or solvent
tray. Solvent is used to separate the components of the mixture. In modern
instruments, the mixture of solvents can be used as mobile phase which is
called gradient elution. Elution is the separation into components.
 Use methanol-water or chloroform-heptane etc as your solvent.

2. Select the types of HPLC based on the relative polarity of the phases

 Normal Phase: Use a comparative polar stationary phase than mobile phase if
doing normal phase HPLC.
 Reverse Phase HPLC: Use a less polar stationary phase as compared to mobile
phase. Generally reverse phase is used.

3. Feed the data into the system or program the HPLC

 Assign the number to the solvents when you are opting for gradient elution.
 Specify the flow rate.
 Regulate the pressure of the pump.
4. Analyse your sample
  Add your sample in the injector system. This is the mechanism which
introduces the sample (the mixture to be separated) to the system.
 Load the stationary phase in the column.
 Start the HPLC by clicking on the start button on the screen of computer
attached to it.
 Wait for some time for the separation of the mixture into components. The
retention time is the time taken by solvent to separate the components, which
is equal to the time when you enter the mixture in the column to when it is
detected/analysed.

5. Develop your components

Wait for your mixture to be separated into its components. This is called
development, when the sample contents will be detected by detector.

6. Observe the result: Look at the separation of components detected and recorded on
graph. There will be various peaks corresponding to the components and their
concentration.

# TYPES OF HPLC

The two most common variants of High Performance Liquid Chromatography:

1. Normal Phase HPLC: The column is filled with tiny silica particles, and a non-
polar solvent, for example, hexane. A typical column has an internal diameter
of 4.6 mm or smaller and a length of 150 to 250 mm. Non-polar compounds in
the mixture will pass more quickly through the column, as polar compounds will
stick longer to the polar silica than non-polar compounds will.
2. Reverse Phase HPLC: The column size is the same. The column is filled with
silica particles which are modified to make them non-polar. This is done by
attaching long hydrocarbon chains (8–18 C atoms) to its surface. A polar
solvent is used, for example, a mixture of water and an alcohol such as
methanol. Polar compounds in the mixture will pass more quickly through the
column because a strong attraction occurs between the polar solvent and the
polar molecules in the mixture.Non-polar molecules are slowed down on their
way through the column. They form varying degrees of attraction with the
hydrocarbon groups principally through van der Waals dispersion forces and
hydrophobic interactions. They are also less soluble in the aqueous mobile
phase components facilitating their interactions with the hydrocarbon groups.

Reversed phase HPLC is the most commonly used form of

HPLC.
# FUNCTIONS OF HPLC

1. Water purification
2. Detection of impurities in pharmaceutical industries
3. Pre-concentration of trace components
4. Ligand exchange chromatography
5. Ion exchange chromatography of proteins
6. High pH anion exchange chromatography of carbohydrates and
oligosaccharides
# APPLICATIONS OF HPLC IN DIFFERENT FIELDS

1. Manufacturing: HPLC has many applications in both laboratory and clinical

science. It is a common technique used in pharmaceutical development, as it
is a dependable way to obtain and ensure product purity.[24] While HPLC can
produce extremely high quality (pure) products, it is not always the primary
method used in the production of bulk drug materials.[25] According to the
European pharmacopoeia, HPLC is used in only 15.5% of syntheses.[26]
However, it plays a role in 44% of syntheses in the United States
pharmacopoeia.[27] This could possibly be due to differences in monetary and
time constraints, as HPLC on a large scale can be an expensive technique. An
increase in specificity, precision, and accuracy that occurs with HPLC
unfortunately corresponds to an increase in cost.

2. Legal: This technique is also used for detection of illicit drugs in urine. The most
common method of drug detection is an immunoassay.[28] This method is
much more convenient. However, convenience comes at the cost of specificity
and coverage of a wide range of drugs. As HPLC is a method of determining
(and possibly increasing) purity, using HPLC alone in evaluating concentrations
of drugs is somewhat insufficient. With this, HPLC in this context is often
performed in conjunction with mass spectrometry.[29] Using liquid
chromatography instead of gas chromatography in conjunction with MS
circumvents the necessity for derivitizing with acetylating or alkylation agents,
which can be a burdensome extra step.[30] This technique has been used to
detect a variety of agents like doping agents, drug metabolites, glucuronide
conjugates, amphetamines, opioids, cocaine, BZDs, ketamine, LSD, cannabis,
and pesticides.[31][32] Performing HPLC in conjunction with mass
spectrometry reduces the absolute need for standardizing HPLC experimental
runs.

3. Research: Similar assays can be performed for research purposes, detecting

concentrations of potential clinical candidates like anti-fungal and asthma
drugs.[33] This technique is obviously useful in observing multiple species in
collected samples, as well, but requires the use of standard solutions when
information about species identity is sought out. It is used as a method to
confirm results of synthesis reactions, as purity is essential in this type of
research. However, mass spectrometry is still the more reliable way to identify
species.

4. Medical: Medical use of HPLC can include drug analysis, but falls more closely
under the category of nutrient analysis. While urine is the most common
medium for analyzing drug concentrations, blood serum is the sample collected
for most medical analyses with HPLC.[34] Other methods of detection of
molecules that are useful for clinical studies have been tested against HPLC,
namely immunoassays. In one example of this, competitive protein binding
assays (CPBA) and HPLC were compared for sensitivity in detection of vitamin
D. Useful for diagnosing vitamin D deficiencies in children, it was found that
sensitivity and specificity of this CPBA reached only 40% and 60%, respectively,
of the capacity of HPLC.[35] While an expensive tool, the accuracy of HPLC is
nearly unparalleled.