HIGH PERFORMANCE LIQUID

CHROMATOGRAPHY

Prepared by:

Runa Akter
Lecturer, UAP
What is HPLC?

• HPLC is an abbreviation for High Performance Liquid Chromatography or High Pressure Liquid
Chromatography.
• HPLC is actually the automation of traditional liquid chromatography under conditions which
provide enhanced separations during shorter periods of time, utilizing very small particles, small
column diameters, and very high fluid pressures.
Principle of HPLC:

Figure: A schematic diagram of HPLC machine

Principle of HPLC continued……

The amount of retention depends on-

1. Nature of the sample,
2. Nature of the composition of the stationary phase, and
3. Nature of the composition of mobile phase.
Stationary phases

• Polar (“Normal” Phase): Silica, alumina etc.

• Non-Polar (“Reversed” Phase): ODS Silica gel, C8 column etc.
Mobile phases
Advantages & disadvantages:

• Advantages:

 Requires a very small amount of sample,

 Provides high accuracy and precision,
 Non-destructed sample during operation compared to GC, etc.
• Disadvantages:

 Need a skilled person to run the instrument,

 Solvent consumption is high, etc.
Applications of HPLC

1. Pharmaceutical industry
2. Analysis of natural contaminations
3. Forensic test
4. Clinical test
5. Food and essence manufacture
Components of HPLC machine

The main components of a HPLC machine are-

1. Solvent reservoir
2. Solvent filtration and degasser
3. Pumps
4. Sample injector
5. Columns
6. Detectors
7. Data processing
8. Waste collection
Solvent reservoir

It contains the solvent used to carry the sample through the column.

Solvent filtration and degasser

• To remove any insoluble particles from the solvent that could potentially damage pump valves,
block the capillaries, reduce column performance.
• To remove air or any other gases.

-Gas bubbles may give ghost peak.

-Dissolved O2 may absorb UV light or quench fluorescence and thus interfere with the detection.
Pumps

A pump produces an appropriate pressure to push solvent through the tightly packed column. Flow rate of
the solvent depends on the pressure. For example, a pump is used to rise the pressure up to 4000 psi and flow
the solvent at 10 ml/min through the column.

Sample Injector
The injector introduces the liquid sample into the flow stream of the mobile phase.
 Manual injection system
 Autoinjector
Fixed-volume loops of between 1 – 200 l is available. 20 l is often used as standard.
Columns

• Considered as the “heart of the chromatograph”. Most HPLC columns are made of stainless steel which is
alloy of chromium-nickel-molybdenum.
• 15 to 150 cm in length; 2 to 3 mm in width.
• Packing - silica gel, alumina, ODS silica gel, etc.

Detectors
The detector can detect the individual molecules that come out (elute) from the column. For example:
 UV/Vis,
 Refractive index,
 FluorescencE
Data processing

 Frequently called the data system.

 Using specific software that is connected to HPLC machine.
 Receive the information from HPLC machine and present it as a graph.
 The graph describes about qualitative data (Retention time) and quantitative data (area under
curve).
Waste collection

HPLC machines produce a huge volume of chemical waste which are collected in the labelled
containers.
Parameters of HPLC column:

 Internal diameter
The internal diameter of an HPLC column is a critical aspect that determines quantity of sample that
can be loaded onto the column and also influences sensitivity. Columns of internal diameter 2-5 mm
are generally used for analytical purposes. Wider columns of internal diameter 10-25 mm are used for
preparative work.
 Length
Columns are generally 5, 10, 15 or 25 cm long are common. For preparative purposes columns up to
1m in length are used.
 Particle size

Most traditional HPLC is performed with the stationary phase of small spherical silica particles. These
particles come in a variety of sizes (3, 5 or 10µm in size) with 5µm beads being the most common.
Smaller particles provide more surface area and better separations, but the pressure required for
optimum linear velocity increases by the inverse of the particle diameter squared (Pa1/d2).
 Pore size
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 sample. Typically, 6 to 10
nm average pore size is widely used.
The factors which influence the HPLC performance

1. Internal diameter of column

- smaller the diameter, higher the sensitivity
2. Pump pressure
- higher the pressure, higher the separation
3. Sample size
- 1 – 200 l (20 l is often used as standard)
4. The polarity of sample, solvent and column
5. Temperature
- higher the temperature, higher the separation
Some commonly used terms:

Isocratic flow and gradient elution

With regard to the mobile phase, a composition that remains constant throughout the procedure is
termed isocratic.
Gradient elution is a separation technique where the mobile phase changes its composition during a
separation process. One example is a gradient in 20 min starting from 10% Methanol and ending up
with 30% Methanol. Such a gradient can be increasing or decreasing. The benefit of gradient elution
is that it helps speed up elution by allowing components that elute more quickly to come off the
column under different conditions than components which are more readily retained by the
column. By changing the composition of the solvent, components that are to be resolved can be
selectively more or less associated with the mobile phase as a result at equilibrium they spend more
time in the solvent and less in the stationary phase therefore they elute faster.
Retention time

The retention time of a solute is taken as the elapsed time between the time of injection of a solute and
the time of elution of the peak maximum of that solute.

Run time
This is a little longer than it takes for the last peak to elute. It is usually from 2-60 min.
How can we analyze HPLC chromatogram?

Figure: Qualitative analysis using retention

 How can we analyze HPLC chromatogram?

Figure: Quantitative analysis using peak height or peak area

Resolution:
Calculation:

It is calculated as the difference in retention time of two eluting peaks divided by the average width of
the two peaks at the baseline.
Column preparation/packing
Pre-columns
HPLC band broadening

Band-broadening is a general term used to describe the overall dispersion or widening of a sample
peak as it passes through a separation system. Band broadening is a phenomenon that reduces the
efficiency of the separation being carried out leading to poor resolution and chromatographic
performance.
Causes:
1. Multiple paths of an analyte through the column packing/ Eddy diffusion
2. Molecular diffusion
3. Effect of mass transfer between phases
1. Eddy diffusion:

Figure: Eddy diffusion

2. Molecular diffusion/ longitudinal diffusion

Figure: Longitudinal diffusion

 2. Molecular diffusion/ longitudinal diffusion Continued….

Figure: Porous particle as stationary phase Figure: Porous layer bead

3. Effect of mass transfer between phases:

Figure: Stagnant mobile phase within the pores

HPLC detectors:

An ideal detector should have the following properties:

(i) It should either be equally sensitive to all eluted peaks or record only those of interest.
(ii) It should not be affected by changes in temperature or in mobile phase composition.
(iii) It should be able to monitor small amounts of compounds (trace analysis).
(iv) It should not contribute to band broadening; hence cell volume should be small.
(v) It should react quickly to pick up correct narrow peaks of components which pass rapidly through
the cell.
(vi) It should be cheap.
Types of HPLC detectors

According to chromatogram presentation and sample recovery, mainly four types

1. Integral
2. Differential
[Link] destructive
4. Destructive
Types of HPLC detectors Continued…….

Figure: Responses from an integral (A) and a differential (B) detector for the
same sample
Types of HPLC detectors Continued…….

Based on mode of operation or mechanism, there are various types-

• Refractive index (IR) detector
• Ultraviolet-visible spectroscopy detector
• Fluorescence spectroscopy detector
• Photodiode array (PDA) detectors
• Electrochemical detectors
• Mass spectrometry detector
Safety measures in HPLC handling

1. Toxic solvents:
2. Pulmonary irritation from the stationary phase:
3. Dangers resulting from the high pressures:
4. Dangers from high voltage:
Gas chromatography (GC) versus HPLC

Factors GC HPLC

Principle Follow partition/adsorption. Follow adsorption/desorption.

Mobile phase Mobile phase is constant which Mobile phase changes which
is a gas. are liquids.
Temperature Increasing temperature. Constant temperature.

Personnel The operation of a gas Need a skilled person to run

chromatography and related the instrument.
calculations does not require
highly
skilled persons.
Gas chromatography (GC) versus HPLC

Factors GC HPLC

Separation Separation of components depends on Separation of components depends on

depends on partition/distribution from the mobile adsorption/desorption phenomenon from
phase based on evaporation/volatility, or the
separations depend on mobile phase based on solubility and
adsorption/desorption phenomenon adsorptivity.
from the mobile phase based on solubility.
Elution Elution is generally temperature Elution is generally
dependent. time/volume dependent.
Sample The sample must be No decomposition of sample
decomposition volatilized/vaporized where occurs during operation
decomposition may occur in some compared to GC.
cases.