HPLC

PHAR 212 LAB COURSE / 2012-2013

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

HIGH PRESSURE LIQUID CHROMATOGRAPHY
HIGH PRICE LIQUID CHROMATOGRAPHY
Chromatography (chroma: color and graphein: to write) is one of the laboratory
techniques that is used for the seperation of mixtures.

Chromatography technique is used for the seperation of chlorophylles (green plant

pigment) for the first time in the early 20th century by a Russian botanist Mikhail
Semyonovich Tsvet.

Later, during the 1940s and 1950s Archer John Porter Martin and Richard Laurence
Millington Synge continued working on and discovered partition technique.
High Performance Liquid Chromatography (HPLC) Harris Chapter 25

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 TYPES OF CHROMATOGRAHY
CHROMATOGRAPHY

LIQUID GAS
GSC GLC
PLANAR COLUMN
TLC PAPER NP RP IEC SEC
By means of these studies various chromatography techniques are developed such
as paper chromatography, column chromatography, gas chromatography and high
performance liquid chromatography.

High performance liquid chromatography (or High pressure liquid

chromatography, HPLC) is a type of column chromatography which is used in
chemistry laboratories for compound separation, identification, and quantification.
High Performance Liquid Chromatography (HPLC) Harris Chapter 25

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The principle of HPLC is;

The analyte that will be examined passes through a stationary phase as dissolved
in a mobile phase. Based on the polarities and interactions between the mobile
and stationary phases, the sample is seperated.
High Performance Liquid Chromatography (HPLC) Harris Chapter 25

http://www.waters.com/waters/nav.htm?cid=10049055&locale=en_US
Parts of a typical HPLC system

 Solvent reservoir
 Inlet solvent filter
 Pump
 Inline solvent filter
 Sample injection valve
 Precolumn filter
 Guard column
 Column
 Detector
 Back pressure regulator
 Waste reservoir
 Recorder / PC

http://www.idex-hs.com/support/upchurch/hplc_center.aspx
Parts of a typical HPLC system

Solvent reservoir, which contains the solvent used to carry the sample through the system. Inlet
solvent filter removes particles that could potentially damage the system's sensitive
components.
Solvent is propelled through the system by the pump.
Sample injector equipped with a sample loop of the appropriate size for the analysis being
performed.
Sample filter or a precolumn filter is used to filter the sample which usually contains
undissolved materials.
Analytical column allows the primary sample separation to occur.
Guard column is often included just prior to the analytical column to chemically remove
components of the sample that would otherwise foul the main column.
Following the analytical column, the separated components pass through a detector flow cell
before they pass into the waste reservoir.
The sample components' presence in the flow cell prompts an electrical response from the
detector, which is digitized and sent to a recorder. The recorder helps analyze and interpret the
data.
As a final system enhancement, a back pressure regulator is often installed immediately after
the detector. This device prevents solvent bubble formation until the solvent is completely
through the detector.
Alternatively, an inert gas sparging system may be installed to force dissolved gasses out of the
solvent being stored in the solvent reservoir.

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 PUMPS
• Pump refers to the device that forces the mobile phase through a liquid
chromatography column at pressures up to (and sometimes above) 10,000 psi.

• Pumps used in liquid chromatography are required to deliver very constant liquid
flows, free of pressure pulses (pressure pulses are one of the sources of detector
noise).

The two basic classifications are;

constant pressure and the constant flow pump
The first is used only for column packing.
The second type is the most widely used in all common HPLC applications.

1 pound per square inch (psi) = 6 894.75729 pascals

http://www.chromatography-online.org/topics/pump.html
http://www.pharm.uky.edu/asrg/hplc/pumps.html
 INJECTORS

Introduces the sample into the liquid stream of the instrument.

Injectors should provide the possibility of injecting the liquid sample within the range
of 0.1 to 100 ml of volume with high reproducibility and
under high pressure (up to the 4000 psi).

Rheodyne injector
 Automatic Injectors

Such equipment is popular for the analysis of routine samples.

Because of their superior characteristics, valves are now used.
With these sampling valves, samples can be introduced reproducibly into pressurized
columns without significant interruption of flow, even at elevated temperatures.

schematic drawings of a six-port Rheodyne valve

 Column

A tube which contains the stationary phase.

The stationary phase differentially interacts with the sample’s constituent

compounds as they are carried along in the mobile phase.
Column, in any chromatographic mode the composition of the mobile phase
provides the chemical environment for the interaction of the solutes
with the stationary phase.
 Column Thermostats
It often is advantageous to run ion-exchange, size-exclusion and reverse-phase
columns at higher temperatures, and to precisely control the temperature of
liquid-liquid columns.
 Reversed Phase Chromatography

In reversed phase separations organic molecules are separated

based on their degree of hydrophobicity.
There is a correlation between the degree of lipophylicity and retention in the column.
The majority of the HPLC separations are done with Reversed Phase separation,
probably over 90%. The rest is divided between the other modes described here.
Normal Phase Chromatography
Elution order in Normal Phase HPLC shows that the polar solutes
elute later than non-polar lypophilic ones.
 Elution
• A term used to describe the emergence of
chemicals from a column in
chromatography.

• The material emerging from the column is

known as the eluate. The eluent is the
carrier that moves the chemicals through
the column. In liquid chromatography, the
eluent is the liquid solvent; in gas
chromatography, it is the carrier gas*.

* IUPAC. Compendium of Chemical Terminology, 2nd ed. (the "Gold Book"). Compiled by A. D. McNaught and A. Wilkinson. Blackwell Scientific
Publications, Oxford (1997). XML on-line corrected version: http://goldbook.iupac.org (2006-) created by M. Nic, J. Jirat, B. Kosata; updates
compiled by A. Jenkins. ISBN 0-9678550-9-8. doi:10.1351/goldbook.
Many of the modes of modern liquid chromatography, it becomes readily apparent
that the most practical system for the delivery of the mobile phase is that which
can combine several liquids in different proportions. This blending capability
greatly speeds the process of selecting the optimum eluent mixture required for
analysis.
• The peaks were monitored in the UV range from 200-400nm.

• Flow rate of 400µl/min. Mobile phase A (acetonitrile) and B (0.3%

formic acid) ratios where changed after 15 minutes from 15:85 to
25:75 and total run time was 33 minutes*.

* M.J. Dubber; I. Kanfer. HIGH-PERFORMANCE LIQUID CHROMATOGRAPHIC DETERMINATION OF SELECTED FLAVONOLS IN GINKGO BILOBA SOLID ORAL DOSAGE FORMS. J. Pharm
Pharmaceut Sci., 7(3):303-309, 2004.
 Detector
• Detector is usually an optical sensor that detects changes in the characteristics of
the solvent stream.

• An appropriate detector has the ability to sense the presence of a compound and
send its corresponding electrical signal to a computer data station.

• A choice is made among many different types of detectors, depending upon the
characteristics and concentrations of the compounds that need to be separated
and analyzed.
 Common detectors are

1. Refractive Index (RI)

2. Ultra-Violet (UV)
3. Fluorescent
4. Radiochemical
5. Electrochemical
6. Near-Infra Red (Near-IR)
7. Mass Spectroscopy (MS)
8. Nuclear Magnetic Resonance (NMR)
9. Light Scattering (LS)

http://chemistry.gsu.edu/CHEM/HPLC_detector.ppt
http://kerouac.pharm.uky.edu/ASRG/HPLC/Detectors.html
 Refractive Index (RI)
Refractive Index (RI) detectors measure the ability of sample molecules
to bend or refract light.

P1

P2

http://chemistry.gsu.edu/CHEM/HPLC_detector.ppt
http://kerouac.pharm.uky.edu/ASRG/HPLC/Detectors.html
 Important advantage of RI detector

Universal (respond to all types of compounds).

Therefore, nonchromophoric analytes (carbohydrates, alcohols and polymers)

can be detected using RI detection.

Disadvantages of RI detector

 Not suitable for gradient elution. Changes in solvent composition change RI.

 Require careful control of the column and detector temperature.

 Moderate sensitivity 10-9 10-10 g. “Not useful for trace analysis”

http://chemistry.gsu.edu/CHEM/HPLC_detector.ppt
http://kerouac.pharm.uky.edu/ASRG/HPLC/Detectors.html
 Fixed Wavelength Detectors is the most common and inexpensive detector.

The use of suitable λ is determined by the nature of the light source use

Variable Wavelength Detectors provide detection of eluted peak at any selected λ .

Less sensitive than fixed wavelength but the detection wavelength can be varied.

http://chemistry.gsu.edu/CHEM/HPLC_detector.ppt
http://kerouac.pharm.uky.edu/ASRG/HPLC/Detectors.html
 Photodiode Array Detectors (PDA or DAD )

Even much more rapid scanning of the

absorption spectra of the eluted peak is possible

using a photodiode array detector.

Advantage: DAD provides absorption spectra of each

peak and can be used for peak purity analysis.

Disadvantage: DAD is less sensitive and more expensive

than single λ detector.

http://chemistry.gsu.edu/CHEM/HPLC_detector.ppt
http://kerouac.pharm.uky.edu/ASRG/HPLC/Detectors.html
 Applications of DAD

3-D spectra of each peak eluting from the column can be obtained

http://chemistry.gsu.edu/CHEM/HPLC_detector.ppt
http://kerouac.pharm.uky.edu/ASRG/HPLC/Detectors.html
 ELECTROCHEMICAL DETECTION

Electrochemical detection (ECD) involves

the application of electric field (via a suitable electrode) to a sample solution,

followed by measurement of resultant current.

ECD includes the technique of Voltammetry, Amperometry and Coulometry

Common characteristics in ECD:

A chemical reaction (e.g., Faradaic oxidation or reduction occurs) during ECD.

To be capable of ECD solutes must be easily oxidized or reduced. One example is:

+
OH O +H + e-

http://chemistry.gsu.edu/CHEM/HPLC_detector.ppt
http://kerouac.pharm.uky.edu/ASRG/HPLC/Detectors.html
 ECD is useful for organic molecule containing functional group
capable of being oxidized or reduced.
Some typical functional groups sensed by electrochemical detectors are shown to the
below.
Disadvantages of ECD
• Technique is not very suitable for electroreducible compounds
• Metal ion impurities interfere for electroreductible compound in ECD.
 FLUORESCENCE DETECTION IN HPLC

• There are two steps in

measurement of fluorescence.

• Fluorescence molecule absorbs

radiation at one λ and emit
radiation at a longer λ.

http://chemistry.gsu.edu/CHEM/HPLC_detector.ppt
http://kerouac.pharm.uky.edu/ASRG/HPLC/Detectors.html
 Advantage;

1. Inherent advantage is higher sensitivity

~2-3 orders of magnitude greater than
UV-Detection.
For example, polycyclic aromatic
hydrocarbons (PAHs) needs to be detected
at low concentration).
Chromatogram on the right
compares the UV and fluorescence
detection.
2. Derivitization with fluorescent
reagent o-phthaldehyde will enhance
the detectability.

Disadvantage;
Non-linear calibration curve results at
higher concentrations of the analyte.
 Mass detector

Universal detectors.

Also can be used to identify analytes with great certainity.

http://chemistry.gsu.edu/CHEM/HPLC_detector.ppt
http://kerouac.pharm.uky.edu/ASRG/HPLC/Detectors.html
http://www.mhhe.com/physsci/chemistry/carey/student/olc/graphics/carey04oc/ref/ch13ms.htm
 SYSTEM SUITABILITY TESTING
An overall system suitability testing should be routinely run to determine if the
operating system is performing properly.

A. System Resolution
B. Column Efficiency
C. Column Capacity
D. Column Selectivity
E. Peak Symmetry

Chromatographic Analysis of Pharmaceuticals, edited by John A. Adamovics, Second Edition, Marcel Dekker, 1997
 System Resolution
Resolution is the parameter describing the separation power of the complete
chromatographic system relative to the particular components of the mixture.

2(t2-t1)
R=
W2 + W1
t2 and t1 are the retention times of the two components
W2 and W1 are the corresponding widths at the peak base

The peak width is measured by drawing tangents from the sides to the base.
image: http://www.lcresources.com/resources/getstart/resolution.gif
text: İlaçların Tanınması ve Kantitatif Tayini, İ.Ü. Yayınları, 1989
 Resolution values are typically equal or
greater than 2
for a suitable chromatographical system.

R≥2

image: http://chemwiki.ucdavis.edu/@api/deki/files/213/=resolution.jpg
text: İlaçların Tanınması ve Kantitatif Tayini, İ.Ü. Yayınları, 1989
 Column Efficiency
Column efficiency refers to the performance of the stationary phase to accomplish
particular separations. This entails how well the column is packed.
Column efficiency is basicly defined as the number of theoretical plates in the
column.
2 A good separation requires narrow peaks in
tR the chromatogram.
N = 16
The plate number is fairly constant for all the
W peaks in a particular chromatogram.

The value of N for a column varies with many factors:

 column length,
 particle size,
 mobile phase flow rate,
 how well the column was made,
 how long the column has been in use.
However in general N value must be equal or greater
than 2000. (N ≥ 2000)
image: http://www.lcresources.com/resources/getstart/resolution.gif
text: İlaçların Tanınması ve Kantitatif Tayini, İ.Ü. Yayınları, 1989
 Column Capacity
For the definition of a peak one should use some retention measurements:

Retention time (tR) = Time passed since the injection of solute until detected with
maximum concentration.
Retention volume (VR) = Solvent volume necessary for elution of solute from the column.

Capacity factor (k’) is basically the measure of balance between the solute and the HPLC
system ( stationary and mobile phase compositions).
Here, V0 is the dead volume, characterized by a material which does not retain in the
column, ex:toluene.
VR1 – V0
k’ =
V0

2 ≤ k’ < 5 k‘ ≥ 2

It is controlled entirely by the temperature and

by the chemical characteristics of both the
stationary phase and the mobile phase.
image: http://www.lcresources.com/resources/getstart/resolution.gif
text: İlaçların Tanınması ve Kantitatif Tayini, İ.Ü. Yayınları, 1989
 Column Selectivity
If a sample has two or more peaks, we can also calculate a retention ratio (α) for
any pair of peaks. This is simply the ratio of capacity factors (k' of the second peak
divided by k' of the first peak).

It gives information about the selectivity of the chromatographic system.

Like the capacity factors that comprise it, the retention ratio is unaffected by flow
rate or column dimensions; it is controlled entirely by the temperature and by the
chemical characteristics of the stationary phase and the mobile phase.

k ’2
α=
k ’1

α > 1  good separation

image,text: http://www.lcresources.com/resources/getstart/3a01.htm
 Peak Symmetry
We can measure peak asymmetry in one of two ways as shown here. The Tailing Factor,
measured at 5% of the peak height, is largely used in the pharmaceutical industry.
The Asymmetry Factor measured at 10% of the peak height is most often used in non-
pharmaceutical analyses.
In most cases, the Asymmetry Factor and Tailing Factor will be roughly the same (although
rarely exactly equal). Values should be equal of smaller than 2 for a new column and the
conditions of the test chromatogram.

T≤2

image,text: http://www.lcresources.com/resources/getstart/3a01.htm
 Mobile Phase Preparation
Calculate and measure appropriate amounts of solvent components,
Mix them thorougly with a magnetic stirrer,
Add buffers and additives, if necessary,
Filter mobile phase with a vacuum filtration equipment,
Degas mobile phase by an appropriate way.

Filtration Apparatus Ultrasonic bath for degassing the solvent

 Sample Preparation

Sample should be dissolved in the mobile phase but could be also dissolved in
a stronger solvent, finally it should be in very dilute concentration and small
volume.

Filtering the sample through a syringe filter with a 0.45µm microporous

membrane filter will remove particulates that can cause premature
instrument failure.

text: http://www.scientistlive.com/European-Science-News/Process_Technology/HPLC_sample_preparation/9446/
 Column Installation

Columns have their own flow direction, shown on the column with an arrow.
Operator should install the column according to this direction,
otherwise it would decrease the column efficiency.

image: http://www.blog.analtech.com/Portals/41906/images//color%20columns-2-resized-600.jpg
 Column Care
For long term storage, silica based columns should be stored in an aprotic
solvent. The water content should not be higher than 50%. The best
storing solvent is Acetonitrile.

For the column to be stored, operator should check the handbook given
by the manufacturer, every column has a particular procedure to be
stored or washed. (Some columns should be stored in 2-propanol).
After the removal of the column one should quickly stopper the both
sides and prevent drying.

Column flow rate is also mentioned on those sheets but generally, one
should operate with 0.25-1.0 ml/min flow rates.

One should flush the column with an appropriate solvent and wait until a
proper baseline occurs. After a proper baseline one can install the mobile
phase and start the analysis.

http://www.nestgrp.com/pdf/colcare.pdf
 Applications of HPLC

Preparative HPLC refers to the process of isolation and purification of compounds.

Chemical Separations can be accomplished using HPLC by utilizing the fact that
certain compounds have different migration rates given a particular column and
mobile phase.

Purification refers to the process of separating the target compound from other
compounds or contaminants.

Identification of compounds by comparing with a standard material.

Quantification of compounds by HPLC is the process of determining the unknown

concentration of a compound in a known solution.

http://kerouac.pharm.uky.edu/asrg/hplc/applications.html
 Separation Mechanism

Separation is determined by column (packing material) and mobile phase.

High amounts of materials (mg) can be separated. Preparative columns are
used.
Mobile phase (solvent)
↓ ↓ ↓ ↓
B
A B C
C
A

Column
time

C>B>A
Packing
material

46
Qualitative analysis
Identification - What is component A?

Comparing Rt values of standard materials and unknown material.

Component A elutes the same time as a caffeine peak.

Component A is identified as caffeine.

47
Quantitative analysis
What is the concentration of component A?

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.
48