The presentation contains basic introduction to mostly used and versatile reversed phase chromatography, its instrumentation, working and applications. It will be useful for you to understand basic concepts about RP-HPLC.

1. BASIC INTRODUCTION
TO RP-HPLC
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Presented by
Mr. Devesh R. Pelagade
Pharmaceutical Quality Assurance
MVP Samaj’s College of Pharmacy,
Nashik.

2. Contents:
1. Introduction
2. Instrumentation
3. Working
4. Applications
5. References
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3.  What Is Chromatography ?
• Chromatography is a separation technique which is used to separate a mixture of
compounds into its individual components based on certain physical and chemical
properties.
• It was developed by Russian botanist M. Tswett, who is referred as ‘Father of
Chromatography’.
 High Pressure Liquid Chromatography(HPLC):
• HPLC is a type of Liquid chromatography where analytes are dissolved in a solution and
are separated by passage through a column packed with micrometer sized particles
(1.5-5 um in diameter).
• HPLC is further divided into Normal phase (NP) and Reversed phase (RP) HPLC based
upon polar and non-polar nature of mobile and stationary phase.

4. • NP-HPLC features a polar column as stationary phase in combination with
non-polar mobile phase.
• RP-HPLC features a non-polar column as stationary phase in combination with
a polar mixture of water plus an organic solvent as mobile phase, thus called
‘Reversed phase’.
• Due to its simplicity and versatility most of HPLC separations are carried out
by Reversed phase mode.
Stationary Phase Mobile Phase
Normal Phase HPLC Polar Non-polar
Reversed Phase HPLC Non-polar Polar
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5. Components of an HPLC system are:
1. Solvent reservoirs and Filters
2. Mobile phase degasser
3. HPLC pump
4. Precolumn/Guard column
5. Sample injector
6. Analytical column
7. Detectors
 Instrumentation:
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7. 1. Solvent reservoir and filters:
• Solvent reservoirs are the containers which contains the solvents used to carry
the sample through the system.
• Most of the reservoir containers are made of glass and are inert to the
solvent.
• In RP-HPLC, the mobile phase is a polar mixture of water plus an organic
solvent.
• Organic solvent is added to lower the polarity of aqueous mobile phase.
e.g. Acetonitrile or Methanol.
• The lower the polarity of mobile phase, the greater its eluting strength in RP-
HPLC.
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8. • The mobile phase used in RP chromatography is generally prepared with strong
acids such as Trifluoroacetic acid or Ortho-phosphoric acid. These acids maintain a
low pH environment and suppresses the ionization of acidic groups in the solute
molecules.
• The pH of mobile phase should be in range of 2.0-8.0 as Siloxane linkages in
stationary phase gets cleaved below pH 2.0 and above pH 8.0 Silica may get
dissolve.
• The several sensitive components of HPLC system could get damaged if particulate
matter is present in solvent, therefore prefiltered HPLC grade solvents are used
which are highly purified.
• If prefiltered HPLC grade solvents are not available then the solvents should be
filtered with an inlet solvent filter.
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9. 2. Mobile Phase degasser:
• The presence of air bubbles in mobile phase is common problem in HPLC system.
• These bubbles can lead to pump-delivery problems and spurious peaks in detector
output.
• These bubbles are eliminated by degassing the mobile phase prior to its use.
Degassing can be done by-
A. Helium Sparging:
• The presence of dissolved Oxygen in the mobile phase degrades detectors
performance, so mobile phase is purged with Helium to remove dissolved Oxygen.
• It is most effective degassing technique and removes upto 80-90% of dissolved air.
• To avoid re-dissolving of air with the solvent, continuous Helium sparging is of
better choice.
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10. B. Vacuum Degassing:
• The partial vacuum is applied to the mobile phase which can remove sufficient
amount of dissolved gas.
• Vacuum degassing for 10-15 minutes will remove 60-70% of dissolved gas.
• It is not quite as effective as Helium sparging, but due to its convenience and high
cost of Helium Vacuum degassing is more preferred.
3. HPLC Pumps:
• The role of pump is to force the mobile phase through the system at specific flow
rate.
• Normal flow rates are in the 1-2 ml/min range.
• Most HPLC system pumps are designed to work at pressures of upto 6000 psi (400
bar).
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11. Reciprocating Piston Pumps:
• They consists of hydraulic chamber where a reciprocating piston is placed.
• When piston moves back, the solvent gets in and is pushed into the column
when piston moves forward.
Main components of a Piston pump are:
1) Motor/Cam - To drives piston back and forth in
pump head.
2) Piston - Usually made of Sapphire, although some
pumps uses ceramic pistons.
3) Pump seal - A polymeric pump seal is used to
prevent mobile phase from leaking out of the pump
head.
4) Check valves - An inlet and outlet valves are present
to control the direction of flow of mobile phase.
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12. Pump-Module types:
A. Isocratic Pump System:
• The composition and concentration of mobile phase is kept constant during
whole HPLC run.
• It is best for simple separations.
B. Gradient Pump System:
• Composition and concentration of mobile phase varies during whole HPLC run.
• Best for complex separations.
• Gradient pump system can be Binary which delivers two solvents or
Quaternary which delivers four solvents.
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13. 4. Precolumn or Guard column:
• Precolumn is placed anterior to the analytical
column and is used to protect the analytical column
thus known as ‘Guard column’.
• It is short in length about 2-10 cm with diameter 4-5
mm and contain same packing as or similar to that
in analytical column.
• Precolumn increases life of analytical column by
capturing strongly retaining sample components and
impurities before it enters into analytical column.
• Replacement of Precolumn at regular intervals must
be done to optimise its protective function.
Precolumn/Guard column
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14. Sample injection can be done by-
A. Manual Injector:
• User manually loads sample into the injector
using a syringe.
• Then turns the handle to inject sample into
beginning of the column which is at high pressure.
• Rheodyne injector is a most popular manual
injector is widely used.
Rheodyne injector
5. Sample Injector:
• The sample injector serves to introduce the liquid sample into the flow system of
mobile phase.
• The injector must be able to withstand high pressure of the liquid system.
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15. B. Autosampler:
• An autosampler is an automatic version for
sample injection when user has mass samples to
analyze.
• User fills loads of vials filled with sample solution
into the autosampler tray (120 samples).
• The autosampler then automatically measures the
appropriate sample volume, injects the sample
and flushes the injector to be ready for next
sample.
Autosampler
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16. 6. Analytical Column:
• It is ‘Heart of an HPLC system’ as it contains the stationary phase that separates
sample components of interest.
• It is placed after the sample injector and resides in a Column oven which maintains
the required temperature.
• In the Reversed phase chromatography, the material packed inside the column is
relatively non-polar and solvent is polar with respect to sample.
• Retention in RP-HPLC results by interaction of non-polar components of solute and
non-polar stationary phase.
• It is about 10-30 cm in length with 4-4.6 mm internal diameter and is packed with
modified non-polar Silica having particle size between 1.5-5 um in diameter.
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17. • C8 or C18 column is mostly used and is generally suitable for all samples in RP
system.
• Generally longer columns with smaller particle size provide better separation as it
gives more surface area for retention.
Analytical column Precolumn
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18. 7. Detectors:
• Detectors are used in detection of the solute present in the eluent coming out
from the Analytical column.
• It measures the amount of molecules for analysis of the sample components.
• Detectors also provide an output to a recorder or computer that results in a
chromatogram that is the graph of detector response.
A large numbers of detectors are used for RP-HPLC analysis. However only five of them
are dominant which are-
a. UV-Visible detector.
b. Fluorescence detector.
c. Conductivity detector.
d. Refractive Index(RI) detector.
e. Mass spectrometric(MS) detector.
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19. A. UV-Visible Detector:
• It is most widely used detector in modern
RP-HPLC and is based on Ultraviolet (UV) and
Visible light absorption.
• These detectors have high sensitivity for
many solutes, but samples must absorb in
the UV (or Visible) region (e.g. 190-600).
• The detector identifies the sample by
measuring its absorption of light at different
wavelength.
• UV-Visible detectors are reliable and easy to
operate and are suitable for use by less
skilled operators.
UV-Visible Detector
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20. B. Fluorescence Detectors:
• These detectors are very sensitive and selective for the
solutes that fluoresce when excited by UV radiation.
• Deuterium lamp or Xenon flash lamp is used as light
source.
• When the compound fluoresces, the emission
wavelength is isolated and directed to photometer
where it is converted to an electronic signal for data
processing.
• Because of its high sensitivity it is particularly used for
trace analysis or when solute concentration is
extremely low.
• The major disadvantage is that compounds that does
not fluoresce, cannot be analysed.
Fluorescence Detector
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21. C. Conductivity Detectors:
• These detectors uses detector cells to measure
change in conductivity of column effluent as it
passes through the cells.
• It is mostly used in Ion chromatography and Ion
exchange applications where analyte does not
absorbs UV light.
• If mobile phase shows conductance then
Conductivity detectors cannot be used.
• Conductivity detectors are ideal for analysis of
inorganic ions present in water samples, plating
bath, power plant cooling fluids etc. Conductivity Detector
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22. D. Refractive Index (RI) Detectors:
• These detectors responds to a difference in
refractive index of column effluent as it
passes through the detector flow cell.
• RI detectors shows excellent versatility, all
solutes can be detected if refractive index of
solute is different from that mobile phase.
• Generally not useful for trace analysis and
Gradient elution.
RI Detector
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23. E. Mass Spectral (MS) Detectors:
• Hyphenated HPLC detectors refer to coupling of an
independent analytical instrument (MS, NMR, FTIR) to
HPLC system to provide detection.
• The Mass Spectrometric (MS) detector id most popular
Hyphenated detector in use today.
• As MS Detectors detect ions in gaseous phase, the
mobile phase must be evaporated to generate the
sample ions during analysis.
• It is standard detector system in Bioanalytical methods
for analysis of Pharmaceutical compounds in biological
system like plasma or urine. MS Detector
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24.  Working of RP-HPLC:
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25. • Solvents are drawn out from the reservoir by HPLC pump towards the
degassers.
• Degasser removes the dissolved air from solvents either by Helium sparging or
by Vacuum degassing method.
• After degassing, solvents are mixed along with modifier (organic solvent)
e.g. Acetonitrile to form a polar mobile phase.
• HPLC pump controls the flow rate and with sufficient pressure pumps the
mobile phase further into Precolumn where strongly retaining components and
impurities are captured.
• Sample injector injects the sample in the flow either by manually or by
autosampling.
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26. • Mobile phase with sample is pumped into analytical column containing non-
polar stationary phase, hydrophobic components of mobile phase retains
whereas hydrophilic ones passes column and get eluted first.
• Detector analyses the eluted mobile phase and responds accordingly to
changes in analyte concentration during the run.
• The data system or computer attached to the detector monitors the detectors
output and generates electronic signals which gives chromatogram.
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27. Applications:
1. It is used in quality control to ensure the purity of raw materials and
formulated products.
2. Used for water purification and for analyzing air and water pollutants.
3. In law enforcement, to compare a sample found at crime scene to samples
from suspects.
4. For detection of illegal drugs and their concentrations in urine and blood.
5. In medical area it can be used for drug analysis or nutrient analysis.
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28.  References:
1) Willard HH, Dean AJ: Instrumental Method of Analysis. CBS Publishers and
distributors, 7th edition 1986.
2) Kumar SD Kumar HDR: Importance of RP-HPLC in Analytical Method
Development- A Review: A Review. Int J Pharm Sci Res. 3(12); 4626-4633.
3) Lloyd Snyder, J.J. Kirkland, J.W. Dolan: Introduction to Modern Liquid
Chromatography, 3rd edition. A John Wiley and Sons, Inc., Publications.
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