All you need to know about the HPLC Chromatography! HPLC (High-performance liquid chromatography) also referred as high-pressure liquid chromatography, is a technique used in analytical labs to separate, identify, and quantify each component in a mixture. HPLC separates and purifies compounds according to their polarity. I have tried to simplify the whole process of HPLC Chromatography and explain in simple terms. Let’s look at the main components involved in HPLC in this video. I discussed about Normal Phase Chromatography, Reverse Phase Chromatography and their comparison and differences. Watch this video for more details. If you have any doubts: Please leave a comment in the comments section below.

1. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
Prepared By,
S.R.Yadav
Assistant Professor

2. CONTENT
▪ WHAT IS HPLC
▪ DISCOVERY OF HPLC
▪ TYPES OF HPLC
▪ PRINCIPLE OF HPLC
▪ PROCEDURE OR MECHANISM
▪ PARTS OF HPLC
▪ ADVANTAGES OF HPLC

4. • WHAT IS HPLC-
High-performance liquid chromatography(HPLC) is a form of column
chromatography that pumps a sample mixture or analyte in a solvent (known as the
mobile phase) at high pressure through a column with chromatographic packing
material (stationary phase).

5. •DISCOVERY OF HPLC

6. • The term HPLC, coined by the Prof. Csaba Horváth for his 1970 Pittcon paper,
originally indicated the fact that high pressure was used to generate the flow required
for liquid chromatography in packed columns.
• In the beginning, pumps only had a pressure capability of 500 psi [35 bar]. This was
called high pressure liquid chromatography, or HPLC. The early 1970s saw a
tremendous leap in technology.
• These new HPLC instruments could develop up to 6,000 psi [400 bar] of pressure,
and incorporated improved injectors, detectors, and columns.
• HPLC really began to take hold in the mid-to late-1970s. With continued advances
in performance during this time [smaller particles, even higher pressure], the term
HPLC remained the same , but the name was changed to high performance liquid
chromatography.

7. • High performance liquid chromatography is now one of the most powerful tools in
analytical chemistry.
• It has the ability to separate, identify, and quantitative the compounds that are present
in any sample that can be dissolved in a liquid. Today, compounds in trace
concentrations as low as parts per trillion (ppt) may easily be identified.
• Now according to its performance and modernization of its part and the able to give
precise result, that’s why today it is also called high precession liquid
chromatography and UPLC.

8. • TYPES OF HPLC
1) Normal Phase HPLC:
This method separates analytes on the basis of polarity. NP-HPLC uses polar stationary
phase and non-polar mobile phase. Therefore, the stationary phase is usually silica and
typical mobile phases are hexane, methylene chloride, chloroform , diethyl ether , and
mixtures of these. Polar samples are thus retained on the polar surface of the column
packing longer than less polar materials.

9. • 2)Reverse Phase HPLC:
The stationary phase is non polar (hydrophobic) in nature, while the mobile phase is a
polar liquid, such as mixtures of water and methanol or acetonitrile. It works on the
principle of hydrophobic interactions hence the more nonpolar the material is, the
longer it will be retained.

10. PRINCIPLE OF HPLC

11. VAN DEEMTER EQUATION
H= HETP=A+ B/ ū + C x ū
A= EDDY’S DIFFUTION,
B= LONGITUDINAL DIFFUTION,
C= RESISTANCE TO MASS TRANSFER,
ū = AVARAGE MOBILE PHASE VELOCITY,

12. • HUBER AND KNOX EQUATION
• Huber pointed out that in relating plate height to mass transfer kinetic factors, need
to add an additional term to account for mass transfer in both the stationary phase
and mobile phase.
• H = A + B/ū + Cm ū + Cs ū
• Then KNOX develop an experimental equation for liquid chromatography that
contain a term useful for correcting the deviation from the VAN DEEMPTER
equation, containing the third root of velocity. The KNOX equation is usually
expressed in the dimensionless reduce from since the physical meaning of the term
is not clear.

13. • h = Av1/3 + B/v + C x v
• ▪ The term A is typically 1 or 2, with a large value representing a poorly
bed; B is about 1.5 and C is 0.1. So a typical good column follows:
• h = v1/3 + 1.5/v + 0.1 x v

14. PROCEDURE OF HPLC

16. • The sample is introduced through an injection system into the entrance
to the column
• The stationary phase is porous silica.
• Mobile phase can either be aq. Or org. solvent.
• In Interaction btw mobile and stationary phase, two types of scenarios
can result.

19. PARTS OF HPLC

21. • SOLVENT RESERVOIR
Mobile Phase in NP-HPLC: –
Typically non polar solvents such as hexane , heptane , iso-octane are used in
combination with slightly more polar solvents such as Isopropanol , ethyl acetate , or
chloroform. Retention increases as the amount of non polar solvent increases in the
mobile phase.
▪ Mobile phase in RP-HPLC: –
In reverse phase water is usually the base solvent. Other polar solvent such as
Methanol , Acetonitrile or Tetrahydrofuran are added . pH is adjusted by buffers to
modify separations of ionizable solutes.

22. • DEGASSER ▪
• Air in the pump will cause the pump to stop delivering mobile phase to the column.
If only an occasional bubble is present, the flow rate will be erratic, causing retention
time problems.
• Air in an optical detector, such as an ultraviolet (UV), fluorescence, or refractive
index detector, will scatter light passing through the flow cell, causing noise spikes
in the chromatogram or an off-scale signal.
• To get rid of these problems eliminated if the air is removed from the mobile phase is
necessary. And that’s why degasser is use.

23. • SOLVENT DELIVERY SYSTEM (PUMP) ▪
• The role of the pump is to force a liquid (called the mobile phase) through the liquid
chromatograph at a specific flow rate, expressed in millilitres per min (mL/min).
• Normal flow rates in HPLC are in the 1-3 mL/min range, pressure is about 2000
psi.
• Typical pumps can reach pressures in the range of 6000-9000 psi (400- to 600-bar).
• During the chromatographic experiment, a pump can deliver a constant mobile
phase composition (isocratic) or an increasing mobile phase composition (gradient).

24. • INJECTOR
• Manual Injector:
1. User manually loads sample into the injector using a syringe
2. and then turns the handle to inject sample into the flowing mobile phase … which
transports the sample into the beginning (head) of the column, which is at high
pressure.
Auto sampler:
▪ User loads vials filled with sample solution into the auto sampler tray (100
samples) and the auto sampler automatically measures the appropriate sample
volume, injects the sample, then flushes the injector to be ready for the next sample,
etc., until all sample vials are processed.

25. • COLUMN
▪ The Columns are called “the heart of HPLC”
• Columns are usually made of polished stainless steel, are between 50 and 300 mm
long and have an internal diameter of between 2 and 5 mm. They are commonly
filled with a stationary phase with a particle size of 3–10 µm. Columns with internal
diameters of less than 2 mm are often referred to as microbore columns. Ideally the
temperature of the mobile phase and the column should be kept constant during an
analysis.
There are several types of column use in HPLC according to their work:
A) Normal phase columns: Pours silica gel, polymer gel.
B) Reverse phase columns: Silica gel is coated with ODS, C18.

26. • DETECTORS ▪
The HPLC detector, located at the end of the column detect the analytes as they elute
from the chromatographic column.
▪ Commonly used detectors are:
A) UV detector: If a compound can absorb ultraviolet light, a UV-absorbance detector
is used.
B) Fluorescence Detector: Fluorescence detectors are very specific and selective
among the others optical detectors. Use in the measurement of specific fluorescent
species in samples.
C) Refractive Index Detector: Refractive index detector (RI or RID) is a detector that measures
the refractive index of an analyte relative to the solvent. They are often used as detectors for
high-performance liquid chromatography and size exclusion chromatography

27. • RECORDER
• A recorder, is a device that draws the chromatogram that results from a
chromatographic process onto chart paper and provides a visual representation of
the separation that has been achieve.
• The time scale of the chart movement normally ranges from about 1 cm per second
to 1 cm per hour which can also be selected to suit the separation that is being
carried out. Most modern chromatographs no longer employ recorders to present
the chromatogram and the results are automatically processed by a computer and
presented on the computer monitor or printed out as required.

28. APPLICATIONS OF HPLC

29. • HPLC can be used in both qualitative and quantitative applications, that is for both
compound identification and quantification.
• ▪ Pharmaceutical Applications
• 1. To control drug stability.
• 2. Tablet dissolution study of pharmaceutical dosages form.
• 3. Pharmaceutical quality control.
• ▪ Environmental Applications
• 1. Detection of phenolic compounds in drinking water.
• 2. Bio-monitoring of pollutants

30. • ▪ Food and Flavour
• 1. Measurement of Quality of soft drinks and water.
• 2. Sugar analysis in fruit juices.
• 3. Analysis of polycyclic compounds in vegetables.
• 4. Preservative analysis.
• ▪ Applications in Clinical Tests
• 1. Urine analysis, antibiotics analysis in blood.
• 2. Analysis of bilirubin, biliverdin in hepatic disorders.
• 3. Detection of endogenous Neuropeptides in extracellular fluid of brain etc

31. • Applications in Forensics
• 1. Quantification of drugs in biological samples.
• 2. Identification of steroids in blood, urine etc.
• 3. Forensic analysis of textile dyes.
• 4. Determination of cocaine and other drugs of abuse in blood, urine etc

32. This Photo by Unknown Author is licensed under CC BY-SA-NC