hplc history, type,principle, procedure, parts, applications

1. HIGH PERFORMANCE
LIQUID
CHROMATOGRAPHY
PARTHA PRATIM KANDAR SEM-218
SWAGATA CHAKRABORTY SEM-217
SHIMALI CHAUHAN SEM- 229

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

3. HPLC DIAGRAM

4. WHAT IS HPLC?
High-performanceliquid 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. DISCOVERYOF 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
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. 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. H= HETP=A+ B/ ū + C x ū
A= EDDY’S DIFFUTION,
B= LONGITUDINAL DIFFUTION,
C= RESISTANCETO MASSTRANSFER,
ū = AVARAGE MOBILE PHASEVELOCITY,
VAN DEEMTER EQUATION

12. ▪ 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 theVAN 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.
HUBER AND KNOX EQUATION

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

15. MECHANISM

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.

17. This has polar stationary phase. It contains silica beads in intact fashion.
POLAR/HYDROPHILLIC

18. HYDROPHOBIC
This is made with hydrophobic molecules.

19. PARTS OF HPLC

20. 1) MOBILE PHASE / SOLVENT RESERVOIR,
2) DEGASSER,
3) PUMP,
4) INJECTOR,
5) COLUMN,
6) DETECTOR,
7) RECORDER.

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 , ethyle actate , 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
Tetrahydrafuran 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.

27. ▪ 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.

28. 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.

29. APPLICATIONS OF HPLC

30. 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.

31. ▪ 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 ClinicalTests
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

32. ▪ 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.