1. Department of Chemistry
Dnyanasadhana College, Thane.
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Department of Chemistry
Dr.Bhagure G.R. T.Y.B.Sc. Analytical Chemistry Paper-IV Sem-VI
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

2. Contents 2.3 High Performance Liquid chromatography (HPLC) (06 L)
2.3.1 Introduction to HPLC
2.3.2 Instrumentation in HPLC
A) Solvent Reservoir
B) Degassing systemFiltration,
Distillation, Sparging inert gas
C) PumpsReciprocating
pumps, Screw driven syringe type
pumps, Pneumatic pumps , advantages and disadvantages of each pump.
D) Precolumns
E) Sample injection system
F) HPLC Columns
G) Detectors: Ultraviolet – Visible detector, Refractive index
detector. Advantages and Limitations of each detector.
2.3.3 Applications of HPLC

3. High
Performance
Liquid
Chromatography

4. High
Pressure
Liquid
Chromatography

5. High
Priced
Liquid
Chromatography

6. *
Principles of HPLC

7. C A B Time Zero Retention time of A Peak area of compound A
Peak area of compound B
Peak area of compound C B C A
Retention time of A
Injection point
Time Zero
Time in Minutes

8. Nature of Stationary phase : more polar
Nature of Mobile phase: Less polar solvent
Solute components are separated on differential solubility in stationary phase
Soluble component in stationary phase carried by Mobile phase to detector

9. Stationary phase
Mobile phase

10. Partitioning Stationary Phase Mobile Phase
Separation is based on the analyte’s relative solubility between two liquid phases
Stationary Phase
Mobile Phase
EQULIBRIUM
Solvent
Bonded Phase

11. C A B Time Zero Retention time of A Peak area of compound A
Peak area of compound B
Peak area of compound C B C A
Retention time of A
Injection point
Time Zero
Time in Minutes

12. HPLC INSTRUMENTATION

13. Sample Injection System
Components of
HPLC INSTUMENT
Solvent system.
Pumps
Pre-column
Analytical Column
Sample Injection System
Detector
Read out Device System

14. Solvent system
K = Cs/Cm
** Differences between the m.p. and s.p. (partitioning)
* Generally we want a significant difference between the polarities of the s.p. and the m.p., the reason being is that separation is based on solubility

15. Phases: Both phases are liquid
Stationary Phases: More polar of two are made as stationary phase
Mobile Phase: Less polar of two is made as mobile phase.

16. HPLC - Modes
Normal phase
Reversed
phase
Polar solvent act as Mobile phase
Polar solvent act as stationary phase
Non Polar solvent act as Mobile phase
Non Polar solvent act as stationary phase
Almost all reversed phase separations (polar m.p. & Non polar s.p.) can be carried out with combination of acetonitrile (CH3CN), and/or methanol, and water as a m.p.

17. Water is the most polar solvents
Pentane
Hexane
Heptane
Trichlorofluroethane
Toluene
Chloroform
Dichloromethane
Diethyl ether
Ethyl Acetate
Methyl t-butyl ether
Dioxane
Acetonirile
WATER P O L A R I T y
Increases

18. Columns: Pre column & Analytical Column
Pre column : Solid Support - Backbone for bonded phases.
Usually 10µ, 5µ or 3µ silica or polymeric particles. b)
Bonded Phases - Functional groups firmly linked (chemically bound) to the solid support.
Extremely stable
Reproducible

19. Functions of Pre-column:1
Its function is to Protects the analytical column. 2
It removes impurities present in solvent system. 3
It saturates the mobile phase with stationary phase. 4
It prolongs the life of the analytical column.

20. Analytical Column- Performs the separation. 1
Length of the 10 to 30 cm in length and 4 to 10 mm. in diameter. 2
It is made-up of stainless steel or glass 3
Column Packing: 1) Porous bead packing; silica gel and alumina are used for the separation of low m.wt. compounds. 4
Pellicular Beads: Its made from glass beads of diameter of about 400 um. Coated with the thin layer of porous material like silica gel , alumina or ion exchange resin.

21. Bonded Phases Name Formula No. of Carbons C-2 C-8 C-18 CN Ethyl Silyl
Octyl Silyl
Octadecyl Silyl Si-(CH2)17-CH3
Cyanopropyl Silyl
Formula
Si-CH2-CH3
-Si-(CH2)7-CH3
-Si-(CH2)17-CH3
Si-(CH2)3-CN

22. Sample Injection System:
• An auto sampler is the automatic version when the user has many samples to analyze or when manual injection is not practical.
• The injector serves to introduce the liquid sample into the flow stream of the mobile phase. Typical sample volumes are 5-to 20-microliters (μL). The injector must also be able to withstand the high pressures of the liquid system.

23. INSTRUMENT for HPLC
Pre column

24. HPLC Instrument

25. WORKING OF HPLC

26. Detectors Detector should have following characteristics.
High sensitivity
Negligible baseline noise
Large linear response range (analyte concentration range over which detector response is proportional to concentration)
Insensitive to temperature changes and solvent composition Universality or predictable specificity
Low dead volume
No sample destruction
Stability over time Reliable
Inexpensive to produce and continuous operation

27. UV Detector
Reference cell

28. Desirable characteristics of detectors
High sensitivity
Negligible baseline noise
Large linear response range (analyte concentration range over which detector response is proportional to concentration)
Insensitive to temperature changes and solvent composition
Universality or predictable specificity
Low dead volume
No sample destruction
Stability over time
Inexpensive to produce and continuous operation
Capable of providing information on solute identity

29. Characteristics of Selected Liquid Chromatography Detectors
Type
Approximate Limit of Detection
Approximate Linear Range
Comments
Ultraviolet and visible absorption
10-11 g
104
Specific for light-absorbing compounds
Differential Refractive Index g
103
Universal detector -measures changes in refractive index. Cannot be used with gradients
Electrochemical Amperometric g
105
Specific detector. Compound must be electroactive
Electrochemical Conductometric
10-8 g/mL
Specific detector, but for all ions
Fluorescence
10-14 g
Specific detector. Compound must be fluorescent
Mass Spectrometry
Universal detector. Also can be used to identify analytes with great certainty
Solution Light Scattering
10-6 g/mL
Used to determine MW’s of polymers as they elute. Concentration usually far above limits of detection
Evaporative Light Scattering
10-9 g
106
Universal except for volatile analytes. Not a linear response

30. HPLC Detectors respond to:
Solute property not exhibited by MP
UV/VIS (0.1-1 ng)
Fixed
Variable (VWD)
Photodiode array (DAD)
FTIR (1mg)
Electrochemical ( pg)
Amperometric
Coulobmetric
Fluorescence (1-10 pg)
Requires fluorophore
Bulk property that changes with eluted solute
Refractive Index
( ng)
Conductivity
( ng)
Direct solute detection
Mass Spectrometry
( pg)
ELSD
( pg)

31. UV Detector
Reference cell

32. U.V. Radiation Source Collimating Lens Photodiode detector
Outlet
Inlet
U.V.filter
U.V. Radiation Source Collimating Lens Photodiode detector
Quartz window
Outlet
Inlet

33. UV-VIS Light Absorption Detectors
Most common HPLC detector
Many solutes absorb ultraviolet (UV) light
Most employ the most intense 254 nm (emission of a mercury lamp)
Modern HPLC instruments have the capability to choose the appropriate wave length for a given analyte.
Use of photodiode arrays which can record the entire UV region at once in a fraction of a second (spectrum of each solute as it is eluted).
Full scale absorbance range absorbance units
Linear range 5 orders of magnitude of solute concentration Good for gradient elution

34. Refractive Index (RI) Detector

35. Refractive Index (RI) Detector
Where solvent passes through one half of the cell and then it passes through another chamber where eluent flows.
Two compartments are separated by a glass plate mounted at an angle such that bending of the incident beam occurs if the two solutions differ in refractive index.
The resulting displacement in beam with respect to the photosensitive surface of a detector causes variation in the output signal.

36. Advantages of Detector
Respond almost to every solute but sensitivity is lowered by a factor of 1000 compared to a UV detector.
Sensitive to changes in pressure and temperature
Unsuitable for gradient elution due to problem of matching sample and reference while the composition is changing.
Its primary appeal is due to a nearly universal response to all solute including those with little UV absorption

37. 5. Computer
Frequently called the data system, the computer not only controls all the modules of the HPLC instrument but it takes the signal from the detector and uses it to determine the time of elution (retention time) of the sample components (qualitative analysis) and the amount of sample (quantitative analysis).

38. Qualitative Analysis
APPLICATIONS OF HPLC
The identification (ID) of individual compounds in the sample; the most common parameter for compound ID is its retention time (the time it takes for that specific compound to elute from the column after injection); •depending on the detector used, compound ID is also based on the chemical structure, molecular weight or some other molecular parameter.

39. Quantitative Analysis
Quantitative Analysis can be performed by two ways
1) Determination of the peak height of a chromatographic peak as measured from the baseline;
2) Determination of the peak area (see figure below);In order to make a quantitative assessment of the compound, a sample with a known amount of the compound of interest is injected and its peak height or peak area is measured. In many cases, there is a linear relationship between the height or area and the amount of sample.

40. C A B Time Zero Retention time of A Peak area of compound A
Peak area of compound B
Peak area of compound C B C A
Retention time of A
Injection point
Time Zero
Time in Minutes

41. Other Applications-1
Separation and analysis of non-volatile or thermally-unstable compounds HPLC is optimum for the separation of chemical and biological compounds that are non-volatile Typical non-volatile compounds are:
Pharmaceuticals like aspirin, ibuprofen, or acetaminophen (Tylenol)
Salts like sodium chloride and potassium phosphate
Proteins like egg white or blood protein.

42. Other Applications-2
Organic chemicals like polymers (e.g. polystyrene, polyethylene)
Heavy hydrocarbons like asphalt or motor oil
Many natural products such as ginseng, herbal medicines, plant extracts
Thermally unstable compounds such as trinitrotoluene (TNT), enzymes
NOTE: If a compound is volatile (i.e. a gas, fragrance, hydrocarbon in gasoline, etc.), gas chromatography is a better separation technique

43. Other Applications-3 1
HPTLC can be used for a large number of applications in food industry for screening purposes, e.g.: 2
Determination adulterants in food products such as argemone in mustard oil. 3
Separation of food colours in food samples. 4
Determination of residues of mycotoxins in food products.

44. Thank you