HIGH PERFORMANCE LIQUID CHROMATOGRAPHY PART-2

INSTRUMENTATION OF HPLC :-

HPLC INSTRUMENT CONSISTS OF FOLLOWING COMPONENTS:-

• PUMP
• MIXING UNIT
• SOLVENT DELIVERY SYSTEM
• INJECTOR
• COLUMN
• DETECTOR

1. PUMP:- The role of the pump is to force a liquid through the liquid chromatography at a specific flow rate expressed in milliliter per min(mL/min).Normal flow rate in HPLC are in the 1-2ml/min range. During the chromatographic experiments a pump can deliver a constant mobile phase composition or an increasing mobile phase composition. best for the analysis of complex sample.

TYPE OF PUMP :-

Constant flow reciprocating pump:-

• the term reciprocating describes any continuously repeated backwards and forward motion.
• widely used pump.
• solvent is sucked during back stroke and gets deliver to the column in forward stroke.

Syringe or displacement type pump:-

• Consists of large syringe like chamber
• suitable for small bore column

Pneumatic pump:-

• Gas is used to pressurize the mobile phase present in a collapsible solvent contanier.

2.  MIXING UNIT :-

• Mixing unit is used to mix solvent in different proportions and pass through the column.
• there are two types of mixing units 1. they are low pressure mixing chamber which uses helium for degassing solvent. 2.High pressure mixing chamber does not require helium for degassing solvents.
• mixing of solvents is done either with a static mixer which is packed with beads or a dynamic mixer which uses magnetic stirrer and operates under high pressure.

3. SOLVENT DELIVERY SYSTEM:-

           REQUIREMENTS :-

• ability to mix solvent and vary polarity of mobile phase during run
• ”unlimited” solvent reservoir
• generation of pressure upto 6000psi
• flow rate ranging from 0.1 to 10 ml/min.
• flow reproducibility of 0.5% or better.
• pulse free output

4. INJECTOR :-

• The injector serves to introduced the liquid sample into the flow stear the mobile phase.
• typical sample volumes are 5-20 microliters.
• the injector must also be able to withstand the high pressure of the liquid system
• An auto sampler is the automatic version for when the user has many sample to analyzer or when manual injection is not practical.

TYPES OF INJECTORS:-

1. SEPTUM INJECTOR:- For injecting sample through a rubber septum. this is not common , since the septum has to withstand high pressure .
2. STOP FLOW :- In which the flow of m.p. is stopped for a while and the sample is injected through a valve device.
3. Rheodyne Injector :-  it is the most popular injector. this has a fixed volume loop like 20μL or 50μL or more. Injector has 2 modes i.e. Load position when the sample is loaded in the loop and inject mode when the sample is injected

COLUMN :-

It is the heart of the chromatograph

COLUMN LENGTH:-5-30 CM

COLUMN DIAMETER :-2-50 MM

PARTICAL SIZE:-1-20μ

PARTICLE NATURE :-  spherical, uniform sized , porous material are used.

MATERIAL OF CONSTRUCTION FOR THE TUBING :-

• Stainless steel (the most popular , give high pressure capability)
• Glass (mostly for biomolecule)
• PEEK (POLY ETHER ETHER KETONE)

PACKING MATERIAL :-

• The packing material is prepared from SILICA particle,aluminium particle and ion exchange resins.
• Porous plug of stainless steels or teflon are used in the end of the column to retain the packing material.

DETECTORS :-

• UV DETECTOR
• REFRACTIVE INDEX DETECTOR
• FLOURIMETRIC DETECTOR
• AMPEROMETRIC DETECTOR

MCQ

1.Which statement is false about pump?

a.Normal flow rate in HPLC are in the 1-2ml/min range

b.During the chromatographic experiments a pump can deliver a constant mobile phase composition or an increasing mobile phase composition.

c.the term reciprocating describes any continuously repeated backwards and forward motion.

d.Normal flow rate in HPLC are in the 6-9ml/min range

2.which statement is true about solvent delivery system ?

a.generation of pressure upto 600psi

b.flow reproducibility of 0.3% or better.

c.flow rate ranging from 10 to 100 ml/min.

d.generation of pressure upto 6000psi

3.Syringe pumps used in High pressure liquid chromatography are most suitable for which of the following columns?
a) Capillary columns
b) Guard columns
c) Short-fast columns
d) Small bore column

4. column length in HPLC is ?

a.15-30 cm

b.20-25 cm

c.35-45 cm

d.5-30 cm

5. which is most  popular injector ?

a.SEPTUM INJECTOR

b.Rheodyne Injector

c. STOP FOLW

d. none of this

6. which detector are not used in HPLC ?

a. REFRACTIVE INDEX DETECTOR

b.FLOURIMETRIC DETECTOR

c.AMPEROMETRIC DETECTOR

d. MASS DETECTOR

7. Column diameter in HPLC is

a.5-9 mm

b.1-3 mm

c.2-50mm

d.10-50mm

ANSWER KEY

1.d

2.d

3.d

4.a

5.b

6.d

7.c

REFERENCE :-

TEXT BOOK OF PHARMACEUTICAL ANALYSIS THIRD EDITION DR.S.RAVI SANKAR.(18.1 TO 18.15)

TEXT BOOK OF DAVID G. WATSON FOURTH EDITION (296 TO 344)