This document discusses various components of HPLC instrumentation including mobile phase reservoirs, pumps, sample introduction systems, columns, and detectors. It describes the basic components of an HPLC system including solvent bottles, pumps, autosamplers, columns, and detectors. It discusses different types of pumps including reciprocating pumps and syringe pumps. It also covers topics like column dimensions, fittings, packing materials, and sample introduction methods like manual injection and autosamplers.

1. HPLC INSTRUMENATION &
TECHNIQUES
SRAVAN KUMAR BUSSA

2. HPLC Instrumentation and Techniques
Instrumentation
Mobile phase reservoirs
Construction pump material
Pumps
Recriprocating
Syringe
Manual Injection
Automated Injection
Packings
Measure column
performance
Column care and use
Solvent
Delivery
Sample
Introduction
Column
Packings
and harware
Detectors
UV-Vis
Fluorescence
Refractive Index
Conductivity
Volatametry/Amperometry

3. Solvent supply bottle
Chromatographic
column
Sample
Pump
Autosampler Detector

4. SOLVENT DELIVERY SYSTEM IN HPLC
A. Mobile Phase Resorvoir. Figure to your right
shows a generic version of mobile phase resorvoir.
The reservoir that holds the mobile phase is often no
more than a glass bottle. Often, the reagent bottle
that holds our HPLC solvent can be used as a
reservoir. Solvent is delivered from the reservoir to
the pump by means of Teflon tubing--called the "inlet
line" to the pump
Q. What is the purpose of a sinker frit or push filter?
--Push filter (10 mm) prevent any dust or particulate matter to enter the pump.
--This is because particulate matter can interfere with pumping action, damage
valves and seals. In addition, it may also damage the column (by collecting at the
top of the column).
Bottle capped on
outside with a
UV- absorbing
plastic
He degasser
Q. Why He degassing of the liquid mobile phase is required?
DEGASSING: the practice of removing air from the mobile phase; degassing
can be achieved by bubbling He gas into the M.P
--He degassing removes dissolve oxygen from the M.P
--The presence of oxygen in mobile phase causes bubble formation resulting in
air in the flow system and pump pressure will change causing spike in the
chromatogram (due to air bubble formation in the detector cell)

5. Requirements for a solvent reservoir are simple:
*The reservoir and its attachment to the pump should be made of materials that
will not contaminate the mobile phase: Teflon, glass, or stainless steel.
*The vessel should have some sort of cap to prevent particulate matter from
contaminating the mobile phase. If you are using a solvent bottle as a reservoir,
the top of the bottle can be wrapped in aluminum foil to keep dust out or the
bottle cap can be drilled to allow inserting the inlet line through the cap.
*Don't close the bottle too tightly or removal of mobile phase by the pump will
create a vacuum. This prevents mobile phase from flowing the pump, creating a
"vapor lock" within the pump.
Instead of He degassing vaccum degassing
method can also be used

6. B. HPLC PUMP
- The function of a HPLC pump is to pass M.P through the column at a
controlled flow rate
Pump hardware are shown in Figure to the right
Important parts are:
Hardware
a. Check valves draws the liquid in and
out of the solvent chamber
Diaphram Coil
b. Pulse dampener prevent pulses
in the baseline created because
of pump strokes

7. (c) Pressure Transducer
Monitor the pressure of the
liquid flow
(d) Flow Controller
Controls the liquid flow rate

8. Construction Material of Solvent Delivery System
Metallic Non-metallic
Steel Titanium PEEK Teflon Ceramic
A variety of options are available depending on
the needs
--Most commonly used pumps are resistant to
chemical attack and leaching off the solvent
(except HCl)
--Low cost, ease of machining and greater
operating pressure limit (6000 psi)
is an advantage with stainless steel pump
--Titanium pump has greater degree of inertness
Useful for biomolecule analysis
---High cost and tendency to act as ion-exchanger
himits its acceptance
--PEEK(polyetherethylketone) and Teflon pumps are
useful when corrosive solutions such as HCl is used
as M.P. Also used for separation of metals

9. Microbore column are used with diameter upto 2 mm (i.d)
Narrow column diameter and small size of the packing
material requires low flow rate. Reliable separation (<10 mL/min)
is difficult unless syringe pump are used.
HPLC Pump Classification based on the Flow Rate
Microbore Standard bore Preparative
(1-250 mL/min) (100mL/min—10 mL/min) (>10 mL/min)
-Standard bore is most commonly used for pumping system for both
analytical and semi-preparative work (2-12 mm i.d) columns
-Preparative column. High flow rate requires special design pump. Upper range
for flow rate is usually around 50 mL/min

10. The elution conditions contain the elution profile (isocratic, linear gradient,
Column diameter determines to what degree samples are diluted by the system.
Smaller diameters result in less dilution and thus higher sensitivity.
Specifically, narrow bore columns are 4 to 6 times more sensitive
(b) using the injection volume required for a standard bore column (a). © ESA Inc.

11. Pump classification based on the mechanism of eluent displacement
Solvent Delivery System
Recriprocating piston pump
Syringe pump
Single Head Multi Head
Syringe Pump
--Syringe pump provides pulseless solvent delivery
(capillary LC) or microbore HPLC hooked to MS.
--Larger barrel syringe (10-50 mL) with plunger connected to a digital stepping
Motor. As plunger moves forward, it drives the eluent through chromatography
with a pulseless flow
--- A design of a simple syringe is shown above. What do you think is the major
problem with the use of syringe pump? (Hint no separate reservoir is used for
M.P)
Run time in syringe pump is limited by the volume of the syringe—no flow
occurs when the syringe is empty

12. Reciprocating Pumps
Q. Why these pumps are called reciprocating?
--The piston is driven in and out of the solvent chamber by a gear
--Forward Stroke ------ inlet check valve closes
and outlet check valve opens and M.P pump to the
flowing system
---Backward Stroke--outlet check valve closes and
inlet check valves is refilled
Piston
Inlet check valve
Outlet check valve
Single piston pump
--Used in HPLC to deliver
solutions (e.g., post
column derivatization)
where pump pulsation
is less critical

14. Another approach is to minimize the pulses associated with reciprocating
pump. Several approaches can be used
•Use of in-line pulse dampeners
•Adjustable spring-loaded dampeners
•Bellows dampeners
Use of two or more pumps, very effective but it is an expensive approach
Advantage/Disadvantage of single piston pump
--Advantage: cheaper than the dual piston pump.
--Disadvantage: rarely used to deliver mobile phase due to the fact that they
produce pulse flow

15. Double Piston Pump in HPLC
As the name indicates it consists of
two pump heads and four check
valves
--The two piston are driven such that
their directions are opposite
--Non-circulating gears are continously
lubricating the oil chamber which drives
the piston so that the forward motion in
expelling the solvent takes more time than
the reverse motion in drawing the solvent
Why do you think the dual piston pump
are better than single piston pump?
Because dual piston pump minimizes pulsations, it uses two
pulgers/two pump heads, which expel solvents alternately
First head-- outlet valve opens/inlet valve closes
Second head- inlet valve opens/outlet valve closes
!st plunger
drawing solvent
in 2nd plunger
drawing solvent
out

16. Left and Right chamber
delivery
•While pump pulsation has no effect on separation (resolution),detectability
at trace levels is often limited by baseline noise from pump pulsation

17. Your company has decided to manufacture HPLC pump and they are trying
to decide what materials to build it with. To a large extent this decision is
influenced by the performance criteria that are set by the intended function of
the unit. What do you think would be the five most important criteria?
1. Materials should be able to withstand pressure in excess of 7000 psi
2. Inertness---non reactive to sample and solvents
3. Precise flow delivery
4. Reliability---leak free connections and long service life
5. Low raw material cost and machining cost

18. Sample Introduction in HPLC
---The function of injector in HPLC is to introduce the sample in the flowing
solvent so that it may be carried to the column
What considerations are necessary in injector design?
a) Minimize dispersion or band-broadening (no extra column tubing should be
used)
b) Allow sample to be introduced without disturbing the solvent flow(otherwise
we get baseline disturbances in RI and conductivity detector
--Injector may be operated either manually or automatically
Manual has two different modes for injecting samples
(i) septum mode injection (b) valve injection
Septum injection
--is the simplest form of injector in the septum device (similar to one used in GC)
This allows injecting sample into pressurized solvent stream using a self-elastic
septum with a microsyringe
Greatest Drawback: limited to max operating pressure of 1500 psi. Therefore, not
widely used in HPLC anymore.
More useful in low pressure HPLC applications (e.g., protein separations)

19. Valve-Type Injection in HPLC
--widely used in HPLC. Allows reproducible introduction of
sample into the pressurized M.P without interruption of
flow even at high temperature
Six-port Valve Type Injector (Used in the laboratory exercise)
--One of the simplest and most common valve injector in HPLC
is the “six-port valco (or rheodyne model) injector as shown below:
Inject valve has two position: (a) Load (b) Inject
Valve in the load position. Before injecting the
sample the valve is turned to the load position.
What is the flow direction in load position?
In load position M.P by-passes through the sample
Loop and flows straight from the pump through 2
and 3 into the column
Why analyte is loaded when the valve is in the load
position? This allows to get the sample ready for
the next injection. It does not interrupt the flow and
will only fill the loop with the new sample.
When the valve is switch to inject position the
sample is introduced into the flowing stream ----
M.P passess through sample loop from the pump through
Port 2 , 1, 4 and 3 into the column
2 3
4
1
5
6
3
1
2
4
5
6

20. What precautions should be taken before the sample is injected?
Loop should be flushed with sample using twice the loop volume--flushing the
loop with sample prevents the sample carry over
What care you must take during HPLC injection using 6-port valve?
--Valve should be rapidly switched from load to inject position. If the rotor is left
Halfway flow to the column is stopped and baseline will be disturbed
Automated Injection
Sample is introduced from a vial
held in a sample carousel using
a syringe assembly.
Valves are automatically
Actuated used to wash syringe
needle and syringe assembly
Benefits of Autoinjector
Wide variability in injection
Volume (0-1 mL-2 mL)
Precision is better than or
Equal to manual injection
High sample throughput and less labor intensive

21. Benefits of Autoinjector
--Wide variability in injection volume (01 mL-2mL)
--Precision is better or equal to manual injection
--High sample throughput and less labor intensive

22. HPLC Columns
Packing Material Container
Length Internal Material
Diameter
Nature Particle
of the particle size
Container Material
--Columns most commonly used at the moment are made with 316 grade stainless
Steel (a Cr-Ni-Mo steel, relatively inert to chemical corrosion)
Dimensions (Length and Diameter)
--Common dimensions are:
4.6 mm (046 cm) internal diameter, 25 cm length, 6.35 mm
(0.25 inch) external diameter
Range of Length and Diameter
Length: 5-30 cm; Internal Diameter: 2-4.6 mm
Act as filter
prevents particulate
to enter the column

23. Fittings from Different Manufactureres
--The following figure shows the appearance of different male nuts and ferrules
obtained from different manufacturer
--The dimension “X” is the distance
between the end of the ferrule and the
tubing which is different for different
manufacturer
What are the consequences of having too long or too short of dimension “X”
Too Long X: Ferrule does not seat properly and fitting leaks
Too Shor X: Get undesirable dead volume in fittings
“Rule of Thumb: Tubing and Fittings has to be Matched”

24. Zero Dead Volume (ZDV)
There is really no way of
knowing whether the tubing
is butting together or not, or
if the tubing is butting in the
center of the union. [Figure
to the right] demonstrates
this difficulty.
A true zero dead-volume union, or ZDV, would
connect two pieces of tubing without any dead-
volume, or mixing chamber, between the tubing.
The tubing would butt perfectly together as shown
to the right. A connection of this type is difficult,
If not impossible to achieve.

25. High Pressure Plastic Fittings
-- A welcome development has been the advent of reliable high pressure plastic fittings
and tubings
---Most chromatography suppliers now offer a range of plastic fittings that can be
tightened to be leak free, by hand
-- Some of the commonly used ones are PEEK (polyethylethyl ketone) as shown below:
These fittings are resistance to
organic and inorganic liquids
except nitric acid, sulfuric acid
and THF

26. The problems posed by a true zero dead-volume union are solved by the
kind of union most manufacturers term a ZDV. Although the two pieces of
tubing do not butt directly together, these unions come closest to providing
a reliable zero dead-volume connection
Zero Dead Volume (ZDV) continued
The union body is designed to have a
thin web of material in the center
A small thru-hole (matching the ID of the
intended tubing) is drilled through the center
of the web allowing fluid to flow from the tubing
on one side of the web into the tubing on the
other side.
Even though this is not a true zero dead-volume
union, it is a practical solution and a generally
accepted alternative to a true zero dead-volume union

27. Types of Packing Material Used in HPLC
Based on the particle size there are three types
(a) Pellicular Particles (20-40 mm) (Avg= 30 mm)
--have solid inner core with a thin outer surface
of S.P
--This thin outer surface (1-2 um) is very porous.
Hence, aka. superficially porous particle
--Used in ion-exchange chromatography
--Give high N then porouse particles of same size
(b) Micro particles (3-10 mm)
--Most popular S.P in modern HPLC
--Role of pore size is critical. Particles with small pores
have high surface area, have greater sample capacity
3-10 mm
(c)Totally porous particles (20-40 mm)
--are very porous, more porus than pellicular, may lead
to irreversible adsorption.
--seldom used as packing material for HPLC due to low N
values
--Provide ease for dry packing and lower cost (used as
guard column and extraction column
•Currently microparticles are most common in HPLC
and are packed with 3-10 mm diameter particles

28. Ethylene
bridges
Modifying silica particles to include an ethylene
bridge gives the particles exceptional chemical
stability. (Suitable for UHPLC)
High Strength Silica (HSS) and Ethylene Bridged
Hybrid (BEH) materials. Particles of both materials,
which were designed for the UPLC systems, have
diameters in the 1.7–1.8-µm range and feature pores
with diameters of either 10 (HSS) or 13 (BEH) nm
The hybrid organic-inorganic particles are prepared
via copolymerization of TEOS and bis(triethoxysilyl)ethane,
are designed to retain silica’s mechanical strength while
overcoming pure silica’s tendency to undergo column-
damaging (i.e., hydrolysis in alkaline environments ,
greater than pH 8).
BEH’s covalently bonded Si–C–C–Si units render the hybrid material
chemically stable up to a pH of 12, which is an ideal condition for
analyzing some pharmaceutical agents and other types of compounds.
Taken from Chem. Eng
News. April-2008, pp 17-23
Packing for Ultra High-Pressure HPLC (U-HPLC)

29. 1-µm particles pack more irregularly in
smaller diameter LC capillary columns than
in larger (50-µm-diameter column, top; 10
µm, bottom). Yet orderly packing may not
yield the best separations.
The biggest problem, is the tendency of tiny
particles to agglomerate. Use of various
solvents and ultrasonication to prepare
well-dispersed slurries and then pack
capillary columns at high pressure.
Packings for UPHPLC

30. Advantage of Using Short Columns with small particle size

31. Effect of Column Internal Diameter on Sensitivity
Advantages of using small diameter column
--Better sensitivity and higher S/N obtained using a column with small i.d
---Flow rate is slow. This means less solvent consumption, less disposal cost
What are the disadvantages of using small i.d columns?
--Less loading capacity

32. What causes extra column band-broadening?

33. Nature of the Particles
a, k’ Rs
Silica based
Resin (polymer based)
Silica Based Particles
Depending on how the silica is treated prior to packing in a column the silanol group (Si—
OH gps) present on the silica surface can adapt a number of configurations as shown above
Si Si
O
Siloxane
When silica is heated at temp > 800 0C, it forms siloxane (devoid of any silanol gps)
When silica is heated in temperature range of 200-3000C it allows formation of:
Free silanol where the surface silicon atom has three bonds into the bulk structure
and the fourth to the OH group
Vicinal or associated silanols where two isolated silanol groups attached to two
different silicon atoms are bridged by H-bond.
Geminal silanols consists of two hydroxyl groups attached to one silicon atom.
Geminal silanols are close enough to have H-bond, whereas the free silanol
are too far separated. The silanol gps with lower pKa values are believed to be
isolated gps with no H-bond with its neighbor

34. Advantages of Silica Packing in HPLC
a) Can withstand high pressure generated when 10-30 cm columns with
3-10 mm particles are used
b) Silica is abundant, inexpensive and available in a variety of shapes, sizes
and degree of porosity
c) Functional group can be readily bonded to silanol and the chemistry of
bonding reactions are well understood
Limitations of Silica Packing in HPLC
--Major limitation is its instability at high and low pH (i.e. above pH 8 or below
pH2)

35. Resin or Polymer-Based Packings in HPLC
Polymethylmethacrylate
based polymers
Predominantly used in size exlusion chromatography or ion-exanchaged HPLC
However, resin based reveresed phase columns are also commercially available
What is the advantage of resin-based packing over silica based packing?
---Stability over a wide pH range (1-13)
Disdvantages are: (a) Unstable in presence of organic solvents
(b) Polymers swell in the organic solvent

36. Column Care and Use
a)The manufacturer’s recommendation regarding M.P pH, flowrates and
organic modifier content, temperature as well as maximum operating pressure,
etc.; should be followed.
General Guidelines
b) Use of HPLC grade water and HPLC grade organic solvents, analytical
reagent grade chemicals (buffers) must be used to prepare solvents and
standards.
c) M.P should always be filtered through an appropriate solvent compatible
filter (e.g., 0.45 or 0.2 um filter) and vacuum degassed before use.
d) M.P flow rate should be altered in small increments (0.2 mL/min- 1mL/min
to avoid column back pressure particularly for softer S.P packings
e) When not in use store column according to manufacturer’s recommendation
Silica based columns

37. Use of Guard Column in HPLC
Guard column is used at two points in the HPLC flow system
(A)Between pump and the injector ------- Remove particulates and contaminants
from mobile phase
Pump
Guard
column
Injector
(B) Between Injector and analytical column -- Remove contaminants
From sample before it enters the column
Injector
Guard
column
Analytical
column
--Guard columns are very short columns (0.5-3.0cm), which are
packed with the same S.P as the analytical column except that the
particles are bgenerally large to minimize pressure drops
Guard columns are used primarily to protect the analytical column from strongly adsorbed
sample and matrix. Over time the packing dissolves, resulting in increased backpressure,
column voids and broad peaks.
Under very harsh mobile phase conditions (above pH 7 and 40°C, and with a buffer salt
concen-tration > 50 mM), a silica saturator column should also be used. As the mobile phase
passes through this guard column, it dissolves enough silica to saturate (or nearly so) the
mobile phase. Then when the mobile phase reaches the analytical column, no additional
packing can dissolve. Therefore, column life is extended at high pH. In addition, some HPLC
columns are designed for operation at higher pH.

38. Good, economical guard column performance can be obtained with a cartridge type system, as shown in
above Figure. The hardware is reused, and only the guard cartridge is replaced. The new, more efficient
design minimizes dead volume so that one holder can be used efficiently with analytical (4.6, 3.0 mm id)
and narrow-bore (2.1 mm id) columns. The simple design assembles quickly and easily, making it more
likely that a guard column will be used and analytical column lifetime extended.
When to replace guard columns?
A guard column is most effective if it is replaced before the chromatography of the
analytical column deteriorates. This can be hard to determine, so many chromatographers
select a specific time interval (every week) or sample interval (every 100 samples) for
changing the guard column. This is probably the most effective way to minimize
contamination of the analytical column. Alternatively, peak shape deterioration and
backpressure increases (no more than 10% - see Section 2) can serve as reminders

39. Column Validation
History
First commercial HPLC column demonstrated poor column-to-column
reproducibility. In addition, there were irreproducible retention time and peak
area (due to variation injection)-due to poor injector and column design.
As the instrument and column technology improved, analysis of non-polar
compounds became routine. Howver,variation in retention time and peak shape
continued to be problematic in pharmaceutical industry where analysis of
polar acidic and basic compounds are required routinely.
(Taken from HPLC column Technical Bulletin (From the web)

40. shows test chromatograms from two reputable column manufacturers. Test chromatograms are run for
each individual column and are intended to provide information regarding that column’s performance,
but give no information about column-to-column or batch-to-batch reproducibility.
This test, using neutral test probes, typically measures column efficiency (theoretical plates) and includes
some measure of peak asymmetry. Tr are given, but no specification for relative retention.
Chromatogram on the right, however, provides some additional information regarding true column
performance as it uses a more varied mixture of test probes.
Neither “test” chromatogram provides any information about the long-term reproducibility of the
column in question. This information is only available with separate validation data.

41. Figure to the right shows a single validation
Test. The test is somewhat effective as
acidic, neutral and basic solutes are used
to probe the column performance, but
carried out using one set of M.P conditions
There is specification given for retention
Relative to acenapthene, but nothing is
Mention about retention of acenapthene
(a hydrophobic compound).
--Basic compounds USP tailing factor are
specified but not for column efficiency
Very little information about consistency
Between batches

42. What is Batch Validation in HPLC Column Production?
The variation between different “batches” or “lots” of packing
material can best be eliminated by strict control of the
manufacturing process and also extensive testing
using tight specifications for each batch. Minimum criteria are
established for retention, selectivity (k´), efficiency (N), asymmetry
and tailing factor. Tight specifications and a greater variety of test
probes (multiple test validation) assures greater reproducibility in
the final product.

43. Test 1 uses five neutral probes in an
unbuffered mobile phase. Unlike the
previous example, hydrophobic
retention is specified for biphenyl and
phenanthrene, as are efficiency (N)
asymmetry and USP tailing factor.
Although neutral molecules do not have
the problems associated with peak
shape as do basic and polar
compounds, hydrophobic retention
is very important in environmental
analyses. This test is very effective
in detecting selectivity differences
between batches due to silane bonding.

44. Test 2 uses a mixture of acidic probes.
Note that mobile phase conditions are
changed to suppress the ionization of
the acids. This mobile phase is typical of
this type of analysis.
The retention of ethyl paraben is specified and
the relative retention of acids is reported. Peak
asymmetry and USP tailing factors are also
specified. There is no measurement of
efficiency, N, since this was specified in test 1.
Hydrophilic retention is the critical parameter
measured in this test. Unlike hydrophobic
retention which relies more on bonded phase
characteristics, the retention of acids depends
greatly on interaction with the silica surface.
Variations in silica activity between batches
will become apparent when using this test.

45. Test 3 uses a mixture of basic drugs using
phosphate buffer and organic solvents at
pH 6.0. A low pH M.P is used so that the
silanol groups. On the silica surface are
protonated. This will give the best peak
shape for basic compounds.
Amitryptline has become a benchmark
compound for defining peak tailing.
Non-ideal conditions are chosen to better
detect variations between batches. Even
under these non-ideal conditions once can
See near perfect peak symmetry.
Hydrophobic retention is again specified
and selectivity values for four basic
compounds are specified based on this
value. The results obtained from these
basic compounds under less than ideal
mobile phase conditions are invaluable in
assuring the robustness of methods
involving basic drugs.