PLC1002

PLC1002
Introduction to
High Performance Liquid Chromatography
(HPLC)
Pharmaceutical Liquid Chromatography – PLC1002
Telephone: 416-502-2277
e-mail: info@aaps.ca
Course Objectives:
This course aims to give students the basic understanding in order to perform pharmaceutical HPLC
analysis as per the United States Pharmacopoeia. Common HPLC testing procedures, techniques,
theory, equipment types, and SOP’s are emphasized and accurately covered through step-by-step
instructions and hands-on experiments.
Using interactive exercises, students are guided through compendial HPLC fundamentals. This
course bridges the gap between the theoretical and the practicality of the pharmaceutical workplace.
Required Text and Readings:
AAPS Hand-outs and material
Mark Allocation
Quizzes 10 %
Experimental and Documentation 40 %
Final Exam 50 %
Class Participation
Due to the practical and participative nature of the learning environment, punctual and regular
attendance is strongly encouraged. Absenteeism will almost guarantee your inability to achieve
satisfactory grades. Some of the progress tests may not be announced in advance and details of
assignment requirements may be explained in class. There is not formal provision for make up tests to
replace tests you miss. If there are any extenuating circumstances concerning a student's absence, the
instructor should be notified as soon as possible. Students are responsible for "catching up" on any info
from missed classes regardless of the reason for absence.
Dishonesty and Plagiarism
Each student should be aware of the College’s policy regarding this subject. AAPS’s Academic policy
will be strictly enforced.
Academy of Applied Pharmaceutical Sciences (AAPS) Page 2

PLC1002 - Manual - Rev 06.0
Grading Policy
A+ = 90
A = 85-89
A- = 80-84
B+ = 75-79
B = 70-74
C+ = 65-69
C = 60-64
F = 59
Discrimination/Harassment
All students and employees have the right to study and work in an environment that is free from
discrimination and/or harassment. Language or activities that defeat this objective violate the College
Policy on Discrimination/Harassment and shall not be tolerated. Information and assistance are
available from the admission office.
Prerequisites
Previous analytical laboratory work experience and / or PLT 1005

Contents
INTRODUCTION TO THE HPLC (Session One) .......................................................................7

1. The Role of HPLC in the Pharmaceutical Industry ........................................................................... 7
1.1 Quality Control (QC) Testing ................................................................................................. 7
1.2 Research and Development (R&D) ........................................................................................ 9
2. Chromatography .............................................................................................................................. 10
2.1 History ..................................................................................................................................... 10
2.2 Liquid Chromatography (LC) in Analytical Analysis ........................................................ 10
3. High Performance Liquid Chromatography (HPLC) ...................................................................... 11
3.1 Development ........................................................................................................................... 11
3.2 Instrumentation ...................................................................................................................... 12
3.3 Pump........................................................................................................................................ 12
3.3.1 Constant Pressure ......................................................................................................................... 13
3.3.2 Constant Flow ............................................................................................................................... 13
3.3.3 Isocratic ......................................................................................................................................... 15
3.3.4 Gradient......................................................................................................................................... 15
3.4 Sample Introduction .............................................................................................................. 17
3.4.1 Rheodyne Type Valve .................................................................................................................... 17
3.4.2 Automatic Samplers ....................................................................................................................... 18
3.4.3 Auto-sampler ................................................................................................................................. 19
3.5 Analytical Columns ................................................................................................................ 20
3.6 Normal Phase .......................................................................................................................... 21
3.7 Bonded Phases ........................................................................................................................ 22
3.7.1 Reversed Phase.............................................................................................................................. 22
3.7.2 Other Bonded Phases .................................................................................................................... 23
3.8 Ion Exchange .......................................................................................................................... 25
3.9 Size Exclusion ......................................................................................................................... 26
4. Detectors .......................................................................................................................................... 27
4.1 Flow-Through Cell ................................................................................................................. 27
4.2 Refractive Index ..................................................................................................................... 28
4.3 UV Detector ............................................................................................................................ 28
4.3.1 Fixed Wavelength .......................................................................................................................... 29
4.3.2 Variable Wavelength ..................................................................................................................... 30
4.3.3 Diode Array/Photo Diode Array (PDA) ........................................................................................ 31
4.4 Fluorescence ............................................................................................................................ 32
4.5 Conductivity............................................................................................................................ 33
4.6 Electrochemical ...................................................................................................................... 34
5. Data Collection and Processing ....................................................................................................... 35
5.1 Computers / Software ............................................................................................................ 35
5.1.1 Network / LIMS Data Storage ....................................................................................................... 36
6. Factors Affecting Performance ....................................................................................................... 36
6.1 Mobile Phase ........................................................................................................................... 36
6.2 Stationary Phases ................................................................................................................... 37
7. GMP and GLP for HPLC Analysis ................................................................................................. 39
8. Session One Assignment ................................................................................................................. 42
KEY CONCEPTS IN HPLC (Session 2) .....................................................................................43
9. The Chromatogram and Performance Measurement....................................................................... 43
9.1 The Chromatogram................................................................................................................ 43
9.2 Retention Time ....................................................................................................................... 43
10. Performance Measurements ............................................................................................................ 45
10.1 Capacity................................................................................................................................... 45
10.2 Selectivity ................................................................................................................................ 46
10.3 Efficiency ................................................................................................................................. 47
10.4 Resolution ................................................................................................................................ 49
10.5 Peak Tailing and Symmetry .................................................................................................. 52
11. System Suitability............................................................................................................................ 53
11.1 Performance Measurements.................................................................................................. 53
11.2 System Reproducibility .......................................................................................................... 53
12. Session Two Assignment ................................................................................................................ 54
Equation for Raw Material Assay .............................................................................................................. 57
Session Two Experiment – I.D. via In-House Method ................................................................................ 58
HPLC Columns (Session Three)...................................................................................................59
13. Role and Effects of the Stationary Phase ........................................................................................ 60
13.1 Column Packing Materials .................................................................................................... 61
13.2 Pore Size .................................................................................................................................. 63
13.3 Dimensions .............................................................................................................................. 64
15. Column Protection........................................................................................................................... 65
15.1.1 Upon Receipt of the Column ..................................................................................................... 66
15.1.2 Mobile Phase Considerations: .................................................................................................. 66
15.1.3 Backpressure and Flow Rates:.................................................................................................. 67
15.2 Column Storage ...................................................................................................................... 68
15.3 Column Cleaning Procedures ............................................................................................... 69
16. Session Three Assignment .............................................................................................................. 70
17. HPLC Known Impurities – External Standard Calculation ............................................................ 71
Session Three Experiment – System Suitability via In-House Method....................................................... 72
Mobile Phase (Session Four) .........................................................................................................73
17.1 Role of the Mobile Phase ....................................................................................................... 73
17.2 Purpose – What to Make ....................................................................................................... 73
17.3 Composition ............................................................................................................................ 74
17.4 Buffers and pH – Analyte Effects ......................................................................................... 74
17.5 Organic Modifiers .................................................................................................................. 75
17.6 Mobile Phase Preparation ..................................................................................................... 75
17.6.1 Choice of Solvents ..................................................................................................................... 76
17.6.2 Buffer Preparation .................................................................................................................... 76
17.6.3 Filtration ................................................................................................................................... 77
17.6.4 Degassing .................................................................................................................................. 79
17.7 Sample Preparation................................................................................................................ 79
18. Good Practices for HPLC Analysis ................................................................................................. 81
18.1 Glassware Labelling ............................................................................................................... 81
18.2 Labelling of Chromatograms ................................................................................................ 81
18.3 Documentation ........................................................................................................................ 81
19. Session Four Assignment ................................................................................................................ 83
Session Four Experiment – In-House Raw Material Assay ........................................................................ 84
Session Five – Assay of a Finished Product .................................................................................85
Session Five Experiment ............................................................................................................................. 85
Acetaminophen Tablets ........................................................................................................ 86
Attachments....................................................................................................................................88
19.1 Membrane Selection Guide ................................................................................................... 88
19.2 Syringe Filter Compatability Guide ..................................................................................... 89
19.3 <621> CHROMATOGRAPHY............................................................................................. 91
19.4 Chromatographic Columns ................................................................................................. 105
19.5 Example USP Monographs ................................................................................................. 111
Fluvastatin Sodium .................................................................................................................................... 111
Ampicillin .................................................................................................................................................. 115
Ampicillin and Sulbactam for Injection ..................................................................................................... 121
Cyclosporine .............................................................................................................................................. 124
Cyclosporine Capsules .............................................................................................................................. 126
19.6 Solvent Polarity .................................................................................................................... 129
19.7 Analyte Functional Group Polarity .................................................................................... 130
19.8 Glossary of HPLC Terms .................................................................................................... 131
Millennium Software Intro Guide .............................................................................................. 152

INTRODUCTION TO THE HPLC (Session One)
1. The Role of HPLC in the Pharmaceutical Industry
High Performance Liquid Chromatography (HPLC) is one of the primary analytical

techniques used today in the Pharmaceutical Industry. The versatility of this procedure
can be seen in several ways:
Sensitive - Can determine accurate and representative amounts of the particular

substance.
Selective - Can be designed to analyze for one or more particular components in a

matrix, without interference.
Stability Indicating - Can determine the amount of a particular component without

interference by its related compounds or degradation products.
Non-selective – can be designed to analyze many different components

simultaneously including the components of interest.
Automation – can be designed to run unattended for long periods of time without
interruption or operator intervention.
Reproducible – analyses can be replicated on different systems, by different operators

or on the same system with the same operator.
1.1 Quality Control (QC) Testing
The Quality Control department assures the quality of pharmaceutical

components, and the quality of finished goods going to the consumer. Routine
testing is performed using validated analytical methods. Testing methods are set
in stone and are never deviated from. Quality Control testing includes:
• Raw Material Testing
• In-process Testing
• Finished Product Testing
• Shelf-life Stability Testing
• Cleaning Validation and
• Package Component Testing

Raw Material Testing
Raw material testing consists of verifying components before they are released
to production. This includes verification of identity, purity and potency.
Finished Product Testing
Finished product testing consists of verifying drugs that are ready to be shipped
to the consumer. Included in Finished Product testing is verification of potency,
bioavailability, and purity.
Shelf-Life Stability
Quality Control Stability is often called “shelf-life stability” or “Follow-Up Stability”

or “Marketed Products Stability”
Quality Control Stability programs verify the shelf life of finished pharmaceutical
products currently residing with pharmacies or consumers. They are used to
verify the continued potency, bioavailability, and purity of a pharmaceutical
product until it reaches its expiry date.
In-Process Testing
In-Process testing is testing that is performed on a pharmaceutical material or

mix of materials before it is finished all production stages. An example of an in-
process test would be an assay to determine uniformity of blend before
compression.
Packaging Component Testing
Labels and components, which come in direct contact with the finished
pharmaceutical product, are checked for suitability and quality.

Cleaning Validation
Manufacturing rooms and equipment must be clean before use. Generally,

production equipment is used for multiple products. Cross contamination can
occur, so equipment and rooms are cleaned, then swabbed, and the Quality
Control laboratory verifies that equipment is suitable before it is released for use.
1.2 Research and Development (R&D)
The Research and Development (R&D) department develops methods, verifies

that methods work reliably and consistently over time (validation). This
department also formulates new drug products, performs stability testing on
potential new drug products and verifies that production is consistent and reliable
in the manufacturing of new drug products (Process Validation).
Formulation
A finished pharmaceutical product contains Active/s and Excipients. The ratio of

Excipients and actives is the formulation. The R&D (product development, PD)
laboratory determines what formulation gives acceptable stability, release
characteristics.
Accelerated Stability (R&D Stability)
Accelerated Stability Studies determine the suitability of new product

formulations over time while under environmental stress.
Analytical Method Development & Validation
Quality Control methods must be created and validated before use. Analytical
Method Development is the process of creation. Analytical Method Validation is
the process of verifying that methods work reliably and consistently over time.
Process Validation
Process validation in the R&D laboratory verifies that production can perform the
manufacture of a specific drug product reliably and consistently over time.

2. Chromatography
Chromatography is defined as “a process in which a chemical mixture carried by a liquid

or gas is separated into components as a result of differential distribution of the solutes
as they flow around or over a stationary liquid or solid phase”.
The process is based on the varying affinities of the components of the chemical
mixture (in the case of liquid chromatography) dissolved in a solvent system (mobile
phase) to the solid phase. As the solvent percolates through the solid material, the rate
at which the components of the mixture pass around or through the solid varies, thus
leading to a separation of the individual components. Components with a higher affinity
to the solid phase move at a slower rate than those with a lower affinity. Continual
flushing of the solvent will eventually lead to each component being flushed out of the
solid phase.
By passing the eluent through a detection device, which can detect the individual
components, the relative amounts of each component can be measured.
2.1 History
The first published works in chromatography were by a Russian born botanist

named Michael Tswett. In 1906 he presented a paper in which he described the
separation and isolation green and yellow pigments of chloroplast by adsorption
chromatography. He used a column packed with a solid absorbent material
(sucrose powder) into a glass column and used petroleum ether to elute the
pigments from the column. Since this early beginning, chromatography has
blossomed into the premier
2.2 Liquid Chromatography (LC) in Analytical Analysis
Liquid Chromatography has been used in analytical analysis since its beginnings
in the early 1900’s. By the 1970’s, it was widely used but mostly as an
identification and purification tool. Thin Layer Chromatography is still used today
in the pharmaceutical industry as a means for identification and for degradation
analysis. Liquid chromatography was not used extensively as a quantitative tool,
due to the inability to regulate liquid flow. Initial gravity feed columns required
large volumes of mobile phase and took a long time to achieve the desired
separations. Pressure was used to increase mobile phase flow rate and thus
decrease analysis time, however, it was difficult to maintain pressure and flow at
a constant rate, thus reproducibility was an issue.
Solid phases initially were silica or alumina gel and were very polar, thus the
procedure was limited to relatively non-polar materials and required non-polar
organic solvents.
The most popular of these are Hexane, Ethyl acetate, Acetone, Chloroform and
Dichloromethane. Unfortunately, most pharmaceutical preparations are water-
soluble or are derivatized to be initially water soluble to allow assimilation in the
body. Thus, to use this type of chromatography required neutralization with acid
or caustic and then extensive extractions into hydrophobic solvents to allow for
chromatographic separation and analysis. This process allows for greater
chances of experimental error and is thus not as reliable as a procedure that
could be developed to analyze the component directly.
Since silica and alumina are fairly reactive, derivatization of the free silanol
groups could be performed to create stable, non-ionizable substrates. By
derivatizing the silica gel with alkyl halides, the solid matrix could then be
rendered hydrophobic, resulting in a solid matrix, which would not be highly
adsorbent to hydrophilic components.
3. High Performance Liquid Chromatography (HPLC)
3.1 Development
HPLC is a development of column chromatography. It was long realized that

using particles with a small particle size (3, 5, 10um) with a very narrow size
distribution would greatly improve resolution, especially if the flow rate and
column dimensions could be adjusted to minimize band broadening. Pumps were
developed that could handle both the chemicals and pressures required.
Traditional column chromatography (nonpolar solvent and polar surface) is
described as "normal" and, as well as silica, there are columns with amino, diol,

and cyano groups. If, the system uses a polar solvent (water, methanol,
acetonitrile, etc.) and a non-polar surface it is described as "reverse-phase".
Common surface treatments of silica include octadecylsilane (a.k.a. ODS or

C18), and it has been the development of “Reverse-phase” HPLC, that has
experienced explosive growth. Reverse-phase HPLC is now the method of
choice for larger non-volatile molecules including hydrophilic and ionic
compounds.
3.2 Instrumentation
Figure 1, shows a typical component HPLC system. The main functional parts are the
pump, injector, column, detector and data station/processor.
Figure 1 – Basic HPLC system
3.3 Pump
The pump system is the heart of any HPLC system. The pump must supply a
constant and reliable flow of mobile phase at all times to ensure the resulting
chromatogram is reproducible. Pumps can be designed to deliver a constant
pressure at the column head, or to generate a constant flow of mobile phase.

3.3.1 Constant Pressure
The advantages of constant pressure pumps are their simplicity and ease of
maintenance. The pressure on any column is dependent on the viscosity of the
mobile phase and the extent of interaction between the solid and mobile phases.
Viscosity can change dramatically throughout the course of an HPLC run by
changes in temperature of the mobile phase. The viscosity will also change upon
changes in mobile phase composition. Thus gradient elution can cause
significant changes in the flow rate in order to keep a constant head pressure.
This type of pump is thus not used much for either qualitative or quantitative
analysis, since changes in retention time can be significant enough to render
peak identification impossible.
3.3.2 Constant Flow
The most common type of pump system used in analytical analysis is the
constant flow pump. A constant flow can be generated by a consistent
displacement of a fixed volume. The most common of these is the reciprocating
pump. This type of pump uses a small bore cylinder that is filled with the mobile
phase either by gravity or by a vacuum generated by the piston action of the
pump. A series of check (one way) valves is used to keep the mobile phase
flowing in one direction. The two main types of these are the single and the dual
piston systems.

Figure 2 - Single Piston Pump
Figure 3 - Dual Piston Pump
The pistons are usually sapphire rods, machined to have a consistent length and
diameter. The advantage of sapphire is the extremely high tensile strength in the
linear direction and the material wets consistently. This allows for a high-
pressure seal around the piston thus eliminating the chance of seepage around
the piston.
Check valves are “ball and seat” type. These are manufactured from ruby, since
when wet, will make an extremely good seal between highly polished surfaces.

When the CAM rotates, the piston moves in and out of the housing chamber. As
it moves out, mobile phase is drawn in and when the piston moves the other
direction, the check valve “seats” itself forcing the mobile phase out to the
column. In the dual piston system, the pistons are aligned on opposite sides of
the CAM. Thus as one chamber is filling the other is emptying, creating a more
constant flow arrangement. The chambers are usually gravity filled and thus
require the mobile phase reservoirs to be at a higher level than the pump.
Both the single and dual piston systems create a “pulse” effect on the pressure,
which can lead to unsteady baselines. These pumps employ a dampener after
the pump.
3.3.3 Isocratic
The reciprocating pump will deliver a consistent reliable flow rate of any one
mobile phase. This is known as isocratic analysis. Isocratic analysis is a very
versatile and well-used type of HPLC analysis. It has the advantages of not
requiring expensive pump systems and is very reproducible, providing the mobile
phase can be prepared the same each time (slight changes in composition
usually does not drastically effect the chromatographic requirements. This is
known as the “ruggedness” of a procedure). The analyses described in the
experimental section of this session will be isocratic.
3.3.4 Gradient
Gradient Elution chromatography is a very versatile procedure. It cannot be

created using one standard reciprocating pump alone. There are two types of
gradient mixing – high pressure and low pressure.
High-pressure gradient is produced with two or more pump systems set in

parallel, each pumping a single mobile phase. The flows from each pump system
are mixed in a high pressure mixing chamber after the pumps and before in
sample injector system. As the flow rate of each pump is varied, the composition
of the final eluent varies depending on the percentage of the total effluent that is
delivered from each pump (see figure 4).

Figure 4 - High-pressure System
The system controller controls the flow from each pump, for example increasing
one as the other is decreased, thus maintaining a steady flow rate. The
advantage of this is the gasses normally expelled during the mixing of two
different solvents are not formed due to the elevated pressure and the gradient
can be controlled and reproduced very precisely. The disadvantage is the
requirement of two highly accurate pump systems and a controller for these, all
of which require maintenance and calibration.
Low pressure gradient mixing is accomplished by a series of valves (usually

solenoid type valves), which mix solvents from different sources prior to the
pump (see figure 5).
Figure 5 – Low-pressure Gradient Mixing System
There can be multiple solenoid valves, which can be controlled to open and close
at various intervals to change the composition of the final effluent. This generally

requires a mixing chamber after the solenoid valves to allow the mixture to
equilibrate prior to the pump. Since the solvents are mixed at a low pressure,
gasses dissolved in the solvents may be expelled during the mixing. This can be
problematic to most pump systems available today, thus solvents must be
thoroughly degassed prior to the mixing process. This can be accomplished
degassing the solvents prior to the initial set-up, by constant helium sparging of
the solvents or by employing an online degassing system.
3.4 Sample Introduction
The sample is generally prepared in a media in which the analyte is soluble and
which is soluble in the mobile phase it is being injected into. Particulates in the
sample can cause problems in the column, causing the column to plug and thus
increasing the backpressure to the point in which the pump can be damaged.
Thus the samples are usually made particulate free by either centrifugation or
filtering through a membrane filter of 0.45 um or less. The sample must be
introduced to the system after the pump and thus the system is under pressure
when the sample is introduced. This requires the use of a valve switching
system, which will not significantly interfere with the flow.
3.4.1 Rheodyne Type Valve
Rheodyne Corporation began developing an injector system for this purpose in

1974. In 1976 the first Rheodyne manual injector system was developed and
marketed. The basic structure was a 6 valve cylindrical system, which would
significantly reduce the interference of normal flow through an HPLC system
(See figure 6).
Figure 6 - Rheodyne Type Valve

With this system, the sample is introduced using a micro liter (ul) syringe in to the
sample port. A “sample loop” of known volume would accept the sample and
allow excess to go to a waste collector. When the loop was filled, the valve
system could be rotated to place the sample loop into the path of the mobile
phase flow. Since the distance the valve needed to move was small, the flow
disruption was minimal and a reproducible amount of sample could be introduced
(See figure 7 and 8).
Figure 7
Figure 8
3.4.2 Automatic Samplers
The Rheodyne sampling system solved a big problem hampering the

development of HPLC. A large variety of materials could now be introduced
reproducibly into an HPLC system. Loops were designed of different sizes from 5
to 1000 ul. They were further streamlined to reduce band broadening and other
effects. The next development in the sampling system was to automate the
process. This was accomplished originally by using air pressure.
The process would begin through a controller, which would operate valves
holding compressed gas (usually air but nitrogen and helium were also used).
Each of these valves was connected to specific portions of the “automatic

sampler”. The first valve would switch the Rheodyne valve from the “inject” to the
“load” position. The next valve would push a syringe into a vial containing the
sample solution. The next valve would create a positive pressure in the vial,
causing the sample to be forced up the syringe and into a line to the Rheodyne
valve. After the sample loop was filled (the sample solution would emerge in the
waste line of the Rheodyne valve), the next valve would cause the Rheodyne
valve to move to the “inject” position and the sample would then be introduced
into the HPLC system. Although this sounds like a cumbersome process, this
was computer controlled and was quite effective at multiple samples. The
software involved was fairly simple and it allowed for unattended processing of a
large number of samples, including multiple injections of the same sample. The
“inject” step in the process would be set up with a signal to the data recorder
(chart recorder or software system) to start collecting the signal from the
detector.
3.4.3 Auto-sampler
With commercially available automatic sampling devices, large numbers of

samples can be routinely analyzed by HPLC without operator intervention. Such
equipment is popular for the analysis of routine samples (e.g., quality control of
pharmaceuticals), particularly when coupled with automatic data-handling
systems. Automatic injectors are indispensable in unattended searching (e.g.,
overnight) for chromatographic parameters such as solvent selectivity, flow rate,
and temperature optimization.

Most modern autosamplers have a piston metering syringe type pump to suck
the pre-established sample volume into a line and than transfer it to the relatively
large loop (~100 ml) in a standard six-port valve. The simplest autosamplers
utilize the special vials with pressurization caps. A special plunger with a needle,
push the cap down to the vial and displace the sample through the needle into
the valve loop.
Most of the auto-samplers are microprocessor controlled and can serve as a

master controller for the whole instrument.
3.5 Analytical Columns
Today, column material is normally Type 316 stainless steel, once again chosen
because it offers the best compromise of cost, workability, and corrosion resistance.
Most commercial columns available today have internal diameters of either 2.6-3 mm or
4.6-5 mm. Most leading manufacturers obtain a great amount of interchangeability
between column sizes without excessive fitting replacement by supplying columns with
a variety of internal diameters, but a uniform ¼ in. outside diameter. Naturally,
preparative columns have larger diameters: usual O.D. values are 3/8 - 5/8 in.,
corresponding to internal diameters of 6.4-12.7 mm, respectively.
Figure 9 - Analytical HPLC Column
Most modern liquid chromatography plumbing is performed with compression fittings

such as Swagelok, Parker-Hannifin, Gyrolok, etc. These offer a great deal of flexibility,
can be connected and disconnected frequently without loss of sealing power, are

commonly available, and may be used without special seating and flanging tools
allowing users to install, maintain, modify, and improvise equipment.
Great care should be taken in the choice of end fittings, connectors, and unions
however, since inclusion of rapid diameter changes corners, and other upswept areas in
a system can destroy a good separation. It is best to obtain the above parts from a
reputable liquid chromatograph manufacturer since most of these companies offer
fittings, which are specifically made for LC.
Many reducing unions and column end fittings incorporate special devices designed
to hold packings in place and/or prevent their flowing into the detector or detector
tubing. The most frequently used of these consist of fine porous stainless steel fritted
filter discs, usually with an average opening of 1 m. These may be forced into the ends
of columns or may be in the connector or end fitting. The important point is that the frit
disc must be tightly fitted to avoid occurrence of large-diameter spaces at the edge
where the disc contacts the wall.
An additional benefit of the frit is derived from the fact that it fills what otherwise might
be a large empty area in conversion zones thus, the sample cannot diffuse, cross mix,
or undergo other efficiency-killing effects when passing through these areas.
3.6 Normal Phase
A normal phase column is an un-derivatized solid phase (generally silica or

alumina). This type of material is polar and is not stable to high aqueous mobile
phases. The solvents of choice for mobile phases are hydrophobic solvents such
a hexane, ethyl acetate, ether, chloroform and dichloromethane. This type of
column is useful to separate non-polar compounds but is not used extensively in
the pharmaceutical industry since most pharmaceuticals are water-soluble or are
soluble in hydrophilic solvents.

3.7 Bonded Phases
3.7.1 Reversed Phase
Reversed phase columns are silica based solid phases derivatized with a
hydrophobic ligand. In the most common case, the free silanol groups are
reacted with an alkyl-silane producing a structure such as:
Si-O-Si-R
Where, R can be an alkyl group of anywhere from 1 to 18 carbon atoms. This

type structure causes a hydrophobic layer on the surface of the silica particles.
The most commonly used alkyl groups are C1, C2, C4, C8 and C18.

3.7.2 Other Bonded Phases
Other forms of the R group are:
Amino – R = -CH2-CH2-CH2-NH2

Amino-phase is a weak anion-exchanger. This type of column is mainly used in
normal-phase mode, especially for selective retention of aromatic compounds.
Cyano – R = CH2-CH2-CN
A cyano-modified surface is very slightly, polar. Columns with this phase are
useful for fast separations of mixtures consisting of very different components.
These mixtures might show very broad range of retention times on the usual
columns.
Phenyl – R = (CH2)3C6H5

Propylphenylsilane ligands attached to the silica gel show weak dipole - induced
dipole interactions with polar analytes. Usually this type of bonded phase is
used for group separations of complex mixtures. Amino-compounds show some
specific interactions with phenyl-modified adsorbent.
Diol – R = CH(OH)2
Diols are a slightly, polar adsorbent for normal-phase separations. These are
useful for separation of complex mixtures of compounds with different polarity,
and which usually show a strong retention on unmodified silica.
3.8 Ion Exchange
Ion exchange columns can be either silica based or polymer-based resins

(usually a polystyrene polymer crosslinked with divinyl benzene), containing
active sites – either anionic or cationic – that will interact with the molecule of
choice.

The reactivity of these is dependent on the mobile phase – i.e. pH and/or ionic
strength. This can be quite useful especially for biochemical and protein analysis.
The polymer based types are quite sensitive to organic solvents and even
methanol can cause extensive swelling of the polymer base, rendering the
column useless. Silica based ion-exchange columns are very sensitive towards
extreme pH’s i.e. <2.0 and >8.0. The smaller pore sizes available in silica gel
bases can limit the usefulness of these types of ion exchange columns for use
with protein analysis, but their resistance to organic solvents makes them useful
for analysis of ionic pharmaceutical products.
3.9 Size Exclusion
Size exclusion chromatography (SEC) is employed to estimate the molecular

weight (MW) of a soluble sample. It can also be useful for sample clean up or
purification. Polymers that are larger than the pores are excluded from the pore
volume inside the porous packing and remain in the volume around the outside
of the particles. Polymers or compounds that are smaller than the pores travel
throughout the pore volume. The rate at which these materials pass through the

pores is inversely proportional to their molecular mass (actually their molecular
volume). This type of chromatography is useful in analysis of polymers - for
molecular weight distributions, and protein and peptide analysis.
4. Detectors
4.1 Flow-Through Cell
The heart of an efficient HPLC detector is the Flow-Through Cell. The figure
below shows the schematic of a modern follow-through cell. The optics provides
focusing of the light beam in the center of the cell where it is virtually unaffected
by the entry-exit-window-interface disturbance, or drift induced by flow,
temperature, or refractive index changes. The short, wide cell assures that
maximum energy is transmitted, and a post-cell collecting lens focuses all of the
existing light from the cell.
Figure 10 - Standard Flow Through Cell
Detectors equipped with the flow-through cell were a major breakthrough in the
development of modern liquid chromatography. The group of Tiselius, in Sweden
in 1940, first applied this by continuously measuring the refractive index of the
column effluent. Current LC detectors have wide dynamic range normally
allowing both analytical and preparative scale runs on the same instrument. They
have high sensitivities often allowing the detection of nanograms of material, and
the better models are very flexible, allowing rapid conversion from one mobile
phase to another and from one mode to another.

4.2 Refractive Index
The refractive index (RI) detector is the only universal detector in HPLC.
The detection principle involves measuring of the change in refractive index of
the column effluent passing through the flow-cell. The greater the RI difference
between sample and mobile phase, the larger the imbalance will become.
Thus, the sensitivity will be higher for the higher difference in RI between sample
and mobile phase. On the other hand, in complex mixtures, sample components
may cover a wide range of refractive index values and some may closely match
that of the mobile phase, becoming invisible to the detector.
RI detector is a pure differential instrument, and any changes in the eluent

composition require the rebalancing of the detector. This factor severely limits
the RI detector’s application in analyses requiring the gradient elution, where
mobile phase composition is changed during the analysis to effect the
separation.
Two basic types of RI detectors are on the market today. Both require the use of
a two-path cell where the sample-containing side is constantly compared with the
non-sample-containing reference side.
4.3 UV Detector
Any chemical compound may interact with the electromagnetic field. A beam of
the electromagnetic radiation passed through the detector flow-cell will
experience some change in its intensity due to this interaction. Measurement of
this change is the basis of the most, optical HPLC detectors.
Radiation absorbance depends on the radiation wavelength and the functional

groups of the chemical compound. Electromagnetic field depending on its energy
(frequency) can interact with electrons causing their excitation and transfer onto
the higher energy level, or it can excite molecular bonds causing their vibration or
rotation of the functional group. The intensity of the beam which energy
corresponds to the possible transitions will decrease while it is passing through
the flow-cell. According to the Bear-Lambert law absorbance of the radiation is

proportional to the compound concentration in the cell and the length of the cell.
The electromagnetic spectrum is traditionally divided into several regions:
Spectrum Wavelength (λ) (nm)

Infrared (IR) 2,500 – 50,000 nm
Near Infrared (Near IR) 800 - 2,500 nm
Visible 400 – 800 nm
Ultraviolet (UV) 190 – 400 nm
The most widely used portion of the electromagnetic spectra for HPLC analysis
is the 190 – 800 nm range, since typical mobile phase solvents are invisible to
much of this range, thus they do not interfere greatly with the detection of
compounds which are detectable in this range. This is the range of the normal
UV-Vis detectors employed, today in HPLC. Four major types of UV-Vis
detectors are available on the market today. Each has applied the UV spectra
absorbance in a similar manner.
4.3.1 Fixed Wavelength
The fixed wavelength detector was the first of its kind. A low-pressure mercury
vapor lamp will emit very intense light at 253.7 nm. By filtering out all other
emitted wavelengths, manufacturers have been able to utilize this 254 nm line to
provide stable, highly sensitive detectors. Most components containing an
aromatic ring structure have a strong absorbance in the 254 nm range, and thus
these detectors are capable of measuring sub-nanogram quantities of these
components. The 254 nm was chosen since the most intense line of mercury
lamp is 254 nm, and most UV absorbing compounds have some absorbance at
254 nm.

Figure 11 - Fixed Wavelength Detector
4.3.2 Variable Wavelength
The major drawback with the fixed wavelength detector is the fact that most
compounds that absorb UV light absorb in the 254 nm region. Thus interference
by other components can be a significant problem. Some compounds are
extremely difficult to separate even with today’s advances in column chemistry.
However, many of these compounds absorb in other regions of the UV-Vis
spectra as well. By employing a detector, which can read at other, unique
wavelengths, complete chromatographic separation is not as necessary. The
sensitivity for a particular component can be enhanced greatly by setting the
wavelength to that in which the component has the maximum absorbance.
Variable wavelength detectors use a deuterium lamp, which gives a much

broader range of UV radiation. The radiation is spit by a prism and mirror system
and the wavelength of choice is focused through the flow cell. The photodiode
then measures the intensity of the specific wavelength compared to the baseline.

Figure 12 - Variable Wavelength Detector
4.3.3 Diode Array/Photo Diode Array (PDA)
The functionality of the variable wavelength detector was enhanced again by the
diode array type source. In diode array, the source light (again from a broad
spectrum source such as a deuterium lamp) is passed through the sample cell
and then is split into its corresponding spectrum. The entire spectrum is then
focused onto an array of photo-diodes. The spacing of the diodes is designed to
measure the intensity at specific wavelengths, usually 1 or 2 nm each. Thus the
electromagnetic spectrum is measured by combining the response from each
individual diode. Since the analyte will absorb the UV radiation whether it is split,
or in combination with other wavelengths, the response at any particular
wavelength will be affected by the sample compared to that of the baseline.
The advantage of this is that the entire spectra can be analyzed instantaneously.
Thus, two, three or many more readings at different wavelengths can be
measured simultaneously. All the measurements can be combined to produce a
three dimensional chromatogram (RT x wavelength x absorbance). This is a very
effective tool for characterization of known and unknown components of a
mixture.

Figure 13 – PDA Detector
4.4 Fluorescence
Compounds having specific functional groups are excited by shorter wavelength

energy and emit higher wavelength radiation. This is known as fluorescence and
this emission is usually measured at right angles to the excitation. About 15% of
all compounds have a natural fluorescence. The presence of conjugated pi-
electrons especially in the aromatic components gives the most intense
fluorescent activity. Also, aliphatic and alicyclic compounds with carbonyl groups
and compounds with highly conjugated double bonds fluoresce, but usually to a
lesser degree. Most un-substituted aromatic hydrocarbons fluoresce with
quantum yield increasing with the number of rings, their degree of condensation
and their structural rigidity. Fluorescence intensity depends on both the excitation
and emission wavelength, allowing selective detection some components while
suppressing the emission of others.
Figure 14, below shows the optical schematic of a typical fluorescence detector
for liquid chromatography. The detectors available on the market differ in the
method in which the wavelengths are controlled. Less expensive instruments

utilize filters; medium priced units offer monochromator control of at least
emission wavelength, and full capability research-grade instruments provide
monochromator control of both excitation and emission wavelengths.
Figure 14 – Fluorescence Detector
4.5 Conductivity
The conductivity of the column effluent is continuously measured, giving a

baseline measurement and the appearance of the analyte in the cell is indicated
by a change in conductivity.
Usually this is a very low volume flow-through capillary equipped with two
electrodes and variations in conductivity of the mobile phase due to the eluted
sample components are continuously recorded. Response is linear with
concentration over a wide range, and quantitation of the output signal is possible
with suitable preliminary calibration. Best use is made of this detector is in
isocratic analysis, since solvent gradients will cause a proportional shift in the
baseline. Such detectors have been used most successfully in ion-exchange
chromatography of anions and cation but generally, they have found only limited
popular acceptance.

Figure 15 – Conductivity Detector
4.6 Electrochemical
The electrochemical detector is also a popular liquid chromatographic detector. It

should be considered by, the chromatographer because of the additional
selectivity and sensitivity for some compounds.
This detector is based on the measurements of the current resulting from

oxidation/reduction reaction of the analyte at a suitable electrode. Since the level
of the current is directly proportional to the analyte concentration, this detector
could be used for quantification.
Figure 16 – Electrochemical Detector

The eluent should contain electrolyte and be electrically conductive. Most of the
analytes to be successfully detected require the pH adjustments.
The areas of application of electrochemical detection are not large, but the
compounds for which it does apply, represent some of the most important drug,
pollutant and natural product classes. For these, the specificity, and sensitivity
make it very useful for monitoring these compounds in complex matrices such as
body fluids and natural products. Sensitivities for compounds such as phenol,
catecholamines, nitrosamines, and organic acids are in the picomole (nanogram)
range. The purity of the eluent is very important, because the presence of
oxygen, metal contamination and halides may cause significant background
current and therefore, noise and drift in the base line.
5. Data Collection and Processing
The output from any detector needs to be collected by a system that can plot the
detector response vs. time. The signal coming from the detector is reading of the
absorbance at regular intervals throughout the run. Detectors will report anywhere from
1 to 20 response readings per second, depending on the settings on the detector. The
greater the number of readings will usually lead to more accurate chromatogram.
However, this can also lead to a huge amount of data points for a chromatogram. The
number of points stored per second is dependent on the type of analysis. With HPLC
runs, tangent peak widths are generally in the 1 to 3 minute range. Thus a “sampling
rate” of 1 to 5 data points per second is usually quite sufficient for most analyses.
5.1 Computers / Software
One of the most significant advances in HPLC analysis is the evolution of

computers and computer software. Over the past 10 years, computers have
become a standard tool in any analytical laboratory. Processor speeds
approaching 3 gigahertz and hard drive storage up to and above several
terabytes have made procedures such as integration, re-integration and
sequence processing fast and easy. Huge data files that would have choked
(and killed) computers of the early 1990’s are easily managed by today’s

personal computers. The data file from a full scan diode array system, running a
15 minute chromatogram can be as much as 50 megabytes in size. Processing
this file on a 66 MHz 486 computer with a 400 megabyte hard drive and 64
megabytes of ram would take about 30 minutes. Two years prior to this, it would
not even have been possible.
5.1.1 Network / LIMS Data Storage

Despite the size and power of today’s desktop computers, storage of numerous
chromatographic files is not an option on a desktop computer. Regulatory
agencies have required the pharmaceutical industry to permanently store and
archive raw data from HPLCs and other equipment.
Large pharmaceutical laboratories may house up to 100 or more HPLC and other
chromatographic equipment, all continuously generating huge files. For this
reason, most companies have moved to network data storage and database
systems that can handle these huge files and very high volumes network traffic.
Files are bundled together in large compressed data files and stored outside of
the main system and copies are stored off-site as well.
6. Factors Affecting Performance
6.1 Mobile Phase
In HPLC type and composition of the mobile phase (eluent) is one of the
variables influencing the separation. Despite of the large variety of solvents used
in HPLC, there are several common properties:
Purity - Always use ultra pure, HPLC grade or glass distilled solvents for mobile
phase preparation. In organic solvents, impurities can be retained at the head of
the column, eventually leading to a build-up of material which will reduce the
efficiency of the column and may result in ghost peaks, band broadening, analyte
interactions or clogging of the column. All mobile phases must be filtered through
a membrane filter with a pore size of 0.45 um or smaller.

Detector Compatibility - The mobile phase should be as undetectable as
possible with the detection system.
Solubility of the Sample - If the sample is not soluble in the mobile phase, it will
not elute from the column.
Low Viscosity - High viscosity mobile phases will lead to excessively high back
pressures and can damage the pump, seals and check valves.
Chemical Inertness - The analyte must be stable in the mobile phase.
Reasonable Price - Many HPLC runs can be up to an hour in length (per

injection). This will use large volumes of mobile phase for analysis of multiple
samples.
Each type HPLC analysis has its own requirements. For normal phase, solvents
are mainly non-polar and for reversed phase, eluents are usually a mixture of
water (sometimes pH adjusted or buffered) with some polar organic solvent such
as acetonitrile, methanol or THF.
6.2 Stationary Phases
Most stationary phases are silica base. These are generally stable to organic
mobile phases but there are some exceptions. Silica has a strong affinity towards
halogens and other strongly electronegative substances. Atoms with lone pairs
such as Nitrogen are also readily attracted to silica. As a result, silica gel tends to
be reactive in low or high pH environments. The best and safest chromatography
with silica gel based columns occurs within the pH range of 2.5 to 7. Mobile
phases that are outside of this range tend to destroy the silica base, thus
reducing particle size and causing loss of bonded substrates.
Other materials that may cause problems are:
Amines - Compounds with primary amine functionalities can be difficult to

chromatograph well since the lone pair of electrons on the Nitrogen can be
attracted to the silica. The result can be excessive tailing of the peak of interest.

This effect has been minimized two ways; addition of triethyl amine to the mobile
phase (pH of the mobile phase should be buffered to between 4 and 6) and/or
using a column which is “deactivated” – i.e. free reactive sites on the silica gel
have been reacted with a small chain alkyl group to increase stearic hindrance
around the reactive sites on the silica gel.
Hydrochloric acid (HCl) - Hydrochloric acid is highly attracted to silica gel and
the chloride may even bond with the silica, displacing bonded substrates. HCl
should never be used in a mobile phase for a silica-based column. HCl salts are
common among many pharmaceutical compounds. Usually the HCl is in a small
enough concentration to have a negligible effect on the stability of the solid
phase, however, repeated introduction of this type of analyte will have an effect
on the performance of the column. This effect can also be minimized by the
addition of a competing base such as triethylamine in the mobile phase (buffered
to between 3.5 and 6). Here the triethylamine will act as an “HCl scavenger”,
complexing the HCl such that it does not come into contact with the silica base.
Columns are available in today’s market that are designed to handle these types
of problems. Some are so well protected that they are stable at pHs from 2.0 to
10.0 without appreciable loss of performance even after hundreds of hours of
use.
Another type of solid phase that has become popular is the polymer-based
column. These have the advantage that they are stable to strong acids (including
up to 2N HCl) and to strong bases (2N NaOH). However, the polymer backbone
is not stable to strong organic solvents. As much as 25% Methanol or Acetonitrile
in water can cause many of these polymer backbones to swell, rendering the
column useless.

7. GMP and GLP for HPLC Analysis
The following are excerpts from the cGMP regulations 21 Code of Federal Regulation,
(CFR) Part 211. These indicate the requirements for records creation and storage. Data
acquired from HPLC analyses fall under this jurisdiction. The requirements are,
basically, that all records must be kept and must be positively identifiable to a specific
batch of the particular product or material. All equipment used must be calibrated such
that the normal mode of operation is within the limits of the calibration. The actual
regulations on Laboratory records are included here for completeness.
CFR 211.194 Laboratory records (from cGMP Regulations 21 Code of Regulations).
(a) Laboratory records shall include complete data derived from all tests necessary
to assure compliance with established specifications and standards, including
examinations and assays, as follows:
Note: important points and points relating to HPLC analysis have been
underlined)
(1) A description of the sample received for testing with identification of

source (that is, location from where sample was obtained), quantity, lot
number or other distinctive code, date sample was taken, and date
sample was received for testing.
(2) A statement of each method used in the testing of the sample.
The statement shall indicate the location of data that establish that the
methods used in the testing of the sample meet proper standards of
accuracy and reliability as applied to the product tested. (If the method
employed is in the current revision of the United States Pharmacopeia,
National Formulary, Association of Official Analytical Chemists, Book of
Methods, {2} or in other recognized standard references, or is detailed in
an approved new drug application and the referenced method is not
modified, a statement indicating the method and reference will suffice).
The suitability of all testing methods used shall be verified under actual
conditions of use.

Copies may be obtained from: Association of Official Analytical Chemists,
2200 Wilson Blvd., Suite 400, Arlington, VA 22201-3301.
(3) A statement of the weight or measure of sample used for each test, where
appropriate.
(4) A complete record of all data secured in the course of each test, including
all graphs, charts, and spectra from laboratory instrumentation, properly
identified to show the specific component, drug product container, closure,
in-process material, or drug product, and lot tested.
(5) A record of all calculations performed in connection with the test, including
units of measure, conversion factors, and equivalency factors.
(6) A statement of the results of tests and how the results compare with
established standards of identity, strength, quality, and purity for the
component, drug product container, closure, in-process material, or drug
product tested.
(7) The initials or signature of the person who performs each test and the
date(s) the tests were performed.
(8) The initials or signature of a second person showing that the original
records have been reviewed for accuracy, completeness, and compliance
with established standards.
(b) Complete records shall be maintained of any modification of an established

method employed in testing. Such records shall include the reason for the
modification and data to verify that the modification produced results that are at
least as accurate and reliable for the material being tested as the established
method.
(c) Complete records shall be maintained of any testing and standardization of

laboratory reference standards, reagents, and standard solutions.
(d) Complete records shall be maintained of the periodic calibration of laboratory

instruments, apparatus, gauges, and recording devices required by CFR
211.160(b)(4).
(e) Complete records shall be maintained of all stability testing performed in
accordance with CFR 211.166.

8. Session One Assignment
Give a detailed description of a reverse phase HPLC system. How would

increasing the non-polar (hydrophobic) component of the mobile phase affect
analyte retention time?
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Give a detailed description of a normal phase system. How would increasing the
non-polar(hydrophobic) component of the mobile phase affect analyte retention
time?
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KEY CONCEPTS IN HPLC (Session 2)
9. The Chromatogram and Performance Measurement
9.1 The Chromatogram
The mobile phase transports sample components to the detector where the
signal produced by the sample components is plotted against time. This output is
called a "Chromatogram".
Figure 17
9.2 Retention Time
The retention of an analyte in a chromatogram can be defined in terms of elution

time or volume of mobile phase required to elute the analyte from the HPLC
column. With a particular column type and mobile phase composition, the
retention is characteristic of the particular analyte and is thus a positive
identification.

The easiest way to find the chromatographic retention is to measure the time
between the injection point and maximum of the detector response for
correspondent compound. This parameter is usually called "Retention Time, tR".
Retention time is inversely proportional to the eluent flow rate.
Retention times provide the qualitative aspect of the chromatogram. The

retention time of a compound will be the same under identical chromatographic
conditions. The chromatographic condition peak height or peak area is related to
the quantity of analyte. For determination of actual amount of the compound, the
area or height is compared against standards of known concentration.
The product of retention time and eluent flow rate, so called "Retention Volume,
VR ", is more of a global retention parameter. Retention volume represents the
volume of the eluent passed through the column while eluting a particular
component.
Component retention volume, VR could be split into two parts:

• Reduced retention volume is the volume of the eluent that passed through the
column while the component was sitting on the surface.
• Dead volume is the volume of the eluent that passed through the column
while the component was moving with the liquid phase.
The second part is equal to the combined volume of the liquid phase in the
column and tubing between the injector and the detector (dead volume, Vo),
and it will be the same for any component eluted on this column and system.
Components that are not retained by the stationary phase but carried through
the column without interaction are said to elute at the dead time "t0" of the
system.

10. Performance Measurements
10.1 Capacity
Figure 18
The elution of the compound is characterized by the partition ratio, k', a

dimensionless quantity also called the Capacity Factor. It is equivalent to the
ratio of the time required for the compound to flow through the column (the
retention time) to the elution time of an un-retained compound.
The value of the capacity factor, k' depends on the chemical nature of the
compound, the nature, amount, and surface area of the liquid phase,
the column temperature, and the gas flow rate.
Under a specified set of experimental conditions, a characteristic capacity factor

exists for every compound. Separation occurs only if the compounds concerned
have different capacity factors.
The Capacity factor, k' is a measure of the retention of the sample molecule on
the column. A high k' value means that the sample is highly retained and has
spent a significant amount of time interacting with stationary phase.
Increase in k', increases the "Resolution" between chromatographic peaks.

• Capacity factor, k' (also known as Retention factor) is characteristic of a
specific compound at a given mobile phase composition, temperature, and
column type.
• Capacity factor is also equal to the number of moles in the stationary phase
divided by the number of moles in the mobile phase
k', can also be calculated as:
t1 − t 0
k `=
t0
10.2 Selectivity
The selectivity is the ability of the stationary phase to distinguish between two
separate sample components. Selectivity, is calculated as the ratio of the
capacity factors k'.
Figure 19
k `2
Selectivity =
k `1

Parameters affecting selectivity are:
• Mobile phase composition

• Mobile phase pH
• Column temperature
• Chemical additives
• Stationary phase
A selectivity value of 1 means that there is no difference in the capacity of the

system for the two peaks and therefore, there is no separation between
components.
The greater separation, the greater selectivity value, however, actual separation
cannot be determined by selectivity alone.
10.3 Efficiency
Figure 20
The efficiency of a column/mobile phase system is a general determination of the

fitness of the particular column/mobile phase system to perform the separation
required for the analysis. The efficiency is defined in terms of "Number of
Theoretical Plates, N" – originally derived from distillation separation.
The sharpness of a chromatographic peak is an indication of the quality of the
chromatographic column.
• Peak sharpness is determined by measurement of the "Peak Width, Wb" at
the baseline.
• Peak width, Wb is dependent on flow rate so measurement of the width alone
is not enough.
A good measure of column efficiency is:
tR / Wb
The actual equation for column efficiency is:
2
 t1 
N = 16   

W
 B1 
The efficiency, N, or plate number is a mathematical measure of the broadening
of the peak as it passes through the column. The relationship is an inverse
relationship

10.4 Resolution
The most important part of chromatography is the resolution (i.e., separation)

between the important chromatographic peaks in the sample.
Figure 21
Resolution can be defined as the extent to which two compounds can be

separated on a chromatographic system. The resolution can be determined by
the following equation:
2  (t 2 − t1 )
R=
WB1 + WB 2
Where:
R = Resolution
tRB = Retention time of component B
tRA = Retention time of component A
W = Peak width at base

A resolution value of  1.5 (equal or greater than 1.5) between two
chromatographic peaks of approximately equal peak height is considered
baseline resolution.
Figure 22
Factors effecting resolution are:
Figure 23
• Capacity Reflects the interaction of the sample molecule with the

stationary and mobile phase.
• Selectivity Describes the ability of chromatographic system to distinguish

(i.e., to separate) between two or more compounds/molecules.
• Efficiency Relates to the width of the peak in the separation.

Figure 24 – Effects of Stationary and Mobile Phase Changes
Figure 25 - Sample chromatogram
Where:
W = Peak width
Wh/2 = Peak width at 1/2 the peak height
h = Peak height
h/2 = Half the peak height
t1 = Retention time for peak 1
t2 = Retention time for peak 2

10.5 Peak Tailing and Symmetry
Chromatographic peaks should be Gaussian or symmetrical. Often, however, the

chromatographic peaks are tailed due to extra-column effects, sample adsorption
on stationary phase or column bed irregularity.
The tailing factor, T, is a measure of peak symmetry showing the unity for
perfectly symmetrical peaks. T value increases as tailing becomes more
pronounced.
The calculation for the Tailing factor (T) is:
W0.05
T=
2 f
Figure 26
Figure 27 - Examples of peak tailing
Excellent Acceptable Unacceptable Poor

11. System Suitability
System suitability tests are an integral part of gas and liquid chromatographic methods.
They are used to verify that the resolution and reproducibility of the chromatographic
system are adequate for the analysis to be done. The tests are based on the concept
that the equipment, electronics, analytical operations, and samples to be analyzed
constitute an integral system that can be evaluated as such.
11.1 Performance Measurements
Performance measurements, capacity, resolution and tailing are integral parts of system
suitability. Values of these measurements are defined in the analytical procedure to
indicate the analytical system is functioning as required to achieve the desired
separation.
11.2 System Reproducibility
Reproducibility is defined as the ability for the HPLC system, as prepared, to reproduce
the injection volumes desired. This is vital for quantitative analysis. The primary
standard is usually injected 5 times in sequence. The relative standard deviation of the
analyte peak area is determined. The standard in the pharmaceutical industry (USP) is
2.0% for 5 replicate injections.
Automated analysis is designed to analyze many samples over an extended period of

time without requiring operator intervention. To ensure the system maintains its
reproducibility, check standards are introduced throughout the sample run to ensure the
systems maintains its integrity. As required by USP. a check standard is injected after
no more than six sample injections. The check standards are analyzed as samples and
the results as a percent recovery should be 100.0  2.0%.
Performance evaluation and calculation of performance measurements is not always a

system suitability requirement in USP methods, however, reproducibility is always a
requirement for quantitative analysis and must be achieved before sample analysis can
begin.

12. Session Two Assignment
1) How does pH play a role in an analyte’s retention on a column?
Explain using different functional groups as examples. How is it
possible to increase (change) resolution by adjusting pH?
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2) Why is the tubing used in an HPLC so small? What is meant by

band broadening?
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3) Draw a simplified diagram of an HPLC system. Include the reservors,
pump, column and detector. Explain the function of each.

4) System Suitability Calculations. Determine how many components
are present in the chromatogram below. Calculate Retention Time,
K', Number of plates (N), Tailing factor, and selectivity values of each
peak.

5) HPLC External Standard Method - Raw Material Assay Calculation
 Perform Calculations
 Weight of Standard A= 98.15 mg
 Weight of Standard B= 99.25 mg
 Purity of Standard = 99.7%
 Weight of Sample A = 101.21mg
 Weight of Sample B = 102.14mg
 Dilution Sample = 1/200 ml x 5.0ml/100ml
 Dilution Standard = 1/200 ml x 5.0ml/100ml
 Areas as detected by HPLC
1. Standard A - 178721
6. Standard B - 178722
7. Sample A - 187211
8. Sample B – 187256
9. Check Standard – 17783
 Does standard A meet a criteria of NMT 2.0% for five injections?
 Does Standard B meet a 98.0-102.0% criterion?
 Does the check standard meet a 97.0-103.0% criteria?
 Does the sample comply with a specification of 98.0-102.0%? If not what should
be done?
Insert tabulated data into the following table:
Sample % Result Complies (y/n)

RSD of Standard A Injections
% Recovery (Standard B)
% Drift (Check Standard)
A
B
Equation for Raw Material Assay
PurityStd (%)
Wt Std (mg )   DilStd (1 / ml )
ASpl 100(%)
% Purity =   100(%)
AStd Wt Spl (mg )  DilSpl (1 / ml )

Session Two Experiment – I.D. via In-House Method
▪ Introduction to and Initial usage of equipment, single injection
▪ Filtration and degassing
▪ Identification via HPLC
Mobile Phase
Prepare 300ml of 50:50 methanol and water. Filter and degas.
Preparation of Standard
Accurately weigh about 10 mg of acetaminophen into a 250ml volumetric flask,

dissolve in about 25 mL MeOH, add 125 mL water and dilute to volume with
MeOH.
Preparation of Sample
Accurately weigh about 10 mg of acetaminophen into a 250ml volumetric flask,

dissolve in about 25 mL MeOH, add 125 mL water and dilute to dilute to volume
with methanol.
Equipment Parameters
▪ Column – 3.9 x 300 mm or 4.6 x 250 mm L1 column (10m)

▪ Flow rate – 1.5 ml/min
▪
▪ Wavelength – 243 nm
▪ Injection volume – 5 µL
▪ Run time - 10 minutes
After allowing the HPLC to come to equilibrium with your mobile phase,
inject your standard and sample. Do you have a positive identification for
your sample. Consider Retention time and peak shape in comparison to
the reference standard.

HPLC Columns (Session Three)
The High Performance Liquid Chromatography (HPLC) column is the most important
part of the HPLC. The separation of sample components occurs in the column. The
most commonly used HPLC columns are between 20 and 30 cm long and have an
internal diameter (i.d.) of 4 to 5 mm.
Most of the HPLC column hardware is constructed of high grade stainless steel, which
is a chromium-nickel-molybdenum steel. The steel tubes are precision drilled and
polished. The columns are closed at both ends with frits that vary in pore size according
to the support particle diameter. The columns are chemically resistant to most HPLC
solvents with exception to chloride ions, which may react with stainless steel surface.
These columns are designed to hold up well under typical HPLC pressures.
To protect a column use of a guard column is recommended. The guard columns,

which are placed between the injector and the analytical column, protect the more
expensive analytical columns from impurities and particulates. Any strongly retained
sample component, which might fowl the analytical column, will be trapped on the guard
column. Guard columns are filled with the same stationary phase as the analytical
column. They have the same i.d. and particle size, but very short in size.
Figure 37
FLOW
Analytical Column Guard Column
Detector Pump

There are also cartridge columns that are popular because of their low cost and ease of
replacement.
The column life may be extended for silica-based columns. Silica dissolves more readily
at extreme pH, high ionic strength, or high mobile phase polarity.
13. Role and Effects of the Stationary Phase
As explained in the chromatography section, chromatographic separation

process based on the difference in the surface interactions of the analyte and
eluent molecules.
Let us consider a separation of a two-component mixture dissolved in the eluent.

Assume that component A has the same interaction with the adsorbent surface
as an eluent, and component B has strong excessive interaction. Being injected
into the column, these components will be forced through by eluent flow.
Molecules of the component A will interact with the adsorbent surface and retard
on it by the same way as an eluent molecules. Thus, as an average result,
component A will move through the column with the same speed as an eluent.
Molecules of the component B being adsorbed on the surface (due to their
strong excessive interactions) will sit on it much longer. Thus, it will move
through the column slower than the eluent flow.
Figure 38 represents the general shape of the chromatogram for this mixture.
Figure 38

Usually a relatively narrow band is injected (5 - 20 l injection volume). During
the run, the original chromatographic band will be spread due to the uneven
flows around and inside the porous particles, and other factors. These processes
together produce band broadening of the chromatographic zone. In general, the
longer the component retained on the column, the more broad its zone (peak on
the chromatogram).
Separation and performance depend on both component retention and band

broadening. Band broadening is, in general, a kinetic parameter; dependent on
the adsorbent particle size, porosity, pore size, column size, shape, and packing
performance. On the other hand, retention does not depend solely on the above-
mentioned parameters, but it reflects molecular surface interactions and depends
on the total adsorbent surface area.
Stationary phases today are designed to minimize these effects as much as

possible.
13.1 Column Packing Materials
Porous silica particles are commonly used as HPLC supports for adsorption
chromatography and forms basis for numerous chemically modified stationary
phases for reversed phase, normal phase, ion exchange, chiral, and size
exclusion chromatography. The chemical composition is SiO2 x H2O. The purity
of the silica can affect the chromatographic reproducibility.
There are two basic shapes are available, irregular and spherical.
Original stationary phases were comprised of irregular shaped silica particles of

various sizes, coated with an octadecylsilyl (C18) bonded phase. Varying the
mobile phase strength and composition minimized band broadening. However,
this was only partially effective for many analytes and thus limiting the variety of
analytes possible for HPLC analysis.

Band broadening, as mentioned is a kinetic parameter. Thus by improving the
way the molecules interact with the stationary phase should reduce the extent of
band broadening.
When a manufacturer quotes a particle size, the quoted number is an average –

and thus there is a distribution of particle sizes packed in the column. The
magnitude of this distribution can also have a large effect on the band
broadening of a peak. If there are many small particles, flow can be impeded,
resulting in high backpressures. A large amount of larger particles can cause
some paths of the flow to be much larger than others, resulting in excessive band
broadening. Irregular particle shape can also cause band broadening. The paths
that the analyte must flow through the particles can vary from short to
excessively long, resulting in a much broader elution of the analyte from the
column.
Figure 39
Spherical particles have the effect of consistency while mobile phase is passing
around them. The paths through the particles are more regular and thus there is
less variation in the paths the analyte can take through the solid phase.
Figure 40

Although irregular particles are somewhat cheaper, they tend to have poor
efficiency than spherical particles and they also tend to produce fines that may
clog the outlet frit of the column.
Analytical columns have particle size of 3 to 10um in diameter. Larger particles

produce more band broadening leading to poor resolution. Smaller particles can
lead to high column pressures. The most popular particle sizes are 3.5 - 5 um.
Distribution is another characteristic of packing material. Most HPLC pickings

have approximate Gaussian distribution around the mean particle diameter.
Figure 41
Number %
Particle Diameter, um
13.2 Pore Size
One of the most important adsorbent parameters is the pore size and pore size
distribution. Adsorbent surface area is a major factor directly affecting the analyte
retention. Pore size defines an ability of the analyte molecules to penetrate inside
the particle and interact with its inner surface. This is especially important
because the ratio of the outer particle surface to its inner one is about 1:1000.
The surface molecular interaction mainly occurs on the inner particle surface.
Pore sizes vary from column type to column type, but control of pore size can
greatly affect the capacity and thus the resolution of a chromatogram.

Pore sizes can range from 40 Å to as much as 1000 Å, however, the desired
size will depend on the analyte of interest, since the accessibility to the pores of
the solid matrix will be limited if the pore size is small and the molecule is large.
Also, small molecules in large pores may not have good interaction with the
bonded surfaces in the pores. Most pharmaceuticals can be successfully
separated using columns with pore sizes of about 60 to 100 Å.
Porosity of the solid phase needs to be consistent in order to achieve good peak
quality. The larger pore size in the solid phase will result in better flow with lower
backpressures. Large variations in pore size will result in various flow rates
through the column, causing larger bandwidths.
13.3 Dimensions
Commercially available columns are generally 4.6 mm in internal diameter;

however, many manufacturers are using various widths that have become almost
trademark dimensions. For example, Waters used 3.9 mm as their standard
(Bondopak columns) and Merck used 4.0 mm as standard for their cartridge
systems (Lichocart). Generally, the larger the width, the more the analyte band is
spread out through the solid phase, thus leading to broader peaks. Narrower
columns usually contain less solid phase and thus the capacity can be reduced.
They are generally more useful for analytes of low concentration, since the
bands are more focussed, thus enhancing detection.
Column lengths vary considerably and some have become “trademark”

dimensions – i.e. Waters – 300 and 150 mm (Bondopak), Merck – 250, 125 and
75 mm cartridges (Lichocart), Brownlee - 220 mm (Brownlee Cartridge).
Recently, with the introduction of 3 um particle size and smaller solid phases,
30mm cartridges and columns are becoming popular.

15. Column Protection
This can be the most critical part of column performance – the next time the
column is to be used. Analytical columns are not inexpensive – they range
anywhere from $300 to $4000 per column. Although they are generally made of
very strong steel, they are fragile. The hardware will not be damaged if it is
dropped on the floor, but the impact may cause voids to be formed in the packing
material, thus leading to band broadening, loss of efficiency or split peaks. Thus,
they must be handled with care.
Whenever a column is used, it should be washed with a solvent system that will
remove any retained material or any buffer that is used in a mobile phase.
Never store silica based, reversed phase columns in buffer! These can cause
degradation of the silica resulting in loss of bonded phase. The buffer may also
precipitate in the column.
Never wash a reversed phase column with 100% water! This can cause the
hydrophobic “branches” of the bonded phase to collapse.
The best wash and storage solution for a reversed phase column is the mobile
phase without any buffer or organic modifier. The column should be rinsed with
this for about 15 minutes at the method flow rate, followed by a wash with a
higher organic version of the same solution for 15 to 60 minutes, then another 15
minutes with the wash solvent. The column should be stored in the wash
solution. If the wash solution contains less than 25% organic solvent, then a
subsequent wash of 25% organic solvent in water should be performed prior to
column storage to prevent microbial growth.
The column wash procedure should be performed on the HPLC that was used
for the analysis, thus washing the HPLC as well.

15.1.1 Upon Receipt of the Column
• Verify the column you received is the column you ordered.

• Check the column for physical damage that may have occurred during
shipping.
• Test the column immediately to verify quality and performance.
• All columns are shipped in the testing solvent, unless otherwise specified.
Each HPLC column is individually packed and tested to ensure outstanding

column quality. Every column is supplied with its Test Chromatogram and a
Specification Sheet that indicates conditions, operating parameters, column
serial number and identity.
Testing is especially important if the column is to be placed in storage. Test the

column using the same conditions in the test chromatogram and determine
column efficiency and peak asymmetry.
Chromatographic performance depends on the entire system, not just the

column. Columns are QC tested using optimum conditions to minimize band
spreading from “Extra Column Effects”.
15.1.2 Mobile Phase Considerations:
• Use only HPLC grade solvents
• Use only highest purity chemicals and reagents
• Degas and filter all mobile phases prior to use
• Make sure all solvents to be used are miscible
• Always check sample solubility
• If possible, use the mobile phase as the diluent (sample solvent)
• Maintain pH between 2.0 and 8.5
• Where possible use guard columns
• Avoid aldehydes and ketones with amino columns

15.1.3 Backpressure and Flow Rates:
• Keep backpressures below 3500psi (245bar), unless otherwise specified
• Avoid any sudden pressure changes
• If high backpressure is observed reverse flush the column
• Use a backpressure regulator if you are experiencing out-gassing problems in

the detector cell
Columns can be operated at any flow rate that is consistent with the
backpressure limitations described below. Flow rates should be optimized to
provide the highest efficiency for your sample.
Typical Column flow Rates & Backpressures
Particle Size Internal Diameter Typical Flow Rate (ml/min) Typical Pressure (psi)
(u) (mm) 150mm* 250mm*
3 4.6 0.5 985 1640

5 1.0 0.1 1500 2500
5 2.0 0.2 750 1250
5 3.2 0.5 732 1226
5 4.6 1.0 710 1180
5 10.0 5.0 750 1250
10 4.6 2.0 355 590
10 21.2 20.0 170 280
*Column length

15.2 Column Storage
Column storage conditions affect column lifetime
• Never store columns with buffers or ion-pairing reagents
• Flush with at least five column volumes of mobile phase without buffer to
remove any buffers or salts
Storage Conditions for Silica-Based HPLC Columns
Column Type Storage Solvent (Examples)
Reversed Phase 25:75
C18, C12, C8, C4, C2, C1, Phenyl MeOH: H2O
MeCN:H2O
Best: Mobile phase with no buffer

or organic modifiers.
Normal Phase 5 – 10% Isopropanol in Hexane
Silica, CN, NH2, PAC, Diol,

Alumina
*Ion-Exchange Follow manufacturer’s instructions.

Will vary with type, matrix and
DEAE, CM, SAX, SCX
usage.
*Size Exclusion 0.05% NaN3 in water or 10%

methanol
*Flush column with 50 ml water prior to storage solvent

15.3 Column Cleaning Procedures
Due to interactions between the stationary phase and sample components,

HPLC columns may occasionally require cleaning or regeneration. The
following conditions apply to silica-based columns with the exception of chiral
columns:
• Flow rates should be 1/5-1/2 of the typical flow rate
• To estimate the column volume, use the following equation:
V=r2L Where:
V = Column volume, ml
r = Column radius, cm
L = Column length, cm
UNBONDED SILICA COLUMN (Si) REVERSED PHASE COLUMNS (C18,

C12, C8, C4, C2, C1, PHENYL, CN,
Rinse with 10 column volumes each of:
NH2)
Hexane
Methylene Chloride
Rinse with 10 column volumes each of:
Isopropanol
Methylene Chloride
90% Water/10% Methanol or Acetonitrile
Mobile Phase
(for buffer removal)
Water Removal: Flush column with 300mL
2.5% 2,2-dimethoxy propane and 2.5%
90% Methanol or Acetonitrile/10% water
glacial acetic acid in hexane
Mobile phase

16. Session Three Assignment
6) What is meant by column particle size? What is meant by pore size?

What impact do these parameters have on analyte retention? How
do these parameters affect resolution?
7) How does column packing material shape affect chromatography

(consider irregular versus spherical particles_? What impact do
these parameters have on analyte retention and peak shape?

17. HPLC Known Impurities – External Standard Calculation
 Weight of Impurity Standard = 102.21mg
 Weight of Sample A= 101.39 mg
 Weight of Sample B = 101.18 mg
 Purity of Impurity Standard = 99.6%
 Standard Dilution Scheme = 200 ml Volumetric flask, 5.0ml to 100ml volumetric
flask
 Sample Dilution Scheme = 200 ml Volumetric Flask
 Areas as detected by HPLC
7. Sample A – 785
8. Sample B - 774
9. Check Standard – 7783
 Does standard "A" meet the criteria of NMT 6.0% for six injections?
 Does the sample comply with a specification of NMT 1.0% for this Impurity?
% Result Complies (y/n)

RSD of Standard Injections
% Impurity in sample A
% Impurity in sample B

Session Three Experiment – System Suitability via In-House
Method
▪ Introduction to System Suitability
▪ Calculation of various system suitability parameters
▪ Two component sample
Initial Mobile Phase
Prepare 300 ml of 50:50 methanol and water. Filter and degas.
Preparation of System suitability Solution (Resolution solution)

Place 5 mg of acetaminophen and 100mg of ASA in a 250ml volumetric flask,
dissolve in 10 mL methanol, add 125 mL water and dilute to volume with
methanol. Filter solution through a 0.45 m filter into an autosampler vial.
▪ Column – 3.9 x 300 mm or 4.6 x 250 mm L1 column (10m)

▪ Flow rate – 1.5ml/min
▪ Run time – Approximately 10 minutes
 After allowing the HPLC to come to equilibrium with your mobile phase,
inject your System Suitability Solution (Label as Sys Suit 1).
 Add one drop of glacial acetic acid to your mobile phase. After allowing
the HPLC to come to equilibrium with your mobile phase, inject your
System Suitability Solution (Label as Sys Suit 2 ).
 Add 100ml of methanol. After allowing the HPLC to come to equilibrium
with your mobile phase, inject your System Suitability Solution (Label as
Sys Suit 3).
 Compare the effect that mobile phase changes have on your
chromatography. Calculate tailing factor, column efficiency and
resolution for all chromatograms produced.

Mobile Phase (Session Four)
The other main phase of chromatographic analysis is the mobile phase. Mobile phases
must be made with as pure materials as possible. Organic impurities can cause high
baselines, noise and eventual decrease in performance of analytical columns. Inorganic
impurities can lead to reactions on the silica supports, causing loss of bonded phase
and eventual decomposition of the column itself. Mobile phases for normal phase HPLC
are usually hydrophobic in nature and are non-polar to slightly polar. Mobile phases for
reversed phase HPLC are generally hydrophilic and can range from moderately polar
(pure solvent such as methanol or acetonitrile) to strongly ionic buffer systems with
small amounts of solvents.
17.1 Role of the Mobile Phase
The mobile phase is the mobility in a chromatographic system. The

analytes must be completely soluble in order to have the analyte elute
from the end of the column. Care must be taken in the preparation of
mobile phases. Poor mixing can cause retention time shifts and inaccurate
preparation will result in unexpected chromatograms with poor resolution,
poor peak shape or very long or short retention times.
17.2 Purpose – What to Make
Mobile phases must be chosen to balance the interaction between the

analyte and the stationary phase. If the mobile phase chosen is too strong
(i.e. less polar for a reversed phase system or too polar for a normal
phase system), the analyte molecules will favour the mobile phase for the
solid phase and not interact with the solid phase resulting in short
chromatograms that do not effectively resolve the analyte(s). If the mobile
phase is too weak (i.e. too polar for a reversed phase system or two non-
polar for a normal phase system) the analyte will favour the solid phase
over the mobile phase resulting in excessively long chromatograms, or
even no chromatogram at all if the analyte(s) do not elute at all. Often,
performance can be enhanced by the use of organic modifiers that inhibit
some unwanted interactions between the analyte and the stationary

phase, or conversely can enhance the interaction of the analyte(s) with
the stationary phase.
17.3 Composition
Variation of the eluent composition allows adjustment of the retention

times of target components in the mixture to the desired values. Retention
times can be varied from elution with the solvent front to never eluting at
all. Thus, there is much flexibility in the elution of different components by
simple alteration of the ratio of organic to aqueous content. The drawback
of this is that the longer an analyte remains on the column, the broader
will be the final peak. Thus, a balance must be determined in which
adequate separation is achieved while keeping the peak shape and run
times within acceptable levels. If the peak elutes too early, there may also
be interference with other materials in the sample matrix and even the
solvent front. When simple changes in composition do not effect the
separation as desired, other means can be employed to increase
separation without sacrificing peak quality.
17.4 Buffers and pH – Analyte Effects
Buffers are quite often used for analysis of pharmaceuticals by HPLC.

Buffers stabilize pH within the chromatography allowing analytes to be
either in their protonated or non-protonated states. This can enhance the
capacity of the chromatography, for example, by allowing normally
ionizable molecules to remain in their non-ionized state – reducing the
polarity and thus enhancing the interaction of the molecule with the
bonded phase in a reversed phase system. Buffers can be selected to
stabilize a pH range that will allow other, interfering materials to be fully
ionized, thus having little retention on a reversed phase column. The most
common buffers used in Pharmaceutical analysis are Phosphate, Acetate
and Citrate buffers.

17.5 Organic Modifiers
Organic modifiers are organic compounds or solvents that are added in

small amounts but usually have a large effect on retention and peak
shape of any particular analyte. Some examples of organic modifiers are
triethylamine (usually with pH adjustment), tetrahydrofuran (THF),
ethylene diamine (with pH adjustment) and ethylenediaminetetracetic acid
(EDTA).
Another class of Organic modifiers are Ion Pair reagents. These are in the
form of sulphonated alkyl chains as shown below:
R – SO3-Na+
where “R” is usually an alkyl group of chain length from 1 to 12. These are
useful reagents when the analyte is an ionic molecule that does not have
good retention on a reverse phase column. The emulsifying action of the
ion pair reagent causes the ionic molecule to slow down in the alkyl end
will be attracted to the solid phase while the polar/ionic end will attract the
ionic analyte. These will be used in conjunction with a buffer and quite
often with triethylamine as well.
17.6 Mobile Phase Preparation
Preparation of the mobile phase is extremely important. Composition

needs to be as specified in the procedure to be followed in order to
achieve the desired separations and peak performances. The more
accurate the preparation can be then the better will be the final
chromatography.
Major steps in mobile phase preparation:
• Measure appropriate volume of each solvent
• Mix solvents
• Add buffers and additives
• Filter mobile phase
• Degas mobile phase

17.6.1 Choice of Solvents
As mentioned previously, all solvents must be high purity with a very low
UV cutoff range. Water for mobile phases must be Type 1 grade, i.e.
purified by reverse osmosis or distillation, ion exchange and filtered to at
least 0.45 µm. This is the USP definition of HPLC grade water:
Water, HPLC Grade, H2O (MW = 18.02, Colorless liquid)
Absorption Characteristics:
Determine the ultraviolet absorbance in a 1 cm cell, using water as
the blank. The maximum absorbances are 0.005, 0.01, and 0.01 at
400 nm to 250 nm, 200 nm, and 190 nm respectively.
Residue on evaporation:
Evaporate an accurately measured volume on a steam bath to

dryness, and dry the residue at 105° for 1 hour: the limit is 3 ppm.
Mobile phase preparation can be divided into the following major steps:
• Mixing (Buffer preparation)

• Filtration
• Degassing
17.6.2 Buffer Preparation
Buffers must be prepared in the aqueous part of the mobile phase.

Calculate required buffer salt to make the desired concentration in prepare
1 or 2 litres of water. Dissolve the salt in approximately 85 % of the total
volume of water (i.e. 850 ml water to make 1 litre or 1700 ml of water to
make 2 litres) in an appropriate sized Erlenmeyer flask. Adjust to the
desired pH and transfer to a 1 or a 2 litre volumetric flask. Rinse the
mixing flask with 2 small portions of water (about 25 ml each) and dilute
almost to volume. Mix the solution by inverting the (stopper) flask several
times. Check the pH of the solution and adjust if necessary. Dilute to
volume and mix.

Most mobile phases are a mixture of at least two solvents. They may also
contain buffers and other additives. Always measure each part of the
mobile phase separately and then combine.
17.6.3 Filtration
All buffers and prepared solvent mixtures must be filtered through a 0.45
um or smaller membrane filter. This will remove any particulates that are
present in the solution. Vacuum filtration also serves a dual purpose in
degassing the mobile phase. Filters available today are made of many
different types of materials for different purposes. Some filters are not
compatible with different solvents, thus care must be taken in choosing
the correct filter type. One of the most versatile materials is Nylon 66.
These filters will handle most of the aqueous mobile phases available for
reverse phase. They are compatible with buffer systems and with many
organic modifiers in dilute concentrations with water (see membrane
selection chart).
Figure 42 - Typical Mobile Phase Filtration Devices

17.6.4 Degassing
Degassing can be performed efficiently using vacuum filtration through a

suitable membrane filter. This will remove all of the dissolved gasses in
the mobile phase, however, air dissolves well in aqueous and hydrophilic
solvents and solutions. Thus as soon as the mobile phase is transferred
out of the filter flask, air is again dissolved in it. Most flasks for storing
mobile phase cannot withstand much vacuum, thus vacuum filtration into
these directly is not an option. Other means include heating and
sonication; however, these can changes to your mobile phase if volatile
organics are present. Bubbling a volatile gas through the media will
displace other dissolved gasses. Since liquids can only dissolve a fixed
amount of any gas at room temperature, a large excess of one gas will
cause other gasses to be expelled. If this gas is very volatile, it will not
remain in solution and the solution will become gas free. The best gas for
this purpose is Helium. A “sparge” of helium gas over a relatively short
period of time will remove any dissolved air in an aqueous system. By
keeping a constant flow of helium gas through the mobile phase, the
mobile phase will remain air-free.
When a mobile phase on the HPLC system has been idle, there is always
the possibility that air has managed to permeate to the felow. Priming the
HPLC system, which involves pimping each channel at 100% composition
and high flow rate until steady pressure and flow are obtained, is highly
recommended.
17.7 Sample Preparation
Analytes must be soluble in the mobile phase. If there is any doubt, a trial
should be performed to ensure the sample solution does not precipitate
the mobile phase or visa versa. The ideal solvent for the analyte is mobile
phase. If this is not feasible for any reason, then the analyte should be
dissolved in a solvent system that is weaker than the mobile phase - i.e.
for a reversed phase system, choose a solvent that is more aqueous and

for a normal phased system, choose a solvent that is less polar or more
hydrophobic than the mobile phase. This will ensure that your analyte will
not be carried through the column on a “plug” of strong solvent passing
through the column.
All samples should be filtered prior to injection onto an HPLC system. The
best filters to use have a nominal pore size of 0.45 um or less. Disposable
syringe filters are available for this purpose and the membranes discussed
for filtering mobile phases are all available (see section 4.6.3) Filters
should be evaluated prior to use to establish saturation levels of the
analyte. Often 5 to 10 ml of sample solution is required to saturate the
filter. Filter studies are a common part of most analytical method
validations. Syringe filters are available in many sizes – from 3 mm to 47
mm – however, the normal size for pharmaceutical analysis is 25 mm.
Filters should not be re-used. Each sample or standard is unique and
should be treated individually in order to avoid cross-contamination
between samples and standards. Smaller filters have an advantage in that
the amount of analyte solution required for saturation is generally less
than a larger filter. However, small filters are usually more difficult to pass
solution through them, since they can become clogged easily. If there are
any visible particulates in the analyte solution, a pre-filter of larger pore
size should be used prior to the final filter. This filtration can be of a gravity
type using filter paper and a funnel, or a larger disposable filter can be
attached in series with the final filter. A common pre-filter is a glass fibre
filter of 1 to 3 um pore size. Any pre-filtration step must be evaluated to
determine the saturation volume required.

18. Good Practices for HPLC Analysis
18.1 Glassware Labelling
All Glassware used must be labeled for safety reasons and for GMP/GLP.
Your labels should contain:
• All components contained in the glassware. If there is methanol, acetonitrile

and sample X, then put it on the label.
• Your initials.
• The testing method.
• Date of Preparation.
• Expiry Date.
Details about the testing itself - Is this a stock solution or a working solution? Is
this the third dilution in a series of five? The more you put on a label, the more
organized you’ll be and the less likely you are to make a mistake.
18.2 Labelling of Chromatograms
All chromatograms must be labeled for correct identification. An HPLC run will
contain standard chromatograms, system suitability chromatograms, sample
chromatograms (often duplicate samples and multiple lots) and check/control
standards. Each of these needs to be clearly identified to avoid the possibility of
mix-ups. Normally this can be accomplished through the chromatographic
software in the sequence table.
18.3 Documentation
Good documentation in a pharmaceutical laboratory (especially a Quality Control

Laboratory) is critical. The basic analogy used by regulatory agencies is “if it is
not documented it was not done”. This implies that every procedure that was
performed must be fully documented at all steps. As mentioned in section 1.9,
records are to be made which are traceable to the batch in question. All weights
must be clearly shown as to the sample origin and all calculations must be
shown up to final concentrations. Standards must be identified as to their origin

and expiry. Again all calculations must be clearly documented to show the final
concentrations being used. If factors in a sequence table are used for
calculations of final results, the source of these factors must be identified and
sample calculations must indicate their production. All units in the calculations
must balance to yield the final desired units.

19. Session Four Assignment
8) HPLC Internal Standard Method – Assay

 Weight of Sample A = 51.21mg
 Weight of Sample B = 52.14mg
 Weight of Standard A = 50.75 mg
 Weight of Standard B = 51.25 mg
 Purity of Standard = 99.8%
 Injections of 5μl
 244nm, flow rate 1.5ml/min
 Sample Dilution Scheme = 200 ml Volumetric flask, 5.0ml to 100ml volumetric
flask
 Standard Dilution Scheme = 200 ml Volumetric Flask, 5.0ml to 100ml volumetric
flask
 Areas as detected by HPLC-UV
Injection Area of Active Area of Internal Std
Standard A 98215 63148
Standard B 69421 43937
Sample A 98725 63218
Sample B 88993 56324
Chk Standard 98552 63981
 Does standard A meet a criteria of an RSD of NMT 2.0% for the area ratios of the
five standard injections?
 Does Standard B meet a 98.0-102.0% criterion?
 Does the check standard meet a 97.0-103.0% criterion?
 Does the sample comply with a specification of 98.0-102.0%? If not, what should
be done?

Sample % Result Complies (y/n)
RSD of Standard A Injections
Recovery Standard (standard B)
Drift (Check Standard)
A
B

Session Four Experiment – In-House Raw Material Assay
▪ Introduction to sample sets via a raw material assay
▪ Preparation of solutions need to run a pharmaceutical sequence
▪ Identification via HPLC
Mobile Phase
Prepare 200ml of 50:50 methanol and water. Filter and degas.
Preparation of Standard (Prepare in Duplicate)
Place 10 mg of acetaminophen in a 250ml volumetric flask, dilute to volume with

50:50 methanol and water.
Preparation of Sample (Prepare in Duplicate)
Place 10 mg of acetaminophen in a 250ml volumetric flask, dilute to volume with

50:50 methanol and water.
▪ Column – 300 mm x 3.9 mm or 250 mm x 4.6 mm ODS, 10 m packing

▪ Flow rate – 1.5ml/min
▪ Run time - 10 minutes
 After allowing the HPLC to come to equilibrium with your mobile

phase, inject your standard A five times, Standard B once and your
Sample A and Sample B once each respectively. At the end of your
run inject your Standard A again as a check standard.
 Does standard A comply with an RSD<2.0% for five injections?
 Does the standard B comply with a specification of 98.0-102.0%?
 Does the check standard comply with a specification of 97.0-
103.0%
 Do Sample A and B comply with a specification of 98.0-101.0%?
Session Five – Assay of a Finished Product
Session Five Experiment
Proceed as per Acetaminophen Tablets USP monograph. You will be assigned
tablets by your instructor.
 After allowing the HPLC to come to equilibrium with your mobile

phase, inject your standard A five times, Standard B Once and your
Sample A and Sample B once each respectively. At the end of your
run inject your Standard A again as a check standard.
 Does standard A comply with an RSD<2.0% for five injections?
 Does the Recovery for standard B comply with a specification of
98.0-102.0%?
 Does the System Drift comply with a specification of 98.0-102.0%
 Does the Product comply with the USP Specification?
Calculations
PurityStd (%)
Wt Std (mg )   Dil Std (1 / ml )
ASpl 100(%) AvgWt(mg / DU )
% LC =    100(%)
AStd Wt Spl (mg )  Dil Spl (1 / ml ) LC (mg / DU )
AStd 2 WtStd1 (mg )

=
%Recovery   100(%)
AStd1 WtStd 2 (mg )
ACheckStd
%Drift=  100(%)
AStd1

Acetaminophen Tablets
» Acetaminophen Tablets contain not less than 90.0 percent and not more than
110.0 percent of the labeled amount of acetaminophen (C8H9NO2).
Packaging and storage—Preserve in tight containers, and store at controlled room
temperature.
Labeling—Label Tablets that must be chewed to indicate that they are to be chewed before
swallowing.
USP Reference standards 11 —
USP Acetaminophen RS .
Identification—
A: The retention time of the major peak in the chromatogram of the Assay preparation
corresponds to that in the chromatogram of the Standard preparation, as obtained in the
Assay.
B: Triturate an amount of powdered Tablets, equivalent to about 50 mg of acetaminophen,
with 50 mL of methanol, and filter: the clear filtrate (test solution) responds to the Thin-layer
Chromatographic Identification Test 201 , a solvent system consisting of a mixture of
methylene chloride and methanol (4:1) being used.
Dissolution 711 —
Medium: pH 5.8 phosphate buffer (see Buffer Solutions in the section Reagents, Indicators,
and Solutions); 900 mL.
Apparatus 2: 50 rpm.
Time: 30 minutes.
Procedure—Determine the amount of C8H9NO2 dissolved by employing UV absorption at the
wavelength of maximum absorbance at about 243 nm on filtered portions of the solution under
test, suitably diluted with Dissolution Medium, if necessary, in comparison with a Standard
solution having a known concentration of USP Acetaminophen RS in the same Medium.
Tolerances—Not less than 80% (Q) of the labeled amount of C8H9NO2 is dissolved in 30
minutes.
FOR TABLETS LABELED AS CHEWABLE—
Medium: pH 5.8 phosphate buffer (see Buffer Solutions in the section Reagents, Indicators,
and Solutions); 900 mL.
Time: 45 minutes.
Procedure—Proceed as directed for Procedure for Acetaminophen Tablets.
Tolerances—Not less than 75% (Q) of the labeled amount of C8H9NO2 is dissolved in 45
minutes.
Uniformity of dosage units 905 : meet the requirements.

Assay—
Mobile phase, Standard preparation, and Chromatographic system—Proceed as directed in
the Assay under Acetaminophen Capsules.
Assay preparation—Weigh and finely powder not fewer than 20 Tablets. Transfer an
accurately weighed portion of the powder, equivalent to about 100 mg of acetaminophen, to a
200-mL volumetric flask, add about 100 mL of Mobile phase, shake by mechanical means for
10 minutes, sonicate for about 5 minutes, dilute with Mobile phase to volume, and mix.
Transfer 5.0 mL of this solution to a 250-mL volumetric flask, dilute with Mobile phase to
volume, and mix. Pass a portion of this solution through a filter having a 0.5-µm or finer
porosity, discarding the first 10 mL of the filtrate. Use the clear filtrate as the Assay
preparation.
Procedure—Proceed as directed for Procedure in the Assay under Acetaminophen Capsules.
Calculate the quantity, in mg, of acetaminophen (C8H9NO2) in the portion of Tablets taken by
the formula:
10,000C(rU / rS)
in which C is the concentration, in mg per mL, of USP Acetaminophen RS in the Standard
preparation; and rU and rS are the acetaminophen peak responses obtained from the Assay
preparation and the Standard preparation, respectively.
Auxiliary Information—Staff Liaison : Clydewyn M. Anthony, Ph.D., Scientist
Expert Committee : (MDCCA05) Monograph Development-Cough Cold and Analgesics
USP31–NF26 Page 1271
Pharmacopeial Forum : Volume No. 27(3) Page 2495
Phone Number : 1-301-816-8139
Chromatographic Reagent—
ACETAMINOPHEN TABLETS
Chromatographic reagents text is not derived from, and not part of, USP 31 or NF 26.

Attachments
19.1 Membrane Selection Guide
This table offers general guidelines for membrane characteristics and applications.
Membrane Type Membrane Characteristics & Application
Regenerated Regenerated Cellulose (RC) is a hydrophilic, solvent resistant, low protein
Cellulose binding membrane. Membrane of choice for low non-specific binding
(biological analysis, protein, peptides). Ideal for removing particulates from
HPLC samples prior to injection. Compatible with all HPLC solvents. Use
for particle removal and degassing of solvents. RC membranes are also
compatible with aqueous solutions in the 3 to 12 pH range. Extractables
with water are less than 1%. When used with a glass pre-filter, RC is ideal
for tissue culture media filtration, as well as general biological sample
filtration.
Nylon A naturally hydrophilic membrane that requires no pre-wetting. Nylon is
extremely low in extractables and is mechnically strong. Can withstand
solution up to 50° C. Can be used with organic or aqueous solutions. Flow
rate is excellent. Not recommended for strong acids or bases.
Highly recommended for general laboratory filtration and filtration of HPLC
samples prior to injection. Nylon binds protein and should not be used
when protein recovery is important.
PTFE PTFE (polytetrafluoroethylene) is hydrophobic and chemically resistant1 to
all solvents, acids, and bases. Membrane is mechnically strong and can
withstand high temperature liquids. Requires prewetting with an alcohol
prior to use with aqueous solutions. Very low in extractables PTFE natural
hydrophobicity blocks water vapor, making it ideal for transducer
protectors. Also ideal for filtering-degassing organic and highly basic
mobile phase solutions for liquid chromatography.
PVDF PVDF (polyvinylidene difluoride) is a hydrophilic, solvent resistant1
membrane that exhibits low levels of UV adsorbing extractables Low non-
specific binding character.
Use for HPLC sample filtration and general biological filtration. PVDF is a
low protein binding membrane.
Polypropylene Polypropylene is a hydrophilic membrane that exhibits a wide range of
chemical compatibility to organic solvents. A good choice for HPLC
sample filtration when performing chromatography protein analysis. Highly
solvent resistant. Low non-specific adsorbing membrane for maximum
protein recovery in critical analysis. Also well suited for biological sample
filtration.
Glass Microfiber (GMF) membranes are commonly used as pre-filters to
Glass Microfiber remove large particulates and to extend the load capacity of the
membrane. Membrane of choice for dissolution testing.
Is a very low protein binding membrane, ideal for aqueous based samples.
CA membranes are an excellent choice when maximum protein recovery
Cellulose Acetate
in filtrate is critical. Laboratory studies show that CA membranes bind less
(CA)
protein than PVDF. CA is ideal for tissue culture media filtration and
sensitive biological samples.
PES (Polyethersulfone) provides high flow rates and good throughput
volume. This membrane is low protein binding and can be used with high
temperature liquids. PES is a mechanically strong membrane and is
Polyethersulfone resistive to a broad range of aqueous solvents. PES is the filter of choice
for tissue culture work‹very low extractables and low binding of protein and
nucleic acids. Good to excellent flow rates. PES is certified for Ion
Chromatography.

19.2 Syringe Filter Compatability Guide
Chart Legend
C = Compatible | LC = Limited Compatibility (membrane swells and shrinks)
NC = Not Compatible | ND = No Data Available | PTFE = Polytetrafluoroethylene
PVDF = Polyvinylidene Difluoride | PES = Polyethersulfone | CA = Cellulose Acetate
RC = Regenerated Cellulose | PP = Polypropylene | GMF = Glass MicroFiber
Chemical Nylon PTFE PVDF PES CA RC PP GMF
ACIDS
Acetic, Glacial LC C C C NC C C C
Acetic, 25% C C C C C C C C
Hydrochloric, Concentrated NC C C C NC NC C C
Hydrochloric, 25% NC C C C NC NC C C
Sulfuric, Concentrated NC C NC NC NC NC C C
Sulfuric, 25% NC C C C NC LC C C
Nitric, Concentrated NC C C NC NC NC C LC
Nitric, 25% NC C C C NC NC C LC
Phosphoric, 25% NC C ND ND C LC C ND
Formic, 25% NC C ND ND LC C C C
Trichloroacetic, 10% NC C ND ND C C C ND
ALKALIES
Ammonium Hydroxide,
C C LC C C LC C C
25%
Sodium Hydroxide, 3
C C C C NC LC C NC
Normal
ALCOHOLS
Methanol, 98% C C C C C C C C
Ethanol, 98% C C C C C C C C
Ethanol, 70% LC C C C C C C C
Isopropanol C C C C C C C C
n-Propanol C C C C C C C C
Amyl Alcohol, Butanol C C C C C C C C
Benzyl Alcohol C C C ND LC C C NC
Ethylene Glycol C C C C C C C C
Propylene Glycol C C C C LC C C C
Glycerol C C C C C C C C
HYDROCARBONS
Hexane, Xylene C C C NC C C NC C
Toluene, Benzene C C C NC C C NC C
Kerosene, Gasoline C C C LC C C LC ND
Tetralin, Decalin ND C C ND C C ND ND
HALOGENATED
HYDROCARBONS
Methylene Chloride LC C C NC NC C LC C
Chloroform C C C NC NC C LC C
Trichloroethylene C C C NC C C C C
Monochlorobenzene C C C LC C C C C
Freon C C C LC C C C C
Carbon Tetrachloride C C C NC LC C LC C
KETONES
Acetone C C NC NC NC C C C

Cyclohexanone C C NC NC NC C C C
Methyl Ethyl Ketone C C LC NC LC C LC C
Isopropylacetone C C NC NC C C ND C
Methyl Isobutyl Ketone ND C LC NC ND C LC C
ESTERS
Ethyl & Methyl Acetate C C C NC NC C LC C
Amyl & Butyl Acetate C C NC NC LC C LC C
Propyl Acetate C C NC NC LC C LC ND
Propylene Glycol Acetate ND C ND NC NC C C ND
2-Ethoxyethyl Acetate ND C ND NC LC C ND ND
Methyl Cellosolve Acetate ND C ND NC NC C C C
Benzyl Benzoate C C ND NC C C ND ND
Isopropyl Myristate C C ND NC C C ND ND
Tricresyl Phosphate ND C ND NC C C ND ND
OXIDES-ETHERS
Ethyl Ether C C C C C C LC ND
Dioxane C C LC NC NC C C C
Tetrahydrofuran C C LC NC NC C C C
Triethanolamine C C ND ND C C ND ND
Dimethylsulfoxide (DMSO) C C NC NC NC C C C
Isopropyl Ether ND C C C C C C ND
SOLVENTS WITH
NITROGEN (AMIDES)
Dimethyl Formamide LC C NC NC NC LC C C
Diethylacetamide C C ND ND NC C ND C
Triethanolamine C C ND ND C C ND ND
Aniline ND C ND ND NC C ND ND
Pyridine C C C NC NC C NC C
Acetonitrile C C LC LC NC C C C
MISCELLANEOUS
Phenol, Aqueous, 10% ND C LC NC NC NC C C
Formaldehyde Solution,
C C C C C LC C C
30%
Hydrogen Peroxide, 30% C C ND ND C C ND ND
Silicone Oil & Mineral Oil ND C C C C C C C
Pyridine C C C NC NC C LC C

19.3 <621> CHROMATOGRAPHY
INTRODUCTION
Chromatographic separation techniques are multistage separation methods in which the components of a sample
are distributed between two phases, of which one is stationary and the other mobile. The stationary phase may
be a solid or a liquid supported on a solid or a gel. The stationary phase may be packed in a column, spread as a
layer, distributed as a film, or applied by other techniques. The mobile phase may be gaseous or liquid or
supercritical fluid. The separation may be based on adsorption, mass distribution (partition), or ion exchange; or it
may be based on differences among the physicochemical properties of the molecules, such as size, mass, and
volume. This chapter contains general procedures, definitions, and calculations of common parameters and
describes general requirements for system suitability. The types of chromatography useful in qualitative and
quantitative analysis employed in USP procedures are column, gas, paper, thin-layer (including high-performance
thin-layer chromatography), and pressurized liquid chromatography (commonly called high-pressure or high-
performance liquid chromatography).
GENERAL PROCEDURES
This section describes the basic procedures used when a chromatographic method is described in a monograph.
The following procedures are followed unless otherwise indicated in the individual monograph.
Paper Chromatography
Stationary Phase: The stationary phase is a sheet of paper of suitable texture and thickness. Development may
be ascending, in which the solvent is carried up the paper by capillary forces, or descending, in which the solvent
flow is also assisted by gravitational force. The orientation of paper grain with respect to solvent flow is to be kept
constant in a series of chromatograms. (The machine direction is usually designated by the manufacturer.)
Apparatus: The essential equipment for paper chromatography consists of a vapor-tight chamber with inlets for
addition of solvent and a rack of corrosion-resistant material about 5 cm shorter than the inside height of the
chamber. The rack serves as a support for solvent troughs and for antisiphon rods that, in turn, hold up the
chromatographic sheets. The bottom of the chamber is covered with the prescribed solvent system or mobile
phase. Saturation of the chamber with solvent vapor is facilitated by lining the inside walls with paper wetted with
the prescribed solvent system.
Spotting: The substance or substances analyzed are dissolved in a suitable solvent. Convenient volumes,
delivered from suitable micropipets, of the resulting solution, normally containing 1–20 µg of the compound, are
placed in 6- to 10-mm spots not less than 3 cm apart.
Descending Paper Chromatography Procedure
1. A spotted chromatographic sheet is suspended in the apparatus, using the antisiphon rod to hold the
upper end of the sheet in the solvent trough. [NOTE—Ensure that the portion of the sheet hanging
below the rods is freely suspended in the chamber without touching the rack, the chamber walls, or the
fluid in the chamber. ]
2. The chamber is sealed to allow equilibration (saturation) of the chamber and the paper with the solvent
vapor. Any excess pressure is released as necessary.
3. After equilibration of the chamber, the prepared mobile phase is introduced into the trough through the
inlet.
4. The inlet is closed, and the mobile solvent phase is allowed to travel the desired distance down the
paper.
5. The sheet is removed from the chamber.
6. The location of the solvent front is quickly marked, and the sheet is dried.
7. The chromatogram is observed and measured directly or after suitable development to reveal the location
of the spots of the isolated drug or drugs.

Ascending Paper Chromatography Procedure
1. The mobile phase is added to the bottom of the chamber.
2. The chamber is sealed to allow equilibration (saturation) of the chamber and the paper with the solvent
vapor. Any excess pressure is released as necessary.
3. The lower edge of the stationary phase is dipped into the mobile phase to permit the mobile phase to rise
on the chromatographic sheet by capillary action.
4. When the solvent front has reached the desired height, the chamber is opened, the sheet is removed, the
location of the solvent front is quickly marked, and the sheet is dried.
5. The chromatogram is observed and measured directly or after suitable development to reveal the location
of the spots of the isolated drug or drugs.
Thin-Layer Chromatography
Stationary Phase: The stationary phase is a relatively thin, uniform layer of dry, finely powdered material applied
to a glass, plastic, or metal sheet or plate (typically called the plate). The stationary phase of TLC plates has an
average particle size of 10–15 µm, and that of high-performance TLC (HPTLC) plates has an average particle
size of 5 µm. Commercial plates with a preadsorbent zone can be used if they are specified in a monograph.
Sample applied to the preadsorbent region develops into sharp, narrow bands at the preadsorbent–sorbent
interface. The separations achieved may be based on adsorption, partition, or a combination of both effects,
depending on the particular type of stationary phase.
Apparatus: A chromatographic chamber made of inert, transparent material and having the following
specifications is used: a flat-bottom or twin trough, a tightly fitted lid, and a size suitable for the plates. The
chamber is lined on at least one wall with filter paper. Sufficient mobile phase or developing solvent is added to
the chamber that, after impregnation of the filter paper, a depth appropriate to the dimensions of the plate used is
available. The chromatographic chamber is closed and allowed to equilibrate. [NOTE—Unless otherwise
indicated, the chromatographic separations are performed in a saturated chamber. ]
Detection/Visualization: An ultraviolet (UV) light source suitable for observations under short- (254 nm) and long-
(365 nm) wavelength UV light and a variety of other spray reagents used to make spots visible are often used.
Spotting: Solutions are spotted on the surface of the stationary phase (plate) at the prescribed volume in
sufficiently small portions to obtain circular spots of 2–5 mm in diameter (1–2 mm on HPTLC plates) or bands of
10–20 mm × 1–2 mm (5–10 mm × 0.5–1 mm on HPTLC plates) at an appropriate distance from the lower edge of
and sides of the plate. [NOTE—During development, the application position must be at least 5 mm (TLC) or 3
mm (HPTLC) above the level of the mobile phase. ] The solutions are applied on a line parallel to the lower edge
of the plate with an interval of at least 10 mm (5 mm on HPTLC plates) between the centers of spots, or 4 mm (2
mm on HPTLC plates) between the edges of bands, then allowed to dry.
Procedure
1. Place the plate in the chamber, ensuring that the spots or bands are above the surface of the mobile
phase.
2. Close the chamber.
3. Allow the mobile phase to ascend the plate until the solvent front has traveled three-quarters of the length
of the plate, or the distance prescribed in the monograph.
4. Remove the plate, mark the solvent front with a pencil, and allow to dry.
5. Visualize the chromatograms as prescribed.
6. Determine the chromatographic retardation factor (RF) values for the principal spots or zones.
7. Presumptive identification can be made by observation of spots or zones of identical R F value and about
equal magnitude obtained, respectively, with an unknown and a standard chromatographed on the same
plate. A visual comparison of the size or intensity of the spots or zones may serve for semiquantitative
estimation. Quantitative measurements are possible by means of densitometry (absorbence or
fluorescence measurements).

Column Chromatography
Solid Support: Purified siliceous earth is used for normal-phase separation. Silanized chromatographic siliceous
earth is used for reverse-phase partition chromatography.
Stationary Phase: The solid support is modified by the addition of a stationary phase specified in the individual
monograph. If a mixture of liquids is used as the stationary phase, mix the liquids before the introduction of the
solid support.
Mobile Phase: The mobile phase is specified in the individual monograph. If the stationary phase is an aqueous
solution, equilibrate with water. If the stationary phase is a polar organic fluid, equilibrate with that fluid.
Apparatus: Unless otherwise specified in the individual monograph, the chromatographic tube is about 22 mm in
inside diameter and 200–300 mm long. Attached to it is a delivery tube, without stopcock, about 4 mm in inside
diameter and about 50 mm long.
APPARATUS PREPARATION: Pack a pledget of fine glass wool in the base of the tube. Combine the specified volume
of stationary phase and the specified amount of solid support to produce a homogeneous, fluffy mixture. Transfer
this mixture to the chromatographic tube, and tamp, using gentle pressure, to obtain a uniform mass. If the
specified amount of solid support is more than 3 g, transfer the mixture to the column in portions of approximately
2 g, and tamp each portion. If the assay or test requires a multisegment column with a different stationary phase
specified for each segment, tamp after the addition of each segment, and add each succeeding segment directly
to the previous one. Pack a pledget of fine glass wool above the completed column packing. [NOTE—The mobile
phase should flow through a properly packed column as a moderate stream or, if reverse-phase chromatography
is applied, as a slow trickle. ]
If a solution of the analyte is incorporated into the stationary phase, complete the quantitative transfer to the
chromatographic tube by scrubbing the beaker used for the preparation of the test mixture with a mixture of about
1 g of Solid Support and several drops of the solvent used to prepare the sample solution before adding the final
portion of glass wool.
Procedure
1. Transfer the mobile phase to the column space above the column packing, and allow it to flow through
the column under the influence of gravity.
2. Rinse the tip of the chromatographic column with about 1 mL of mobile phase before each change in
composition of mobile phase and after completion of the elution.
3. If the analyte is introduced into the column as a solution in the mobile phase, allow it to pass completely
into the column packing, then add mobile phase in several small portions, allowing each to drain
completely, before adding the bulk of the mobile phase.
4. Where the procedure indicates the use of multiple chromatographic columns mounted in series and the
addition of mobile phase in divided portions is specified, allow each portion to drain completely through
each column, and rinse the tip of each with mobile phase before the addition of each succeeding
portion.
Gas Chromatography (GC)
Liquid Stationary Phase: This type of phase is available in packed or capillary columns.
Packed Column GC: The liquid stationary phase is deposited on a finely divided, inert solid support, such as
diatomaceous earth, porous polymer, or graphitized carbon, which is packed into a column that is typically 2–4
mm in internal diameter and 1–3 m in length.
Capillary Column GC: In capillary columns, which contain no packed solid support, the liquid stationary phase is
deposited on the inner surface of the column and may be chemically bonded to it.
Solid Stationary Phase: This type of phase is available only in packed columns. In these columns the solid phase
is an active adsorbent, such as alumina, silica, or carbon, packed into a column. Polyaromatic porous resins,
which are sometimes used in packed columns, are not coated with a liquid phase. [NOTE—Packed and capillary
columns must be conditioned before use until the baseline and other characteristics are stable. The column or
packing material supplier provides instructions for the recommended conditioning procedure. ]

Apparatus: A gas chromatograph consists of a carrier gas source, injection port, column, detector, and recording
device. The injection port, column, and detector are temperature controlled and may be varied as part of the
analysis. The typical carrier gas is helium, nitrogen, or hydrogen, depending on the column and detector in use.
The type of detector used depends on the nature of the compounds analyzed and is specified in the individual
monograph. Detector output is recorded as a function of time, and the instrument response, measured as peak
area or peak height, is a function of the amount present.
Temperature Program: The length and quality of a GC separation can be controlled by altering the temperature of
the chromatographic column. When a temperature program is necessary, the individual monograph indicates the
conditions in table format. The table indicates the initial temperature, rate of temperature change (ramp), final
temperature, and hold time at the final temperature.
Procedure
1. Equilibrate the column, injector, and detector with flowing carrier gas until a constant signal is received.
2. Inject a sample through the injector septum, or use an autosampler.
3. Begin the temperature program.
4. Record the chromatogram.
5. Analyze as indicated in the monograph.
Liquid Chromatography (LC)
The term liquid chromatography, as used in the compendia, is synonymous with high-pressure liquid
chromatography and high-performance liquid chromatography. LC is a separation technique based on a solid
stationary phase and a liquid mobile phase.
Stationary Phase: Separations are achieved by partition, adsorption, or ion-exchange processes, depending on
the type of stationary phase used. The most commonly used stationary phases are modified silica or polymeric
beads. The beads are modified by the addition of long-chain hydrocarbons. The specific type of packing needed
to complete an analysis is indicated by the “L” designation in the individual monograph (see also the section
Chromatographic Columns, below). The size of the beads is often described in the monograph as well. Changes
in the packing type and size are covered in the System Suitability section of this chapter.
Chromatographic Column: The term column includes stainless steel, lined stainless steel, and polymeric columns,
packed with a stationary phase. The length and inner diameter of the column affects the separation, and therefore
typical column dimensions are included in the individual monograph. Changes to column dimensions are
discussed in the System Suitability section of this chapter. Compendial monographs do not include the name of
appropriate columns; this omission avoids the appearance of endorsement of a vendor’s product and natural
changes in the marketplace. See the section Chromatographic Columns for more information.
Mobile Phase: The mobile phase is a solvent or a mixture of solvents, as defined in the individual monograph.
Apparatus: A liquid chromatograph consists of a reservoir containing the mobile phase, a pump to force the
mobile phase through the system at high pressure, an injector to introduce the sample into the mobile phase, a
chromatographic column, a detector, and a data collection device.
Gradient Elution: The technique of continuously changing the solvent composition during the chromatographic run
is called gradient elution or solvent programming. The gradient elution profile is presented in the individual
monograph as a gradient table, which lists the time and proportional composition of the mobile phase at the
stated time.
Procedure
1. Equilibrate the column and detector with mobile phase at the specified flow rate until a constant signal is
received.
2. Inject a sample through the injector, or use an autosampler.
3. Begin the gradient program.
4. Record the chromatogram.
5. Analyze as directed in the monograph.
CHROMATOGRAPHIC COLUMNS
A complete list of packings (L), phases (G), and supports (S) used in USP–NF tests and assays is located in USP–
NF and PF, Reagents, Indicators, and Solutions—Chromatographic Columns. This list is intended to be a
convenient reference for the chromatographer in identifying the pertinent chromatographic column specified in the
individual monograph.
DEFINITIONS AND INTERPRETATION OF CHROMATOGRAMS

Chromatogram: A chromatogram is a graphical representation of the detector response, concentration of analyte
in the effluent, or other quantity used as a measure of effluent concentration versus effluent volume or time. In
planar chromatography, chromatogram may refer to the paper or layer with the separated zones.
Figure 1 represents a typical chromatographic separation of two substances, 1 and 2. t R1 and tR2 are the respective
retention times; and h is the height, h/2 the half-height, and Wh/2 the width at half-height, for peak 1. W1 and W2
are the respective widths of peaks 1 and 2 at the baseline. Air peaks are a feature of gas chromatograms and
correspond to the solvent front in LC. The retention time of these air peaks, or unretained components, is
designated as tM.
Figure 1. Chromatographic separation of two substances.

Dwell Volume (D): The dwell volume (also known as gradient delay volume) is the volume between the point at
which the eluents meet and the top of the column.
Hold-Up Time (tM): The hold-up time is the time required for elution of an unretained component (see Figure 1,
shown as an air or unretained solvent peak, with the baseline scale in min).
Hold-Up Volume (VM): The hold-up volume is the volume of mobile phase required for elution of an unretained
component. It may be calculated from the hold-up time and the flow rate F, in mL/min:
VM = tM × F
In size exclusion chromatography, the symbol VO is used.
Number of Theoretical Plates (N)1: N is a measure of column efficiency. For Gaussian peaks, it is calculated by:
N = 16(tR/W)2
where tR is the retention time of the substance, and W is the peak width at its base, obtained by extrapolating the
relatively straight sides of the peak to the baseline. The value of N depends upon the substance being
chromatographed as well as the operating conditions, such as the flow rate and temperature of the mobile phase
or carrier gas, the quality of the packing, the uniformity of the packing within the column, and, for capillary
columns, the thickness of the stationary phase film and the internal diameter and length of the column.
Where electronic integrators are used, it may be convenient to determine the number of theorical plates, by the
equation:

where Wh/2 is the peak width at half-height. However, in the event of dispute, only equations based on peak width
at baseline are to be used.
Peak: The peak is the portion of the chromatographic recording of the detector response when a single
component is eluted from the column. If separation is incomplete, two or more components may be eluted as one
unresolved peak.
Peak-to-Valley Ratio (p/v): The p/v may be employed as a system suitability criterion in a test for related
substances when baseline separation between two peaks is not achieved. Figure 2 represents a partial
separation of two substances, where Hp is the height above the extrapolated baseline of the minor peak and Hv is
the height above the extrapolated baseline at the lowest point of the curve separating the minor and major peaks:
p/v = Hp/Hv
Figure 2. Peak-to-valley ratio determination.

Relative Retardation (Rret): The relative retardation is the ratio of the distance traveled by the analyte to the
distance simultaneously traveled by a reference compound (see Figure 3) and is used in planar chromatography.
Rret = b / c
Figure 3. Typical planar chromatography.

Relative Retention (r)1:
Is the ratio of the adjusted retention time of a component relative to that of another used
as a reference obtained under identical conditions:
r = tR2 tM/tR1 tM
where tR2 is the retention time measured from the point of injection of the compound of interest; t R1 is the retention
time measured from the point of injection of the compound used as reference; and t M is the retention time of a
nonretained marker defined in the procedure, all determined under identical experimental conditions on the same
column.
Relative Retention Time (RRT): Also known as unadjusted relative retention. Comparisons in USP are normally
made in terms of unadjusted relative retention, unless otherwise indicated.
RRT = tR2/tR1
The symbol rG is also used to designate unadjusted relative retention values.
Relative Standard Deviation in Percentage

Retardation Factor (RF): The retardation factor is the ratio of the distance traveled by the center of the spot to the
distance simultaneously traveled by the mobile phase and is used in planar chromatography. Using the symbols
in Figure 3:
RF = b/a
Retention Factor (k)1: The retention factor is also known as the capacity factor (k¢). Defined as:
or
The retention factor of a component may be determined from the chromatogram:
k = (tR tM)/tM
Retention Time (tR): In liquid chromatography and gas chromatography, the retention time, t R, is defined as the
time elapsed between the injection of the sample and the appearance of the maximum peak response of the
eluted sample zone. tR may be used as a parameter for identification. Chromatographic retention times are
characteristic of the compounds they represent but are not unique. Coincidence of retention times of a sample
and a reference substance can be used as a partial criterion in construction of an identity profile but may not be
sufficient on its own to establish identity. Absolute retention times of a given compound may vary from one
chromatogram to the next.
Retention Volume (VR): The retention volume is the volume of mobile phase required for elution of a component.
It may be calculated from the retention time and the flow rate in mL/min:
VR = tR × F
Resolution (RS): The resolution is the separation of two components in a mixture, calculated by:
RS = 2(tR2 tR1)/(W1 + W2)

where tR2 and tR1 are the retention times of the two components; and W2 and W1 are the corresponding widths at
the bases of the peaks obtained by extrapolating the relatively straight sides of the peaks to the baseline.
Where electronic integrators are used, it may be convenient to determine the resolution, by the equation:
RS = 1.18(tR2 tR1)/(W1,h/2 + W2,h/2)
Separation Factor ( ): The separation factor is the relative retention calculated for two adjacent peaks (by
convention, the value of the separation factor is always >1):
= k2/k1

Symmetry Factor (AS)2: The symmetry factor (also known as the tailing factor) of a peak (see Figure 4) is
calculated by:
AS = W0.05/2f
where W0.05 is the width of the peak at 5% height and f is the distance from the peak maximum to the leading
edge of the peak, the distance being measured at a point 5% of the peak height from the baseline.
Figure 4. Asymmetrical chromatographic peak.

Tailing Factor (T): See Symmetry Factor.
SYSTEM SUITABILITY
System suitability tests are an integral part of gas and liquid chromatographic methods. These tests are used to
verify that the chromatographic system is adequate for the intended analysis.
The tests are based on the concept that the equipment, electronics, analytical operations, and samples analyzed
constitute an integral system that can be evaluated as such.
Factors that may affect chromatographic behavior include the following:
• Composition, ionic strength, temperature, and apparent pH of the mobile phase
• Flow rate, column dimensions, column temperature, and pressure
• Stationary phase characteristics, including type of chromatographic support (particle-based or
monolithic), particle or macropore size, porosity, and specific surface area
• Reverse-phase and other surface modification of the stationary phases, the extent of chemical
modification (as expressed by end-capping, carbon loading, etc.)
The resolution, RS, is a function of the number of theoretical plates, N (also referred to as efficiency), the
separation factor, , and the capacity factor, k. [NOTE—All terms and symbols are defined in the preceding
section Definitions and Interpretation of Chromatograms. ] For a given stationary phase and mobile phase, N may
be specified to ensure that closely eluting compounds are resolved from each other, to establish the general
resolving power of the system, and to ensure that internal standards are resolved from the drug. This is a less
reliable means to ensure resolution than is direct measurement. Column efficiency is, in part, a reflection of peak
sharpness, which is important for the detection of trace components.
Replicate injections of a standard preparation or other standard solutions are compared to ascertain whether
requirements for precision are met. Unless otherwise specified in the individual monograph, data from five

replicate injections of the analyte are used to calculate the relative standard deviation, %RSD, if the requirement
is 2.0% or less; data from six replicate injections are used if the relative standard deviation requirement is more
than 2.0%.
For the Assay in a drug substance monograph, where the value is 100% for the pure substance, and no maximum
relative standard deviation is stated, the maximum permitted %RSD is calculated for a series of injections of the
reference solution:
%RSD = KB n/t90%, n 1
where K is a constant (0.349), obtained from the expression K = (0.6/ 2) × (t 90%,5/ 6), in which 0.6/ 2
represents the required percentage relative standard deviation after six injections for B = 1.0; B is the upper limit
given in the definition of the individual monograph minus 100%; n is the number of replicate injections of the
reference solution (3 n 6); and t90%, n 1 is the Student’s t at the 90% probability level (double sided) with n
1 degrees of freedom.
Unless otherwise prescribed, the maximum permitted relative standard deviation does not exceed the appropriate
value given in the table of repeatability requirements. This requirement does not apply to tests for related
substances.
Relative Standard Deviation Requirements
Number of Individual Injections
3 4 5 6
B (%) Maximum Permitted RSD
2.0 0.41 0.59 0.73 0.85
2.5 0.52 0.74 0.92 1.06
3.0 0.62 0.89 1.10 1.27

The symmetry factor, AS, a measure of peak symmetry, is unity for perfectly symmetrical peaks; and its value
increases as tailing becomes more pronounced (see Figure 4). In some cases, values less than unity may be
observed. As peak symmetry moves away from values of 1, integration, and hence precision, become less
reliable.
The signal-to-noise ratio (S/N) is a useful system suitability parameter. The S/N is calculated as follows:
S/N = 2H/h
where H is the height of the peak measured from the peak apex to a baseline extrapolated over a distance 5
times the peak width at its half-height; and h is the difference between the largest and smallest noise values
observed over a distance 5 times the width at the half-height of the peak and, if possible, situated equally
around the peak of interest (see Figure 5).

Figure 5. Noise and chromatographic peak, components of the S/N ratio.
These system suitability tests are performed by collecting data from replicate injections of standard or other
solutions as specified in the individual monograph.
The specification of definitive parameters in a monograph does not preclude the use of other suitable operating
conditions. Adjustments are permitted only when
• Suitable standards (including Reference Standards) are available for all compounds used in the suitability
test; and
• Those standards show that the adjustments improved the quality of the chromatography with respect to
the system suitability requirements.
Adjustments to chromatographic systems performed in order to comply with system suitability requirements are not
to be made in order to compensate for column failure or system malfunction.
If adjustments of operating conditions are necessary in order to meet system suitability requirements, each of the
items in the following list is the maximum variation that can be considered, unless otherwise directed in the
monograph; these changes may require additional validation data. To verify the suitability of the method under the
new conditions, assess the relevant analytical performance characteristics potentially affected by the change.
Multiple adjustments can have a cumulative effect on the performance of the system and are to be considered
carefully before implementation. Adjustments to the composition of the mobile phase in gradient elution are not
recommended. If adjustments are necessary, only column changes (same packing material) or dwell volume
adjustments are recommended.
pH of Mobile Phase (HPLC): The pH of the aqueous buffer used in the preparation of the mobile phase can be
adjusted to within ±0.2 units of the value or range specified.
Concentration of Salts in Buffer (HPLC): The concentration of the salts used in the preparation of the aqueous
buffer employed in the mobile phase can be adjusted to within ±10% if the permitted pH variation (see above) is
met.
Ratio of Components in Mobile Phase (HPLC): The following adjustment limits apply to minor components of the
mobile phase (specified at 50% or less). The amounts of these components can be adjusted by ±30% relative.
However, the change in any component cannot exceed ±10% absolute (i.e., in relation to the total mobile phase).

Adjustment can be made to one minor component in a ternary mixture. Examples of adjustments for binary and
ternary mixtures are given below.
Binary Mixtures
SPECIFIED RATIO OF 50:50: 30% of 50 is 15% absolute, but this exceeds the maximum permitted change of
±10% absolute in either component. Therefore, the mobile phase ratio may be adjusted only within the range of
40:60 to 60:40.
SPECIFIED RATIO OF 2:98: 30% of 2 is 0.6% absolute. Therefore the maximum allowed adjustment is within the
range of 1.4:98.6 to 2.6:97.4.
Ternary Mixtures
SPECIFIED RATIO OF 60:35:5: For the second component, 30% of 35 is 10.5% absolute, which exceeds the
maximum permitted change of ±10% absolute in any component. Therefore the second component may be
adjusted only within the range of 25% to 45% absolute. For the third component, 30% of 5 is 1.5% absolute. In all
cases, a sufficient quantity of the first component is used to give a total of 100%. Therefore, mixture ranges of
50:45:5 to 70:25:5 or 58.5:35:6.5 to 61.5:35:3.5 would meet the requirement.
Wavelength of UV-Visible Detector (HPLC): Deviations from the wavelengths specified in the procedure are not
permitted. The procedure specified by the detector manufacturer, or another validated procedure, is used to verify
that error in the detector wavelength is, at most, ±3 nm.
Stationary Phase
COLUMN LENGTH (GC, HPLC): Can be adjusted by as much as ±70%.
COLUMN INNER DIAMETER (HPLC): Can be adjusted if the linear velocity is kept constant. See Flow Rate (HPLC)
below.
COLUMN INNER DIAMETER (GC)— Can be adjusted by as much as ±50% for GC.
FILM THICKNESS (CAPILLARY CG)— Can be adjusted by as much as 50% to 100%.

Particle Size (HPLC): The particle size can be reduced by as much as 50%, but cannot be increased.
Particle Size (GC): Changing from a larger to a smaller or from a smaller to a larger particle size GC mesh
support is acceptable if the chromatography meets the requirements of system suitability and the same particle
size range ratio is maintained. The particle size range ratio is defined as the diameter of the largest particle
divided by the diameter of the smallest particle.
Flow Rate (GC): The flow rate can be adjusted by as much as ±50%.
Flow Rate (HPLC): When column dimensions have been modified, the flow rate can be adjusted using:
in which F1 is the flow rate indicated in the monograph, in mL/min; F2 is the adjusted flow rate, in mL/min; l1 is the
length of the column indicated in the monograph; l2 is the length of the column used; d1 is the column inner
diameter indicated in the monograph; and d2 is the internal diameter of the column used. Additionally, the flow
rate can be adjusted by ±50%.
Injection Volume (HPLC): The injection volume can be reduced as far as is consistent with accepted precision
and detection limits; no increase is permitted.
Injection Volume and Split Volume (GC): The injection volume and split volume may be adjusted if detection and
repeatability are satisfactory.
Column Temperature (HPLC): The column temperature can be adjusted by as much as ±10 . Column
thermostating is recommended to improve control and reproducibility of retention time.

Oven Temperature (GC): The oven temperature can be adjusted by as much as ±10%.
Oven Temperature Program (GC): Adjustment of temperatures is permitted as stated above. When the specified
temperature must be maintained or when the temperature must be changed from one value to another, an
adjustment of up to ±20% is permitted.
Unless otherwise directed in the monograph, system suitability parameters are determined from the analyte peak.
Measured values of Rr or RF or tR for the sample substance do not deviate from the values obtained for the
reference compound and mixture by more than the statistically determined reliability estimates from replicate
assays of the reference compound. Relative retention times may be provided in monographs for informational
purposes only to aid in peak identification. There are no acceptance criteria applied to relative retention times.
Suitability testing is used to ascertain the effectiveness of the final operating system, which should be subjected to
this testing. Make injections of the appropriate preparation(s) as required in order to demonstrate adequate
system suitability (as described in the Chromatographic system section of the method in a monograph)
throughout the run.
The preparation can be a standard preparation or a solution containing a known amount of analyte and any
additional materials (e.g., excipients or impurities) useful in controlling the analytical system. Whenever there is a
significant change in the chromatographic system (equipment, mobile phase component, or other components) or
in a critical reagent, system suitability is to be reestablished. No sample analysis is acceptable unless the
suitability of the system has been demonstrated.
QUANTITATION
During quantitation, disregard peaks caused by solvents and reagents or arising from the mobile phase or the
sample matrix.
In the linear range, peak areas and peak heights are usually proportional to the quantity of compound eluting. The
peak areas and peak heights are commonly measured by electronic integrators but may be determined by more
classical approaches. Peak areas are generally used but may be less accurate if peak interference occurs. The
components measured are separated from any interfering components. Peak tailing and fronting is minimized,
and the measurement of peaks on tails of other peaks are avoided when possible.
Although comparison of impurity peaks with those in the chromatogram of a standard at a similar concentration is
preferred, impurity tests may be based on the measurement of the peak response due to impurities and
expressed as a percentage of the area of the drug peak. The standard may be the drug itself at a level
corresponding to, for example, 0.5% impurity, assuming similar peak responses. When impurities must be
determined with greater certainty, use a standard of the impurity itself or apply a correction factor based on the
response of the impurity relative to that of the main component.
External Standard Method: The concentration of the component(s) quantified is determined by comparing the
response(s) obtained with the sample solution to the response(s) obtained with a standard solution.
Internal Standard Method: Equal amounts of the internal standard are introduced into the sample solution and a
standard solution. The internal standard is chosen so that it does not react with the test material, is stable, is
resolved from the component(s) quantified (analytes), and does not contain impurities with the same retention
time as that of the analytes. The concentrations of the analytes are determined by comparing the ratios of their
peak areas or peak heights and the internal standard in the sample solution with the ratios of their peak areas or
peak heights and the internal standard in the standard solution.
Normalization Procedure: The percentage content of a component of the test material is calculated by
determining the area of the corresponding peak as a percentage of the total area of all the peaks, excluding those
due to solvents or reagents or arising from the mobile phase or the sample matrix and those at or below the limit
at which they can be disregarded.
Calibration Procedure: The relationship between the measured or evaluated signal y and the quantity (e.g.,
concentration, mass) of substance x is determined, and the calibration function is calculated. The analytical
results are calculated from the measured signal or evaluated signal of the analyte and its position on the
calibration curve.

In tests for impurities for both the External Standard Method, when a dilution of the sample solution is used for
comparison, and the Normalization Procedure, any correction factors indicated in the monograph are applied
(e.g., when the response factor is outside the range 0.8–1.2).
When the impurity test prescribes the total of impurities or there is a quantitative determination of an impurity,
choice of an appropriate threshold setting and appropriate conditions for the integration of the peak areas is
important. In such tests the limit at or below which a peak is disregarded is generally 0.05%. Thus, the threshold
setting of the data collection system corresponds to at least half of this limit. Integrate the peak area of any
impurity that is not completely separated from the principal peak, preferably by valley-to-valley extrapolation
(tangential skim).
1 The parameters k, N, r, and rG were developed for isothermal GC separations and isocratic HPLC separations.
Because these terms are thermodynamic parameters, they are valid only for separations made at constant
temperature, mobile phase composition, and flow rate. However, for separations made with a temperature
program or solvent gradient, these parameters may be used simply as comparative means to ensure that
adequate chromatographic conditions exist to perform the methods as intended in the monographs.
2 It is also a common practice to measure the Asymmetry Factor as the ratio of the distance between the vertical
line connecting the peak apex with the interpolated baseline and the peak front, and the distance between that
line and the peak back measured at 10% of the peak height (see Figure 4), would be (W0.10 f0.10)/f0.10. However,
for the purposes of USP, only the formula (As) as presented here is valid.
Auxiliary Information— Please check for your question in the FAQs before contacting USP.
Topic/Question Contact Expert Committee
General Chapter Horacio N. Pappa, Ph.D. (GCCA2010) General Chapters - Chemical Analysis
Principal Scientific Liaison
1-301-816-8319
Pharmacopeial Forum: Volume No. 38(2)

19.4 Chromatographic Columns
USP36–NF31
The following list of packings (L), phases (G), and supports (S) is intended to be a convenient reference for the
chromatographer. [NOTE—Particle sizes given in this listing are those generally provided. Where other, usually
finer, sizes are required, the individual monograph specifies the desired particle size. Within any category of
packings or phases listed below, there may be a wide range of columns available. Where it is necessary to define
more specifically the chromatographic conditions, the individual monograph so indicates. ]
Packings
L1 —Octadecyl silane chemically bonded to porous or nonporous silica or ceramic microparticles, 1.5 to 10 µm in
diameter, or a monolithic silica rod.
L2 —Octadecyl silane chemically bonded to silica gel of a controlled surface porosity that has been bonded to a
solid spherical core, 30 to 50 µm in diameter.
L3 —Porous silica particles, 1.5 to 10 µm in diameter, or a monolithic silica rod.
L4 —Silica gel of controlled surface porosity bonded to a solid spherical core, 30 to 50 µm in diameter.
L5 —Alumina of controlled surface porosity bonded to a solid spherical core, 30 to 50 µm in diameter.
L6 —Strong cation-exchange packing–sulfonated fluorocarbon polymer coated on a solid spherical core, 30 to 50
µm in diameter.
L7 —Octylsilane chemically bonded to totally porous or superficially porous silica particles, 1.5–10 µm in
diameter, or a monolithic silica rod.
L8 —An essentially monomolecular layer of aminopropylsilane chemically bonded to totally porous silica gel
support, 1.5 to 10 µm in diameter.
L9 —Irregular or spherical, totally porous silica gel having a chemically bonded, strongly acidic cation-exchange
coating, 3 to 10 µm in diameter.
L10 —Nitrile groups chemically bonded to porous silica particles, 1.5 to 10 µm in diameter.
L11 —Phenyl groups chemically bonded to porous silica particles, 1.5 to 10 µm in diameter.
L12 —A strong anion-exchange packing made by chemically bonding a quaternary amine to a solid silica
spherical core, 30 to 50 µm in diameter.
L13 —Trimethylsilane chemically bonded to porous silica particles, 3 to 10 µm in diameter.
L14 —Silica gel having a chemically bonded, strongly basic quaternary ammonium anion-exchange coating, 5 to
10 µm in diameter.
L15 —Hexylsilane chemically bonded to totally porous silica particles, 3 to 10 µm in diameter.
L16 —Dimethylsilane chemically bonded to porous silica particles, 5 to 10 µm in diameter.
L17 —Strong cation-exchange resin consisting of sulfonated cross-linked styrene–divinylbenzene copolymer in
the hydrogen form, 6 to 12 µm in diameter.
L18 —Amino and cyano groups chemically bonded to porous silica particles, 3 to 10 µm in diameter.
L19 —Strong cation-exchange resin consisting of sulfonated cross-linked styrene-divinylbenzene copolymer in
the calcium form, about 9 µm in diameter.
L20 —Dihydroxypropane groups chemically bonded to porous silica or hybrid particles, 1.5 to 10 µm in diameter.
L21 —A rigid, spherical styrene-divinylbenzene copolymer 3 to 10 µm in diameter.
L22 —A cation-exchange resin made of porous polystyrene gel with sulfonic acid groups, about 10 µm in size.
L23 —An anion-exchange resin made of porous polymethacrylate or polyacrylate gel with quaternary ammonium
groups, 7–12 µm in size.

L24 —A semi-rigid hydrophilic gel consisting of vinyl polymers with numerous hydroxyl groups on the matrix
surface, 32 to 63 µm in diameter. [NOTE—Available as YMC-Pack PVA-SIL manufactured by YMC Co., Ltd. and
distributed by Waters Corp. (www.waters.com). ]
L25 —Packing having the capacity to separate compounds with a molecular weight range from 100–5000 (as
determined by polyethylene oxide), applied to neutral, anionic, and cationic water-soluble polymers. A
polymethacrylate resin base, cross-linked with polyhydroxylated ether (surface contained some residual carboxyl
functional groups) was found suitable.
L26 —Butyl silane chemically bonded to totally porous silica particles, 1.5 to 10 µm in diameter.
L27 —Porous silica particles, 30 to 50 µm in diameter.
L28 —A multifunctional support, which consists of a high purity, 100 , spherical silica substrate that has been
bonded with anionic exchanger, amine functionality in addition to a conventional reversed phase C8 functionality.
L29 —Gamma alumina, reverse-phase, low carbon percentage by weight, alumina-based polybutadiene spherical
particles, 5 µm in diameter with a pore volume of 80 .
L30 —Ethyl silane chemically bonded to totally porous silica particles, 3 to 10 µm in diameter.
L31 —A hydroxide-selective, strong anion-exchange resin-quaternary amine bonded on latex particles attached
to a core of 8.5-µm macroporous particles having a pore size of 2000 and consisting of ethylvinylbenzene
cross-linked with 55% divinylbenzene.
L32 —A chiral ligand-exchange packing–L-proline copper complex covalently bonded to irregularly shaped silica
particles, 5 to 10 µm in diameter.
L33 —Packing having the capacity to separate dextrans by molecular size over a range of 4,000 to 500,000 Da. It
is spherical, silica-based, and processed to provide pH stability. [NOTE—Available as TSK-GEL G4000SWxl from
Tosoh Bioscience (www.tosohbioscience.com). ]
L34 —Strong cation-exchange resin consisting of sulfonated cross-linked styrene–divinylbenzene copolymer in
the lead form, 7 to 9 µm in diameter.
L35 —A zirconium-stabilized spherical silica packing with a hydrophilic (diol-type) molecular monolayer bonded
phase having a pore size of 150 .
L36 —A 3,5-dinitrobenzoyl derivative of L-phenylglycine covalently bonded to 5-µm aminopropyl silica.
L37 —Packing having the capacity to separate proteins by molecular size over a range of 2,000 to 40,000 Da. It
is a polymethacrylate gel.
L38 —A methacrylate-based size-exclusion packing for water-soluble samples.
L39 —A hydrophilic polyhydroxymethacrylate gel of totally porous spherical resin.
L40 —Cellulose tris-3,5-dimethylphenylcarbamate coated porous silica particles, 5 to 20 µm in diameter.
L41 —Immobilized 1-acid glycoprotein on spherical silica particles, 5 µm in diameter.

L42 —Octylsilane and octadecylsilane groups chemically bonded to porous silica particles, 5 µm in diameter.
L43 —Pentafluorophenyl groups chemically bonded to silica particles by a propyl spacer, 1.5 to 10 µm in
diameter.
L44 —A multifunctional support, which consists of a high purity, 60 , spherical silica substrate that has been
bonded with a cationic exchanger, sulfonic acid functionality in addition to a conventional reversed phase C8
functionality.
L45 —Beta cyclodextrin bonded to porous silica particles, 5 to 10 µm in diameter.
L46 —Polystyrene/divinylbenzene substrate agglomerated with quaternary amine functionalized latex beads,
about 9 to 11 µm in diameter.

L47 —High-capacity anion-exchange microporous substrate, fully functionalized with trimethlyamine groups, 8 µm
in diameter. [NOTE—Available as CarboPac MA1 and distributed by Dionex Corp. (www.dionex.com). ]
L48 —Sulfonated, cross-linked polystyrene with an outer layer of submicron, porous, anion-exchange
microbeads, 10 to 15 µm in diameter.
L49 —A reversed-phase packing made by coating a thin layer of polybutadiene onto spherical porous zirconia
particles, 3 to 10 µm in diameter. [NOTE—Available as Zirchrom PBD from www.zirchrom.com. ]
L50 —Multifunction resin with reversed-phase retention and strong anion-exchange functionalities. The resin
consists of ethylvinylbenzene, 55% cross-linked with divinylbenzene copolymer, 3 to 15 µm in diameter, and a
surface area not less than 350 m2 per g. Substrate is coated with quaternary ammonium functionalized latex
particles consisting of styrene cross-linked with divinylbenzene. [NOTE—Available as OmniPac PAX-500 and
distributed by Dionex Corp. (www.dionex.com). ]
L51 —Amylose tris-3,5-dimethylphenylcarbamate-coated, porous, spherical, silica particles, 5 to 10 µm in
diameter. [NOTE—Available as Chiralpak AD from Chiral Technologies, Inc., (www.chiraltech.com). ]
L52 —A strong cation-exchange resin made of porous silica with sulfopropyl groups, 5 to 10 µm in diameter.
[NOTE—Available as TSK-GEL IC-Cation-SW from Tosoh Bioscience (www.tosohbioscience.com). ]
L53 —Weak cation-exchange resin consisting of ethylvinylbenzene, 55% cross-linked with divinylbenzene
copolymer, 3 to 15 µm diameter. Substrate is surface grafted with carboxylic acid and/or phosphoric acid
functionalized monomers. Capacity not less than 500 µEq/column. [NOTE—Available as IonPac CS14 distributed
by Dionex Corp. (www.dionex.com). ]
L54 —A size exclusion medium made of covalent bonding of dextran to highly cross-linked porous agarose
beads, about 13 µm in diameter.
[NOTE—Available as Superdex Peptide HR 10/30 from www.gelifesciences.com. ]
L55 —A strong cation-exchange resin made of porous silica coated with polybutadiene–maleic acid copolymer,
about 5 µm in diameter. [NOTE—Available as IC-Pak C M/D from Waters Corp. (www.waters.com). ]
L56 —Propyl silane chemically bonded to totally porous silica particles, 3 to 10 µm in diameter. [NOTE—Available
as Zorbax SB-C3 from Agilent Technologies (www.agilent.com/chem). ]
L57 —A chiral-recognition protein, ovomucoid, chemically bonded to silica particles, about 5 µm in diameter, with
a pore size of 120 . [NOTE—Available as Ultron ES-OVM from Agilent Technologies (www.agilent.com/chem).
]
L58 —Strong cation-exchange resin consisting of sulfonated cross-linked styrene-divinylbenzene copolymer in
the sodium form, about 6 to 30 µm in diameter. [NOTE—Available as Aminex HPX-87N from Bio-Rad
Laboratories, (2000/01 catalog, #125-0143) www.bio-rad.com. ]
L59 —Packing for the size-exclusion separation of proteins (separation by molecular weight) over the range of 5
to 7000 kDa. The packing is a spherical 1.5- to 10-µm silica or hybrid packing with a hydrophilic coating.
L60 —Spherical, porous silica gel, 10 µm or less in diameter, the surface of which has been covalently modified
with alkyl amide groups and endcapped. [NOTE—Available as Supelcosil LC-ABZ from Supelco
(www.sigmaaldrich.com/supelco). ]
L61 —A hydroxide selective strong anion-exchange resin consisting of a highly cross-linked core of 13-µm
microporous particles having a pore size less than 10 units and consisting of ethylvinylbenzene cross-linked
with 55% divinylbenzene with a latex coating composed of 85-nm diameter microbeads bonded with alkanol
quaternary ammonium ions (6%). [NOTE—Available as Ion Pac AS-11 and AG-11 from Dionex
(www.dionex.com). ]
L62 —C30 silane bonded phase on a fully porous spherical silica, 3 to 15 µm in diameter.
L63 —Glycopeptide teicoplanin linked through multiple covalent bonds to a 100- units spherical silica.
[NOTE—Available as Astec Chirobiotic T from Supelco (www.sigmaaldrich.com). ]

L64 —Strongly basic anion-exchange resin consisting of 8% cross-linked styrene-divinylbenzene copolymer with
a quaternary ammonium group in the chloride form, 45 to 180 µm in diameter. [NOTE—A suitable grade is
available as AG 1-X8 resin chloride form from www.discover.bio-rad.com. ]
L65 —Strongly acidic cation-exchange resin consisting of 8% sulfonated cross-linked styrene-divinylbenzene
copolymer with a sulfonic acid group in the hydrogen form, 45 to 250 µm in diameter. [NOTE—A suitable grade is
available as AG 50W-X2 resin hydrogen form from www.discover.bio-rad.com. ]
L66 —A crown ether coated on a 5-µm particle size silica gel substrate. The active site is (S)-18-crown-6-ether.
[NOTE—Available as Crownpak CR(+) from Daicel (www.daicel.com). ]
L67 —Porous vinyl alcohol copolymer with a C18 alkyl group attached to the hydroxyl group of the polymer, 2 to
10 µm in diameter. [NOTE—Available as apHera C18 from Supelco (www.sigmaaldrich.com). ]
L68 —Spherical, porous silica, 10 µm or less in diameter, the surface of which has been covalently modified with
alkyl amide groups and not endcapped. [NOTE—Available as SUPELCOSIL SUPLEX pKb-100 from Supelco
(www.sigmaaldrich.com). ]
L69 —Ethylvinylbenzene/divinylbenzene substrate agglomerated with quaternary amine functionalized 130-nm
latex beads, about 6.5 µm in diameter. [NOTE—Available as CarboPac PA20 from www.dionex.com. ]
L70 —Cellulose tris(phenyl carbamate) coated on 5-µm silica. [NOTE—Available as Chiralcel OC-H from
www.chiraltech.com. ]
L71 —A rigid, spherical polymetacrylate, 4 to 6 µm in diameter. [NOTE—Available as RSpak DE-613 from
www.shodex.com. ]
L72 —(S)-phenylglycine and 3,5-dinitroanaline urea linkage covalently bonded to silica.
[NOTE—Available as Sumichiral OA-3300, distributed by www.phenomenex.com. ]
L73 —A rigid spherical polydivinylbenzene particle, 5 to 10 µm in diameter. [NOTE—Available as Jordi-Gel DBV
from www.jordiflp.com. ]
L74 —a strong anion-exchange resin consisting of a highly cross-linked core of 7- µm macroporous particles
having a 100- average pore size and consisting of ethylvinylbenzene cross-linked with 55% divinylbenzene and
an anion-exchange layer grafted to the surface, which is functionalized with alkyl quaternary ammonium ions.
[NOTE—Available as IonPac AS14A from Dionex (www.dionex.com). ]
L75 —A chiral-recognition protein, bovine serum albumine (BSA), chemically bonded to silica particles, about 7
µm in diameter, with a pore size of 300 .
Phases
G1 —Dimethylpolysiloxane oil.
G2 —Dimethylpolysiloxane gum.
G3 —50% Phenyl-50% methylpolysiloxane.
G4 —Diethylene glycol succinate polyester.
G5 —3-Cyanopropylpolysiloxane.
G6 —Trifluoropropylmethylpolysiloxane.
G7 —50% 3-Cyanopropyl-50% phenylmethylsilicone.
G8 —80% Bis(3-cyanopropyl)-20% 3-cyanopropylphenylpoly-siloxane (percentages refer to molar substitution).
G9 —Methylvinylpolysiloxane.
G10 —Polyamide formed by reacting a C36 dicarboxylic acid with 1,3-di-4-piperidylpropane and piperidine in the
respective mole ratios of 1.00:0.90:0.20.
G11 —Bis(2-ethylhexyl) sebacate polyester.
G12 —Phenyldiethanolamine succinate polyester.

G13 —Sorbitol.
G14 —Polyethylene glycol (av. mol. wt. of 950 to 1050).
G16 —Polyethylene glycol compound (av. mol. wt. about 15,000). A high molecular weight compound of
polyethylene glycol with a diepoxide linker. [NOTE—Available commercially as Polyethylene Glycol Compound
20M, or as Carbowax 20M, from suppliers of chromatographic reagents. ]
G18 —Polyalkylene glycol.
G19 —25% Phenyl-25% cyanopropyl-50% methylsilicone.
G21 —Neopentyl glycol succinate.
G22 —Bis(2-ethylhexyl) phthalate.
G23 —Polyethylene glycol adipate.
G24 —Diisodecyl phthalate.
G25 —Polyethylene glycol compound TPA. A high molecular weight compound of a polyethylene glycol and a
diepoxide that is esterified with terephthalic acid.
[NOTE—Available commercially as Carbowax 20M-TPA from suppliers of chromatographic reagents. ]
G26 —25% 2-Cyanoethyl-75% methylpolysiloxane.
G29 —3,3¢-Thiodipropionitrile.
G30 —Tetraethylene glycol dimethyl ether.
G31 —Nonylphenoxypoly(ethyleneoxy)ethanol (av. ethyleneoxy chain length is 30); Nonoxynol 30.
G32 —20% Phenylmethyl-80% dimethylpolysiloxane.
G33 —20% Carborane-80% methylsilicone.
G34 —Diethylene glycol succinate polyester stabilized with phosphoric acid.
G35 —A high molecular weight compound of a polyethylene glycol and a diepoxide that is esterified with
nitroterephthalic acid.
G36 —1% Vinyl-5% phenylmethylpolysiloxane.
G37 —Polyimide.
G38 —Phase G1 containing a small percentage of a tailing inhibitor.
[NOTE—A suitable grade is available commercially as “SP2100/0.1% Carbowax 1500” from Supelco, Inc.
(www.sigmaaldrich.com/supelco). ]
G39 —Polyethylene glycol (av. mol. wt. about 1500).
G40 —Ethylene glycol adipate.
G41 —Phenylmethyldimethylsilicone (10% phenyl-substituted).
G42 —35% phenyl-65% dimethylpolysiloxane (percentages refer to molar substitution).
G43 —6% cyanopropylphenyl-94% dimethylpolysiloxane (percentages refer to molar substitution).
G44 —2% low molecular weight petrolatum hydrocarbon grease and 1% solution of potassium hydroxide.

G45 —Divinylbenzene-ethylene glycol-dimethylacrylate.
G46 —14% Cyanopropylphenyl-86% methylpolysiloxane.
G47 —Polyethylene glycol (av. mol. wt. of about 8000).
G48 —Highly polar, partially cross-linked cyanopolysiloxane.
Supports
[NOTE—Unless otherwise specified, mesh sizes of 80 to 100 or, alternatively, 100 to 120 are intended. ]
S1A —Siliceous earth for gas chromatography has been flux-calcined by mixing diatomite with Na2CO3 flux and
calcining above 900 . The siliceous earth is acid-washed, then water-washed until neutral, but not base-washed.
The siliceous earth may be silanized by treating with an agent such as dimethyldichlorosilane [NOTE—Unless
otherwise specified in the individual monograph, silanized support is intended. ] to mask surface silanol groups.
S1AB —The siliceous earth as described above is both acid- and base-washed. [NOTE—Unless otherwise
specified in the individual monograph, silanized support is intended. ]
S1C —A support prepared from crushed firebrick and calcined or burned with a clay binder above 900 with
subsequent acid-wash. It may be silanized.
S1NS —The siliceous earth is untreated.
S2 —Styrene-divinylbenzene copolymer having a nominal surface area of less than 50 m 2 per g and an average
pore diameter of 0.3 to 0.4 µm.
S3 —Copolymer of ethylvinylbenzene and divinylbenzene having a nominal surface area of 500 to 600 m 2 per g
and an average pore diameter of 0.0075 µm.
S4 —Styrene-divinylbenzene copolymer with aromatic –O and –N groups, having a nominal surface area of 400
to 600 m2 per g and an average pore diameter of 0.0076 µm.
S5 —40- to 60-mesh, high-molecular weight tetrafluorethylene polymer.
S6 —Styrene-divinylbenzene copolymer having a nominal surface area of 250 to 350 m2 per g and an average
pore diameter of 0.0091 µm.
S7 —Graphitized carbon having a nominal surface area of 12 m2 per g.
S8 —Copolymer of 4-vinyl-pyridine and styrene-divinylbenzene.
S9 —A porous polymer based on 2,6-diphenyl-p-phenylene oxide.
S10 —A highly polar cross-linked copolymer of acrylonitrite and divinylbenzene.
S11 —Graphitized carbon having a nominal surface area of 100 m2 per g modified with small amounts of
petrolatum and polyethylene glycol compound.
S12 —Graphitized carbon having a nominal surface area of 100 m2 per g.

19.5 Example USP Monographs
Fluvastatin Sodium
(floo'' va stat' in soe' dee um).
C24H25FNNaO4 433.45
6-Heptenoic acid, 7-[3-(4-fluorophenyl)-1-(1-methylethyl)-1H-indol-2-yl]-3,5-dihydroxy-, monosodium salt, [R*,S*-
(E)]-(±)-;
Sodium (±)-(3R*,5S*,6E)-7-[3-(p-fluorophenyl)-1-isopropylindol-2-yl]-3,5-dihydroxy-6-heptenoate [93957-55-
2].
DEFINITION
Fluvastatin Sodium contains NLT 98.0% and NMT 102.0% of C24H25FNNaO4, calculated on the anhydrous basis.
IDENTIFICATION
• A. INFRARED ABSORPTION 197K

[NOTE—If a difference appears in the IR spectra of the analyte and the Standard, dissolve equal portions of the
sample specimen and the USP Reference Standard in equal volumes of methanol. Evaporate the solutions to
dryness on a steam bath, protecting the solutions from light, and dry at 105 for 30 min. Repeat the test on the
residues. ]
• B. IDENTIFICATION TESTS—GENERAL, Sodium 191 : Meets the requirements for the pyroantimonate precipitation
test
ASSAY
• PROCEDURE
Solution A: Add 20 mL of 25% aqueous tetramethylammonium hydroxide solution to 880 mL of water. Adjust with
about 2.3 mL of phosphoric acid to a pH of 7.2 ± 0.2. Add 100 mL of a mixture of methanol and acetonitrile (3:2).
Solution B: Add 20 mL of 25% aqueous tetramethylammonium hydroxide solution and 80 mL of water to 900 mL of
a mixture of methanol and acetonitrile (3:2). Adjust with about 2.3 mL of phosphoric acid to a pH of 7.2 ± 0.2.

Mobile phase: See the gradient table.
Time Solution A Solution B

(min) (%) (%)
0 60 40
6 60 40
20 18 82
20.1 60 40
25.1 60 40
System suitability solution: 0.5 mg/mL of fluvastatin sodium from USP Fluvastatin for System Suitability RS,
dissolved first in Solution B, using 40% of the final volume, then diluted with Solution A to volume
Standard solution: 0.5 mg/mL of USP Fluvastatin Sodium RS, dissolved first in Solution B, using 40% of the final
volume, then diluted with Solution A to volume
Sample solution: 0.5 mg/mL of Fluvastatin Sodium, dissolved first in Solution B, using 40% of the final volume,
then diluted with Solution A to volume
Chromatographic system
(See Chromatography 621 , System Suitability.)

Mode: LC
Detector: UV 305 nm
Column: 4.6-mm × 5-cm; 5-µm packing L1
Column temperature: 35
Flow rate: 3 mL/min
Injection size: 20 µL
[NOTE—Adjust the start time of the gradient step and the equilibration time for each instrument. ]
System suitability
Samples: System suitability solution and Standard solution
[NOTE—The relative retention times for fluvastatin and fluvastatin anti-isomer are about 1.0 and 1.2, respectively. ]
Suitability requirements
Resolution: NLT 1.6 between fluvastatin anti-isomer and fluvastatin, System suitability solution
Column efficiency: NLT 700 theoretical plates for the fluvastatin peak, System suitability solution
Tailing factor: NMT 3.0 for the fluvastatin peak, System suitability solution
Relative standard deviation: NMT 1.0%, Standard solution
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of C24H25FNNaO4 in the portion of Fluvastatin Sodium taken:
Result = (rU/rS) × (CS/CU) × 100
rU = = peak response from the Sample solution

rS = = peak response from the Standard solution
CS = = concentration of USP Fluvastatin Sodium RS in the

Standard solution (mg/mL)
CU = = concentration of Fluvastatin Sodium in the Sample

solution (mg/mL)
Acceptance criteria: 98.0%–102.0% on the anhydrous basis
IMPURITIES
Inorganic Impurities
• HEAVY METALS, Method II 231 : 20 ppm

Organic Impurities
• PROCEDURE
[NOTE—Protect all solutions from light, and use amber autosampler vials and low-actinic glassware. ]
Solution A, Solution B, Mobile phase, Standard solution, and Sample solution: Proceed as directed in the Assay.
System suitability solution A: Prepare as directed for the System suitability solution in the Assay.
System suitability solution B: 0.1 mg/mL of USP Fluvastatin Related Compound B RS in a mixture of methanol and
acetonitrile (3:2). Transfer about 0.5 mL of this solution to a 10-mL volumetric flask, and dilute with System
suitability solution A to volume. [NOTE—System suitability solution B is stable for up to 6 months if stored in a
refrigerator. ]
Chromatographic system: Proceed as directed in the Assay, except to use a liquid chromatograph equipped with
either a programmable variable-wavelength detector or two separate detectors capable of monitoring at 305 and
365 nm.
System suitability
Samples: Standard solution and System suitability solution B
[NOTE—Record the peak responses at 305 nm as directed for Analysis. Identify the peaks corresponding to
fluvastatin, fluvastatin anti-isomer, and fluvastatin t-butyl ester. ]
Resolution: NLT 1.6 between fluvastatin anti-isomer and fluvastatin, System suitability solution B
Column efficiency: NLT 700 theoretical plates for the fluvastatin peak, System suitability solution B
Tailing factor: NMT 3.0 for the fluvastatin peak, System suitability solution B
Relative standard deviation: NMT 1.0% at 305 nm, Standard solution
Analysis
Record the chromatograms at 305 and 365 nm, identify the impurities listed in Impurity Table 1, and measure the
peak responses. [NOTE—3-Hydroxy-5-keto fluvastatin is monitored using a wavelength of 365 nm, and all other
compounds are monitored at 305 nm. ]
Calculate the percentage of each impurity, except for 3-hydroxy-5-keto fluvastatin, in the portion of Fluvastatin
Sodium taken:
Result = (rU/rS) × (CS/CU) × (1/F) × 100
rU = = peak response at 305 nm of each impurity from the
Sample solution

rS = = peak response at 305 nm of fluvastatin from the
Standard solution


solution (mg/mL)
F = = relative response factor (see Impurity Table 1)

Calculate the percentage of 3-hydroxy-5-keto fluvastatin in the portion of Fluvastatin Sodium taken:
Result = (rU/rS) × (CS/CU) × (1/F) × 100
rU = = peak response at 365 nm of 3-hydroxy-5-keto
fluvastatin from the Sample solution
rS = = peak response at 365 nm of fluvastatin from the

Standard solution


solution (mg/mL)
F = = relative response factor (see Impurity Table 1)

Acceptance criteria
Individual Impurities: See Impurity Table 1.
Total impurities: NMT 1.0%
Impurity Table 1
Relative Relative Acceptance
Retention Response Criteria,
Name Time Factor NMT (%)
Fluvastatin N-ethyl analoga 0.7 1.2 0.1
Fluvastatin anti-isomerb 1.2 1.0 0.8
3-Hydroxy-5-keto fluvastatinc 1.5 27.0d 0.1
Fluvastatin
hydroxydienee 2.0 0.92 0.1
Fluvastatin short-chain aldehydef 3.0 1.4 0.1
Fluvastatin t-butyl ester (fluvastatin related compound B)g 3.4 0.94 0.2
Any other
individual
impurity — 1.0 0.1
a [R*,S*-E]-(±)-7-[3-(4-Fluorophenyl)-1-ethyl-1H-indol-2-yl]-3,5-dihydroxy-6-heptenoic acid monosodium salt.
b [R*,R*-E]-(±)-7-[3-(4-Fluorophenyl)-1-(methylethyl)-1H-indol-2-yl]-3,5-dihydroxy-6-heptenoic acid monosodium
salt.

Relative Relative Acceptance
Retention Response Criteria,
Name Time Factor NMT (%)
c E-(±)-7-[3-(4-Fluorophenyl)-1-(methylethyl)-1H-indol-2-yl]-3-hydroxy-5-oxo-6-heptenoic acid monosodium salt.
d At 365 nm.
e [E,E]-(±)-7-[3-(4-Fluorophenyl)-1-(methylethyl)-1H-indol-2-yl]-3-hydroxy-4,6-heptadienoic acid monosodium salt.
f 3-(4-Fluorophenyl)-1-(methylethyl)-1H-indole-2-carboxaldehyde.
g [R*,S*-E]-(±)-7-[3-(4-Fluorophenyl)-1-methylethyl-1H-indol-2-yl]-3,5-dihydroxy-6-heptenoic acid 1,1-
dimethylethyl ester.
SPECIFIC TESTS
• PH 791 : 8.0–10.0, in a 10-mg/mL solution. Perform the test immediately after preparation.
• WATER DETERMINATION, Method I 921 : NMT 4.0%; if labeled as a hydrated form, NMT 12.0%. [NOTE—For this
monograph, the term “hydrated form” refers to several known forms of fluvastatin sodium that are not
stoichiometric hydrates. ]
ADDITIONAL REQUIREMENTS
• PACKAGING AND STORAGE: Preserve in tight, light-resistant containers, protected from moisture. Store at a
temperature not exceeding 40 .
• LABELING: Where it is a hydrated form, the label so indicates.
• USP REFERENCE STANDARDS 11
USP Fluvastatin Sodium RS

USP Fluvastatin Related Compound B RS
Fluvastatin t-butyl ester.
USP Fluvastatin for System Suitability RS

Fluvastatin sodium, containing 1% to 2% of the fluvastatin sodium anti-isomer.
Monograph Elena Gonikberg, Ph.D. (SM32010) Monographs - Small Molecules 3

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Pharmacopeial Forum: Volume No. 36(4) Page 921
Chromatographic Column—
FLUVASTATIN SODIUM
Chromatographic columns text is not derived from, and not part of, USP 36 or NF 31.
Ampicillin
(am'' pi sil' in).
C16H19N3O4S (anhydrous) 349.40

4-Thia-1-azabicyclo[3.2.0]heptane-2 carboxylic acid, [6-(aminophenylacetyl)amino]-3,3-dimethyl-7-oxo-, [2S-[2
,5 ,6 (S*)]]-;
(2S,5R,6R)-6-[(R)-2-Amino-2-phenylacetamido]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-
carboxylic acid [69-53-4].
Trihydrate 403.46 [7177-48-2].
DEFINITION
Ampicillin is anhydrous or contains three molecules of water of hydration. It contains NLT 900 µg and NMT 1050 µg
of C16H19N3O4S per mg, calculated on the anhydrous basis.
IDENTIFICATION
• INFRARED ABSORPTION 197K : Except that where the specimen under test is the trihydrate, both it and the USP
Ampicillin Trihydrate RS are undried.
ASSAY
• PROCEDURE
[NOTE—The Standard solution and the Sample solution should be analyzed immediately after preparation. ]
Solution A: 6.54 mg/mL of monobasic potassium phosphate and 0.34 mg/mL of dibasic potassium phosphate,
adjusted with 1 N sodium hydroxide or 1 N phosphoric acid to a pH of 5.5 before final dilution
Solution B: Acetonitrile and Solution A (2:23)
Solution C: Acetonitrile and Solution A (3:7)
Mobile phase: See gradient table below.
Time (min) Solution B (%) Solution C (%)
0 100 0
6 100 0
15 0 100
16 0 100
18 100 0
20 100 0
Solution D: 46.3 mg/mL of monobasic potassium phosphate and 27.8 mg/mL of dibasic potassium phosphate,
adjusted with 1 N sodium hydroxide or 1 N phosphoric acid to a pH of 6.5 before final dilution

System suitability solution: 0.5 mg/mL of USP Ampicillin RS and 0.1 mg/mL of USP Amoxicillin RS in acetonitrile,
water, and Solution D (4:91:5). [NOTE—Dissolve first in a mixture of acetonitrile, water, and Solution D (4:30:5),
sonicating if necessary, and dilute with water to volume. ]
Standard solution: 0.5 mg/mL of USP Ampicillin RS in acetonitrile, water, and Solution D (4:91:5). [NOTE—
Dissolve first in a mixture of acetonitrile, water, and Solution D (4:30:5), sonicating if necessary, and dilute with
water to volume. ]
Sample solution: 0.5 mg/mL of Ampicillin in acetonitrile, water, and Solution D (4:91:5). [NOTE—Dissolve first in a
mixture of acetonitrile, water, and Solution D (4:30:5), sonicating if necessary, and dilute with water to volume. ]
Chromatographic system
(See Chromatography 621 , System Suitability.)

Mode: LC
Detector: UV 230 nm
Column: 4.6-mm × 15-cm; 5-µm packing L1
Flow rate: 1.5 mL/min
Injection size: 20 µL
System suitability
Samples: System suitability solution and Standard solution
Resolution: NLT 10 between the ampicillin and amoxicillin peaks, System suitability solution
Tailing factor: NMT 1.4, System suitability solution
Analysis
Calculate the quantity, in µg, of C16H19N3O4S in each mg of Ampicillin taken:
Result = (rU/rS) × (CS/CU) × P
rU = = peak response from the Sample solution
rS = = peak response from the Standard solution
CS = = concentration of USP Ampicillin RS in the Standard

solution (mg/mL)
CU = = nominal concentration of Ampicillin in the Sample

solution (mg/mL)
P = = potency of USP Ampicillin RS (µg/mg)

Acceptance criteria: 900 µg–1050 µg on the anhydrous basis
IMPURITIES
Organic Impurities
• PROCEDURE 1
[NOTE—The Standard solution and the Sample solution should be analyzed immediately after preparation. ]
Solution A, Solution B, Solution C, Mobile phase, Solution D, System suitability solution, Sample solution, and
Chromatographic system: Prepare as directed in the Assay.

Standard stock solution: Prepare as directed for Standard solution in the Assay.
Standard solution: 0.005 mg/mL of ampicillin in Solution D and water (1:19) from Standard stock solution. [NOTE—
Transfer an aliquot of the Standard stock solution to a suitable volumetric flask, add Solution D, using about 5% of
the final volume, dilute with water to volume. ]
Sensitivity solution: 0.0005 mg/mL of ampicillin in Solution D and water (1:19) from Standard solution. [NOTE—
Transfer an aliquot of the Standard solution to a suitable volumetric flask, add Solution D, using about 5% of the
final volume, and dilute with water to volume. ]
System suitability
Samples: Sensitivity solution, System suitability solution, and Standard solution
Signal-to-noise ratio: NLT 3, Sensitivity solution
Resolution: NLT 10 between ampicillin and amoxicillin, System suitability solution
Tailing factor: NMT 1.4, System suitability solution
Analysis
Calculate the percentage of each impurity in the portion of Ampicillin taken:
Result = (rU/rS) × (CS/CU) × P × F × 100
rU == peak response of each impurity from the Sample solution
rS == peak response of ampicillin from the Standard solution
CS == concentration of USP Ampicillin RS in the Standard solution

(mg/mL)
CU == nominal concentration of Ampicillin in the Sample solution

(mg/mL)
P == potency of USP Ampicillin RS (µg/mg)
F == conversion factor, 0.001 mg/µg

Acceptance criteria
Individual impurities: See Impurity Table 1.
Total impurities: NMT 3.0%
Impurity Table 1
Relative Acceptance
Retention Criteria,
Name Time NMT (%)
D-Phenylglycinea 0.27 0.5
6-Aminopenicillanic acidb 0.31 0.5
Ampicilloic acidc 0.45 1.0
Ampicillin thiazepine analogd 0.65 0.3
Ampicillin 1.0 —

Relative Acceptance
Retention Criteria,
Name Time NMT (%)
Ampicillin rearrangement product (isomer 1)e 1.8 0.4
Ampicillin rearrangement product (isomer 2)e 2.0 0.3
Ampicillin oligomer 2f 2.2 0.6
D-Phenylglycylampicilling 2.5 0.8
Ampicillin oligomer 1 (dimer)h 2.6 1.0
Ampicillin oligomer 1 (trimer)i 2.9 0.4
Any individual unspecified impurity — 0.25

a (R)-2-Amino-2-phenylacetic acid.
b (2S,5R,6R)-6-Amino-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid.
c (4S)-2-{[(R)-2-Amino-2-phenylacetamido](carboxy)methyl}-5,5-dimethylthiazolidine-4-carboxylic acid.
d (S)-6-[(R)-2-Amino-2-phenylacetamido]-2,2-dimethyl-7-oxo-2,3,4,7-tetrahydro-1,4-thiazepine-3-carboxylic acid.
e (4S)-2-(3,6-Dioxo-5-phenylpiperazin-2-yl)-5,5-dimethylthiazolidine-4-carboxylic acid.
f (4S)-2-{1-[(R)-2-Amino-2-phenylacetamido]-2-[(1R)-2-{carboxy[(4S)-4-carboxy-5,5-dimethylthiazolidin-2-
yl]methylamino}-2-oxo-1-phenylethylamino]-2-oxoethyl}-5,5-dimethylthiazolidine-4-carboxylic acid.
g (2S,5R,6R)-6-{(R)-2-[(R)-2-Amino-2-phenylacetamido]-2-phenylacetamido}-3,3-dimethyl-7-oxo-4-thia-1-
azabicyclo[3.2.0]heptane-2-carboxylic acid.
h (2S,5R,6R)-6-[(2R)-2-{2-[(R)-2-Amino-2-phenylacetamido]-2-[(4S)-4-carboxy-5,5-dimethylthiazolidin-2-
yl]acetamido}-2-phenylacetamido]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid.
i (4S,4'S)-2,2'-{(1R,7R,13R)-1-Amino-14-[(2S,5R,6R)-2-carboxy-3,3-dimethyl-7-oxo-4-thia-1-
azabicyclo[3.2.0]heptan-6-ylamino]-2,5,8,11,14-pentaoxo-1,7,13-triphenyl-3,6,9,12-tetraazatetradecane-4,10-
diyl}bis(5,5-dimethylthiazolidine-4-carboxylic acid).
• PROCEDURE 2: DIMETHYLANILINE 223 : Meets the requirements

SPECIFIC TESTS
• STERILITY TESTS 71 : Where the label states that Ampicillin is sterile, it meets the requirements when tested as
directed for Test for Sterility of the Product to be Examined, Membrane Filtration, except to dissolve 6 g in 800 mL
of Fluid D containing sufficient sterile penicillinase to inactivate the ampicillin and to swirl the vessel until solution
is complete before filtering.
• CRYSTALLINITY 695 : Meets the requirements
• PH 791 : 3.5–6.0
Sample solution: 10 mg/mL
• WATER DETERMINATION, Method I 921 : NMT 2.0% where it is labeled as Ampicillin (anhydrous); between 12.0%
and 15.0% where it is labeled as Ampicillin (trihydrate)
• BACTERIAL ENDOTOXINS TEST 85 : Where the label states that Ampicillin is sterile or must be subjected to further
processing during the preparation of injectable dosage forms, it contains NMT 0.15 USP Endotoxin Unit/mg of
ampicillin.

ADDITIONAL REQUIREMENTS
• PACKAGING AND STORAGE: Preserve in tight containers.
• LABELING: Label to indicate whether it is anhydrous or is the trihydrate. Where the quantity of ampicillin is indicated
in the labeling of any preparation containing Ampicillin, this shall be understood to be in terms of anhydrous
ampicillin (C16H19N3O4S). Where it is intended for use in preparing injectable dosage forms, the label states that it
is the trihydrate and that it is sterile or must be subjected to further processing during the preparation of injectable
dosage forms.
• USP REFERENCE STANDARDS 11
USP Amoxicillin RS
USP Ampicillin RS
USP Ampicillin Trihydrate RS

USP Endotoxin RS
Monograph Ahalya Wise, M.S. (SM12010) Monographs - Small Molecules 1

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AMPICILLIN

Ampicillin and Sulbactam for Injection
» Ampicillin and Sulbactam for Injection is a sterile, dry mixture of Ampicillin Sodium and Sulbactam Sodium. It
contains the equivalent of not less than 90.0 percent and not more than 115.0 percent of the labeled amounts of
ampicillin (C16H19N3O4S) and sulbactam (C8H11NO5S), the labeled amounts representing proportions of ampicillin
to sulbactam of 2:1. It contains not less than 563 µg of ampicillin and 280 µg of sulbactam per mg, calculated on
the anhydrous basis.
Packaging and storage— Preserve in Containers for Sterile Solids as described under Injections 1 .
USP REFERENCE STANDARDS 11 —
USP Ampicillin RS
USP Endotoxin RS
USP Sulbactam RS
Constituted solution— At the time of use, it meets the requirements for Constituted Solutions under Injections
1 .
Identification— The retention times of the major peaks in the chromatogram of the Assay preparation
correspond to those in the chromatogram of the Standard preparation, as obtained in the Assay.
BACTERIAL ENDOTOXINS 85 — It contains not more than 0.17 USP Endotoxin Unit in a portion equivalent to 1
mg of a mixture of ampicillin and sulbactam (0.67 and 0.33 mg, respectively).
STERILITY 71 — It meets the requirements when tested as directed for Membrane Filtration under Test for
Sterility of the Product to be Examined.
PH 791 : between 8.0 and 10.0, in a solution containing 10 mg of ampicillin and 5 mg of sulbactam per mL.
WATER, Method I 921 : not more than 2.0%.
PARTICULATE MATTER 788 : meets the requirements for small-volume injections.
Other requirements— It meets the requirements for Uniformity of Dosage Units 905 and for Labeling under
Injections 1 .
Assay—
0.005 M Tetrabutylammonium hydroxide— Dilute 6.6 mL of a 40% solution of tetrabutylammonium hydroxide with
water to obtain 1800 mL of solution. Adjust with 1 M phosphoric acid to a pH of 5.0 ± 0.1, dilute with water to 2000
mL, and mix.
Mobile phase— Prepare a filtered and degassed mixture of 0.005 M Tetrabutylammonium hydroxide and
acetonitrile (1650:350). Make adjustments if necessary (see System Suitability under Chromatography 621 ).
Standard preparation— Quantitatively dissolve accurately weighed quantities of USP Ampicillin RS and USP
Sulbactam RS in Mobile phase to obtain a solution having known concentrations of about 0.6 mg of ampicillin per
mL and 0.3 mg of sulbactam per mL. [NOTE—Inject this solution promptly. ]
Resolution solution— Prepare a solution of USP Sulbactam RS in 0.01 N sodium hydroxide containing 0.3 mg per
mL, and allow to stand for 30 minutes. Adjust with phosphoric acid to a pH of 5.0 ± 0.1. Transfer 5 mL of the
solution to a 25-mL volumetric flask, add 4.25 mL of acetonitrile, dilute with 0.005 M Tetrabutylammonium
hydroxide to volume, and mix. Transfer 1 mL of this solution to a second 25-mL volumetric flask, add 15 mg of
USP Ampicillin RS, dilute with Mobile phase to volume, and mix. [NOTE—Inject this solution promptly. ]

Assay preparation 1— Mix the contents of a container of Ampicillin and Sulbactam for Injection. Quantitatively
dissolve an accurately weighed portion of the powder in Mobile phase to obtain a solution having a concentration
of about 1 mg of the powder per mL. [NOTE—Inject this solution promptly. ]
Assay preparation 2 (where it is represented as being in a single-dose container)— Constitute a container of
Ampicillin and Sulbactam for Injection with a volume of water, accurately measured, corresponding to the volume
of solvent specified in the labeling. Withdraw the total withdrawable contents from the container, using a suitable
hypodermic needle and syringe, and dilute quantitatively, and stepwise if necessary, with Mobile phase to obtain
a solution containing about 0.6 mg of ampicillin per mL and 0.3 mg of sulbactam per mL. [NOTE—Inject this
solution promptly. ]
Assay preparation 3 (where the label states the quantities of ampicillin and sulbactam in a given volume of
constituted solution)— Constitute a container of Ampicillin and Sulbactam for Injection with a volume of water,
accurately measured, corresponding to the volume of solvent specified in the labeling. Dilute an accurately
measured volume of the constituted solution quantitatively, and stepwise if necessary, with Mobile phase to
obtain a solution containing about 0.6 mg of ampicillin per mL and 0.3 mg of sulbactam per mL. [NOTE—Inject
this solution promptly. ]
Chromatographic system (see CHROMATOGRAPHY 621 )— The liquid chromatograph is equipped with a 230-
nm detector and a 4-mm × 30-cm column containing packing L1. The flow rate is about 2 mL per minute.
Chromatograph the Resolution solution, and record the responses as directed for Procedure: the relative
retention times are about 0.7 for ampicillin and 1.0 for sulbactam alkaline degradation product; and the resolution,
R, between ampicillin and sulbactam alkaline degradation product is not less than 4.0. Chromatograph the
Standard preparation, and record the responses as directed for Procedure: the relative retention times are about
0.35 for ampicillin and 1.0 for sulbactam; the column efficiency determined from the sulbactam peak is not less
than 3500 theoretical plates; the tailing factor is not more than 1.5; and the relative standard deviation for
replicate injections is not more than 2.0%.
Procedure— Separately inject equal volumes (about 10 µL) of the Standard preparation and the appropriate
Assay preparation into the chromatograph, record the chromatograms, and measure the areas for the major
peaks. Calculate the quantities, in µg, of ampicillin (C16H19N3O4S) and of sulbactam (C8H11NO5S) in the portion of
Ampicillin and Sulbactam for Injection taken by the same formula:
(CSP / CU)(rU / rS)
in which CS is the concentration, in mg per mL, of the appropriate USP Reference Standard in the Standard
preparation; P is the assigned content, in µg per mg, of the appropriate USP Reference Standard; CU is the
concentration, in mg per mL, of Ampicillin and Sulbactam for Injection in Assay preparation 1, based on the
weight, in mg, of powder removed from the container and the extent of dilution; and rU and rS are the peak areas
for the appropriate analyte obtained from Assay preparation 1 and the Standard preparation, respectively.
Calculate the quantities of ampicillin (C16H19N3O4S) and of sulbactam (C8H11NO5S) withdrawn from the container,
or in the volume of constituted solution taken by the same formula:
(L / D)(CSP)(rU / rS)
in which L is the labeled quantity, in mg, of ampicillin or sulbactam, as appropriate, in the container or in the
volume of constituted solution taken; D is the concentration, in mg per mL, of ampicillin or sulbactam in Assay
preparation 2 or Assay preparation 3, on the basis of the labeled quantity, in mg, of ampicillin or sulbactam, as
appropriate, in the container and the extent of dilution; rU and rS are the peak areas for the appropriate analyte
obtained from Assay preparation 2 or Assay preparation 3 and the Standard preparation, respectively; and the
other terms are as defined above.


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AMPICILLIN AND SULBACTAM FOR INJECTION

Cyclosporine
(sye'' kloe spor' een).
C62H111N11O12 1202.61
Cyclo[[(E)-(2S,3R,4R)-3-hydroxy-4-methyl-2-(methylamino)-6-octenoyl]-L-2-aminobutyryl-N-methylglycyl-N-
methyl-L-leucyl-L-valyl-N-methyl-L-leucyl-L-alanyl-D-alanyl-N-methyl-L-leucyl-N-methyl-L-leucyl-N-methyl-L-valyl].
[R-[R*,R*-(E)]]-Cyclic(L-alanyl-D-alanyl-N-methyl-L-leucyl-N-methyl-L-leucyl-N-methyl-L-valyl-3-hydroxy-N,4-
dimethyl-L-2-amino-6-octenoyl-L- -aminobutyryl-N-methylglycyl-N-methyl-L-leucyl-L-valyl-N-methyl-L-leucyl)
[59865-13-3].
» Cyclosporine contains not less than 98.5 percent and not more than 101.5 percent of cyclosporine A
(C62H111N11O12), calculated on the dried basis.
Packaging and storage— Preserve in tight, light-resistant containers.
USP REFERENCE STANDARDS 11 —
USP Cyclosporine RS
USP Cyclosporine Resolution Mixture RS
This material is a 100:1 mixture of cyclosporine and cyclosporine U.
Identification— The chromatogram of the Assay preparation obtained as directed in the Assay exhibits a major
peak for cyclosporine, the retention time of which corresponds to that exhibited in the chromatogram of the
Standard preparation, as obtained in the Assay.
LOSS ON DRYING 731 — Dry about 100 mg, accurately weighed, in a capillary-stoppered bottle in vacuum at a
pressure not exceeding 5 mm of mercury at 60 for 3 hours: it loses not more than 2.0% of its weight.
HEAVY METALS, Method II 231 : 0.002%.

Related compounds— Using the chromatograms obtained from Standard preparation 2 and the Assay
preparation in the Assay, calculate the percentage of each impurity by the formula:
2000(C / W)(ri / rS2)
in which C is the concentration, in mg per mL, of USP Cyclosporine RS in Standard preparation 2; W is the
weight, in mg, of Cyclosporine taken to prepare the Assay preparation; ri is the response of an individual impurity
observed in the chromatogram of the Assay preparation; and rS2 is the response of the main cyclosporine peak in
the chromatogram obtained from Standard preparation 2: not more than 0.7% of any individual impurity is found,

and the sum of all such impurities is not more than 1.5%, any impurities corresponding to less than 0.05% being
disregarded.
Assay—
Mobile phase— Prepare a mixture of water, acetonitrile, tert-butyl methyl ether, and phosphoric acid
(520:430:50:1). Make adjustments if necessary (see System Suitability under Chromatography 621 ).
Diluent— Prepare a mixture of acetonitrile and water (1:1).
Standard preparation 1— Dissolve an accurately weighed quantity of USP Cyclosporine RS in Diluent to obtain a
solution having a known concentration of about 1.25 mg per mL.
Standard preparation 2— Transfer 2.0 mL of Standard preparation 1 to a 250-mL volumetric flask, dilute with
Diluent to volume, and mix. This solution contains about 0.01 mg of USP Cyclosporine RS per mL.
Assay preparation— Dissolve about 25 mg of Cyclosporine, accurately weighed, in Diluent, dilute with Diluent to
20.0 mL, and mix.
Resolution solution— Prepare a solution of USP Cyclosporine Resolution Mixture RS in Diluent having a
concentration of about 1.25 mg per mL.
nm detector, a 0.25-mm × 1-m stainless steel tube connected to a 4-mm × 25-cm column that contains 3- to 5-µm
packing L1. The tube and column are maintained at 80 . The flow rate is about 1.2 mL per minute.
Chromatograph the Resolution solution, and record the responses as directed for Procedure: the cyclosporine U
peak and the main cyclosporine peak are resolved from each other. Chromatograph Standard preparation 1, and
record the responses as directed for Procedure: the relative standard deviation for replicate injections is not more
than 1.0%. Chromatograph Standard preparation 2, and record the responses as directed for Procedure: the
relative standard deviation for replicate injections is not more than 10%.
Procedure— [NOTE—Use peak areas where peak responses are indicated. ] Separately inject equal volumes
(about 20 µL) of Standard preparation 1, Standard preparation 2, and the Assay preparation into the
chromatograph, record the chromatograms, and measure the peak responses. Calculate the percentage of
cyclosporine A (C62H111N11O12) in the Cyclosporine taken by the formula:
(CP/10U)(rU / rS)
in which C is the concentration, in mg per mL, of USP Cyclosporine RS in Standard preparation 1; P is the
specified purity, in µg per mg, of USP Cyclosporine RS; U is the concentration, in mg per mL, of specimen in the
Assay preparation; and rU and rS are the main cyclosporine peak responses obtained from the Assay preparation
and Standard preparation 1, respectively.

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CYCLOSPORINE

Cyclosporine Capsules
» Cyclosporine Capsules contain not less than 90.0 percent and not more than 110.0 percent of the labeled amount
of cyclosporine (C62H111N11O12).
Packaging and storage— Preserve in tight containers, and store at controlled room temperature.
Identification— The retention time of the major peak in the chromatogram of the Assay preparation corresponds
to that in the chromatogram of the Standard preparation, as obtained in the Assay.
DISSOLUTION 711 —
WHERE CAPSULES CONTAIN LIQUID—
Medium: water; 500 mL.
Time: 15 minutes.
Procedure— Place 1 Capsule in each vessel, and allow the Capsule to sink to the bottom of the vessel before
starting rotation of the blade. Observe the Capsules, and record the time taken for each Capsule shell to rupture.
Tolerances— The requirements are met if all of the Capsules tested rupture in not more than 15 minutes. If 1 or 2
of the Capsules rupture in more than 15 but not more than 30 minutes, repeat the test on 12 additional Capsules.
Not more than 2 of the total of 18 Capsules tested rupture in more than 15 but not more than 30 minutes.
WHERE CAPSULES CONTAIN POWDER—
Medium: 0.1 N hydrochloric acid containing 0.5% of sodium lauryl sulfate; 1000 mL.
Time: 90 minutes.
Determine the amount of C62H111N11O12 dissolved by employing the following method.
Mobile phase— Prepare a filtered and degassed mixture of acetonitrile, water, methanol, and phosphoric acid
(900:450:50:0.5). Make adjustments if necessary (see System Suitability under Chromatography 621 ).
Standard solution— Quantitatively dissolve an accurately weighed quantity of USP Cyclosporine RS in
Dissolution Medium to obtain a solution having a known concentration of about 0.001L mg per mL, L being the
labeled quantity, in mg, of cyclosporine in each Capsule. Transfer 25.0 mL of this solution to a 50-mL volumetric
flask, dilute with acetonitrile to volume, and mix. This solution contains about 0.0005L mg of USP Cyclosporine
RS per mL.
Test solution— Filter a portion of the solution under test. Transfer 5.0 mL of the filtrate to a 10-mL volumetric
flask, dilute with acetonitrile to volume, and mix.
nm detector and a 4.6-mm × 25-cm column that contains packing L1 and is maintained at a constant temperature
of about 80 . The flow rate is about 2 mL per minute. Chromatograph the Standard solution, and record the peak
areas as directed for Procedure: the column efficiency is not less than 700 theoretical plates; and the relative
standard deviation for replicate injections is not more than 2.0%.
Procedure— Separately inject equal volumes (about 10 µL) of the solution estimated to contain 0.1 mg of
cyclosporine per mL, or 40 µL of the solution estimated to contain 0.025 mg of cyclosporine per mL) of the
Standard solution and the Test solution into the chromatograph, record the chromatograms, and measure the
areas for the major peaks. Calculate the quantity, in mg, of C62H111N11O12 dissolved by the formula:
2000C(rU / rS)
in which C is the concentration, in mg per mL, of USP Cyclosporine RS in the Standard solution; and rU and rS are
the cyclosporine peak areas obtained from the Test solution and the Standard solution, respectively.
Tolerances— Not less than 80% (Q) of the labeled amount of C62H111N11O12 is dissolved in 90 minutes.
UNIFORMITY OF DOSAGE UNITS 905 : meet the requirements.
WATER, Method I 921 — For Capsules that contain powder, not more than 3.5% is found, using finely ground
Capsule contents.
Assay—
WHERE CAPSULES CONTAIN LIQUID—
Mobile phase and Chromatographic system— Proceed as directed in the Assay under Cyclosporine Injection.
Standard preparation— Dissolve an accurately weighed quantity of USP Cyclosporine RS in dehydrated alcohol
to obtain a solution having a known concentration of about 1 mg per mL. Use this solution promptly after
preparation.
Assay preparation— Using a sharp blade, carefully cut open not fewer than 20 Capsules, and with the aid of
dehydrated alcohol transfer the contents of the Capsules to a suitable volumetric flask. Wash the blade with
dehydrated alcohol, and transfer the washings to the volumetric flask. Dilute the contents of the volumetric flask
with dehydrated alcohol to volume, and mix. Quantitatively dilute an accurately measured volume of this solution
with dehydrated alcohol to obtain a solution having a concentration of about 1 mg of cyclosporine per mL.
Procedure— Separately inject equal volumes (about 20 µL) of the Standard preparation and the Assay
preparation into the chromatograph, record the chromatograms, and measure the areas for the major peaks.
Calculate the quantity, in mg, of cyclosporine (C62H111N11O12) in each Capsule taken by the formula:
(L/D)(CP/1000)(rU / rS)
in which L is the labeled quantity, in mg, of cyclosporine in each Capsule taken; D is the concentration, in mg per
mL, of the Assay preparation, based on the labeled quantity of cyclosporine in the Capsules taken and the extent
of dilution; C is the concentration, in mg per mL, of USP Cyclosporine RS in the Standard preparation; P is the
purity, in µg per mg, of USP Cyclosporine RS; and rU and rS are the peak areas obtained from the Assay
preparation and the Standard preparation, respectively.
WHERE CAPSULES CONTAIN POWDER—
Mobile phase— Prepare a filtered and degassed mixture of acetonitrile, water, methanol, and phosphoric acid
(605:400:50:0.5). Make adjustments if necessary (see System Suitability under Chromatography 621 ).
Diluting solvent— Prepare a mixture of acetonitrile, tetrahydrofuran, and dehydrated alcohol (9:5:4).
Standard preparation— Transfer about 25 mg of USP Cyclosporine RS, accurately weighed, to a 25-mL
volumetric flask. Add 2.5 mL of water, and sonicate for 10 minutes. Add about 10 mL of Diluting solvent, sonicate
for 5 minutes, dilute with Diluting solvent to volume, and mix.
Assay stock preparation— Transfer the contents of 20 Capsules to a volumetric flask of such capacity, V, in mL,
to make a final concentration of 10 mg of cyclosporine per mL. Add 0.1V mL of water to the flask, and sonicate for
10 minutes. Add 0.4V mL of Diluting solvent to the flask, and sonicate for 5 minutes. Dilute with Diluting solvent to
volume, and mix.
Assay preparation— Transfer 5.0 mL of Assay stock preparation to a 50-mL volumetric flask, add 5 mL of water,
dilute with Diluting solvent to volume, and mix.
nm detector and a 4.6-mm × 25-cm column that contains packing L13 and is maintained at a constant
temperature of about 70 . The flow rate is about 2 mL per minute. Chromatograph the Standard preparation, and
record the peak areas as directed for Procedure: the column efficiency is not less than 700 theoretical plates; the
tailing factor is not more than 1.5; and the relative standard deviation for replicate injections is not more than
2.0%.
Procedure— Separately inject equal volumes (about 20 µL) of the Standard preparation and the Assay
preparation into the chromatograph, record the chromatograms, and measure the areas for the major peaks.
Calculate the quantity, in mg, of cyclosporine (C62H111N11O12) in each Capsule taken by the formula:

10CV(rU / rS)
in which C is the concentration, in mg per mL, of USP Cyclosporine RS in the Standard preparation; V is the
volume, in mL, of the volumetric flask used to prepare the Assay stock preparation; and rU and rS are the
cyclosporine peak areas obtained from the Assay preparation and the Standard preparation, respectively.

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CYCLOSPORINE CAPSULES

19.6 Solvent Polarity
The relative polarity of a solvent is a useful guide to solvent selection in partition chromatography. The relative
polarities of the listed solvents may differ slightly depending on the literature source, since the scale used to
measure polarity may be different. The following should suffice as a general reference for relative solvent polarity.
Polarity is increasing from top to bottom.
• Fluoroalkanes (least polar)
• Isooctane
• Hexane
• Carbon tetrachloride
• Toluene
• Chloroform
• Methylene chloride
• Diethyl ether (ether)
• Ethyl acetate
• Dioxane
• Acetone
• Tetrahydrofuran (THF)
• Isopropanol
• Ethanol
• Acetonitrile
• Methanol
• Acetic acid
• Water
• Buffer Solutions (most polar)

19.7 Analyte Functional Group Polarity
The various analytes to be separated may also be arranged based on the polarities of their functional groups. A
general guide to relative solute polarity going from non-polar to the most polar group is as follows:
• Hydocarbons (least polar)
• Amides
• Ethers
• Esters
• Ketones
• Aldehydes
• Amines
• Alcohols
• Carboxylic Acids

19.8 Glossary of HPLC Terms
A
Activity
In adsorption chromatography, this is the relative strength of the surface of the packing. For
silica gel, the more exposed the silanol groups, the more active the surface. Activity can be
controlled by adding water or another polar modifier, which is hydrogen bonded to the active
sites, thereby reducing the surface activity.
Adsorbent
Packing used in adsorption chromatography. Silica gel and alumina are the most frequently used
adsorbents in HPLC.
Adsorption
The process of interaction between the solute and the surface of an adsorbent. The forces
involved can be strong such as hydrogen bonds, or weak such as van der Waals forces. For silica
gel, the silanol group is the driving force for adsorption, and any solute functional group that can
interact with this group can be retained by liquid-solid chromatography on silica.
Adsorption chromatography
One of the basic LC modes which relies on the adsorption process to effect the separation. Silica
gel and alumina are the most frequently used supports. The molecules are retained by the
interaction of their polar functional groups with the surface functional groups such as silanols of
silica.
Adsorption isotherm
In adsorption chromatography, this is a plot of the equilibrium concentration of sample in the
mobile phase per unit volume verses the concentration in the stationary phase per unit weight.
The shape of the adsorption isotherm can determine the chromatographic behavior of the solute
such as tailing, fronting, and sample overload.
Affinity chromatography
A technique in which a biospecific adsorbent is prepared by coupling a specific ligand (such as
an enzyme, antigen, or hormone) for the macromolecule of interest to a solid support (or
carrier). This immobilized ligand will interact only with molecules that can selectively bind to it.
Molecules that will not bind elute unretained. The retained compound can later be released in a
purified state. Affinity chromatography is not a chromatographic technique but selective
filtration.
Alumina
An adsorbent sometimes used in adsorption chromatography. Aluminum oxide (AI203) is a
porous adsorbent that is available with a slightly basic surface. For this reason, it can have
advantages over silica, which is considered to have an acidic surface.
Amino phase
A propylamino stationary phase used mostly in normal bonded phase chromatography. It is
somewhat reactive for any solute molecule or mobile phase additive that can react with amines.
The amino phase has found some applications as a weak anion exchanger.
Asymmetry
A factor describing the shape of a chromatographic peak. Theory assumes a Gaussian shape
peak that is symmetrical. The peak asymmetry factor is the ratio (at 10 percent of the peak
height) of the distance between the peak apex and the back side of the chromatographic curve to
the distance between the peak apex and the front side of the chromatographic curve. A value >1
is a tailing peak, while a value <1 is a fronting peak.

B
Backflushing
A column switching technique in which a four-way valve placed between the injector and the
column allows the mobile phase to flow in either direction. Backflushing is used to elute
strongly held compounds at the head of the column. It can be used to analyze these compounds
or merely to remove them from the column.
Backpressure
The pressure above gravity at the head of the column. Expressed in psig, bar, atm., or MPa.
Bandspreading
The dilution of the chromatographic band as it moves down the column. The peak is injected as
a slug, and, if not for the process of band broadening, each separated component would elute as
a narrow slug of pure compound. The measure of band broadening is band width, tw, or more
correctly, the number of theoretical plates in the column, N.
Baseline
The baseline is the line drawn by the data system when the only signal from the detector is from
the mobile phase.
Baseline noise
Interferences to the detector and data system caused by electrical noise or environmental effects.
This interference keeps the baseline from being perfectly flat.
Baseline-resolved peaks
When both sides of a peak reach the baseline without interfering with other peaks.
BET method
A method developed by Bruner, Emmett, and Teller for measuring surface area by using
nitrogen adsorption condensation in pores at liquid nitrogen temperature. Pore volume and pore
size distribution can also be obtained by this method.
Bonded-phase chromatography (BPC)
A stationary phase chemically bonded to a support that is used for the separation. It is the most
commonly used LC mode. The most popular support used is microparticulate silica gel. An
organosilane, such as octadecyl (for reversed-phase chromatography), is the most accepted type
of bonded phase. Approximately 70 percent of all HPLC is carried out on chemically bonded
phases.
Breakthrough volume
The volume at which a particular solute pumped continuously through a column begins to elute.
The breakthrough volume is useful in determining the total sample capacity of the column for a
particular solute.
C
Calibration standards
These are standards of known quantities and substances which assist in peak identification or
quantitation. Calibration standards can be external standards or internal standards.
Capacity factor
A chromatographic parameter that measures the degree of retention. See k’ for calculation
method.
Capping
Same as Endcapping.
Carrier
A term used most often in affinity chromatography, which refers to the support that is used to
carry the active ligand, usually by a covalent bond. It can also refer to the support in other

chromatographic modes.
Cartridge column
A type of column with no endfittings which is held in a cartridge holder. The column is an open
tube with the packing contained by frits in each end. Cartridges are easy to change, less
expensive, and more convenient than conventional columns with endfittings.
Chain length
The length of carbon chain in the hydrocarbon portion of a reversed-phase packing. It is
expressed as the number of carbon atoms such as C8 and C18.
Channeling
This occurs when voids are created in the packing material of a column. It results in the mobile
phase and accompanying solutes moving more rapidly than the average flow velocity producing
band broadening. These voids are created by poor packing or by erosion of the packed bed.
Chemisorption
This is sorption caused by a chemical reaction with the packing. Most of these interactions are
irreversible. They usually occur on packings with reactive functional groups such as silanol or
bonded amino phases.
Chiral stationary phases
Stationary phases that are designed to separate enantiomeric compounds. They can be bonded to
solid supports, created in situ on the surface of the solid support, or they can be surface cavities
that allow specific interactions with one enantiomeric form.
Chromatography
A chemical separation technique based on the differential distribution of the constituents of a
mixture between two phases, one of which moves relative to the other.
Chromatographic methods
A record of the parameters used in a separation that yields a particular result. It allows another
analyst following the method and conditions to reproduce the separation, and achieve the same
results.
Chromatogram
The electronic result of a chromatographic separation which is a plot of detector signal output
versus time or elution volume. It is represented as a series of peaks from the data system.
Chromatographic conditions
Parameters used in an analysis such as column type, mobile phase, and wavelength.
Column
A tube which contains the stationary phase. The stationary phase differentially interacts with the
sample’s constituent compounds as they are carried along in the mobile phase.
Column backpressure
See Backpressure.
Column chromatography
Any form of chromatography that uses a column to hold the stationary phase. Open column
chromatography, HPLC and open tubular capillary chromatography are all examples.
Column-dependent chemical factors
Chemical factors associated with column packing materials including the nature of the base
material, surface activity of the bonded phase, and degree of interference from silanol groups.
Column performance
The efficiency of a column which is measured as the number of theoretical plates for a given
test compound.

Column switching
The use of multiple columns connected by switching valves. Fractions from a primary column
can be switched to two or more secondary columns, which in turn can be further diverted to
additional columns or to the detector. This process is used for better chromatographic
separations or for sample cleanup.
Conductivity detectors
These detectors identify changes in the conductivity of the mobile phase as it passes through a
flow cell. They are used to detect a wide variety of ionized species separated by ion
chromatography.
Connecting tube
A tube that connects the column to the injector and detector. Diffusion within connecting tubing
broadens the peaks, but does not contribute to the separation.
Counterion
In an ion-exchange process, this is the ion in solution which is used to displace the ion of
interest from the ionic site. In ion pairing, it is the ion of opposite charge added to the mobile
phase to form a neutral ion pair in solution.
Coupled columns
A form of column switching. This uses a primary column connected to two secondary columns
via a selector valve. Fractions from the first column can be selectively transferred to the other
two columns for additional separation. This term is also used to describe two or more columns
connected in series to provide increased plate numbers.
Coverage
The amount of bonded phase on a silica support in bonded phase chromatography. The coverage
is usually described in mmol/m2 or in terms of percent C.
Cross-linking
During the process of copolymerization of resins to form a three dimensional matrix, and a
difunctional monomer is added to form cross-linkages between adjacent polymer chains. The
degree of cross-linking is determined by the amount of this monomer added to the reaction. For
example, divinylbenzene is a typical cross-linking agent for polystyrene ion-exchange resins.
The swelling and diffusion characteristics of a resin are governed by its degree of cross-linking.
Cyano phase
A stationary phase that usually consists of cyanopropylsilyl groups. It is used in both normal and
reversed-phase chromatography.
D
Dead volume (Vd)
The volume outside of the column packing itself. The interstitial volume (intraparticle and
interparticle volume) plus extracolumn volume (contributed by injector, detector, connecting
tubing, and endfittings) all combine to create the dead volume. This volume can be determined
by injecting an inert compound. For example, a compound that does not interact with the
column packing. It is also abbreviated Vo or Vm.
Degassing
The process of removing dissolved gas from the mobile phase before or during use. Dissolved
gas may come out of solution in the detector cell and cause baseline spikes and noise. Dissolved
air can affect electrochemical detectors by reaction or fluorescence detectors by quenching.
Degassing is carried out by: heating the solvent, by vacuum ( in a vacuum flask), on-line using
evacuation of a tube made from a gas permeable substance such as PTFE, or by helium

sparging.
Desalting
A technique in which low molecular weight salts and other compounds are removed from non-
ionic and high molecular weight compounds.
Detector
An electronic device that quantitatively discerns the presence of the separated components as
they elute. There are different types of detectors. Some of the common detector types are:
UV/Visible light absorbance, differential refractive index, electrochemical, conductivity, and
fluorescence.
Detection limits
The post-column concentration of a compound and not the concentration in the sample. A
detector specified to detect a compound at 0.1 ppm may be also acceptable for a method that
tests a water sample containing the compound at a much lower concentration. This is because
the compound can be concentrated on the column or sample preparation techniques may used to
preconcentrate the sample as well.
Detection threshold
The point at which the software determines the beginning and the end of the peak will shift
depending on the threshold. The threshold should be set as low as possible. If it is set too low,
the software will interpret baseline noise as peaks. If it is set too high, a portion of the bottom of
the peak may be cut off and is not quantitated.
Detector Linearity
The linearity of the response over a range of sample concentrations controlled by a response
factor setting on the detector, or in the detector controller software.
Detector Sensitivity
The sensitivity setting is the line between normal background noise and a true peak.
Perturbations from the baseline that fall below the sensitivity setting are considered noise, and
are filtered out. Setting the sensitivity too high may result in missing small peaks, while setting
it too low may result in a lot of spurious raw data as the software tries to integrate peaks out of
the noise.
Differential Refractive Index (RI)
Detectors that identify the difference in the mobile phase’s refractive index when it contains
dissolved sample. RI detectors are universal detectors which respond to the presence of all
compounds. Because the RI detector responds to changes in mobile phase composition, they are
not suitable for gradient methods. RI detectors are less sensitive than UV/Vis detectors, but are
useful when the sample molecule does not have a chromophore.
Diffusion along the flow path
The coefficient for molecular diffusion along the flow path. It is represented as the B term in the
van Deemter equation. The greater the diffusion coefficient of the component in the mobile
phase, the more the narrow band of separated component diffuses into the surrounding mobile
phase before going to the detector. This results in band broadening. As linear velocity increases,
the effect of axial diffusion on column efficiency decreases. The van Deemter equation reflects
this effect by dividing B by u.
Diffusion coefficient
(DM or DS)
A fundamental parameter of a molecule in solution (Dm) or stationary phase (Ds) expressed in
cm2/sec. Dmdepends on molecular weight of the solute, temperature, solvent viscosity, and
molar volume of the solute. A typical value of a small molecule in RPC at room temperature

would be 5 x 106 cm2/sec.
Diode Array Detection (DAD) – See entry under Photodiode Array Detection (PDA
Diol phase
A stationary phase useful in both normal and reversed phase chromatography. It consists of a
diol structure (two OH groups on adjacent carbon atoms in an aliphatic chain). It is less polar
than silica stationary phases used in normal-phase chromatography but it has been used for the
reversed-phase separation of proteins and polypeptides.
Displacement chromatography
A chromatographic process in which the sample is placed onto the head of the column and is
then displaced by a compound that is more strongly sorbed than the compounds of the original
mixture. Sample molecules are displaced by each other and by the more strongly sorbed
compound. The result is that the eluting sample solute zones may be sharpened. Displacement
techniques have been used mainly in preparative EPLC applications.
Distribution coefficient
(D or KD)
See Partition coefficient
E
Eddy diffusion term
This is represented by the A term in the van Deemter equation. It is the contribution to plate
height that is due to molecules traveling along different paths through the column and it
depends on the particle size and geometry of the packing. It is also called the multipath term.
See van Deemter equation.
Effective theoretical plates (Neff)
The true number of plates in a column that corrects the number of theoretical plates for dead
volume. , where tm is the void time.
Efficiency (N or H)
A measure determined by the number of theoretical plates calculated from the equation:
This can be visually represented by the following figure.
Measure peak width at 4.4% of peak height
Electrochemical detector
Al detector which monitors an oxidation or reduction reaction between the detector’s working
electrode and the sample. Electrochemical detectors are characterized by high sensitivity, with
detection limits in the low ppb range common for electroactive compounds.
Eluate
Combination of mobile phase and solute exiting column.
Eluent
Mobile phase used to carry out a separation.
Eluotropic series
A series of solvents with an increasing degree of polarity, generally used to explain solvent
strength in liquid-solid or adsorption chromatography. A nonpolar solvent such as pentane
would be at one end of the scale; dichloromethane would be an intermediate solvent; a strongly
polar solvent, such as water, would be at the other end of the scale. Thus, when developing a
method or running a gradient, an eluotropic series is useful for selecting solvents.
Elution

The process of passing mobile phase through the column to transport solutes.
Elution order
The order in which the separated compounds comes off of the column (elutes) and through the
detector.
Elution chromatography
The most commonly used chromatographic method. The sample is injected at the head of the
column, and the individual compounds are separated and eluted at the end of the column.
Elution volume (VR)
The volume of mobile phase required to elute a solute from the column at maximum
concentration (apex).
Endcapping
A column is said to be endcapped when a small silylating agent, such as trimethylchlorosilane,
is used to bond residual silanol groups on a packing surface. It is most often used with reversed-
phase packings and may cut down on undesirable adsorption of basic or ionic compounds.
Endfitting
The fitting at the end of the column that connects the column to the injector or detector. Most
HPLC endfittings contain a frit to hold the packing and have a low dead volume for minimum
band spreading and usually made of stainless steel.
Exchange capacity
See Ion-exchange capacity.
Exclusion chromatography
See Steric-exclusion chromatography (SEC).
Exclusion limit
In SEC, this is the upper limit of molecular weight (or size), beyond which molecules will elute
at the same retention volume. Many SEC packings are referred to by their exclusion limit. For
example, a 105 column of porous silica gel will exclude any compounds with a molecular
weight higher than 100,000, based on a polystyrene calibration standard.
Exclusion volume (VC)
The retention volume of a molecule on SEC packing. All molecules larger than the size of the
largest pore are totally excluded and elute at the interstitial volume of the column.
Extracolumn effects
The band broadening effects of parts of the chromatographic system outside of the column
itself. Areas of band broadening can include the injector, connecting tubing, endfittings, frits,
detector cell volume, and internal detector tubing. The variances of all of these contributions are
additive. These extracolumn effects should be minimized in order to maintain the efficiency of
the column.
External standards
A separate sample containing known quantities of the same compounds of interest. External
standards are used primarily for peak identification by comparing elution times.
F
Fast LC
The use in BPLC of short columns (3 to 7 cm in length) with conventional internal diameters (2
to 6 mm) packed with small particles (3 or 5 mm dp). The separation times are commonly in
minutes and sometimes seconds.
Flow rate (F)
The volumetric rate of flow of mobile phase through an LC column. For a conventional HPLC
column of 4.6 mm i.d., typical flow rates are 1 to 2 mL/min.

Fluorescence detectors
Fluorescence detectors project a specified wavelength of light into the sample, causing the
component of interest to fluoresce and the emitted light is detected. Fluorescence detectors are
commonly used with derivatization methods. Fluorescence detectors are very selective and very
sensitive.
Fractionation range
This is the range in which the packing can separate molecules based on their size. Molecules
that are too large to diffuse into the pores are excluded. Molecules that can diffuse into all of
the pores totally permeate the packing, eluting (unseparated) at the permeation volume. In SEC,
it is the operating range of a gel or packing.
Frit
The porous component at either end of a column that serves to contain the column packing. It is
placed at the very ends of the column tube or, more commonly, in the endfitting. Frits are made
from stainless steel or other inert metal or plastic, such as porous PTFE or polypropylene.
Frontal analysis
A chromatographic technique that involves continuous addition of sample to the column. The
result is that only the least sorbed compound, which moves at the fastest rate, is obtained in a
pure state. The second least sorbed compound elutes with the first eluting compound, the third
least sorbed compound with the first and second compound and so forth. This continues until
the original sample is eluting at the column exit. Frontal analysis is seldom used and is mainly a
preparative technique.
Fronting
This is an asymmetrical peak shape in which the front part of a peak (before the apex) in a
chromatogram tapers in advance of the remainder of the peak. There is an asymmetric
distribution with a leading edge. The asymmetry factor for a fronting peak has a value <1. The
opposite effect is tailing. Fronting is related to the shape of the sorption isotherm.
G
Gaussian curve
A standard error curve, based on a mathematical function, that is a symmetrical, bell shaped
band, or peak. Most chromatographic theory assumes a Gaussian peak.
Gel
The solid packing used in gel permeation chromatography. A gel actually consists of two parts:
the dispersed medium (solid portion) and the dispersing medium (the solvent).
Gel filtration chromatography (GFC)
This is size-exclusion chromatography carried out with aqueous mobile phases, also known as
aqueous GPC. It generally refers to separations carried out on soft gels such as polydextrans.
Most gel filtration separations involve biopolymers and are used for the separation and
characterization of polymers.
Gel permeation chromatography (GPC)
See GFC.
Gradient elution
A technique for decreasing separation time by increasing mobile phase strength over time
during a chromatographic separation. It is also known as solvent programming. Gradients can

be continuous or step-wise. Binary, ternary, and quaternary solvent gradients are used routinely
in HPLC.
Guard column
A small column placed between the injector and the analytical column. It protects the analytical
column against contamination by sample particulates, and by strongly retained species. The
guard column is usually packed with the same material as the analytical column and is often of
the same i.d. It is much shorter, costs less, and is usually discarded when it becomes
contaminated.
H
HETP
The height equivalent of a theoretical plate. It is a carryover from distillation theory which is a
measure of a column’s efficiency. For a typical HPLC column packed with 5 mm particles,
HETP (or H) values are usually between 0.01 and 0.03 mm. HETP = L/N, where L is column
length, and N is the number of theoretical plates.
Hydrophilic
It is often referred to as water loving. It adverts both to water compatible stationary phases, and
to water soluble molecules. Most columns used to separate proteins are hydrophilic in nature
and should not sorb or denature protein in the aqueous environment.
Hydrophobic
It is often referred to as water hating. It adverts both to stationary phases not compatible with
water and molecules with little affinity for water. Hydrophobic molecules have few polar
functional groups and are mostly hydrocarbons or have high hydrocarbon content.
Hydrophobic interaction chromatography
A technique in which reversed-phase packings are used to separate molecules by the
interactions between their hydrophobic moieties and the hydrophobic sites on the surface. High
salt concentrations are used in the mobile phase and separations are effected by changing the
salt concentration. The technique is analogous to "salting out" molecules from solution.
Gradients are run by decreasing the salt concentration over time.
I
Injector
A mechanism for accurately injecting a predetermined amount of sample into the mobile phase
stream. The injector can be a simple manual device, or a sophisticated autosampler that permits
automated injections of many different samples for unattended operation.
In-line filter
A device that prevents particulate matter from damaging the column by filtration. Modem in-
line filters can be placed between the injector and the column without contributing to band
broadening. A filter in this position is used to prevent sample particulates from entering the
packed bed or the inlet frit.
Inlet
The initial part of the column, where the solvent and sample enter. There is usually an inlet frit
that holds the packing in place and, in some cases, protects the packed bed.
Internal standards
Internal standards consist of a specific quantity of a compound that is known not to be in the
sample, but that exhibits the same characteristics under the separation conditions as the sample
components. Internal standards are used primarily to calibrate quantitation, especially for

methods susceptible to volumetric error resulting from sample preparation.
Interstitial volume (VO)

The total volume of mobile phase within the length of the column. It is made up of the
intraparticle volume (inside the packing itself) and interparticle volume (between the packing
particles). Same as Void volume. It is also abbreviated Vi or Vm.
Ion chromatography (IC)
An ion-exchange technique in which low concentrations of anions or cations are determined
using low capacity ion exchangers with weak buffers. Conductivity detectors are often used.
Ion chromatography is practiced in two forms: suppressed IC, and non-suppressed IC.
Ion-exchange chromatography (IEC)
A mode of chromatography in which ionic substances are separated on cationic or anionic sites
of the packing. The sample ion (and usually a counterion) will exchange with ions already on
the ionogenic group of the packing. Retention is based on the affinity of different ions for the
site and on a number of other solution parameters such as, pH, ionic strength, and counterion
type.
Ion-exchange capacity
The number of ionic sites on the packing that can take part in the exchange process. The
exchange capacity is expressed in mequiv/g. Typical strong anion-exchange resin may have 3 to
5 mequiv/g capacity.
Ion exclusion
A process in which ionized solutes can be separated from un-ionized or partially ionized solutes
using ion-exchange resins. Separation results from Donnan membrane potential. This is where
ionic solutes exist at a higher concentration in solution than in the resin but nonionic solutes are
evenly distributed between the mobile phase and resin. Therefore, ionic solutes will elute faster
from the column than the nonionic solutes.
Ion-pair chromatography
A form of chromatography in which ions in solution can be "paired" or neutralized and
separated as an ion pair on a reversed-phase column. Ion-pairing agents are usually ionic
compounds that contain a hydrocarbon chain which imparts a certain hydrophobicity so that the
ion pair can be retained on a reversed-phase column. Ion-pairing can also occur in normal-
phase chromatography when one part of the pair is loaded onto a sorbent, but this technique is
not as popular as the RPC technique.
Ion suppression
This involves buffering in an aqueous mobile phase at a particular pH to suppress solute
ionization. For example, the ionization of weak carboxylic acids can be suppressed by adjusting
the pH below their ionization constant. This technique is useful for improving the peak shape of
weak acids and bases in RPC.
Irregular packing
The shape of a silica gel-based packing. Irregular silicas are available in microparticulate sizes.
The packings are made by grinding silica gel into small particles, sizing them into small
particles, and into narrow fractions using classification machinery. While spherical packings are
now used more often than irregular packings in HPLC, the less expensive irregular packings are
still widely used in prep LC.
Irreversible adsorption

A state when a compound with a very strong affinity for the adsorbent is injected into a column,
it is so strongly adsorbed that it cannot be eluded from the column. An example of irreversible
adsorption is a chemical reaction between the sample and the surface of the adsorbent.
Isocratic
A constant composition mobile phase used in liquid chromatography.
K
k or k'
The capacity factor. It can be calculated from the equation, where tR is retention time for the
sample peak, and to is the retention time for an unretained peak.
L
Ligand
In ligand-exchange chromatography, it is the molecule added to the mobile phase that acts as
the chelating agent. In affinity chromatography, it is the biospecific material (enzyme, antigen,
or hormone) coupled to the support (carrier) to form the affinity column.
Ligand-exchange chromatography
A technique in which chelating ligands are added to the mobile phase and undergo sorption
onto a packing. These sorbed molecules, can act as chelating agents with solutes. For example,
using of copper amine chelates for the separation of amino acids. Chelating resins function in a
similar manner. The chelating groups are chemically bonded to the polystyrene backbone.
Linearity
A measurement process which ensures accurate quantitation. In chromatography it is important
that the detector provide a linear response over the range of sample concentrations it
encounters.
Linear elution adsorption chromatography (LEAC)
A term coined by Lloyd Snyder, which refers to adsorption chromatography carried out in the
linear portion of an adsorption isotherm.
Linear velocity(u)
A measure of the speed with which an unretained compound moves through the column. Linear
velocity is represented by the letter u and is reported in cm/min or mm/sec. Linear velocity is
used to calculate flow rate based on the cross sectional area of a column. Linear velocity is
useful for adapting methods from one column diameter to another.
Liquid-liquid chromatography (LLC)
This is the same as Partition chromatography. It was the earliest form of HPLC, which was
replaced with chemically bonded phases in the early 1970s.
Liquid-solid chromatography (LSC)
Same as Adsorption chromatography.
Loading
The amount of stationary phase coated or bonded onto a solid support. In liquid-liquid
chromatography, it is the milligram amount of liquid phase per gram of packing. In BPC, the
loading may be expressed in mmol/m2 or in percent C. See Coverage.
M
Macroporous resin
Cross-linked ion-exchange resins that have both micropores of molecular dimensions and
macropores several hundred Å wide. These are highly porous resins with large internal surface
areas accessible to large molecules.

Mass transfer
The process of solute movement into and out of the stationary phase or mobile phase. It is
represented by the C term of the van Deemter equation and is referred to as the mass transfer
term. The faster the process of mass transfer, the better the efficiency of the column. In HPLC,
mass transfer is the most important factor affecting column efficiency. It is increased by the use
of small particle packings, thin layers of stationary phase, low viscosity mobile phases, and
high temperatures.
Mean pore diameter
The average pore diameter in a porous packing. The pore diameter is important because it
allows free diffusion of solute molecules so they can interact with the stationary phase. In SEC,
the packings have different pore diameters, allowing molecules of different sizes to be
separated. For a typical adsorbent such as silica gel, 60 Å and 100 Å pore diameters are most
popular. For packings used for the separation of biomolecules, pore diameters > 300 Å are used.
Method-dependent chemical factors
The variables in the chromatographic method which include the choice of mobile phase,
column temperature, sample preparation method, sample size, flow rate, reagent quality, and
laboratory technique.
Micellar chromatography
The addition of micelles to the mobile phase to effect separations. The micelles act as
displacing or partitioning agents and provide another parameter that can be used to change
selectivity.
Microbore
Columns with smaller than usual internal diameters ( < 2 mm) used in HPLC.
Microparticulate
Small particles used as HPLC stationary phases. These packings generally have a particle
diameter <10 mm are considered microparticles.
Microporous resin
Same as Microreticular resin.
Microreticular resin
Cross-linked synthetic ion-exchange resins with pore openings that correspond to molecular
sizes. Diffusion into the narrow pores can be impaired, and low exchange rates, as well as poor
performance, can occur, especially for large molecules.
Minimum plate height
The minimum of the curve that results from a plot of H vs. u. This value represents the most
theoretical plates that can be obtained for a certain column and mobile phase system. It usually
occurs at very low flow rates.
Mixed-bed column
Combination of two or more stationary phases in the same column. It is used most often in IEC
and SEC. The advantage in IEC is the total removal of ionic compounds. It is useful in SEC
because a wider molecular weight range can be accommodated by the column.
Mobile phase
The solvent that moves the solute through the column.
Modifier
An additive that changes the character of the mobile phase. For example, in reversed phase
water is the weak solvent and methanol is the strong solvent. Methanol is known then as the
modifier.
Molecular weight distribution

The distribution of molecular weight of molecules in a polymer sample. Distribution can be
defined as weight average and number average.
Monomeric phase
A bonded phase in which single molecules are bonded to a support. For silica gel, monomeric
phases are prepared by the reaction of an alkyl or arylrnonochlorosilane. Polymeric phases are
generally prepared from a di or trichlorosilane reactant.
Multidimensional chromatography
The use of two or more columns or chromatographic techniques which enable a better
separation. It is useful for sample cleanup, increased resolution, and increased throughput. It
can be used off-line by collecting fractions and reinjecting onto a second column or on-line by
the use of a switching valve. It also called coupled column chromatography, column switching,
multicolumn chromatography, or boxcar chromatography.
N
N
The number of theoretical plates. A measure of the efficiency of a column. , where tR is
retention time, and wb is the base width of the peak. Sometimes it is measured as , where wl/2 is
the peak width at half height.
Narrow-bore column
Columns of < 0.5 mm i.d. used in HPLC.
Nonporous particle
A solid particle used as a support for a porous coated or bonded phase.
Non-suppressed Ic
A type of ion chromatography were weakly conducting buffers at low concentration are
carefully selected, and the entire effluent is passed through the detector. The ions are detected
above the background signal.
Normal-phase chromatography
A mode of chromatography carried out with a polar stationary phase and a nonpolar mobile
phase. Adsorption on silica normal phase system. It also refers to the use of polar bonded
phases, such as CN or NH2. It is sometimes referred to as straight phase chromatography.
O
Octadecylsilane (ODS)
The most popular reversed phase in HPLC. Octadecylsilane phases are bonded to silica or
polymeric packings. Both monomeric and polymeric phases are available.
Open-tubular columns
Columns of small internal diameter. Stationary phases can be bonded on the internal walls of
these columns. The most common type is the fused silica tubing made for capillary GC. These
columns are currently being investigated for HPLC, SFC, and capillary electrophoresis.
Organic modifier
A water miscible organic solvent which is added to an aqueous mobile phase to effect
separations in reversed-phase HPLC.
Overload
The increased mass of sample injected onto a column which begins to affect efficiency and
resolution. See Sample capacity.
P
PEEK
Polyetheretherketone - semicrystalline thermoplastic with extraordinary mechanical properties.
The material is also resistant to both organic and aqueous environments, and is used in

bearings, piston parts, pumps, compressor plate valves, and cable insulation applications and
HPLC fittings and high pressure tubing.
Packed bed quality.
The coefficient that reflects the quality of packed bed. It is represented as the A term in the
expanded van Deemter equation. This term is determined by the size and distribution of voids,
channels, and other nonuniformities in the packed bed. It represents a practical lower limit for
H, and can be changed by using a better packed bed.
Paired-ion chromatography
Same as Ion-pair chromatography.
Partially-resolved peaks
When two or more adjacent peaks run together and are not baseline resolved.
Particle size (dp)
The average particle size of the packing in an LC column.
Particle-size distribution
A measure of the distribution of the particles used to pack the LC column. In HPLC, a narrow
particle size distribution is desirable. A particle size distribution of dp ±10 percent would mean
that 90 percent of the particles fall between 9 and 11 mm for an average 10 mm dp packing.
Partition chromatography
A separation process in which one of the liquid phases is held stationary on a solid support
while the other is allowed to flow freely down the column. Solutes partition themselves
between the two phases based on their individual partition coefficients. Liquid-liquid
chromatography is an example.
Partition coefficient (K)
The amount of solute in the stationary phase relative to the amount of solute in the mobile
phase. It can also be the distribution coefficient, KD.
Peak
When the detector registers the presence of a compound, the normal baseline signal it sends to
the data system changes, resulting in a deflection from the baseline called a peak. Well resolved
peaks are symmetrical, touch the baseline, and do not interfere with other peaks.
Peak Identification
Peak identification is usually performed by comparing the sample chromatogram to a
chromatogram of a standard solution separated under the same conditions. Peaks that appear at
the same elution time as peaks in the standard are identified as the same component.
Peak Quantitation
Correctly detecting the beginning and end of a peak.
Peak shape
The profile of a chromatographic peak. Theory assumes a Gaussian peak shape (perfectly
symmetrical). A peak asymmetry factor is used to describe a shape as a ratio. See Asymmetry.
Peak tailing
Same as tailing peaks.
Peak width (wb)
Same as Band width.
Permeation
In SEC, this is the process in which a solute can enter a mobile phase filled pore of the packing.
Phenyl phase
A non polar bonded phase prepared by the reaction of dimethylphenylchlorosilane with silica
gel. It is claimed to have an affinity for aromatic compounds.

Photodiode Array (PDA)
PDA detectors are UV/Vis detectors that record the absorbance of light at many different
wavelengths simultaneously.
Physical factors
Variables such as particle size, particle surface area, column dimensions, leaks in the fluid path,
system bandspreading, and detector cell design.
Plates
The theoretical plates in a packed column. See Theoretical plate.
Polyacrylamide gel
Neutral hydrophilic polymeric packings used in aqueous SEC. It is prepared by the
copolymerization of acrylamide with (N, N'-methylene) bisacrylamide.
Polymeric packings
Packings based on polymeric materials, usually in the form of spherical beads. The common
polymers used in LC are polystyrene-divinylbenzene, polyacrylamide, polymethylacrylate,
polyethyleneoxide, polydextran, and polysaccharide.
Polystyrene-divinylbenzene resin (PS-DVB)
The most common polymer base for ion-exchange chromatography. Ionic groups are
incorporated by various chemical reactions. Neutral PS-DVB beads are used in reversed-phase
chromatography. Porosity and mechanical stability can be altered by varying the cross-linking
through the variation of the DVB content.
Pore diameter
Same as Mean pore diameter.
Pore volume
The total volume of the pores in a porous packing that is usually expressed in mL/g. It is
measured by the BET method of nitrogen adsorption or by mercury intrusion, where Hg is
pumped into the pores under high pressure.
Porosity
The ratio of the volume of the interstices, to the volume of the solid particles. The pore volume
is also used as a measure of porosity.
Precolumn
A small column placed between the pump and the injector. It removes particulate matter which
could be in the mobile phase, chemically sorbs substances that might interfere with the
separation, or acts as a saturator column presaturating the mobile phase with stationary phase to
prevent stripping of the column. Its volume has little effect on isocratic elution but contributes a
delay to the gradient elution.
Preparative chromatography
The process of using liquid chromatography to isolate a sufficient amount of material for other
experimental or functional purposes. For pharmaceutical or biotechnological purifications,
columns several feet in diameter can be used for multiple grams of material. For isolating just a
few micrograms of a valuable natural product, an analytical column can be used. Both are
preparative chromatographic approaches.
Pulsating flow
Flow originating from a reciprocating pump. Normally, the pulses are dampened out by pulse
damper, by an electronic pressure feedback circuit, or by an active damper pump head. Some
detectors, such as electrochemical, are affected by flow pulsations.
Q
Qualitation

An analysis process which is designed to identify the components of a substance or mixture.
Quantitation
An analysis process which is designed to determine the amounts or proportion of the
components of a substance.
R
Radial compression
The use of radial pressure applied to a flexible wall column to lessen wall effects.
Radial diffusion
Diffusion across the LC column in a radial direction. If the sample is injected into the exact
center of a column, it will spread not only in a longitudinal direction as it flows, but radially as
well.
Recovery
The amount of solute (sample) that elutes from a column relative to the amount injected. This is
most often used with protein separations in which proteins "hang up" on active sites of the
packing in certain columns.
Recycling
A technique in which the column effluent is recirculated onto the head of the column in an
attempt to gain the advantage of an extended column length. It can be carried out on a single
column by passing the effluent back through the pump. An alternative technique uses two
columns connected by a switching valve. This technique is very seldom used in HPLC, and
only in size-exclusion chromatography.
Reduced plate height (h)
A calculated value used to measure efficiencies of columns. A BPLC column with an h value
<2 is considered to be well packed.
Reduced velocity (v)
A calculated value used to compare different chromatographic columns. It relates the solute
diffusion coefficient (Dm) in the mobile phase to the particle size of the column packing (dp).
Regeneration
A process of restoring the packing in the column to its initial state after a gradient elution. The
mobile phase is passed through the column step-wise or in a gradient solvating the stationary
phase to its original condition. In ion-exchange chromatography, regeneration involves
replacing ions taken up in the exchange process with the original ions that occupied the
exchange sites. Regeneration can also refer to bringing back any column to its original state by
removing impurities with a strong solvent.
Relative retention
Relative retention is a measure of the difference of affinities of two compounds for the
stationary phase. Mathematically it is the ratio of the retention factors of two compounds, one
of which is usually a standard. It is represented by the letter r, and is reported in dimensionless
units. Relative retention is used in quality control, reproducibility, and method validation
calculations.
Relative retention ratio
Same as Separation factor or Selectivity.
Residual silanols
The silanol (SiOH) groups that remain on the surface of a packing after a phase is chemically

bonded onto its surface. These silanol groups may not be accessible to the reacting bulky
organosilane such as octade- cyldimethylchlorosilane, but may be accessible to small polar
compounds. Often they are removed by endcapping with a small organosilane such as
trimethylchlorosilane. See Endcapping.
Resolution (Rs)
The ability of a column to separate chromatographic peaks. It is usually expressed in terms of
the separation of two peaks.
Retention factor
Retention factor is how long a compound is retained by the stationary phase relative to the time
it resides in the mobile phase. In IUPAC nomenclature, the retention factor is represented by
the letter k and is dimensionless. In former usage, k was often represented as k’, and was
termed the capacity factor.
Retention time (tR’)
The time between injection and the appearance of the peak maximum. The adjusted retention
time tR’ adjusts for the column void volume.
Retention volume (VR)
The volume of mobile phase required to elute a substance from the column. This is where Vm is
the void volume, KD the distribution coefficient, and Vs the stationary phase volume.
Reversed-phase chromatography (RPC)
The most common HPLC mode. Uses hydrophobic packings such as octadecyl or octysilane
phases bonded to silica or neutral polymeric beads. The mobile phase used is usually water and
a water miscible organic solvent such as methanol or acetonitrile. There are many variations of
RPC in which various mobile phase additives are used to impart a different selectivity. For
example, in the RPC of anions, the addition of a buffer and tetraalkylammonium salt would
allow ion pairing to occur, resulting in separations that rival ion-exchange chromatography.
S
Sample capacity
The amount of sample that can be injected onto an LC column without overload. It is often
expressed as grams of sample per gram of packing. Overload is defined as the sample mass
injected at which the column efficiency falls to 90 percent of its normal value.
Sample diffusion (within the column)

As a band of sample migrates along the column, diffusion causes it to broaden proportionally to
the square root of the distance it has traveled.
Sampling rate
The frequency with which the detector checks the flow cell. Sampling rate is often called time
constant. If it is set too fast too much raw data will be generated. If it is set too slow, a narrow
peak could be missed.
Selectivity (a)
The same as Separation factor or Relative retention ratio. A thermodynamic factor that is a
measure of relative retention of two substances. Fixed by a certain stationary phase and mobile
phase composition.
Semipreparative chromatography
Preparative liquid chromatography carried out on an analytical size (4 to5 mm i.d.) or slightly
larger (6 to 10 mm i.d.) column. Normal injection size is in the milligram to low gram range.
Separation factor

The separation factor, sometimes called selectivity, is the relative retention measured for two
adjacent peaks. The separation factor is represented by the Greek letter a, and is reported in
dimensionless units.
The serparation factor for two compounds is calculated using the formula .
Separation can be thought of as measuring either the difference between the centers of each
peak, or the distance betwee the tops of the peaks. Two compounds with a high a can appear far
apart on the chromatogram, though they are not resolved.
Silanol
The SiOH group found on the surface of silica gel. There are different strengths of silanols
depending on their location and relationship to each other. The strongest silanols are acidic and
often lead to undesirable interactions with basic compounds during chromatography.
Siloxane
The Si-O-Si bond. A principal bond found in silica gel, for attachment of a silylated compound,
or bonded phase. It is stable except at high pH values.
Silylation
The reaction of an organochloro- or organoalkoxysilane with a compound containing a reactive
group. In liquid chromatography, it refers to the process of derivatizing the solute before
chromatography in order to make it detectable or to prevent unwanted stationary phase
interactions. It can also refer to the process of adding a chemically bonded phase to a solid
support or to deactivating the packing to cut down on surface activity.
Size-exclusion chromatography (SEC)
Same as Steric exclusion chromatography.
Slurry packing
The technique most often used to pack HPLC columns with microparticles. The packing is
suspended in a slurry (10 percent wt/vol) and is rapidly pumped into the empty column. Special
high pressure pumps are used.
Soap chromatography
An early name for ion-pair chromatography. Long chain soaps or detergents were used as
mobile phase additives.
Solid-phase extraction (SPE)
A sample preparation technique that uses a solid phase packing contained in a small plastic
cartridge. The solid stationary phases are the same as HPLC packings but the principle is
different from HPLC. It is sometimes referred to as digital chromatography. This process as it is
most often practiced, requires four steps: conditioning the sorbent, adding the sample, washing
away the impurities, and eluting the sample in as small a volume as possible with a strong
solvent.
Solid support
Same as Support.
Solute
The dissolved component of a mixture that is to be separated in the chromatographic column.
Solvent strength
The ability of a solvent to elute a particular solute or compound from a column. Lloyd Snyder
described it for LEAC (LSC) adsorption chromatography on alumina and solvents were
quantitatively rated in an eluotropic series. No eluotropic series exists for other modes.
Sorbent
An adsorption packing used in liquid chromatography. A common sorbent is silica gel.
Specific surface area

The surface area of an LC packing based on a measurement by an accepted technique, such as
the BET method that uses nitrogen adsorption.
Spherical packing
A spherical solid packing material. Spherical packings are generally preferred over irregular
particles.
Stationary phase
The immobile phase involved in the chromatographic process. The stationary phase in liquid
chromatography can be a solid, a bonded or coated phase on a solid support, or a wall coated
phase. The stationary phase used often characterizes the LC mode. For example, silica gel is
used in adsorption chromatography, an octadecylsilane bonded phase in reversed-phase
chromatography, etc.
Stepwise elution
This process uses eluents of different compositions during the chromatographic run. These
eluents are added in a stepwise manner with a pump, or by a selector valve. Gradient elution
incorporates continuous changing of solvent composition.
Steric exclusion chromatography (SEC)
A major LC mode in which samples are separated by virtue of their size in solution. Also
known as size-exclusion, gel permeation, gel filtration, or gel chromatography.
Straight-phase chromatography
Same as Normal-phase chromatography.
Supercritical fluid chromatography (SFC)
A technique that uses a supercritical fluid as the mobile phase. The technique has been applied
to the separation of substances that cannot be handled effectively by liquid chromatography
(because of detection problems) or gas chromatography (because of the lack of volatility).
Examples are separations of triglycerides, hydrocarbons, and fatty acids. GC detectors and
HPLC pumps have been used together in SFC.
Support
The solid particles in a column. The support can be naked, coated, or can have a chemically
bonded phase in HPLC.
Suppressed IC
A second column is used to remove the buffer ions so that sample ions can be more easily
detected; membrane separator is sometimes used.
Suppressor column
In ion chromatography, it is the column placed after the ion-exchange column. Its purpose is to
remove or suppress the ionization of buffer ions so that sample ions can be observed in a
weakly conducting background with a conductivity detector.
Surface area
In an adsorbent, it is the total area of the solid surface as determined by an accepted
measurement technique such as the BET method using nitrogen adsorption. The surface area of
a typical porous adsorbent such as silica gel can vary from 100 to 600 m2/g.
Surface coverage
The mass of stationary phase per unit area of an LC support. It is often expressed in mmol/m2
of surface. Sometimes the percent C is given as an indicator of surface coverage.
Swelling
A process in which resins and gels increase their volume because of their solvent environment.

The solvent enters the ion-exchange resin to dilute ions, where in gels the solvent penetrates the
pores. If swelling occurs in packed columns, blockage or increased back pressure can occur. In
addition, column efficiency can be affected.
T
Tailing
A peak with an asymmetrical factor of >1. An asymmetrical peak is the result of a component
that is excessively retarded in eluting. Tailing is caused by sites on the packing that have a
stronger than normal retention for the solute. A typical example of a tailing phenomenon is the
strong adsorption of amines on the residual silanol groups of a low coverage reversed-phase
packing.
Temperature programming
A technique that changes the column temperature as a function of time during the separation. It
is rarely used in HPLC, then it is done in a stepwise manner.
Tortuosity
A property of a packed column that indicates the degree of unevenness of the path followed by
the solute molecule as it passes down the column. The A term in the van Deemter equation
takes this into consideration.
Total permeation volume (Vp)
The retention volume on an SEC packing in which all molecules small than the smallest pore
will elute. Therefore, at Vp, all molecules totally permeate all of the pores and elute together.
Trace enrichment
A technique in which trace amounts of compounds from a weak mobile phase or solution are
retained on an HPLC packing. They are then eluted by the addition of a stronger mobile phase
in a concentrated form. The technique has been most successfully applied in the concentration
of trace amounts of hydrophobic compounds, such as polynuclear aromatic hydrocarbons, out
of water using a reversed-phase column. A strong solvent such as acetonitrile serves to elute the
enriched compounds.
U
Ultraviolet/Visible Light (UV/Vis)
The tunable or variable wavelength UV/Vis detector is the most popular form of detector. For
methods involving organic compounds in aqueous mobile phases, the UV/Vis detector takes
advantage of compounds’ varying absorptivities of ultraviolet and visible light.
Unretained compounds
These compounds are not retained at all on the column but elute at the beginning of the
chromatogram immediately after the void volume.
V
Vacancy chromatography
A technique in which a mobile phase additive causes a positive detector signal output. When a
solute elutes from the column, it dilutes the signal and gives a negative peak or a vacancy. The
technique has been most recently applied to single column ion chromatography, in which
mobile phases that absorb in the UV region, such as citrate and phthalate buffers, are used.
When a nonabsorbing anion elutes, it dilutes the UV absorbing background and causes a
negative peak. The detector output leads are usually reversed to make the chromatogram look
normal.
van Deemter equation
An equation used to explain band broadening in chromatography. This equation represents the
height equivalent of a theoretical plate and has three terms. The A term is used to describe eddy

diffusion, which considers the different paths a solute may follow when passing over particles
of different sizes. The B term is for the contribution caused by molecular diffusion or
longitudinal diffusion of the solute while passing through the column. The C term is the
contribution of mass transfer and allows for the finite rate of transfer of the solute between the
stationary phase and mobile phase.
Velocity (u)
Same as Linear velocity.
Void
The formation of a space, usually at the head of the column, caused by a settling or dissolution
of the packing. A void in the column leads to decreased efficiency and loss of resolution. Even
a small void can be disastrous for small microparticulate columns. The void can sometimes be
removed by filling it with glass beads or porous packing.
Void time (tm or to)
The time for elution of an unretained peak.
Void volume (Vi)
The total volume of mobile phase in the column with the remainder of the column taken up by
packing material. It can be determined by injecting an unretained substance that measures void
volume plus extracolumn volume. It also is referred to as interstitial volume. Vo or Vm are
sometimes used as symbols.
W
Wall effect
The consequence of the looser packing density near the walls of the rigid HPLC column.
Mobile phase has a tendency to flow slightly faster near the wall because of the decreased
permeability. The solute molecules that happen to be near the wall are carried along faster than
the average of the solute band and band spreading results.
Waste container
At the end of the fluid path, the mobile phase and separated sample components are collected
into a waste container. This container is suitable for safely collecting and disposing of the
solvents used in the separation.
X
Xerogels
Gels that are used in size-exclusion chromatography. They have the ability to swell and shrink
in different solvents.
X-axis
The X-axis of the chromatogram records the time or volume of mobile phase that passes though
the detector.
Y
Y-axis
The Y-axis of the chromatogram records the strength of the detector signal, which is usually
proportional to the concentration of sample in the eluent passing through the detector. The units
depend on the type of detector being used.
Z
Zwitterions
Compounds that carry both positive and negative charges in solution.

Millennium Software Intro Guide
Enter User Name and Password
User Name = “analyst”

Password = “aaps”

Right Click “Configure System” and select “Projects”

Click upper left icon (New Project)

New Project Table Space – Maximum 15MB

Grant Access to all

Copy “View Filters”, “Custom Fields” and “Methods” from the Defaults method. Click
“Next”.

Give Your Project a name!
Use the format “plcX_grpY_mmddyy
Where: X = Class number (1, 2, 3 or 4 for PLC1002, PLC2002, PLC3002 or

PLC4002)
Y = group number (1, 2, 3 or 4)
mmddyy = the date the project was created in the format of Month
(mm - 01) Day (dd - 12) and Year (yy - 07) = 011207 = January 12,
2007.
Enter “New” for the Audit Trail Comment and click “Finish”

Select your project! Click on the “Create Date” header twice. This will re-order the
database in the date order of the creation date of the project.

Right Click on your project and choose “Open”. This will open the Browse Project
window for your project.

Click on the “Run Samples” Icon to open the Instrument Control screen (“Run
Samples”).

Select Instrument and Click “OK”. The computer will now connect to the HPLC.
This may take a few moments – be patient!

When your system is connected, set up to condition your column with mobile phase.
Click on the blue triangle & Tap in the lower centre of the screen.

Flow should be the same as in your instrument method. Set the Ramp for 1.0
minutes (on the right hand side of the Change Flow window under Ramping)

Click Develop Methods.

Click “Create New” to create a new Instrument Method

Select the icon for the detector (W486). Set Wavelength to the wavelength specified
in the method.

Select the Pump icon (W600). Set Flow and Mobile Phase Line (Usually “%A”)
under the “Flow” tab.

Save Your Instrument Method under a name you’ll remember.
You can use the format “mmddyy_im”. Enter a comment for the Audit Trail; for a new
instrument method, you can enter
“New”
When the dialogue box disappears, close the instrument method window.

In the “New Method Set” dialogue box, select the instrument method you just built
and click “Next”.

Click the “Next” button. We will not be processing the chromatogram, generating a
report or exporting the results at this time.

Use the instrument method name for the name of your Method Set (it will already
be entered for you). Enter a comment for the audit trail (enter “New” for the audit
trail for any new procedure etc. you build)

You have two options to monitor the baseline (See the signal from the detector).
You can go “Actions” and then “Monitor Baseline” or Select “Monitor” button,
lower centre of the screen.

You will see the baseline. The signal is self-scaling. If you have numerous zeros,
then your baseline is relatively flat.

Make sure that the “Vial”, “Injection Volume” and “Run Time” fields are correctly
filled in.
** Note – The “samples” tab allows for a sample set…. a group of injects from
different vials. Your instructor will assist you with sample sets in later sessions. **

Stop monitoring the baseline by pushing the abort icon . Then click the “Inject”
icon when it becomes highlighted as in the above illustration.

You will see your chromatogram develop on the screen.

Go to the main screen. You can browse your project by selecting “Browse Project”
and right clicking your project name.

Select “Injections”, and choose the injection you want to examine. Double-click on
it.

You’ll see your stored chromatogram. Ask your instructor to illustrate integration
techniques.


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AAPS Hand-outs and material

Dishonesty and Plagiarism

Academy of Applied Pharmaceutical Sciences (AAPS) Page 2

Previous analytical laboratory work experience and / or PLT 1005

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INTRODUCTION TO THE HPLC (Session One) .......................................................................7

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1. The Role of HPLC in the Pharmaceutical Industry

High Performance Liquid Chromatography (HPLC) is one of the primary analytical

Sensitive - Can determine accurate and representative amounts of the particular

Selective - Can be designed to analyze for one or more particular components in a

Stability Indicating - Can determine the amount of a particular component without

Non-selective – can be designed to analyze many different components

Reproducible – analyses can be replicated on different systems, by different operators

1.1 Quality Control (QC) Testing

The Quality Control department assures the quality of pharmaceutical

Academy of Applied Pharmaceutical Sciences (AAPS) Page 7

Finished Product Testing

Quality Control Stability is often called “shelf-life stability” or “Follow-Up Stability”

In-Process testing is testing that is performed on a pharmaceutical material or

Packaging Component Testing

Academy of Applied Pharmaceutical Sciences (AAPS) Page 8

Manufacturing rooms and equipment must be clean before use. Generally,

1.2 Research and Development (R&D)

The Research and Development (R&D) department develops methods, verifies

A finished pharmaceutical product contains Active/s and Excipients. The ratio of

Accelerated Stability (R&D Stability)

Accelerated Stability Studies determine the suitability of new product

Analytical Method Development & Validation

Academy of Applied Pharmaceutical Sciences (AAPS) Page 9

Chromatography is defined as “a process in which a chemical mixture carried by a liquid

The first published works in chromatography were by a Russian born botanist

2.2 Liquid Chromatography (LC) in Analytical Analysis

3. High Performance Liquid Chromatography (HPLC)

HPLC is a development of column chromatography. It was long realized that

Academy of Applied Pharmaceutical Sciences (AAPS) Page 11

Common surface treatments of silica include octadecylsilane (a.k.a. ODS or

Figure 1 – Basic HPLC system

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3.3.2 Constant Flow

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Figure 3 - Dual Piston Pump

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Gradient Elution chromatography is a very versatile procedure. It cannot be

High-pressure gradient is produced with two or more pump systems set in

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Low pressure gradient mixing is accomplished by a series of valves (usually

Figure 5 – Low-pressure Gradient Mixing System

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3.4 Sample Introduction

3.4.1 Rheodyne Type Valve

Rheodyne Corporation began developing an injector system for this purpose in

Figure 6 - Rheodyne Type Valve

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3.4.2 Automatic Samplers

The Rheodyne sampling system solved a big problem hampering the

Each of these valves was connected to specific portions of the “automatic