Welcome to Class!
Kevin Olsen
359 Richardson Hall
973-655-4076
OlsenK@Mail.Montclair.Edu
1
Class Expectations and Grading
• Grades will be awarded based on laboratory
reports and homework assignments. There may be
quizzes given in class depending on time
limitations.
• Some homework assignments will require an oral
presentation to the class.
• Unless specifically stated otherwise, the standards
of documentation required on laboratory reports
will be those found in a GMP / GLP regulated
environment.
2
Laboratory Reports
• All paperwork is a permanent record.
Nothing will be discarded.
• All chromatograms will include:
1.
2.
3.
4.
5.
Student name
Date
Sample ID
Chromatographic conditions
System ID number and calibration dates
3
Laboratory Reports (continued)
• Laboratory reports will be checked by a
second person before being turned in. The
person checking the report will sign it.
• Any cross outs will consist of a single line,
initials and date. In some instances such as
a faulty injection, an explanation for the
cross out will be included.
• No records will be kept in pencil.
4
Laboratory Reports (continued)
• The identity and calibration data of each
and every instrument used will be included
on the laboratory report, this includes
balances and pH meters. If an instrument is
not calibrated, that fact will be noted.
• The manufacturer and lot number of each
reagent used will be recorded. (Mobile
phases are exempt from this rule.)
5
The Cardinal Rules of GMP / GLP
• If it was not documented, it did not happen.
• To err is human, to destroy the evidence is a
felony.
• Maintain every record as if it were going to
be reviewed by the FDA.
• Your signature is precious, never sign,
approve, or authorize anything you are not
absolutely sure is correct.
6
Lesson 1
• Review of HPLC components
• Operating your HPLC
7
A Generic HPLC System
8
Generic HPLC Pump
9
Controlling your HPLC Pump
•
•
•
•
Flow rate
Minimum and maximum pressure
Purge valves
Mobile phase mixtures
10
Injector
11
Variable Wavelength &
Photodiode Array Detectors
12
Column Heaters
Some separations of complex mixtures must be temperature
optimised to achieve separations of overlapping peaks.
Increased column temperature will also shorten retention times
for a given column dimension.
Selectivity of the column is also a function of temperature and
elution orders of peaks may change and even reverse - some
chiral and amino acid separations are very sensitive to column
temperature effects.
Eliminates retention time variation due to room temperature
13
fluctuations.
Column Heaters
14
Bits & Bobs
15
Bits and Bobs
16
If Your Fittings Leak
•
Check to make sure your tubing is seated properly
• The fitting may not be tightened enough
• You may be using incompatible fittings
• Check the condition of the nut and ferrule
• Sometimes a leaking connection has nothing at all to do with the
nut and ferrule, but with the receiving port
• NOTE: Using fittings made of material that is incompatible with
your mobile phase is a sure way of creating leaks
17
Bits & Bobs
18
Lesson 2
Running a simple separation
19
“Normal Phase”
+ - + - + - + - + - + - + - + - + - + - + - +
Mobile Phase -NON POLAR
+ - + - + - + - + - + - + - + - + - + - + - +
Stationary Phase - POLAR
20
“Reverse Phase”
Mobile Phase - POLAR
+
+
+
-
Stationary Phase - NON POLAR
21
Reverse Phase
Silica
Stationery
Phase
support
The longer the alkyl chains, the longer the
retention time in a reversed phase column.
22
Why Degas the Mobile Phase?
Actual nitrogen
concentration
Theoretical saturation
23
100% water-----------------------------------------------100% Methanol
What is a Solvent Front?
24
How to Degas the Mobile Phase
•
•
•
•
Sonicate.
Apply partial vacuum while agitating.
Helium sparge.
In-line degasser.
25
Gradient
Methanol ammonium
acetate
Acetonitrile water
Ethyl acetate
26
20 minutes
Things to try….
• Mobile phase composition. What happens to
retention times and peak width?
• Pump speed. What happens to peak width?
• Column temperature. What happens to retention
times?
• For Homework: Prepare a written report
explaining your findings and include neatly
tabulated data. Calculate column efficiency using
the same analyte on each first run. You do not
have to submit your chromatograms.
27
Homework Report Format
Parameter
that was
changed
Retention
time.
(analyte
#1)
Peak width
Retention
at half
time.
height
(analyte #2)
(analyte #1)
Column
Peak width
efficiency
at half
(analyte #1)
height
(analyte #2)
28
Peak Width at Half-height
• Peak width, or
sharpness, is an
indication of column
efficiency.
• An ideal peak is a
Gaussian distribution.
• The ratio of standard
deviation to retention
time is independent of
flow rate.
tr
29
Calculating Column Efficiency
• Peak width at half height is used to calculate
column efficiency.
• N = 5.54 ( T / W )2
Where N = theoretical plates
W = Width at half height T = Retention time
5.54 is a constant based on the normal distribution
30
Lesson 3
Changing Columns
Sample Loops
Care and Maintenance of Injectors
31
Installing a Sample Loop
32
Routine Care of Injectors
• Never use a pointed or
bevel tip needle.
• Rinse after the use of
buffer solutions.
• Avoid abrasive particles
by filtering samples before
injection.
• Use burr-free tubing to
avoid metals shavings
from getting into the
injector.
33
Routine Care (continued)
34
Leak Checking the Injector
• Leaks between stator
and stator ring?
• Leaks in needle port
ONLY when loading?
• Fluid escapes by
siphoning out?
• Continual leak out
needle port or vent
lines?
• Loosen set screws or
tighten stator screws.
• Tighten needle seal by
pushing in the guide.
• Adjust the height of
the outlet tubes.
• Surface of the rotor is
scratched, replace.
35
Load Volume
• For a partial fill, use
no more than 50% of
the loop volume. The
sample liquid tends to
expand and fills the
loop completely.
• To completely fill the
sample loop, use at
least 200% of the loop
volume.
36
Flushing the Needle Port Tube
• Use the manufacturer’s recommended
adapter and connect it to a luer lock type
syringe.
• Flush in the INJECT position only so that
the fluid flows out the vent line.
• The sample loop will be flushed with
mobile phase.
37
How Do I Know My Injector is
Clogged?
38
Flushing the Stator and Stator
Face
• With the pump turned off, disconnect the
one sample loop fitting and the column
connection.
• Using a syringe, gently pump a cleaning
fluid (high purity methanol or water)
through the ports.
39
Lesson 4
Care of HPLC Pumps
40
Pump Care
• Flush with water after running a buffer, (note
there are special procedures when using reverse phase
columns.)
• Replace seals in a timely manner.
• Maintain check valves.
• Do not allow solids in the mobile phase.
41
Removing Buffers from a
Reverse Phase Column
• DO NOT FLUSH WITH 100% WATER AS
YOUR FIRST STEP • Substitute water for the buffer but leave the
remaining proportions the same. Run through
about 5 column volumes.
• Wash through 10 column volumes of a strong
organic solvent, example - Methanol.
• If you plan to store the column, read the
directions.
• If the phase collapses, a 50-50 water, organic
solvent wash for 30 minutes can restore it.
42
Lesson 5
HPLC Calibration
43
Why Calibrate?
From an FDA Warning Letter of June 29, 2002
issued to a Medical Gas supplier:
• 3. Failure to routinely calibrate mechanical and
electronic equipment or keep records of calibration
according to a written program designed to assure
proper performance as required by 21 C.F.R. 211.68.
For example, your electronic thermometer was last
calibrated in June 1999, the pressure gauge for the
manifold filler was last calibrated in February 1997,
and the vacuum gauge has never been calibrated.
44
Why Calibrate?
From an FDA Warning Letter of June 29, 2004 issued to a
company that makes metals used in dental implants:
…not in conformity with the Current Good Manufacturing Practice (CGMP) requirements of the
Quality System (QS) regulation found at Title 21, Code of Federal Regulations (CFR), Part 820.
Significant violations include, but are not limited to, the following:
1. Failure to establish and maintain procedures to ensure that equipment is routinely calibrated,
inspected, checked, and maintained, as required by 21 CFR 820.72(a). For example, your written
procedures require that equipment be calibrated two times a year. The following equipment has not
been calibrated or checked two times a year: the…machine used to test the yield strength, ultimate
tensile strength, and elongation on the has not been calibrated. The next calibration date was
supposed to have been January 28, 2004.
2. Failure to document the equipment identification, calibration dates, the individual performing each
calibration, and the next calibration date, as required by 21 CFR 820.72(b)(2). For example, the
calibration data sheet dated August 26, 2003, for the spectrophotometer used to analyze other
metals was not signed and dated by the individual who performed the calibration and had no
specified test value for the “Std. Dev. Sample Units”.
45
Calibration
•
•
•
•
wavelength accuracy,
wavelength precision (repeatability)
absorbance linearity
absolute absorbance accuracy.
46
Automated Detector Calibration
Filter with a known
Absorbance(s)
Flow cell
To waste
From column
47
Automated Detector Calibration
Filter with a known
Absorbance(s)
Flow cell
To waste
From column
48
Holmium Oxide
49
Agilent 1100 Wavelength
Calibration
486 nm
656.1 nm
1 nm slit
Deuterium lamp
50
Manual Calibrations
450 nm
410 nm
640 nm
Linearity with a solution
of potassium dichromate,
0.01 to 0.20 mg/mL
Wavelength accuracy
with a solution of
Holmium oxide
51
Compendial Requirements for
Calibration.
• There must be written SOP’s.
• A calibration schedule must be maintained.
• All standards and meters must be NIST traceable.
Any electrical meters, stopwatches, thermometers,
also require calibration!
• Persons performing the calibration must be trained
and this training must be documented.
• All equipment must be tagged with the date the
calibration was performed, the person performing
the calibration, and the date of the next calibration.
52
Laboratory Exercise
• Remove the connection from the head of the
column and cap the column end. Calibrate the
flow of the pump at 1, 3, and 5 mL/min. using a
stopwatch and graduated cylinder.
• Calibrate the injector by performing ten replicate
injections of caffeine, calculate the average peak
area and the relative standard deviations of all
injections. Use enough standard to completely fill
the sample loop.
53
Homework for Calibration
•
•
1.
2.
3.
4.
Prepare a calibration report.
Include, the following information:
The serial number, date calibrated, and date of next calibration
for the stopwatch.
The peak areas for the replicate caffeine injections, the average
peak area, and the relative standard deviation for all ten
injections.
The manufacturer, lot number, and purity of the caffeine standard.
Your report must be checked by another member of the class
before it is submitted and that person must sign off on the report.
54
Lesson 6
Examples of HPLC in the
Pharmaceutical Industry
55
Some Important Concepts
• What is a check standard?
• What is a system suitability standard?
• What is a five-point calibration?
• How does an internal standard work?
You will not be able to complete your HPLC
lab reports unless you can answer these
questions!
56
“The most important assets of any
business are intangible: its company
name, brands, symbols, and slogans, and
their underlying associations, perceived
quality, name associations, customer
base and proprietary resources such as
patents, trademarks, and channel
relationships.”
-David Aaker, author of several books on branding including:
Managing Brand Equity, 1991
Courtesy Dr. Mark Kay, MSU Dept. of Business.
57
Intangible Assets as Percentage
of Value.
Courtesy Dr. Mark Kay, MSU Dept. of Business.
58
Intangible Assets and Brand
Courtesy Dr. Mark Kay, MSU Dept. of Business.
59
Flu Vaccine and the Winter of
2004 - 2005
THE CHIRON PLAN
1.
2.
3.
Chiron acquires British vaccine maker
Powder Ject Pharmaceuticals.
Efficiency at the Liverpool plant is
increased by 50%.
Sales of flu vaccine will finance an
expansion into new vaccines and biotech
drugs.
60
Flu Vaccine and the Winter of
2004 - 2005
THE CHIRON REALITY
1.
2.
3.
4.
5.
Chiron acquires British vaccine maker Powder Ject
Pharmaceuticals.
Efficiency at the Liverpool plant is increased by 50%.
CHIRON FUMBLES QUALITY
Citing contamination problems, British regulators
suspend the plant’s license in October 2004.
Without sales of flu vaccine Chiron loses $300 million
in revenues. Earnings fall 50% from 2003. The stock
price falls 30%.
61
Chief Executive Officer Howard Pien said his company was
surprised when British regulators suspended the Liverpool plant’s
license.
• Inspectors from the British
Health Authority found
contamination from
“dangerous bacteria”
• FDA inspections revealed
similar problems in 1999,
2001, 2002, and 2003
• Chiron failed to correct
the problems volentarily.
62
Or as convicted counterfeiter
Elsworth Roston once
observed:
“Quality is your security.”
63
Examples of HPLC in the
Pharmaceutical Industry
• Manufacturing:
• Discovery:
• Content Uniformity
• Degradation products
and related substances
• Dissolution
• Stability Studies
•
•
•
•
Purity and concentration
Redox potential
Structural confirmation
Development and
clinical trials
• Metabolite monitoring
• Half life in the body
64
Content Uniformity
65
Content Uniformity
Exampleamine HCl
Exampleamine HCl
Exampleamine HCl
Exampleamine HCl
Exampleamine HCl
Exampleamine HCl
Exampleamine HCl
Exampleamine HCl
Exampleamine HCl
Exampleamine HCl
66
Content Uniformity
• From an FDA Warning Letter of December
2003 issued to a manufacturing facility:
• Regarding your Advicor product, your QCU failed to property
investigate Content Uniformity failures found in lots of Advicor
tablets. Specifically, lots of Advicor 500mg/20mg and lots of
Advicor 750mg/20mg failed Content Uniformity release testing
and were rejected. Your QCU attributed the failures to low "dew
points". However, your "dew point" specification is no more than
___. The investigation into these failures did not evaluate lots
released to the market that had been produced under similar
conditions with similar dew points
67
Degradation and Related
Compounds
• Degradation products may be formed from heat,
bacterial action, or excessive moisture.
• Related compounds are known impurities created
as artifacts of manufacturing.
68
Dissolution
Exampleamine HCl
69
Dissolution
• From an FDA Warning Letter of December 2003
issued to a manufacturing facility:
•
Your QCU attributed the Niaspan dissolution failures seen in lots of
finished product to a low Hydroxypropyl content in the Methocel E10M
raw material. Additionally, Niaspan lots produced using Methocel E10M
lot #0207200002 also failed dissolution and these failures were also
attributed to a low Hydroxypropyl content in the raw material. The
Hydroxypropyl contents in both circumstances were within your NDA
listed specification. This is of particular concern since the Methocel raw
material helps to control the rate of release of the drug in your Niaspan
product. It is also unclear if your QCU has assessed all of the
manufacturing variables needed to optimize your production
process…your Process Evaluation Summary also identified an increase
in the operating temperature as a contributing factor to the dissolution
failures.
70
Dissolution
71
Automated Dissolution
HPLC with autosampler
Fiber optic system
72
Stability Assays
• The FDA Defines a Stability Assay as a:
“Validated quantitative procedure that can detect
changes with time in the pertinent properties of a
drug substance and drug product. A stabilityindicating assay accurately measures the active
ingredients without interference from degradation
products, process impurities, excipients, or other
potential impurities.”
Federal Register, 65, August 2000.
73
Stability Studies
3 months 6months one year
three years
five years
• All pharmaceutical manufacturers are required to
periodically test stored samples of their products
(sometimes they are subjected to high
temperatures and moist environments) in order to
determine their stability over long periods of time.
74
Levothyroxine sodium is a synthetic
hormone used to treat thyroid disorders.
Hyperpotent dosage - cardiac pain
palpitations, cardiac arrhythmias,
Increased risk of osteoporosis
IDEAL DOSAGE LEVEL
Subpotent dosage - depression,
lack of concentration, fatigue, weight gain,
edema, loss of concentration, and constipation
75
Levothyroxine sodium
1 962 - FDA requires NDA filings for all new drug products
1982 - One manufacturer removes two inert ingredients
and changes the coating process. Potency increases by
22 to 30%.
1982 - FDA sets up a commission to develop “stability
indicating” HPLC methods.
1982 - 1994 - FDA receives 58 adverse drug experience reports.
1991 - The first of ten recalls involving 150 lots and more than
100,000,000 tablets. All recalls were due to subpotency or
because potency could not be guaranteed through the expiration
date.
76
Levothyroxine sodium
1993 - FDA issues Warning Letter to manufacturer. Five lots of
tablets failed stability testing. Another lot was recalled because
potency could not be guaranteed through the expiration date.
1993 - Another manufacturer recalls six lots. Recalled tablets
tested 75 to 90% potency (USP requirements are 90 to 110%).
Another lot was recalled after failing routine stability testing.
1994 - Seven lots recalled because potency could not be assured
through the expiration date.
1995 - 60 lots and 50,436,000 tablets recalled
1995 - 22 lots recalled because potency could not be assured
through the expiration date.
77
Levothyroxine sodium
The customary 2 year shelf life of most pharmaceuticals is
inappropriate for Levothyroxine sodium because:
1. It is unstable in the presence of warm temperatures, light, air,
and humidity. Some of these stability problems can be
corrected through better control of the manufacturing
environment.
2. Some of the excipients used in the tablets can catalyze
degradation reactions.
3. The drug’s degradation occurs in two phases. An initial high
degradation rate is followed by a slower degradation. To
compensate, some manufacturers increase the tablet’s potency.
This sometimes leads to hyperpotent doses.
78
Example applications in Drug
Discovery
• Compound purity and concentration prior to
testing against a therapeutic target.
• Redox potential to estimate solubility and
biological availability.
• Molecular weight confirmation
79
Purity and Concentration
80
Redox Potential
• Electrochemical detectors are
used when the analytes do not
have good chromophores.
• This detector measures 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 is used for
quantification
81
Redox Reactions
•
The pKa of a molecule is the pH at which half of the molecules
(dissolved in water) are in one ionized form, with the other half being
unionized or in a higher ionized form. pKa Therefore indicates the form
a molecule will take at a given pH value, for example stomach or blood
pH.
•
Some properties are pKa-dependent, such as lipophilicity, solubility and
permeability.
•
pKa is important for understanding how a charged drug interacts with a
receptor. When drugs metabolize they form ionized, hydrophilic
metabolites which are excreted at physiological pH.
82
“Structural” Confirmations
• Many large compound
libraries have been created
for drug discovery programs.
• Robot assisted synthesis or
Combinatorial Chemistry
can create hundreds or even
thousands of compounds in a
short time.
• There is therefore a need to
rapidly confirm the identity
of these new compounds.
83
High Throughput HPLC
• Drug discovery costs are now topping
2000$ per minute
• In a typical screen, as many as 250,000
compounds are tested against a disease
target
• Most drug discovery operations perform an
HPLC analysis of every compound in their
collection
84
Example
85
Example
86
The BRIO System
24 lanes with reversed
phase packing.
Eight channel
autosampler for
loading
Reusable
87
LC/MS Monitoring of Drugs in
Blood
• Clinical trials for schizophrenia medications
and other anti-psychotic drugs
• Half life in blood from 6 to 36 hours
• Agilent LC/MS systems with Zorbax C-18,
30 mm X 4.6 mm columns. 3.5 micrometer
stationary phase.
88
LC/MS Monitoring of Drugs in
Blood
• Start with 0.5 mL serum
• Add internal standards
• Add organic solvents (hexane, n-butanol,
acetonitrile,93:5:2)
• Evaporate
• Reconstitute in 50 uL MeOH (a ten-fold increase
in concentration)
• Run in SIM mode (target masses between 313 and
388, run times under four minutes.)
89
The Future of Instrumentation and
the FDA: 21CFR11
or…
Is a Paperless Laboratory Really
Practical?
90
21CFR11
Beyond the buzzwords there is one simple concept:
SECURITY.
1. If users can save data, they can delete data
2. Centralized networks have centralized security
3. Central data stores can have different levels of
access for different persons, chemists,
supervisors, metrologists, and maintenance.
91
The Big Issues with 21CFR11
1. Computer system security
2. Backup and restoration
3. Automated audit trails
capturing the date, time, and
user’s name any time that data
is generated.
4. Secure electronic signatures
92
“Simple” Steps to Implement
21CFR11
• Be honest with people, explain to them at the start
of the project that they will be monitored and set
down clear rules about what will and will not be
allowed with the new system.
• One mechanism from one vendor, not multiple
systems from multiple vendors.
• No more individual access levels on individual
computers.
93
Software Mission Statements
• When starting any software project, write a onepage, easy to understand, simple, and concise
explanation of what the software is supposed to
do.
• Compare the real world performance of the system
against the expectations set down in this
document.
• Make this document available to the FDA should
they ever want to audit or inspect your data
systems.
94
Homework for Lesson 6
• Read a journal article that describes the use
of HPLC in a pharmaceutical application. It
can be from any sector of the industry but
should describe a specific application in
some detail.
• Write a one page summary of the article.
95
Lesson 7
Introduction to the USP
96
Lesson 7
Retention factors
Dead volume
Resolution
Tailing Factor
System suitability
97
Retention Factor k
Retention
=
Factor
retention time - dead time
-------------------------------------------dead time
Dead time = dead volume / flow rate
(for a 4.6 mm ID column dead volume is approximately:
0.1 X L where L is the column length in centimeters.
98
Using the Retention Factor
• The retention factor is a measure of the how much
the analyte is retained on the column compared to
an unretained analyte.
• Ideally k should be between 2 and 20, and not less
than 1.
• Values less than 1 usually indicate that the analysis
is too sensitive to mobile phase organic
composition, impurities, and injection effects.
99
An LC/MS Example of the
Importance of Retention Factors
• Problem: Suppression of early eluting
compounds by the solvent front and the
early eluting impurities.
K < 1, BAD
100
An LC/MS Example of the
Importance of Retention Factors
K < 2, GOOD
101
Calculating Resolution
Selectivity = k (analyte 2) / k (analyte 1)
2 (RT B - RT A)
Resolution = -----------------------------------Peak width A + Peak Width B
Baseline resolution has a value of 1.5 or
higher.
102
Tailing Factor (Tf)
• Tf = (peak width) / 2 (front’s half width)
• All widths measured at 5% peak height.
• Values greater than 1.5 generally indicate that
unwanted interactions are occurring.
103
Tailing Factor
Front width
Peak width
5% height
104
Asymmetry Factor (AS)
Back half width
AS = ---------------------Front half width
10% height
105
Why System Suitability?
“They are used to verify that the
resolution and reproducibility of
chromatographic system are adequate
for the analysis to be done. The tests
are based upon the concept that the
equipment, analytical operations, and
the samples to be analyzed constitute
an integral system that can be
evaluated as such.”
106
Why System Suitability?
• In November of 1998, the FDA issued this warning to a cosmetics /
skin care products firm in California:
• 5. Your firm failed to establish and document the
accuracy, sensitivity, specificity, and reproducibility of
test methods used for raw materials, in process, and
finished product testing. Specifically, your company has
not validated laboratory methods used on various tests
used in testing raw materials and sunscreen finished
product. In addition, system suitability testing is not
performed on the HPLC testing performed on the drug
products. [CFR 211.165 (e)]
107
System Suitability is Taken
Seriously by the FDA
From an FDA Warning Letter sent to a pharmaceutical
manufacturing company in New Jersey:
• 12. Written procedures containing provisions for
remedial action in the event of laboratory
instrumentation failing to meeting accuracy and
precision limits are not followed (21 CFR 211.160(b)(4)).
For example, on three occasions, corrective actions
following HPLC system suitability failures were not
recorded in the instrument maintenance logs as
required by your SOP.
108
More System Suitability
• From a Warning Letter to a company the manufactures
sunscreen and vitamins dated November 1998:
•
5. Your firm failed to establish and document the accuracy, sensitivity,
specificity, and reproducibility of test methods used for raw materials, in
process, and finished product testing. Specifically, your company has
not validated laboratory methods used on various tests used in testing
raw materials and sunscreen finished product. In addition, system
suitability testing is not performed on the HPLC testing performed on
the drug products. [CFR 211.165 (e)]
109
Lab Exercise for Lesson 7
• Run five injections of a two component mixture,
each at a decreasing concentration. Example: 100
mg/ml - 10 mg/ml - 1 mg/ml etc. etc.
• Measure the resolution, tailing factor, peak width
at half-height.
• Report your results using the accepted standards
of documentation.
110
Lesson 8
Introduction to FDA Regulation
111
HPLC Problems Mentioned on
FDA Warning Letters.
• No identification of mobile phase or solution.
• No identification of raw data and chromatograms.
• No identification of actual method used, especially
when multiple methods are available.
• Procedures that did not test for degradation
products or impurities.
• Failure to implement changes when HPLC
methods have been found deficient.
• Failed system suitability with no records of the
corrective action.
112
Poor HPLC Methods, Example #1
• From a Warning Letter to Searle, December 1992:
• According to some records reviewed during the
inspection, you developed a stability indicating method in
1981 for Winstrol and in 1988 for Plaquenil capable of
detecting degradation products. However, neither of the
methods have been used for this even though you have
data to show that degradation products and/or impurities
can elute at long retention times. Both aforementioned
methods are only used for assay determination with a
limited run time of 9 minutes
113
Changing a Bad Method.
• Changes are always tightly controlled to insure that
data is valid from year to year and batch to batch.
• Proposed method changes are submitted in writing
to a change control committee.
• The changes to the method are tested. Sometimes
this is done in the same lab that uses the method
every day, other companies have a separate group
that performs this function.
• If approved, the new method is issued. Old copies
of the method are collected and destroyed.
114
Out of Specification (OOS)
Upper control limit
+ 2 standard deviations
Mean value
- 2 standard deviations
Lower control limit
115
Other Topics From the CFR
•
•
•
•
•
Calibration procedures, records, and intervals.
OOS Results (Testing into compliance).
Uncontrolled changes to methods.
Old procedures not collected and destroyed.
Incomplete or absent review of data and
records.
• No signatures on laboratory reports.
116
Lab Exercises for Lesson 8
Content Uniformity Assay
117
Lesson 9
Ion Pairing
118
Ion Pairing
In running HPLC analyses of ionized molecules,
the mobile phase pH is often used to control the
amount of charge on the molecule,(and thus the
polarity). This is sometimes called “ion
suppression.”
119
Ion Suppression
Moderate pH
H+
H+
H+
H+
H+
High pH
H+
Low pH
H+
120
Ion Pairing
In ion pairing, a molecule with a long alkyl chain
and terminal ionization sites is introduced into
the mobile phase.
121
Ion Pairing
Silica
Stationery
Phase
support
The longer the alkyl chains, the longer the
retention time in a reversed phase column.
122
Ion Pairing
Courtesy Regis Technologies 1998.
123
Lesson 10
Performing a Tablet Assay
124
Lesson 11
Troubleshooting
125
Baseline Noise
• During the injection, there
is a slight interruption in
mobile phase flow. (A)
• Air bubbles in the system
may cause irregular flow,
in which case the size of
the noise will be
proportional to the flow
rate. (B)
• If electronic noise in the
detector is the cause, then
the noise level will be
independent of the flow
rate. (B)
126
Example of an Air Bubble
Problem
• Dr. Toney’s sample
number 9746.
• Running a molecular
weight confirmation
• Using 01. ml/min on
the LC/MS system
• A leaking fitting
failed, causing
pressure to drop and
allowing air into the
system.
127
Example of an Air Bubble
Problem
Fitting failure
Good, stable baseline
Air bubbles in system.
128
•
Periodic baseline fluctuations, especially if
accompanied by pressure fluctuations, may be
caused by an air bubble in the pump or some
other pump malfunction.
If the pump speed is
changed does the
fluctuation frequency
change?
1. Prime the pump again
2. Degas the mobile
phase
3. Perform routine pump
maintenance
• Does the problem
persist even if there is
no flow?
• Check detector
• Check for
environmental
influences such as
power supplies, radios,
etc.
• Is the column
temperature uniform?
129
Peak Fronting
• Overloaded column.
• Channels in the solid
phase.
130
Peak Tailing
• Silanol interactions
• Degradation of stationary
phase
• Unswept void volume, or
void formation at head of
column,
• Co-eluting material
• POOR MATCH
BETWEEN ANALYTE,
MOBILE PHASE, AND
COLUMN POLARITIES
131
Overloading the Column
•
•
•
•
Loss of resolution
Tailing
Broadening
Retention time
decreases
• Injection volume less
than 15% of peak
volume.
0.5 min X 0.3 ml/min = 0.15 ml
0.5 min X 3 ml/min = 15 ml
• 1 uG of sample per cm
of column length for a
4.6 mm ID column
132
Matrix Overload
• In this situation,
impurities in the
sample matrix coat the
column packing and
reduce the resolution
and the retention
times.
• This can occur when
samples are extracted
from biological fluids.
133
Ghost Peaks
• Column
Contamination
• Elution of analytes
from a previous run
First run
• Reverse and flush
column
• Use a longer run time,
a different clean up, or
a strong solvent at the
end of the run
Second run
134
Ghost Peaks from Ion Pairs
• Sometimes ghost
peaks can appear from
having a sample
prepared in one
solvent and using
another solvent as
your mobile phase.
• This results in an ion
pair formation that
causes the analyte to
remain on the
stationary phase too
long.
• Ion pairing is a
technique that
increases retention
time.
135
Ghost Peaks or Random Baseline
Noise?
• Sometimes seemingly random baseline
noise can sometimes have the same cure as
ghost peaks, turn the column around and
flush it.
136
Peak doubling or splitting
Sample volume too large Inject sample prepared in mobile phase with total
sample volume <15% of volume of first peak
Injection solvent too strong Use weaker injection solvent
Column void or channeling Replace column; use less-aggressive conditions
Blocked column frit Replace frit; add in-line filter; filter samples
Unswept injector flow path Replace injector rotor
137
Drifting Baselines
One run
Days or weeks
• A suddenly change in the baseline (hours) during an
isocratic separation usually indicates column or detector
contamination.
• A slowly changing baseline (days or weeks) usually
indicates a detector or a source problem.
138
Lesson 11 Continued
More Troubleshooting
139
High Backpressure
• All HPLC pumps have a pressure gauge
• If pressure rises it usually means a clogged
frit or column
• The inlet frit on most columns is about 2
microns.
• Reverse column and flush it.
140
Column Care Tips
Q: How do I now which solvents are safe to use on
my column?
A: Read the directions
Q: How do I select a good solvent to flush my
column?
A: Read the directions
Q: I want to store my column, what should I fill it
with?
A: Read the directions
This may be a hard lesson for us guys to understand.141
Air Bubbles Revisited
• Symptoms that might appear
Irregular baselines
Increasing run times
Low to moderate pressure drop
• Corrective actions
Degas mobile phase prior to a run and purge all air
bubbles from lines.
Use an in-line degasser.
If problems persist, open purge valve and run
system.
142
Pump Care
• Flush with water after running a buffer, (note
there are special procedures when using reverse phase
columns.)
• Replace seals in a timely manner.
• Maintain check valves.
• Do not allow solids in the mobile phase.
143
Removing Buffers from a
Reverse Phase Column
• DO NOT FLUSH WITH 100% WATER AS
YOUR FIRST STEP • Substitute water for the buffer but leave the
remaining proportions the same. Run through
about 5 column volumes.
• Wash through 10 column volumes of a strong
organic solvent, example - Methanol.
• If you plan to store the column, read the
directions.
• If the phase collapses, a 50-50 water, organic
solvent wash for 30 minutes can restore it.
144
Drifting Retention Times
•
•
•
•
•
•
•
Flow rate changes (see pump care)
Improperly calibrated gradient controllers
Air bubbles
Temperature changes
Leaks
Column damage
Changes to mobile phase composition
145
Drifting Retention Times
Changes to mobile phase composition
• Improper mixing either preparing the mixture
in a batch or with a proportioning pump.
• Evaporation of a volatile component (very
rare.)
• Incorrect pH
146
Strategy for Reducing HPLC Analysis Time
and Maintaining High Resolution
Courtesy Agilent Technologies
1. Reduce column length.
2. Select a shorter column that has a smaller particle size.
(In 1969 particle sizes were typically 100 microns which gave 170
plates per 5 cm., by 2003 particle sizes were down to 1.8 microns
which gave 12,000 plates per 5 cm.)
3. Increase mobile phase temperature
(Increasing temperature reduces analysis time by as much as 40%
and pressure as much as 25%)
4. Increase flow rate.
147
Column Packing, Extracolumn
Effects and Peak Resolution
LOSS of
resolution
due to
tubing
volume
Column packing particle size
148
Peak Broadening as a Result of
Extracolumn Volume
• Made worse by
short and narrow
columns
• Smaller packings
are more
vulnerable
149
Homework for Lesson 11
• Read one Troubleshooting column from
LC-GC Magazine.
• Write a summary of the article.
• Be prepared to present your summary to the
class.
150
Student Presentations
151
Semester Review
152
What I have Done Wrong May
‘05
• Biggest mistake was not reviewing all 150 slides
before doing the lesson plans, forgetting key
points for resolution and column efficiency.
Sometimes things planned for later in the class
were needed sooner and I did not remember them
correctly.
• Do not assume that, students know how an
internal standard works, they can do a calibration
curve, they can understand normal distribution.
• Explain system suitability and check standards
slowly and carefully before the labs begin.
153