1

Welcome to Class!

Kevin Olsen

359 Richardson Hall

973-655-4076

OlsenK@Mail.Montclair.Edu

2

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.

3

Laboratory Reports

• All paperwork is a permanent record. Nothing will be discarded.

• All chromatograms will include:1. Student name2. Date3. Sample ID4. Chromatographic conditions5. System ID number and calibration dates

4

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.

5

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.)

6

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.

7

Lesson 1

• Review of HPLC components

• Operating your HPLC

8

A Generic HPLC System

9

Generic HPLC Pump

10

Controlling your HPLC Pump

• Flow rate

• Minimum and maximum pressure

• Purge valves

• Mobile phase mixtures

11

Injector

12

Variable Wavelength & Photodiode Array Detectors

13

Column HeatersSome 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 fluctuations.

14

Column Heaters

15

Bits & Bobs

16

Bits and Bobs

17

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

18

Bits & Bobs

19

Lesson 2

Running a simple separation

20

“Normal Phase”

Stationary Phase - POLAR

Mobile Phase -NON POLAR

+ - + - + - + - + - + - + - + - + - + - + - +

+ - + - + - + - + - + - + - + - + - + - + - +

21

“Reverse Phase”

Stationary Phase - NON POLAR

Mobile Phase - POLAR

-+

--+

+

22

Reverse Phase

The longer the alkyl chains, the longer the retention time in a reversed phase column.

Silica Stationery Phase support

23

Why Degas the Mobile Phase?

Theoretical saturation

Actual nitrogen concentration

100% water-----------------------------------------------100% Methanol

24

What is a Solvent Front?

25

How to Degas the Mobile Phase

• Sonicate.

• Apply partial vacuum while agitating.

• Helium sparge.

• In-line degasser.

26

Gradient

Methanol - ammonium acetate

Acetonitrile - water

Ethyl acetate

20 minutes

27

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.

28

Homework Report Format

Parameter that was changed

Retention time. (analyte #1)

Peak width at half height (analyte #1)

Retention time. (analyte #2)

Peak width at half height (analyte #2)

Column efficiency

(analyte #1)

29

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

30

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

31

Lesson 3

Changing Columns

Sample Loops

Care and Maintenance of Injectors

32

Installing a Sample Loop

33

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.

34

Routine Care (continued)

35

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.

36

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.

37

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.

38

How Do I Know My Injector is Clogged?

39

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.

40

Lesson 4

Care of HPLC Pumps

41

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.

42

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.

43

Lesson 5

HPLC Calibration

44

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.

45

Why Calibrate?

…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”.

From an FDA Warning Letter of June 29, 2004 issued to a company that makes metals used in dental implants:

46

Calibration

• wavelength accuracy, • wavelength precision (repeatability)• absorbance linearity• absolute absorbance accuracy.

47

Automated Detector Calibration

From column

To waste

Filter with a knownAbsorbance(s)

Flow cell

48

Automated Detector Calibration

From column

To waste

Filter with a knownAbsorbance(s)

Flow cell

49

Holmium Oxide

50

Agilent 1100 Wavelength Calibration

656.1 nm

486 nm

1 nm slit

Deuterium lamp

51

Manual Calibrations

Linearity with a solution of potassium dichromate, 0.01 to 0.20 mg/mL

Wavelength accuracy with a solution of Holmium oxide

450 nm

640 nm

410 nm

52

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.

53

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.

54

Homework for Calibration

• Prepare a calibration report.

• Include, the following information:

1. The serial number, date calibrated, and date of next calibration for the stopwatch.

2. The peak areas for the replicate caffeine injections, the average peak area, and the relative standard deviation for all ten injections.

3. The manufacturer, lot number, and purity of the caffeine standard.

4. Your report must be checked by another member of the class before it is submitted and that person must sign off on the report.

55

Lesson 6

Examples of HPLC in the Pharmaceutical Industry

56

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!

57

“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.

58

Intangible Assets as Percentage of Value.

Courtesy Dr. Mark Kay, MSU Dept. of Business.

59

Intangible Assets and Brand

Courtesy Dr. Mark Kay, MSU Dept. of Business.

60

Flu Vaccine and the Winter of 2004 - 2005

THE CHIRON PLAN1. Chiron acquires British vaccine maker

Powder Ject Pharmaceuticals.

2. Efficiency at the Liverpool plant is increased by 50%.

3. Sales of flu vaccine will finance an expansion into new vaccines and biotech

drugs.

61

Flu Vaccine and the Winter of 2004 - 2005

THE CHIRON REALITY1. Chiron acquires British vaccine maker Powder Ject

Pharmaceuticals.

2. Efficiency at the Liverpool plant is increased by 50%.

3. CHIRON FUMBLES QUALITY

4. Citing contamination problems, British regulators suspend the plant’s license in October 2004.

5. Without sales of flu vaccine Chiron loses $300 million in revenues. Earnings fall 50% from 2003. The stock price falls 30%.

.

62

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.

63

Or as convicted counterfeiter Elsworth Roston once observed:

“Quality is your security.”

64

Examples of HPLC in the Pharmaceutical Industry

• Manufacturing:• Content Uniformity

• Degradation products and related substances

• Dissolution

• Stability Studies

• Discovery:• Purity and concentration

• Redox potential

• Structural confirmation

• Development and clinical trials

• Metabolite monitoring

• Half life in the body

65

Content Uniformity

66

Content Uniformity

Exampleamine HCl Exampleamine HCl

Exampleamine HCl Exampleamine HCl

Exampleamine HCl Exampleamine HCl

Exampleamine HCl Exampleamine HCl

Exampleamine HCl Exampleamine HCl

67

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

68

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.

69

Dissolution

Exampleamine HCl

70

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.

71

Dissolution

72

Automated Dissolution

HPLC with autosampler

Fiber optic system

73

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 stability-indicating assay accurately measures the active ingredients without interference from degradation products, process impurities, excipients, or other potential impurities.”

Federal Register, 65, August 2000.

74

Stability Studies

• 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.

3 months 6months one year three years five years

75

Levothyroxine sodium is a synthetic hormone used to treat thyroid disorders.

Hyperpotent dosage - cardiac painpalpitations, cardiac arrhythmias, Increased risk of osteoporosis

Subpotent dosage - depression,lack of concentration, fatigue, weight gain, edema, loss of concentration, and constipation

IDEAL DOSAGE LEVEL

76

Levothyroxine sodium

1 962 - FDA requires NDA filings for all new drug products1982 - 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.

77

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 recalled1995 - 22 lots recalled because potency could not be assured through the expiration date.

78

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.

79

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

80

Purity and Concentration

81

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

82

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.

83

“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.

84

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

85

Example

86

Example

87

The BRIO System

24 lanes with reversed phase packing.

Eight channel autosampler for loading

Reusable

88

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.

89

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.)

90

The Future of Instrumentation and the FDA: 21CFR11

or…Is a Paperless Laboratory Really

Practical?

91

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.

92

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

93

“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.

94

Software Mission Statements

• When starting any software project, write a one-page, 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.

95

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.

96

Lesson 7

Introduction to the USP

97

Lesson 7

Retention factors

Dead volume

Resolution

Tailing Factor

System suitability

98

Retention Factor k

Retention retention time - dead time=

--------------------------------------------Factor 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.

99

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.

100

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

101

An LC/MS Example of the Importance of Retention Factors

K < 2, GOOD

102

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.

103

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.

104

Tailing Factor

Front width

Peak width

5% height

105

Asymmetry Factor (AS)

10% height

Back half width AS = ----------------------

Front half width

106

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.”

107

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)]

108

System Suitability is Taken Seriously by the FDA

• 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.

From an FDA Warning Letter sent to a pharmaceutical manufacturing company in New Jersey:

109

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)]

110

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.

111

Lesson 8

Introduction to FDA Regulation

112

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.

113

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

114

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.

115

Out of Specification (OOS)

Mean value

+ 2 standard deviations

- 2 standard deviations

Lower control limit

Upper control limit

116

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.

117

Lab Exercises for Lesson 8

Content Uniformity Assay

118

Lesson 9

Ion Pairing

119

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.”

120

Ion Suppression

H+

H+

H+

H+

H+

H+

H+

Low pH

Moderate pH

High pH

121

Ion Pairing

In ion pairing, a molecule with a long alkyl chain and terminal ionization sites is introduced into the mobile phase.

122

Ion Pairing

The longer the alkyl chains, the longer the retention time in a reversed phase column.

Silica Stationery Phase support

123

Ion Pairing

Courtesy Regis Technologies 1998.

124

Lesson 10

Performing a Tablet Assay

125

Lesson 11

Troubleshooting

126

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)

127

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.

128

Example of an Air Bubble Problem

Fitting failure

Air bubbles in system.Good, stable baseline

129

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?

130

Peak Fronting

• Overloaded column.• Channels in the solid

phase.

131

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

132

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

133

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.

134

Ghost Peaks

• Column Contamination

• Elution of analytes from a previous run

• Reverse and flush column

• Use a longer run time, a different clean up, or a strong solvent at the end of the run

First run Second run

135

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.

136

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.

137

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

138

Drifting Baselines

• 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.

One run Days or weeks

139

Lesson 11 Continued

More Troubleshooting

140

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.

141

Column Care Tips

Q: How do I now which solvents are safe to use on my column?

A: Read the directionsQ: How do I select a good solvent to flush my column?A: Read the directionsQ: I want to store my column, what should I fill it with?A: Read the directionsThis may be a hard lesson for us guys to understand.

142

Air Bubbles Revisited

• Symptoms that might appearIrregular baselinesIncreasing run timesLow to moderate pressure drop• Corrective actionsDegas 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.

143

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.

144

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.

145

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

146

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

147

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.

148

Column Packing, Extracolumn Effects and Peak Resolution

LOSS of resolution due to tubing volume

Column packing particle size

149

Peak Broadening as a Result of Extracolumn Volume

• Made worse by short and narrow columns

• Smaller packings are more vulnerable

150

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.

151

Student Presentations

152

Semester Review

153

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.