Welcome to Class Kevin Olsen 359 Richardson Hall

Welcome to Class! Kevin Olsen 359 Richardson Hall 973 -655 -4076 Olsen. K@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 p. H 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 Peak width Retention at half time. height (analyte #1) (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 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 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/m. L 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 m. L/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 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 500 mg/20 mg and lots of Advicor 750 mg/20 mg 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 E 10 M raw material. Additionally, Niaspan lots produced using Methocel E 10 M 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 6 months 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 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 p. Ka of a molecule is the p. H 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. p. Ka Therefore indicates the form a molecule will take at a given p. H value, for example stomach or blood p. H. • Some properties are p. Ka-dependent, such as lipophilicity, solubility and permeability. • p. Ka 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 p. H. 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 m. L serum • Add internal standards • Add organic solvents (hexane, n-butanol, acetonitrile, 93: 5: 2) • Evaporate • Reconstitute in 50 u. L Me. OH (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: 21 CFR 11 or… Is a Paperless Laboratory Really Practical? 90

21 CFR 11 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 21 CFR 11 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 21 CFR 11 • 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 - 1 mg/ml 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 p. H 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 p. H H+ H+ H+ High p. H H+ Low p. H 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 • Injection volume less than 15% of peak Tailing volume. 0. 5 min X 0. 3 ml/min = 0. 15 ml Broadening 0. 5 min X 3 ml/min = 15 ml Retention time • 1 u. G of sample per cm of column length for a decreases 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 • This results in an ion • Sometimes ghost pair formation that peaks can appear from causes the analyte to having a sample remain on the prepared in one stationary phase too solvent and using long. another solvent as your • Ion pairing is a mobile phase. 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 p. H 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 LCGC 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