Introduction to Modern Liquid Chromatography, Third Edition part 19 pdf

Malfunctioning check valves and worn pump seals often show up as deviations in expected values of mixing accuracy, gradient linearity, and flow rate.. An additional test of the outlet che

Figure3.28 Results of a gradient proportioning valve (GPV) test (a) Gradient proportioning valve test results; (b) detail showing±2% allowed difference between plateaus Baseline is generated by 50:50 A:B; the remaining plateaus are 90:10 A:C, A:D, B:C, and B:D from left to right Solvents: A= B = water; C = D = 0.1% acetone in water

Table 3.4

Typical System Performance Parameters for Tests of Section 3.10.1 Parameter/Test Typical Specification Typical Acceptance Criteria

GPVatest Not specified ≤2% plateau rangeb

Flow rate (collect 10 mL at 1 mL/min) ±1% ±2% (±12 sec on 10 mL at

1 mL/min) Pressure bleed-down from 4000 psi Not specified 15% in 10 min

Retention reproducibility Not specified ±0.05 min

Area reproducibility (10 μL injection) ±0.3% ±1%

aGradient proportioning valve.

B-solvent, as in Figure 3.28.

the acceptance criterion is set to±2%, since the combination of measurement errors (volumetric glassware) and the timing start/stop errors will add somewhat to the overall measured error

Pressure Bleed-Down Malfunctioning check valves and worn pump seals often

show up as deviations in expected values of mixing accuracy, gradient linearity, and flow rate An additional test of the outlet check valves can be made with a pressure bleed-down test For this test the outlet tubing from the pump is blocked (by a union and a plug) The high-pressure shutoff limit for the pump is set near its maximum

value, for example, 5000 psi (350 bar) for a system capable of 6000 psi (400 bar) The pump is then turned on and allowed to shut off at the upper pressure limit The maximum pressure is recorded, and 10 minutes later the pressure is recorded again

A pressure drop of≤15% indicates that the check valves are working properly A larger drop suggests that the outlet check valve(s) should be cleaned or replaced

or that the pump seal(s) should be replaced if no outlet check valve is used (e.g., accumulator-piston pump, Section 3.5.1.2) A pressure drop to atmospheric pressure over the 10-minute test is more indicative of a leaky fitting

Retention Reproducibility A check of retention reproducibility is an overall

check of on-line mixing and pump performance Although this check can be done with any sample, it is wise to use a sample that can be formulated easily under conditions that can be reproduced at any time This allows an independent check

of system performance to be made for a specific method, and it is a good tool for troubleshooting Example test conditions are listed in Table 3.5 It is important

to use a sample concentration such that the peak is within the detector’s linear range and is sufficiently large that baseline noise does not affect the precision of the measurements For example, a sample that generates a peak height of 0.1 to 0.8 AU would be a good choice for UV detection Retention-time variation of no more than

≈0.05 min (1 S.D.) is acceptable for six replicate injections Larger variations are an

indication of problems If the other performance tests prove to be okay, but retention reproducibility is poor, leaks or air bubbles are the most likely problem sources An alternative test, especially if the system is used only for isocratic methods, is to run the retention test under the isocratic conditions listed in Table 3.5 (80% B)

Table 3.5

Retention-Time and Peak-Area Test Conditions

Gradient/isocratic 5–95% B in 20 mina/80% Bb

Retention (typical) gradient/isocratic 14 mina/3 minb (with VD = 2.2 mL d)

aGradient test conditions.

bIsocratic test conditions; adjust as necessary for 2< k < 10.

d For other values of the dwell-volume V D; gradient retention times will change by the difference in dwell-volumes divided by the flow rate (1.5 mL/min).

Peak-Area Reproducibility The same chromatograms run for retention

repro-ducibility can be used to determine peak-area reprorepro-ducibility, which is primarily a measure of the autosampler’s performance The variation in peak areas should be

<1% RSD based on six injections Most modern autosamplers will generate values

of≤0.5% RSD for injection volumes of ≥5 μL when operated under standardized

conditions (e.g., those of Table 3.5) Poor area reproducibility usually can be traced

to an autosampler problem Peak-area reproducibility should be checked for both the isocratic and gradient operation; similar results should be obtained under both conditions

New-Column Test Repeating the column manufacturer’s test procedure on a

new column is a quick procedure to show that the HPLC hardware is working properly This test should be done as part of the operational qualification (OQ) or performance qualification (PQ) testing (Section 3.10.1.1) to show that the system

is functioning well before its first use The new-column test also serves as a tool in the ‘‘divide-and-conquer’’ strategy (Section 17.3.1) of problem isolation Just install

a new column and repeat the manufacturer’s performance test The test conditions should be similar to the isocratic conditions listed in Table 3.5, and should be listed

in the column test sheet included with the column literature If the test results are within ≈10% of the column manufacturer’s tests (retention times, plate number), the system is working well—the flow rate, column temperature, and mobile-phase composition are correct, and extra-column effects are minimal (It is rare to obtain

a plate number as large as that reported in the manufacturer’s test; their column testing systems are optimized for column performance, not routine operation as in

a user’s laboratory.)

3.10.2 Preventive Maintenance

A good way to improve the reliability of an HPLC system is to anticipate and prevent problems before they occur Problems with HPLC systems can result from normal wear of components or from the way the system is used Many problems can therefore be minimized by performing periodic maintenance so that normal-wear items are serviced or repaired before they fail In addition there are a number of simple laboratory practices that can be undertaken to minimize user-caused system failure

3.10.2.1 Periodic Maintenance

Regular cleaning and/or component replacement will help make the HPLC system work more reliably Some of these periodic maintenance tasks are listed in Table 3.6 The recommended frequency of the various actions is typical for HPLC systems used several times a week; the experience in each laboratory may vary from these recommended intervals The important concept is that a disciplined approach be taken so that every instrument receives regular maintenance For further details on the parts and modules mentioned below, consult the descriptions in the first part of this chapter

Reservoir The reservoir that contains the aqueous solvent (A-solvent) should

be cleaned and replenished, so that microbial growth does not occur Replacement

Table 3.6

Recommended Maintenance Intervals

Reservoir Replace buffer, wash reservoir Weekly

Replace organic, wash reservoir Monthly Filter mobile phase Every batch Replace inlet-line frit 6 months Degas mobile phase Daily Tubing and fittings Inspect for leaks Weekly

Pump(s) Sonicate or replace check valves As needed

Replace pump seal(s) 6 months Flush to remove buffers Daily Inspect for leaks Daily Autosampler Replace wash solvent and clean

Reservoir

Weekly Replace valve rotor seal See manufacturer’s

recommendations Replace needle seal 6 months

Inspect for leaks Daily Column oven Calibration check Annually

Inspect for leaks Daily

Additional detector checks See detector manual Waste container Check capacity; empty Daily

Data system Per experience

LC method System suitability Daily

of the A-reservoir and its contents on a weekly basis should avoid this, although some laboratories stretch this to two weeks It is a good idea never to ‘‘top off’’ the aqueous solvent, but instead to use a clean reservoir for each new batch of aqueous solvent The organic reservoir (B-solvent) has a longer cleaning and replacement cycle, because the probability of microbial growth in>30% organic is considerably

diminished compared with the aqueous phases If the A- or B-solvent comprises only HPLC-grade solvents, mobile-phase filtration is not required; otherwise, all mobile phases should be filtered through a membrane filter of ≤0.5-μm porosity The

inlet-line frit can be an inadvertent source of re-inoculation of the reservoir contents once it becomes contaminated The frit is inexpensive, so it should be replaced every six months as a precautionary measure Mobile-phase degassing is one of the easiest ways to improve system reliability, so all solvents used for reversed-phase HPLC should be degassed (Section 3.3)

Tubing and Fittings If tubing and fittings are properly assembled, they should

work indefinitely without problems However, leaks do occur occasionally, so it is a good idea to check all tubing and fittings for leaks once a week A drop of moisture

or a small bit of white buffer residue may be the only sign of a leak If a leak is found, the pump should be shut off, the fitting tightened, and the pump turned back on again When PEEK fittings and/or tubing are used, the tube end should be reseated

in the fitting port prior to tightening the fitting Sometimes PEEK tubing will slip

in the fitting when it is tightened under pressure, so the pump should be turned off during this operation

Pumps Sticky or leaky check valves are the most common pump problem

in most systems This is indicated most commonly by system-pressure fluctuations Many times a leaky check valve can be reconditioned by sonication in MeOH for a few minutes Prior to sonication, it is best to inspect the check valve to ensure that the components will not fall out and get mixed up If the valve requires reassembly, dust-free gloves should be used, so as to avoid recontamination of the check valve (See Section 17.2.5.4 for additional information on check-valve sonication.) Pump seals may last for a year or more, but they are inexpensive and when pump-seal failure occurs, other problems may arise, such as blocked frits For this reason it

is prudent to replace the pump seals twice a year, that is, before significant wear occurs Buffers left in the pump when it is not used can precipitate and leave abrasive deposits that increase pump-seal wear or cause check-valve problems It is a good practice to flush all buffers out of the HPLC system before shutting it off for more than an hour or two If the system must be ready to run at a moment’s notice (‘‘stat’’ conditions), the pump can be set to its minimum flow rate (e.g., 0.1 mL/min), so the mobile phase is always flowing As with tubing and fittings, pump leaks occur periodically, so it is a good idea to inspect the pump for leaks each day during startup

Autosampler Just as the mobile phase and the reservoirs need to be cleaned

or replaced to avoid microbial contamination, the autosampler wash-solvent and its reservoir should be replaced periodically If an aqueous wash-solvent is used, it should be replaced and the reservoir cleaned each week; if organic solvent is used, monthly service should suffice The injector rotor seal has a long life—100,000 cycles or more if treated properly Check the manufacturer’s recommended service interval in the preventive maintenance section of the operator’s manual The needle seal will wear with continued use If there are no other guidelines in the operator’s manual, replacement of the needle seal every six months will help to avoid problems The autosampler is the component of the HPLC system that is most likely to leak; it

is wise to inspect it daily for leaks

Column Oven The column oven should be a trouble-free component The oven

calibration should be checked when the oven is first installed, then on an annual basis A flow-through thermocouple can be used to check the temperature of the mobile phase exiting the column as a check of the oven temperature and temperature equilibration within the column During system startup each day or when a new method is started, the column endfittings should be checked for leaks

Detector HPLC detectors operate at pressures significantly lower than the

column, so leaks at the detector are less common than elsewhere However, a weekly check is wise Other detector checks will vary depending on the detector; consult the detector manual for more information

Waste Container The waste container is a system component that is easy to

overlook It is wise to check it daily to ensure that it has sufficient capacity for the day’s runs Placement of the waste container in a plastic dishpan or other safety container will help protect against spills when the inevitable overflow occurs

HPLC System-Check The system performance checks described in Sections 3.10.1.2 and 3.10.1.3 should be performed once or twice a year A semiannual check will often catch pending problems that might occur if the check is done on an annual basis Check the system on a yearly basis if a semiannual check cannot be justified

HPLC Method-Check Before samples are run with an analytical method, a

system suitability check should be made This may be the most important test that one can perform for an HPLC system because it shows that both the system and method are suitable for carrying out the desired analysis

3.10.2.2 Suggestions for Routine Applications

To obtain high-quality data, the HPLC system must perform in a reliable and repro-ducible manner This section lists and reviews some additional tips and techniques that will help ensure high quality results

Reagent Quality Gradient elution tends to concentrate nonpolar impurities in

the A- and B-solvents at the head of the column, followed by their release as the gradient progresses These impurities can show up as peaks in both blank and sample runs (e.g., Fig 17.12 and Sections 17.2.5.9, 9.6.2) For this reason it is essential to use HPLC-grade reagents for gradient work Lower quality reagents may be suitable for isocratic applications, but even the most minor impurities can cause problems with gradient elution For best results one should use only HPLC-grade reagents for all HPLC work Aqueous reagents and buffers should be discarded frequently (e.g., weekly) to avoid contamination by microbial growth Water impurities can be especially problematic

System Cleanliness As important as reagent quality in minimizing artifactual

peaks is maintaining a clean instrument The recommendations of Section 3.10.2.1 (reservoirs and pumps) should be followed Spills, leaks, and other potential sources

of contamination should be cleaned up promptly

Degassing Although some HPLC systems will operate without degassing

the mobile phase, every system will operate more reliably with degassed solvents (Section 3.3) Trapped air bubbles and solvent outgassing are common problems that can be largely avoided by solvent degassing It is a good idea to purge the pump(s) and solvent inlet lines daily by opening the purge valve(s) and operating the pump at an elevated flow rate (e.g., 5 mL/min) for a few minutes to remove any air bubbles

Dedicated Columns Each analytical method should have a column dedicated

to that method It is not a good idea to share columns between methods, since peaks that are not of concern in one method (e.g., late-eluting peaks) may cause interferences in a second method Sample components may change the selectivity or degrade the column for one method but not another

Equilibration Prior to each run, the column should be equilibrated to the same

extent as the other runs in the run sequence Complete equilibration may or may not be necessary for gradient methods (Section 9.3.7)

Priming Injections Some methods give better results if several ‘‘priming’’

injections are made before the first sample is injected These injections of standards

or mock samples can help saturate slowly equilibrating active sites on the column

so that more reproducible separations can be obtained Priming injections are more often useful for separations of biological samples (Section 13.3.1.4) Sometimes the system suitability injections serve as priming injections

Ignore the First Injection Because some methods require a priming process

and the first injection may be equilibrated differently than subsequent injections, it

is best to avoid use of the first injection for quantitative analysis The second and subsequent runs will be more reliable than the first injection; a preceding system suitability test (see following) also serves this purpose

System Suitability Many methods that run under the oversight of regulatory

agencies (FDA, EPA, OECD, USP, etc.) will require a system suitability test prior

to sample analysis System suitability serves as a confirmation that the equipment and analytical method are operating in a fashion that will produce reliable results Requirements for system suitability tests vary, so the regulatory guidelines should be consulted to help select appropriate tests (Section 12.3) Many workers use retention time and area reproducibility, peak response (detection sensitivity), peak width, peak tailing, resolution, and column pressure, either alone or in combination, as part of the system suitability test The system suitability sample may be a diluted pure standard, a mock sample in extracted matrix, or some other sample selected

to demonstrate system performance The important concept is to select system suitability samples that test the ability of the method to perform its desired function Whether or not a system suitability test is required, it is wise to run such a test prior

to routine analysis, even if it is just an injection of a standard to see if the retention and peak size are as expected

Standards and Calibrators For quantitative analysis (Chapter 11), the response

of unknown samples is compared to the response for standards of known concen-tration The range of standard concentrations, number of replicates, and sequence

of injection may depend on the specific application Either external or internal standardization may be used In any event, at least one standard should be injected prior to the analysis of unknown samples, to ensure that the analytical method is working properly before potentially valuable samples are injected

Table 3.7

Repair Recommendations

Novice/operator Replace mobile phase, inlet-line filter, in-line filter, guard column, column Experienced Check valves, pump seals, proportioning-manifold replacement, injector rotor,

detector lamp, tubing and fittings Factory-trained Proportioning-manifold repair, electronics, repairs involving module

subassemblies, detector flow cell

3.10.3 Repairs

Sooner or later, no matter how much preventive maintenance is practiced, problems will be encountered on every HPLC system Chapter 17 is dedicated to troubleshoot-ing In this section, some of the more philosophical aspects of instrument repair are considered

3.10.3.1 Personnel

Some laboratories rely completely on a service contract from the equipment man-ufacturer for all maintenance and repairs, whereas other laboratories perform all these tasks themselves Most laboratories, however, rely on something between these two extremes Even in the case of a 100% service-contract arrangement, there are repairs that make sense to accomplish with laboratory staff—for time savings, if nothing else It is a good idea to have a plan in place that determines what repairs are to be made by which personnel Some recommendations are listed in Table 3.7 and discussed in Section 3.10.3.3

3.10.3.2 Record Keeping

Laboratories working under the oversight of regulatory agencies may be required to keep maintenance and repair records, but even laboratories for which such records are not required should maintain some level of equipment records One of the simplest record-keeping techniques is to prepare a three-ring binder with sections for different maintenance activities and records A separate binder is kept with each HPLC system where it can be referenced easily and is most likely to be used during a maintenance session It is convenient to organize this record book in three sections:

System Configuration A record should be made of all the components in

a given system configuration, including the model and serial numbers of each component As changes are made, a fresh copy can be completed This will facilitate communications with the instrument vendors when service is needed, and will help confirm for regulatory auditors the specific system configuration used with a specific sample

Maintenance Records Repair records can be simplified if a template is made

that contains blanks for the who, what, why, when, and how questions of a maintenance session Several blank copies of these pages can be kept in the notebook

so that one is available to complete for each maintenance session Once a year,

or after a number of sessions, these pages can be sorted into failure categories (e.g., pump-seals, check valves) and can serve to help develop a system-specific or laboratorywide preventive maintenance program

System Checks A third section can contain the records from the periodic

instrument system checks (Sections 3.10.1.2, 3.10.1.3) With a readily available historic record, trends from one system check to another can be tracked These also can provide data that help establish preventive maintenance programs

Record-keeping technologies could also be used Computer databases or elec-tronic notebooks are two good options But, it is important that they are sufficiently convenient, so that data can be easily entered at the time of maintenance or repair; otherwise, valuable records will be lost

3.10.3.3 Specific Repair Recommendations

Table 3.7 summarizes various repairs that are appropriate for users of different skill levels:

Novice/Operator Personnel just learning how to run an HPLC system, or

workers in a routine environment that ‘‘just’’ run samples, may have limited skills

or authority to make repairs However, little training is needed to replace mobile phases, filters, guard columns, and columns A system of double-checking or sign-off

by a second person will help avoid errors in such activities

Experienced As the user gains experience, additional troubleshooting and

maintenance skills will be developed Some people have a natural tendency to become more skilled than others, but most workers can learn how to change check valves, pump seals, and tubing and fittings More complex problems, such

as replacement of detector lamps or low-pressure gradient-proportioning manifolds, can be reserved for more skilled workers

Factory-Trained Certain activities should be reserved for specially trained

per-sonnel These may be normal laboratory workers with an excellent troubleshooting and maintenance aptitude, a service group within the company, or a technician con-tracted from the instrument manufacturer Electronics problems generally require advanced skills and/or equipment to fix Other problems that involve disassembly of modules or repair of components that are delicately tuned—such as proportioning valves—may also call for special training

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