Introduction to Modern Liquid Chromatography, Third Edition part 92 doc

Table 17.3Leaks Section 17.4.1: Symptoms and Sources Pre-pump 17.4.1.1 Fitting Loose fitting Tighten or replace Proportioning manifold low-pressure mixing Loose or damaged valve Tighten v

Table 17.3

Leaks (Section 17.4.1): Symptoms and Sources

Pre-pump

(17.4.1.1)

Fitting Loose fitting Tighten or replace Proportioning

manifold (low-pressure mixing)

Loose or damaged valve

Tighten valve or replace manifold

Pump (17.4.1.2) Fittings Loose or damaged Tighten or replace

Cross-threaded Replace; may require new

pump head Seal damaged Replace Pump seal Worn seal Replace Auxiliary component Loose fitting;

damaged component

Tighten or replace

High-pressure

fitting (17.4.1.3)

Stainless steel Loose or

contaminated

Tighten 1/4-turn; clean or replace

contaminated

Turn off pump, reseat tube, retighten; not for use

>6000 psi

Autosampler or

manual injector

(17.4.1.4)

Fittings Loose or damaged See fittings adjustments,

Sections 17.4.1.1, 17.4.1.3 Low-pressure needle

seal

Worn or loose seal Adjust or replace High-pressure needle

seal

Worn or damaged seal

Adjust or replace

Cross-port leakage Damaged rotor seal Clean, replace rotor seal;

may require stator replacement

External leaks not at fittings

Worn rotor seal Clean, replace rotor seal Insufficient

rotor-to-seal pressure

Adjust

Seal-pack failure (Waters only)

Worn or damaged seals

Service or replace

Table 17.3

(Continued)

Column (17.4.1.5) Tube connections Loose or damaged See fittings adjustments,

Section 17.4.1.3 End-fitting Loose or damaged Tighten; may require new

column End-fitting on

cartridge column

Loose or damaged fitting; damaged seal

Tighten; replace seal; may require new assembly Detector (17.4.1.6) Connecting fittings Loose or damaged See fittings adjustments,

Sections 17.4.1.1, 17.4.1.3 Cell leaks Loose fitting Tighten

Over-pressure damage

Remove source of excessive pressure

Failed window seal Adjust or replace Non-UV detector See Chapter 4 and

operator’s manuals

Table 17.4

Pressure Problems (Section 17.4.2): Symptoms and Sources

Pressure too high

(17.4.2.1)

General Wrong mobile phase;

temperature too low; flow rate too high; wrong column; wrong particle size

Use correct conditions

Blocked tubing Buffer precipitation;

sample particulates

Replace tubing; remove problem source Blocked or partially

blocked frit

In-line filter Replace frit; consider

additional sample cleanup

Guard column Replace guard column Analytical column Reverse column (if

permitted); replace column; consider using in-line filter

Upper pressure limit mid-gradient

Normal gradient pressure changes

Adjust upper pressure limit to accommodate

(continued overleaf)

Table 17.4

(Continued)

Pressure too low

(17.4.2.2)

General Wrong mobile phase;

temperature too high; flow rate too low; wrong column

Use correct conditions

Lower pressure limit Insufficient mobile

phase

Refill reservoir Overly sensitive limit

switch

Disable lower pressure limit

Massive leak Isolate and fix

(Section 17.3.1) High-pressure fitting Loose or

contaminated fitting

Tighten, clean, or replace Low-pressure fitting Loose or

contaminated fitting

Tighten, clean, or replace Low-pressure mixing

manifold

Air leak from unused solvent line; leaky proportioning valve

Prime unused lines with organic solvent; replace faulty proportioning valve

Inlet check valve Sticking with ACN

mobile phases

Sonicate in MeOH; replace

Pump starvation (failed siphon test)

Blocked inlet-line frit Replace Faulty proportioning

valve

Replace Pinched or blocked

tubing

Clear or replace Bubbles in pump Inadequate degassing

or purging; degasser failure

Degas mobile phase and purge pump; replace degasser

Pump seal Worn pump seal Replace Pump piston Broken or scratched

piston

Replace Pressure too

variable

(17.4.2.3)

Bubbles in pump Inadequate degassing

or purging

Degas mobile phase and purge pump

Inlet check valve Sticking with ACN

mobile phases

Sonicate in MeOH; replace

High-pressure fitting Loose or

contaminated fitting

Tighten, clean, or replace Low-pressure fitting Loose or

contaminated fitting

Tighten, clean, or replace

Table 17.4

(Continued)

Low-pressure mixing manifold

Air leak from unused solvent line; leaky proportioning valve

Prime unused lines with organic solvent; replace faulty proportioning valve

Pump starvation (failed siphon test)

Blocked inlet-line frit Replace Faulty proportioning

valve

Replace Pinched or blocked

tubing

Clear or replace Pump seal Worn pump seal Replace Pump piston Broken or scratched

piston

Replace

Table 17.5

Interpreting Retention-Factor k and Retention-Time t RChanges (Section 17.4.3)

k Changes t RChanges Possible Cause More Information

No Yes Flow rate (settings, leaks, bubbles, pump problems) Section 17.4.3.1

Yes Yes Mobile-phase error (%B, pH, additives) Section 17.4.3.3 Yes Yes Stationary-phase problem (aging, dirty samples) Section 17.4.3.4 Yes Yes Temperature problem (poor control) Section 17.4.3.5

Table 17.6

Retention-Time Problems (Section 17.4.3.6): Symptoms and Sources

Abrupt change

in t R

When column changed Wrong column; poor

column-to-column reproducibility (especially type-A columns)

Use correct column; use columns from same batch; adjust method for more robust separation; use more reproducible columns (e.g., type-B); Section 2.5.4.6, Table 17.5 When mobile phase

changed

Improperly formulated mobile phase

Make new batch of mobile phase; Table 17.5

(continued overleaf)

Table 17.6

(Continued)

In gradient elution, with change in instrument

Different dwell volume Adjust or compensate dwell

volume; Section 9.3.8.2

No apparent change in conditions

Equipment, bubble, or temperature problem

Check for leaks (Table 17.3, Section 17.4.1); check for pressure problems (Table 17.4,

Section 17.4.2); check k

changes (Table 17.5, Section 17.4.3); Section 2.5.4.6

Retention drifts During first few

injections only

Slow column equilibration;

column ‘‘loading’’

for sample

Normal for some columns and samples; allow longer equilibration; ignore first few injections (Section 3.10.2.2); Sections 2.7.1 (general), 7.4.3.2 (ion-pair), 8.5 (normal-phase), 9.3.7 (gradient elution) Over entire sample

batch

Unstable mobile phase; mobile-phase evaporation;

excessive helium sparging

Use stable mobile phase; cover reservoir to reduce evaporation; reduce helium sparging time or change to different degassing technique (Section 3.3) Retention too

small

Only for large-mass injections for some

or all peaks, with right-triangle peak shape

Mass overload Reduce mass of sample

injected; Table 17.11, Sections 17.4.5.3, 2.6.2

All peaks Mobile phase error;

flow rate, column,

or temperature change

See Table 17.5

Some peaks pH problem or other

mobile-phase error;

column aging

Make appropriate adjustments; Section 7.3 (pH); replace column Only for polar solutes Ionic samples Change mobile-phase pH

(Section 7.3); use ion pairing (Section 7.4)

Table 17.6

(Continued)

Neutral or non-ionic samples

Try EPG or AQ column; use normal phase (Chapter 8)

or HILIC (Section 8.6) Retention too

large

All peaks Mobile-phase error;

flow rate, column,

or temperature change; leak

See Tables 17.5, 17.3

Some peaks pH problem or other

mobile-phase error;

column aging

Make appropriate adjustments; Section 7.3 (pH); replace column

Table 17.7

Peak-Area Problems (Section 17.4.4): Symptoms and Sources

Peak area to large

(17.4.4.1)

All peaks larger by same proportion

Injection volume too large

Adjust Sample preparation errors

Use correct procedure Detector settings

wrong

Adjust Different peaks in

sample larger by different amounts

Detector settings wrong

Adjust

Peak(s) present in blank

Late elution from prior injection

Extend run; add strong-solvent flush; modify sample pretreatment Carryover Add autosampler wash steps;

change wash solvent; adjust connections; change sample loop material; rearrange injection order Peak area too small

(17.4.4.2)

All peaks smaller by same proportion

Injection volume too small

Adjust Sample preparation errors

Use correct procedure

(continued overleaf)

Table 17.7

(Continued)

Detector settings wrong

Adjust Different peaks in

sample smaller by different amounts

Detector settings wrong

Adjust

Peak area too

variable

(17.4.4.3)

Replicate injections give constant areas

Problem prior to injection

Check sampling and sample pretreatment steps Replicate injections

give inconsistent areas

Problem from injector onward

Check autosampler, pump, detector, and integration

Table 17.8

Baseline-Drift Problems (Section 17.4.5.1): Symptoms and Sources

Periodic drift 1 cycle/run Normal with

gradient elution

Ignore; use higher wavelength; change solvent; add UV absorber Several hours or 1

cycle/day

Laboratory temperature cycle

Thermostat oven; move system from drafts; adjust lab HVAC

Other drift Baseline drifts, then

stable

Normal equilibration;

detector warm-up

Wait until baseline stabilizes

Table 17.9

Baseline-Noise Problems (Section 17.4.5.2): Symptoms and Sources

Short-term noise High-frequency (50 or

60 Hz)

‘‘Dirty’’ electrical supply; too small

of detection time constant; too high of data rate

Use UPS to cleanup supply; use larger time constant or

RC filter; use slower data collection rate

50–60 Hz< noise <

peak width

Inadequate mobile-phase degassing

Use better degassing technique

Table 17.9

(Continued)

Long-term noise Cyclic Bubble in pump;

piston seal or piston damage;

sticking or leaking check valve

Degas mobile phase; service pump; clean or replace check valve

Appears as chromatographic peak

Late-eluted solute Extend run; add

strong-solvent flush to method; change sample cleanup

Random, irregular baseline disturbances

Accumulated late-eluted non-polar materials from sample

Add strong-solvent flush to method; replace column; change sample cleanup

Peaks appear in blank gradients (‘‘ghost’’

peaks, Figs 17.11, 17.12)

Contaminated mobile phase

Replace mobile-phase reagents with fresh and/or higher purity reagents

Table 17.10

Peak-Shape Problems (Section 17.4.5.3): Symptoms and Sources

Source

Solution

Peak tailing TF ≤ 1.2 Normal None required

1.2 < TF ≤ 2 If increased over time:

column aging or contamination;

mobile phase error

Flush or replace column; prepare new batch of mobile phase

If constant, may be normal

No action or explore

solutions for TF > 2

TF > 2 For all peaks Also see split or distorted

peaks Early peaks tail more

than later peaks (Fig 17.13):

extra-column effects

Reduce extra-column volume (Sections 2.4.1, 3.9)

(continued overleaf)

Table 17.10

(Continued)

Source

Solution

Worse for bases and ionic compounds

(Fig 5.9a): silanol

effects; trace metal contamination

Adjust pH; use higher purity or less-active column; see Sections 5.4.4.1, 7.3.4.2 Inadequate buffering

or mobile-phase additive concentration

Increase buffer or additive concentration; see Section 7.2.1.1 Right-triangle peak

tailing accompanied

by earlier retention

(Fig 17.15a)

Mass overload of the column

Reduce injection volume

or dilute sample for smaller mass on column (Section 2.6.2) Peak fronting TF ≥ 0.9 Normal None required

TF < 0.9 For ion pairing,

especially with type-A columns

Change temperature

±5−10◦C may help; change to type-B column

Column void or bed collapse

Replace column; operate column below high pH-limit

Broad peaks N > 75% of

manufacturer’s test

May be normal No action required;

compare to new column test with sample solutes

High-molecular-weight compounds (proteins, polymers, etc.)

Some broadening normal

No action required

Gradual broadening over>500

injections

Normal column aging Flush column with strong

solvent; replace column

In the presence of narrower peaks (Fig 17.5)

Late elution Extend run; adjust run

length; use gradient flush; improve sample cleanup

Accompanied by longer retention times

Column temperature too low

Increase temperature

Table 17.10

(Continued)

Source

Solution

Narrower peaks broadened more than normally wider peaks

Detector time constant too large; integrator data-rate too low;

excessive smoothing (especially MS detection)

Use time constant≤1/10 peak width; collect a minimum of 20 points across a peak; use less smoothing

early peaks broad or flat-topped

(Fig 17.16b)

Too large an injection volume

Reduce injection volume; dilute injection solvent (Sections 2.6.1, 3.6.3) Split or distorted

peaks

All peaks distorted in same manner (Figs

17.17, 17.19)

Partially blocked column inlet-frit;

column void

Reverse-flush column; use in-line filter or better cleanup; replace column (Section 5.8) Broad or distorted

peaks, especially at end of run

(Fig 17.20b),

accompanied by change in retention time

Mismatch of mobile phase and column temperature

Use better temperature control; use mobile phase pre-heater (Section 3.7.1)

Early peaks distorted

(Fig 17.16c),

usually accompanied by earlier retention

Too large a volume of too strong a sample-injection solvent

Use more dilute injection solvent; reduce injection volume (Sections 2.6.1, 3.6.2.2)

Table 17.11

Failed System Performance Tests (Section 17.4.6): Symptoms and Sources

Source

Solution

Gradient linearity Steps in gradient Blocked reservoir

frit; bad proportioning valve

Check/replace frit; replace proportioning manifold

Segmented appearance

in linear gradient

Software control error

Adjust control parameters (see service manual)

(continued overleaf)