From Instrument to Column

Tracking Down the Problem

Melissa Goodlad
Applications Engineer
Columns and Supplies Technical Support

1 5/27/2022 From Instrument to Column: Tracking Down the Problem DE27255094

 Troubleshooting Topics

System pressure Peak shape

• Increased pressure • Tailing
• Low pressure 1 • Broadening
• Pressure fluctuations • Fronting
2• Peak splitting and doubling

Separation Detection
• Changing retention time • Noisy baseline
• Loss of resolution • Reduced intensity or
sensitivity
• Drifting baseline

2 5/27/2022 From Instrument to Column: Tracking Down the Problem DE27255094

 Agilent Lab Advisor

• Tools for calibration, diagnosis,

and maintenance
• Daily instrument tests
• General calibration and
maintenance procedures
• Advanced version also available
for expert level troubleshooting
• EMF (Early Maintenance
Feedback) shows the number of
valve switches or pumped
solvent

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 System Pressure

4 5/27/2022 From Instrument to Column: Tracking Down the Problem DE27255094

 Understand Your LC System and Follow the Flow Path

Detector

Column compartment

Autosampler

Pump

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 Changes in System Pressure
Increased Pressure/Overpressure and Blockages

Potential cause Recommended action

Clogging of filter frits in the high- • Identify the culprit by logical

pressure flow path elimination process and replace
affected part.
• Use clean, prefiltered solvent
Plugging of capillaries, needles, and • Prevent algae growth in water
needle seats

Wrong solvent • Check for correct mobile phase

• Check solvent reservoir and tube
connections

6 5/27/2022 From Instrument to Column: Tracking Down the Problem DE27255094

 Blockages and Clogging
P
Characteristics
Parts affected Blockages:
• Capillaries, needle, and needle seat
• Detector flow cells
Clogging:
• Filter frits (inline filter, column filter) t
Characteristic Blockages: Instant pressure
increase step
Identification • Check easily accessible points: needle seat, purge valve, column
• Disconnect capillaries one-by-one, starting at detector and moving P
back toward the pump
Possible root • Debris from mechanically worn parts (needle seat material, rotor
cause seal at injection valve)
• Coring of vial septa material
Instant • Replace part
action/First aid • Backflush affected part
t
Preventive • Proper preventive maintenance schedules, replace worn parts Clogging: Constant
measures regularly pressure increase over
• Use high quality septa time
• Install inline filters

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 Checking for Blocked Needle Seat

8 5/27/2022 From Instrument to Column: Tracking Down the Problem DE27255094

 Checking for Blocked Needle Seat
Track the typical operating pressure for a given application
To troubleshoot:
• Create high pressure on the system by turning on flow
• While the pressure is climbing, move the sampler to the “Bypass”
position
• Watch the pressure when the valve switches to “Bypass”
• If the pressure drops immediately, then the source of the high pressure
is in the portion of the flow path specific to the bypass position
─ Needle Seat
• Where the sample first meets the mobile phase
• Most commonly clogged piece of tubing in an LC
─ Needle
• Less commonly clogged
• Watch for issues with septa

9 5/27/2022 From Instrument to Column: Tracking Down the Problem DE27255094

 Checking for Blocked Needle Seat

Loop
• Not commonly clogged
• Watch for issues with sample

Metering head
• Never exposed to sample
• Consider solvent issues

Injection valve
• Most common issue is with rotor seal
• Look for scratches on stator face

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 Checking for Blocked Needle Seat
Driving the needle into debris may result in a clogged
needle or seat

• In well plate samplers, bottom sensing gives the

most consistent position
• But this isn’t recommended if there’s debris in the
bottom of the sample vial
• For vial samplers, a zero offset is approximately
2 mm from the bottom of a 2 mL vial

Needle height position without bottom sensing:

G1367E/G4226A 54 vial tray = 4 mm
G1367E/G4226A 100 vial tray = 2.5 mm
G7167X 54 vial tray = 5 mm

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 Locating a Clog

If the pressure doesn’t drop when the valve switches to “Bypass”, the
issue is likely outside the sampler.

• Purge valves:
- With 1260 Infinity II model pumps, open the
manual purge valve. The pressure should drop to
between 0 and 5 bar. If the pressure is higher
than this, the PTFE filter may be clogged.
- With 1290 Infinity II model pumps, purging is done
through an automated valve, activated using
software. 1290 binary pumps have the same
PTFE filter, 1290 quaternary pumps have a 5 µm
filter frit.

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 Locating a Clog

PTFE replacement on a 1260 pump:

1. Remove pump outlet and purge waste tubing

2. Unscrew the purge valve using a 14 mm
wrench
3. Remove the gold seal cap
4. Remove the frit
5. Install the new frit, slot side up
6. Replace the gold seal cap
7. Reinstall the valve
Re-align the waste tubing in the correct orientation
during installation.

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 Locating a Clog

If the pressure doesn’t drop when the valve switches to “Bypass”, the issue is likely outside
the sampler.

Column:
• Open the fitting at the inlet of the column.
• Pumping 1 mL/min of water through an Agilent LC with 0.17 mm
id tubing typically shows a pressure of 40 bar.
• If the pressure is much higher than this, a capillary may be
clogged. If the pressure appears “normal” the issue may be with
the column.

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 Locating a Clog

If the pressure doesn’t drop when the valve switches to “Bypass”, the issue is likely outside
the sampler.

Working backwards from detectors:

• Clogs are located by opening a fitting, typically at
most a half turn.
• If the pressure drops, the clog is downstream from
the fitting or towards the detector. If pressure remains
high, the clog is upstream or towards the pump.

15 5/27/2022 From Instrument to Column: Tracking Down the Problem DE27255094

 Changes in System Pressure
Low pressure

Potential cause Recommended action

Leak in high-pressure flow path • Visual inspection of flow path

• Instrument diagnostic tests
Wrong mobile phase • Check for correct mobile phase
• Check solvent reservoir and tube
connections

16 5/27/2022 From Instrument to Column: Tracking Down the Problem DE27255094

 Leaks
Characteristics

Parts affected • Potentially all parts in the flow path P

• High potential at frequently operated fitting connections
(such as the column inlet) and parts with high
mechanical stress (rotor seal, needle, and needle seat)
Characteristic • Lower pressure
• Potentially impacting retention times and peak shape
Identification • Drops of solvent or residues of salt
• System diagnostic tests t
Possible root cause • Loose or bad fitting connections
• Cracked capillaries
• Worn needle and needle seat
Instant action/first aid • Replace affected parts
• Renew or redo fitting connection
Preventive measures • Use proper fitting connections
• Replace fittings and wear parts in time

17 5/27/2022 From Instrument to Column: Tracking Down the Problem DE27255094

 How Do I Locate a Leak?

• Each Agilent LC module is equipped with a leak sensor

• If liquid is detected, the entire LC stack will shut down
• The LC will not start up again until the sensor has been dried and returned to temperature

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 Overtightened Fittings

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 Changes in System Pressure
Pressure fluctuations
Potential Cause Recommended Action
Air in the system • Prime and flush instrument
• Check for sufficient solvent supply
• Check for correct plumbing (SSV/MCGV)
• Check for correct degassing
Malfunctions at pump • Perform pump head diagnostic tests LA
head • Replace defective parts
• Implement proper maintenance schedule
Cavitation effects • Check for flow restrictions (solvent bottle to pump head
inlet)
• Clean or replace parts
• Verify that solvent supply is positioned above pump inlet

Important to know
Pressure fluctuations typically also impact the UV signal due to
!
refractive index effects.

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 Peak Shape

21 5/27/2022 From Instrument to Column: Tracking Down the Problem DE27255094

 Changes in Peak Shape
Peak tailing
I
If applicable to some peaks Recommended Action
Secondary interactions • Check pH of mobile phase (most likely)
Small peak eluting on tail of • Pump malfuntion
larger peak

t
If applicable to all peaks Recommended Action
One bad connection
Poor tubing connections; high • Minimize number of connections mAU

dispersion volume • Check connections/fitting condition and proper

120
80

seat of fittings 40
0
0 0.1 0.2 0.3 0.4 min
• Use fittings with spring-loaded function mAU
180
Column damage • Use specialty, polymeric or sterically protected 120
Fixed
column 60
0
• Column cleaning 0 0.1 0.2 0.3 0.4 min

22 5/27/2022 From Instrument to Column: Tracking Down the Problem DE27255094

 InfinityLab Quick Connect and Quick Turn Fittings

• Spring-loaded design
• Easy to use
• Works for all column types
• Reusable
• Consistent ZDV connection

Quick Connect Fitting

• Finger tight up to 1300 bar
• Hand tighten the nut, then depress the
lever

Quick Turn Fitting

• Finger tight up to 400 bar
• Up to 1300 bar with a wrench
• Compact design

23 5/27/2022 From Instrument to Column: Tracking Down the Problem DE27255094

 Peak Tailing: Column Contamination
Column: StableBond SB-C8, 4.6 x 250 mm, 5 mm Mobile phase: 20% H2O : 80% MeOH Flow rate: 1.0 mL/min
Temperature: R.T. Detection: UV 254 nm Sample: 1. Uracil 2. Phenol 3. 4-Chloronitrobenzene 4. Toluene

QC test forward QC test after cleaning

QC test reverse direction 100% IPA, 35 °C
direction
Plates TF 3
Plates TF Plates TF 3

3
1. 7629 2.08 1. 7906 1.43 1. 7448 1.06
2. 12043 1.64 2. 12443 1.21 2. 12237 1.21
3. 13727 1.69 2 3. 17999 1.19 3. 15366 1.11 2
4 13355 1.32 4 17098 1.25 2 4 19067 1.17

4
1
4
1 1 4

0.0 2.5 5.0 0.0 2.5 5.0 0.0 2.5 5.0

Time (min) Time (min) Time (min)

24 5/27/2022 From Instrument to Column: Tracking Down the Problem DE27255094

 Changes in Peak Shape
Peak splitting/doubling

Potential Cause Recommended Action

Partially plugged column frit • Backflush column (if applicable)
• Use inline filter I
• Use guard column
Column void • Replace column
• Use guard column
• Use less aggressive mobile phase
conditions
Sample volume overload • Use smaller injection volume
Sample solvent incompatibility with • Use mobile phase or weaker miscible
mobile phase solvent as injection solvent t
Issues with injection valve • Check injector valve parts
• Replace worn parts

25 5/27/2022 From Instrument to Column: Tracking Down the Problem DE27255094

 Changes in Peak Shape
Fronting
I
Potential Cause Recommended Action
Channeling in column • Replace column
• Use guard columns
Column overload • Use higher capacity column (increase
length, diameter, or change to high-capacity
material)
• Decrease sample amount t

2 µL 10 µL

26 5/27/2022 From Instrument to Column: Tracking Down the Problem DE27255094

 Changes in Peak Shape
Peak broadening

Potential Cause Recommended Action

Injection volume too large • Decrease injection volume
Long retention times • Use gradient elution or stronger mobile
phase I
System settings • Check data collection rate
• Adjust the detector setting or time constant
to the fastest possible value without
compromising signal-to-noise.
Viscosity of mobile phase too high • Increase column temperature
Detector cell volume too large • Use smallest possible cell volume
Improper fittings and connections • Ensure that your fitting connections are
correct t
Extra tubing volume on system • Ensure that the tubing is narrow and as
short as possible to avoid extra volume
Sample diluent too strong • Reduce diluent strength

27 5/27/2022 From Instrument to Column: Tracking Down the Problem DE27255094

 Strong Diluents Can Disrupt Equilibration – Isocratic Method

Isocratic method – all peaks are affected by diluent mismatch

28 5/27/2022 From Instrument to Column: Tracking Down the Problem DE27255094

 Strong Diluents Can Disrupt Equilibration – Gradient Analysis
Gradient method – early eluting peaks are most affected by diluent
mismatch

29 5/27/2022 From Instrument to Column: Tracking Down the Problem DE27255094

Comparison of Peak Shape at Low and High Loads Broadening and
Tailing
High sample loads give broad or broad and tailing
mAU peaks
0.001 mg/mL
60
0.003 mg/mL
0.005 mg/mL
Dextromethorphan is 35% broader at high load
0.01 mg/mL
0.05 mg/mL
0.1 mg/mL Pw = 0.2481 0.005 mg/mL dextromethorphan (4.1 uL injection
volume)

40

Low sample loads provide symmetrical,

Pw = 0.2062 nontailing peaks with narrow peak widths

20
2.7 um, Poroshell 120 EC-C18
mAU
3
Pw = 0.1827 2.5
Pw = 0.1840
2
1.5
Pw = 0.1835
1
0 0.5
0
8 8.5 9 9.5 10 min
min
8 8.2 8.4 8.6 8.8 9 9.2 9.4 9.6

30 5/27/2022 From Instrument to Column: Tracking Down the Problem DE27255094

 Changes in Separation

31 5/27/2022 From Instrument to Column: Tracking Down the Problem DE27255094

 Changes in Separation
Retention time changing
Potential Cause Recommended Action
Inconsistent online mobile phase mixing Ensure gradient system is delivering constant
composition check vs. manual preparation of
mobile phase
Flow rate changing Check ‘pressure fluctuation' I
Column temperature varying Thermostat column and
ensure constant lab temperature
Equilibration time insufficient with gradient Flush with at least 10 column volumes after
run or change in isocratic mobile phase solvent change or gradient conclusion
Selective evaporation of mobile phase Keep solvent reservoirs covered
component prepare fresh mobile phase
Buffer capacity insufficient Use > 20 mM concentration of buffer
Contamination buildup Occasionally flush column with strong solvent t
to remove contaminants
First few injections – adsorption on active Condition column by initial injection of
sites concentrated sample
Column overloaded with sample Decrease injection volume or concentration
Mobile phase composition changing Follow ‘best practices'

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Mobile Phase Preparation

33 5/27/2022 From Instrument to Column: Tracking Down the Problem DE27255094

Retention Time Shift – Selectivity Differences Due to Incorrect pH

pH 4.5 shows
pH 3.0 shows no
selectivity change
selectivity change
from lot-to-lot for
from lot-to-lot
basic compounds

• For method ruggedness

– Test three different column lots
– Compare Rs for the three lots
•If ∆Rs is too large, modify method

34 5/27/2022 From Instrument to Column: Tracking Down the Problem DE27255094

 Changes in Separation
Ghost peaks, carry over

Potential Cause Recommended Action

Peaks from previous injection • Flush column to remove
contaminants
• Check with blank injection
Specific interaction with metal surfaces • Passivate instrument
• Use InfinityLab Deactivator Additive
• Use bio-inert LC equipment
Contamination or unknown interferences • Proper sample cleanup
in samples

35 5/27/2022 From Instrument to Column: Tracking Down the Problem DE27255094

 Mobile Phase Hygiene
Contaminated mobile phases can cause:
- Lower sensitivity
- Rising/drifting baselines
- Higher noise
- Ghost peaks with gradient separations

Often the issue is confused with autosampler

carryover.
It can be identified by repeating the gradient run
without sample injection or increasing the pre-run
equilibration.
Always run multiple blanks before standards or
samples to distinguish gradient artifacts from
possible carryover.

36 5/27/2022 From Instrument to Column: Tracking Down the Problem DE27255094

 Mobile Phase Hygiene: Glassware

Improper cleaning of solvent bottles can cause contamination of mobile phases and result in
gradient artifacts
• Wash solvent bottles with hot water, deionized water, and organic solvent (IPA or acetonitrile).
• Leave glassware inverted on paper towels on a bench or clean pegboard dowels to dry.
• Avoid using detergents. If it is necessary to use detergents to get glassware clean, rewash with
plenty of hot and cold water so that all detergent residues are removed. Follow with deionized water
and organic (IPA or acetonitrile) rinses.
• Store glassware inverted on shelves or in drawers, or cover openings.

System blank injection,

water/ACN gradient on a C18 column,
PEG contamination

MS response
37 5/27/2022 From Instrument to Column: Tracking Down the Problem DE27255094
 Mobile Phase Hygiene: Solvent Purity and Buffer Preparation
- Use HPLC grade organic mobile phases
- Use HPLC grade water or Milli-Q DI water
- Use HPLC grade reagents, including salts, ion pair reagents, and base and acid modifiers
- Always rinse pH electrodes thoroughly when measuring/adjusting the pH of the mobile phase
- Prepare fresh buffers to avoid contaminants from the growth of bacteria or algae
- Filter your mobile phase buffer with 0.45 µm filter before use
- Solvent filters installed at the end of solvent lines should be replaced periodically

100% ACN 90%ACN+10% buffer

(10 mM phosphate)
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 Autosampler Carryover

Common sources:
• Exterior of needle (use needle wash)
• Worn needle seat
• Worn rotor seal
• Poorly made fitting

39 5/27/2022 From Instrument to Column: Tracking Down the Problem DE27255094

 Autosampler Carryover

40 5/27/2022 From Instrument to Column: Tracking Down the Problem DE27255094

 Changes in Detection

41 5/27/2022 From Instrument to Column: Tracking Down the Problem DE27255094

 Changes in Detection
Noisy baseline

Potential Cause Recommended Action

Gas bubbles in mobile phase • Apply degassing
• Check degasser performance
Low difference between sample and • Check absorbance values of sample vs.
mobile phase absorbance mobile phase
Contamination • Use degassed HPLC-grade solvents
• Flush system
• Clean up the sample
Detector optics • Perform intensity test
• Check signal with flow cell removed if
possible
• Replace lamp
Pressure instability • Check ‘pressure fluctuation'

42 5/27/2022 From Instrument to Column: Tracking Down the Problem DE27255094

 UV Lamp Tests
How do I know if my UV lamp is good?
• Visual inspection of an equilibrated baseline
• Accumulated UV lamp on-time from RFI tag or Lab Advisor
• Lab Advisor intensity test
• Lab Advisor ASTM drift and noise test
• Lab Advisor cell test

43 5/27/2022 From Instrument to Column: Tracking Down the Problem DE27255094

 UV Lamp Tests

Diode array and

multiple wavelength

Counters and hours

The useable lifetime of a deuterium lamp will depend on it’s use:

• How many hours has it been on?
• How many times has it been ignited?
• What wavelength is being used?

44 5/27/2022 From Instrument to Column: Tracking Down the Problem DE27255094

 UV Lamp Tests

Diode array and

multiple wavelength

Intensity test

45 5/27/2022 From Instrument to Column: Tracking Down the Problem DE27255094

 UV Lamp Tests

Diode array and

multiple wavelength

Intensity test

The profile of the intensity scan changes as a lamp ages

46 5/27/2022 From Instrument to Column: Tracking Down the Problem DE27255094

 UV Lamp Tests

Diode array and

multiple wavelength

ASTM drift and noise

47 5/27/2022 From Instrument to Column: Tracking Down the Problem DE27255094

 UV Lamp Tests

Diode array and

multiple wavelength

ASTM drift and noise

Run on a monthly basis, this test

can help track the natural
decline of the lamp and perhaps
raise awareness of a dirty cell.

48 5/27/2022 From Instrument to Column: Tracking Down the Problem DE27255094

 UV Lamp Tests

Diode array and

multiple wavelength

Cell test

Diode array detectors with the fiber optic style flow cell require a Max Light test cell for this
test (part number G4212-60011).

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 UV Lamp Tests

Diode array and

multiple wavelength

Cell test

Example of scans with and without the cell installed.

50 5/27/2022 From Instrument to Column: Tracking Down the Problem DE27255094

 How Do I Know if My UV Lamp is Good?

Diode array and

multiple wavelength
195 nm

Baseline inspection

350 nm

Various factors contribute to the specific amplitude and pattern of baseline noise and drift,
including the specific wavelength, mobile phase, room temperature, and data rate.

51 5/27/2022 From Instrument to Column: Tracking Down the Problem DE27255094

 How Do I Know if My UV Lamp Is Good?
Long cycle wave
Diode array and This is a rhythmic change in the baseline where the periodicity may be
multiple wavelength hours.
• Environmental influences

Baseline inspection Short cycle wave

This is a rhythmic change in the baseline where the periodicity may be
seconds or minutes.
• Solvent mixing noise
• Mechanical issue in pump

If the cycle of the wave does not appear to be mixing noise, evaluate the health of the lamps through Lab
Advisor intensity, noise and drift tests. Also, the cleanliness of the flow cell can be evaluated through the
cell test.
Excessive drift
In a UV baseline, light scattering shows up as drift. If the baseline is drifting more than expected, empty
and rinse the solvent bottles, refilling with fresh solvent. Perform a cell test to check the cleanliness of the
flow cell.
52 5/27/2022 From Instrument to Column: Tracking Down the Problem DE27255094
 Useful Parts
Parts that address potential issues and help to ease your daily tasks
Part Description Information Part number

InfinityLab Stay Safe caps Prevents solvent evaporation; changes in mobile phase Various
concentration www.agilent.com/chem/staysafecaps
InfinityLab Quick Connect and Quick With spring-loaded function for optimized dead volume Various
Turn fittings reduction www.agilent.com/chem/infinitylabfittings
Blank nut, long, 10-32 Blank nut, PEEK with steel core; for system diagnostic 5043-0277
tests; finger tight up to 1300 bar, easy to use and gentle on
receiving port
Agilent Captiva syringe filters Solve issues like inlet clogging, increased backpressure, Various www.agilent.com/chem/filtration
and retention time shift by filtering your samples

InfinityLab Poroshell 120 columns High efficiency and high resolution; available in 18 Various
chemistries www.aglient.com/chem/discoverporoshell

InfinityLab Poroshell 120 columns InfinityLab Stay Safe InfinityLab Quick Connect fitting InfinityLab Quick Turn fitting Blank nut, 5043-0277
cap on solvent bottle

53 5/27/2022 From Instrument to Column: Tracking Down the Problem DE27255094

 LC Troubleshooting Poster Available

Request yours today at

www.agilent.com/chem/troubleshootLC

54 5/27/2022 From Instrument to Column: Tracking Down the Problem DE27255094

May 27, 2022
Resources for Support

• New! HPLC Advisor App: HPLC Advisor app | Agilent

• LC Troubleshooting poster: 5994-0709EN
• Resource page: http://www.agilent.com/chem/agilentresources
– Quick reference guides
– Catalogs, column user guides
– Online selection tools, how-to videos
• InfinityLab Supplies catalog: 5991-8031EN
• LC handbook: 5990-7595EN
• YouTube – Agilent channel (maintenance videos)
• Agilent service contracts

55 5/27/2022 From Instrument to Column: Tracking Down the Problem DE27255094

 Contact Agilent Chemistries and Supplies Technical Support

1-800-227-9770 option 3, option 3:

Option 1 for GC and GC/MS columns and supplies
Option 2 for LC and LC/MS columns and supplies
Option 3 for sample preparation, filtration and QuEChERS
Option 4 for spectroscopy supplies
Option 5 for chemical standards
Available in the U.S. and Canada 8-5 all time zones

gc-column-support@agilent.com
lc-column-support@agilent.com
spp-support@agilent.com
spectro-supplies-support@agilent.com
chem-standards-support@agilent.com

56 5/27/2022 From Instrument to Column: Tracking Down the Problem DE27255094