Agilent LC User Maintenance and Troubleshooting

Agilent Confidential
25 October 2018
1
Agilent 1260 Infinity II LC

Maintenance
Confidentiality Label
October 25, 2018
3
Confidentiality Label
October 25, 2018
4
Confidentiality Label
October 25, 2018
5
Confidentiality Label
October 25, 2018
6
Confidentiality Label
October 25, 2018
7
Confidentiality Label
October 25, 2018
8
Confidentiality Label
October 25, 2018
9
Confidentiality Label
October 25, 2018
10
Confidentiality Label
October 25, 2018
11
Confidentiality Label
October 25, 2018
12
Confidentiality Label
October 25, 2018
13
Pump Maintenance

Confidentiality Label
October 25, 2018
14
Confidentiality Label
October 25, 2018
15
Confidentiality Label
October 25, 2018
16
Confidentiality Label
October 25, 2018
17
Confidentiality Label
October 25, 2018
18
Auto sampler inject sequence

Confidentiality Label
October 25, 2018
19
Confidentiality Label
October 25, 2018
20
Sampler Maintenance

Confidentiality Label
October 25, 2018
21
Sampler Maintenance

Confidentiality Label
October 25, 2018
22
Confidentiality Label
October 25, 2018
23
Confidentiality Label
October 25, 2018
24
HPLC connectors from different manufacturers.

Confidentiality Label
October 25, 2018
25
Confidentiality Label
October 25, 2018
26
Detector Maintenance

Confidentiality Label
October 25, 2018
27
Confidentiality Label
October 25, 2018
28
Confidentiality Label
October 25, 2018
29
Confidentiality Label
October 25, 2018
30
Cleaning a reversed phase silica column
Sometimes you can alleviate a high pressure or peak tailing issue by cleaning your column. Before cleaning your
column, we recommend that you disconnect it from your detector and run your wash solvents into a beaker or other
container. In some cases, it is advisable to backflush the column while cleaning it. This keeps the column
contamination from flowing through the column. Check with your column manufacturer, or review the instructions that
came with your column before doing this.
Cleaning the column requires solvents that are stronger than your mobile phase. You should use at least ten column
volumes (see figure 29) of each solvent for cleaning analytical columns. See the following procedure for cleaning
reversed phase columns. When finished cleaning your column, return your column to the flow direction recommended
by the manufacturer.

Confidentiality Label
October 25, 2018
31
Agilent 1260 Infinity II LC

Troubleshooting
LC possible troubles

Confidentiality Label
October 25, 2018
33
Troubleshooting Steps

• Did System Suitability or Sample Fail?

• Review Method for Compliance

- Is The Procedure Being Followed Properly?
- Are Instrument Settings Correct?

• Ask More Questions!

- When Did the System Last Function Properly?
- Has Anything Been Changed?

• Review ALL parameters!

- The Obvious Is Not Always the Cause
- Was There More Than One Change?

Confidentiality Label
October 25, 2018
34
System Check
• Solvent filters and Bottle heads installed?

• All tubing as per Agilent recommendation?

• Seal wash properly setup (10% IPA) and running?

• Signs for leakage or buffer deposited (pump heads or needle/seat)?

• Sample loop properly guided and not blocking arm movement?

• No vials stuck in sampler blocking the movement?

• Samplers waste tube guided correctly?

• Proper waste tubing for VWD & DAD?

Confidentiality Label
October 25, 2018
35
System Check
Often a simple operator error is the root cause of a problem: gather information!
• Has something been changed on the LC system?
• Is the correct column installed?
• Are the correct solvents being used (including modifier)?
• Is the flow rate set properly?
• Is it the right sample in the vial, dissolved in the correct solvent?
• Has the method been changed?
• ...
Visual inspection for
• Correct connections
• Air bubbles
• Leaks
•…
Check pressure: increased pressure points to a restriction of flow in the system, most
commonly a blocked frit
8

Confidentiality Label
October 25, 2018
36
Troubleshooting: Leaks

Confidentiality Label
October 25, 2018
37
Troubleshooting: Leaks

Confidentiality Label
October 25, 2018
38
Troubleshooting: Frits Blocking

• Absence/dirty solvent filters. Never use system without solvent filters. This
will clog degasser and will create issue in the gradient valve.

• Dirty solvents Filter solvent before usage.

• Degasser Do not leave water in degasser, this will result in algae growth and
contamination will result in frit blocking.

• Never use old\regenerated frits. Always use a new frit. Otherwise particles
will block tubing after the pump.

• Frits getting black Defective pump seals.

• Frits getting yellow Algae growth.

Confidentiality Label
October 25, 2018
39
Troubleshooting: Fluctuating Pressure

Confidentiality Label
October 25, 2018
40
Troubleshooting: Degasser

Pressure Fluctuations
• Solvent Inlet Filters – Block or contaminated
• Change degasser channels/ check for solvent flow
• Bypass degasser

Confidentiality Label
October 25, 2018
41
 Troubleshooting: Pump
Pressure Fluctuations:
• Use pressure plot
• Use pump leak test (system flushed?!)
• Check purge valve frits
• Binary pump: check channel by channel
• Pump maintenance – All parts should be clean and leak free
• Leak
• Symptomes:
• Ripple > 10% → AIV cartridge
• Ripple 3% to 4% → Outlet ball valve/pump seal

Gradient Problem:
• Pump – Mixing issue (Gradient valve)
• Filters – Blocked or contaminated
• Pressure variation
• Column stabilization
• Change of solvent brand

Confidentiality Label
October 25, 2018
42
 Troubleshooting: Autosampler
Pressure Fluctuations
• Switch between main pass and bypass. Pressure difference
should be 3 to 5 bar.
• Use needle-up in main pass (needle vs. needle seat)
• Check waste volume corresponding to injection volume
• Use Agilent materials (vials, plates, mats)
• Perform a tray alignment/autoreferencing
Carry Over
• Washing vial Should be without septa
• Washing solvent Choose correct solvent
• Needle seat Should be clean, leak free and without
scratches
• Injection valve parts Rotor seal age, number of
injections
Area variation
• ALS maintenance Inj. Vol. variation/carry over
• Waste line location
• Sample degration, check cooling performance
• Injection volume Check injection volume by calibration
Confidentiality Label
October 25, 2018
43
Troubleshooting: Detector

• Leak from flow cell Look for visual leaks

• Temperature variation Ambient temperature should be stable
• Lamp warm-up time Need at least 30 min for stable
• Lamp intensity Intensity Test Performed every week or 15 days
• Contaminated or dirty cell Cell Test
• Remove cell or use Test cell to exclude external influence

Confidentiality Label
October 25, 2018
44
Overview poor retention reproducibility

Confidentiality Label
October 25, 2018
45
Troubleshooting: Decreasing Retention Time

Confidentiality Label
October 25, 2018
46
Troubleshooting: Increasing Retention Time

Confidentiality Label
October 25, 2018
47
Troubleshooting: Fluctuating Retention Time (1)

Confidentiality Label
October 25, 2018
48
Troubleshooting: Fluctuating Retention Time (2)

Confidentiality Label
October 25, 2018
49
Troubleshooting: Temperature and pH

• Change in column temperature can change retention time and peak spacing
• Causes for fluctuating temperature:
• Temperature in lab circulates in course of the day (without column oven)
• Increased solvent temperature due to friction through clogged frit
• Peltier elements, sensors, heaters, board… fail (with column oven)
• Change in temperature can lead to change in pH of buffer
• Small changes in pH lead to huge effects on tr

Confidentiality Label
October 25, 2018
50
 Peak Shape: Peak Splitting\Tailing\Fronting

• Additional void volume between injection point to column

• Sample preparation is wrong, diluent strength
• Bad column

All Peaks Broadened:

• Loss of Column Efficiency
• Column Void
• Large Injection Volume

Some Peaks Broadened:

• Late Elution from Previous Sample (Ghost Peak)
• High Molecular Weight or Polymer Sample

Confidentiality Label
October 25, 2018
51
Confidentiality Label
October 25, 2018
52
Agilent Lab Advisor

Confidentiality Label
October 25, 2018
53
Confidentiality Label
October 25, 2018
54