HPLC User Maintenance

& Troubleshooting

Lori Sandford
Applications Chemist
Agilent Technologies
Schaumburg, IL
• Delay volume • Stainless steel or Titanium
• Fittings • Glass versus Stainless Frits
• Column volume
Hardware • Mobile phase pH

Physics Chromatography Chemistry

• Data Sampling  Sample Prep

• Type of Mixing Application  Mobile phase UV cutoff
• Column Pressure  Sample UV Maximun
What are Chromatographers Looking for?
Best performance
•Resolution
•Highest throughput
•Reproducibility
•Accuracy of results
•Sensitivity
•Standard, narrow bore and capillary column
capability
Resolution
N = Number of theoretical. plates Column length, particle size
α = Selectivity Stationary and mobile phase, temperature
k = Retention Stationary and mobile phase
Know your HPLC System:

Detector
Column Comp.

Autosampler

Pump
Vacuum Degasser (integrated in Infinity II and Quat systems)

Page 6
Know Your HPLC Flow Path:

Where are the moving parts?

Where can blockages to flow occur?
Where are the consumables that
need to be replaced on a regular
basis (PM)?
Where can leaks occur ?

What can I do to eliminate, reduce or anticipate

potential problems with the LC ?

Page 7
1290 Infinity Binary Pump
– the flow path

Jet weaver
Flow path in binary pump Both tested flow paths
end inside the purge
valve rotor seal
pressure
sensor
x same for
channel B...

filter assembly purge

valve

waste
heat exch.
Channel A

Secondary pump head starts at OV

Primary pump head starts at PIV

inlet, to SSV/degasser/bottle
Flow path in Jet
Weaver

quaternary pump
(opt.)

multi-
purpose
valve
pressure x Both tested flow paths
end inside the m.p.
sensor
inline valve rotor seal
filter

Secondary pump head starts at OV

to
degasser
inlet weaver Primary pump head starts at PIV
MCGV
Agilent 1200 Series Pump Models - Analytical

Isocratic Pump Quaternary Pump Std Binary Pump

Page 11
Pump Head – Main Components
Outlet ball valve

Purge valve

Plunger housing

Active inlet valve

Pump head

Page 12
Pump and Degasser Maintenance

• Clean the degasser lines by flushing with isopropanol.

• When using buffers, flush with water, then with isopropanol.
• Check for air bubbles in outlet lines.
• Be aware of the possibility of microbial growth in aqueous
phases.
• Check for solvent compatibility.
• Unused channels should be left in isopropanol.
• May have need to exchange the vacuum pump, sensor,
solenoid valve, or vacuum chamber.

Page 13
Overall Routine Pump Maintenance
1. Remove and disassemble the 8. Replace PTFE frit in the purge
pump head. valve.
2. Remove and clean pistons. 9. Clean or replace the outlet ball
valve.
3. Replace piston seals.
10. Replace the AIV cartridge.
4. If seal wash option is installed, 11. Flush with isopropanol.
replace wash seals and gaskets.
12. Clean or replace solvent inlet
filters.
5. Inspect the springs.
13. Clean the leak sensor.
6. Reassemble pump head and
14. Make certain the waste tube is
reinstall.
in place.
7. Perform seal wear-in procedure. 15. Test the pump (Pressure and
Leak Test).
- Covered by an annual Agilent LC PM contract
Page 14
When to use purge, prime, condition ?
Purge
Change solvents
When pump is refilled with new/different mobile phase the purge valves allows
both pump heads (binary pump) to be connected to waste at the same time

Prime
When the pump is dry
When Purge and Condition still show exhausted pressure ripple

Condition
When first starting up for the day or after changing solvents
When pump pressure ripple or composition ripple is too high (mixing noise) air
bubble is hidden in pump head (listen)
best once a day to condition for smooth operation

October 15, 2015

Confidentiality Label

15
 Filters and Bottle necks for blockages
• Solvent inlet filters in solvent bottles  Flow path before pressure sensor
glass: 20um – replace if needed! is uncontrolled !
SST: 12-14um – replace, opt sonicate
• Inlet weaver (mixer) between MCGV &
prim head (quat only)
• Heat exchanger (bent? Connection?)

• High pressure filter assemblies at outlet

of secondary pump heads: 5um – replace
(see cleaning procedure)
• Quaternary pump: inline filter at multiple
purpose valve MPV: 0.3um – replace
• Jet weaver (optional with 35um, 100ul,
380ul)
Troubleshoot: Disconnect other modules
behind pump

October 15, 2015

Confidentiality Label

18
Examples: used / unused Filters
Glass filters: 3150 - 0944 Stainless Steel Filters: 01018 – 60025
(less volume, no Na+ ions)

October 15, 2015

Confidentiality Label

19
Example: Jet Weaver & Inlet Weaver

• Multi-layer technology
Diffusion bonded stainless steel, etched structures

• Two mixers in one cartridge

Standard mixer with 35 µl volume & 100ul
adds delay volume of 45ul and 75ul

•Optional mixer in quat pump with V380

which adds 150ul delay volume

•Mixer for TFA applications with

adds 380 µl volume delay volume

April 6th, 2009

1290 Infinity Liquid Chromatograph
Agilent Restricted
Page 20
Considerations for HPLC systems
Gradient Delay or Dwell Volume
The volume between the point of mixing of
solvents (usually in the mixing chamber or at
the proportioning valves in the liquid
chromatograph) and the head of an LC
column.

Beginning of Mixing

End of Mixing Extracolumn Volume

The volume between the effective injection
point and the effective detection point,
excluding the part of the column containing
the stationary phase. It comprises the
volumes of the injector, connecting lines and
frits, and the detector. It determines the
extracolumn effects.
System – Signal height
System dispersion
• “Dispersion is the sample bandspreading or dilution which
occurs in connecting tubing, sample valves, flow cells and in
column end-fittings.”

Peak height: Loss of sensitivity

Dispersion
Peak width: Loss of resolution

• Capillaries (Inner diameter, length)

 r F L
4
  2

24  Dm Aris-Taylor Equation

Page 22
System – Signal Height

Resolution 0.961
mAU
System dispersion not optimized
400
310 mAU Peak width 0.037 min
300

200

Peak width 0.038 min

100

0
0.5 1 1.5 2 2.5 min

mAU
Resolution 1.902
600
System dispersion optimized
550
380 mAU Peak width 0.019 min
400

350

200
Peak width 0.018 min
100

0
0.5 1 1.5 2 2.5 min
Influence post-column capillary
connections
mAU
140 130 mAU One bad capillary connection!
120
100
80
60
40
20
0
0 0.1 0.2 0.3 0.4 min

mAU

210 160 mAU Fixed!

180
150
120
90
60
30
0

0 0.1 0.2 0.3 0.4 min

Page 26
System – Signal height
System volumes – Delay volume
mAU
400

Delay volume ~ 700 μL

300

200
120 mAU
100

0
0.5 1 1.5 2 2.5 3 3.5 4 4.5 min

mAU Column: ZORBAX SB-C18 2.1 x 50 mm, 1.8 μm

Flow: 0.42 mL/min
400

300 Delay volume ~ 120 μL

220 mAU
200

100

0
0.5 1 1.5 2 2.5 3 3.5 4 4.5 min
Delay volume
Impact of low delay volume

Programmed Low delay High delay volume

gradient step volume
90 %
response

What happens with a

programmed gradient?
Delay volume

10 %
time
High delay vol. Total delay volume of the system (sum
of capillaries, mixer, cells, valves..)
Low delay vol.

Page 28
Effects of Delay Injection Program
Delay volume
Impact of low delay volume
Column: ZORBAX SB-C18 2.1 x 50 mm, 1.8 μm
Flow: 0.42 mL/min
Delay volume ~ 700 μL
mAU
400

300

200

100

0
0.5 1 1.5 2 2.5 3 3.5 4 4.5 min

mAU
Change in
400
selectivity Decrease in
retention time
300

200

100

0
Delay volume ~ 120 μL
0.5 1 1.5 2 2.5 3 3.5 4 4.5 min

Page 30
Performance Characteristics of an HPLC System

Influenced by one module... Influenced by several modules...

Flow: accuracy, precision Pump Repeatability of retention times

Composition: accuracy, precision Delay volume

Injection volume precision Injector Repeatability of peak areas

Linearity, dynamic range Dead volume
Carry over

Column temperature accuracy Column

compartment Peak elution order
Column temperature precision
Wavelength: accuracy, precision Detector Baseline: noise, drift and wander
Signal linearity
Spectral resolution (DAD only)
Schematic of Injection System

Sample loop

Metering
device

Injection valve

Page 32
Principle of Operation

Pump Pump Pump

Column Column
Column

Prior to Injection Draw Sample Injection and Run

Valve in Mainpass Position Valve in Bypass Position Valve in Mainpass Position

Page 33
Exchanging the Needle/Needle Seat – Standard
Autosampler G1329A.

Needle/Needle seat

Parts:
Tools:
Needle G1313-87201
Wrench ¼ inch
Needle seat G1313-87101 (0.17 mm i.d.)
Hexagonal key 2.5 mm
or G1313-87103 (0.12 mm i.d.)

Page 34
Autosampler Maintenance Functions
Before beginning needle or needle seat replacement:
Select "Change Needle” in the autosampler maintenance function.
Instant Pilot: ChemStation:

Page 35
Thermostatted Column Compartment

 Important
 Influence on...
performance
characteristics

 Elution order
 Excellent temperature  Peak identification
accuracy
 Elution order
 Excellent temperature  Retention time precision
precision  Peak identification
Effect of Temperature on Separation
Salicylic acid 20°C

0 1 2 3 4 min

Salicylic acid 30°C

0 1 2

Salicylic acid 40°C

Phenacetin

0 1 2

Salicylic acid
60°C

0 1 2
Salicylic acid Column: RRHT SB-C18
4.6 x 50mm, 1.8um 90°C

5
4
3
0 1 2
Conditions: A: 0.1% formic acid B: ACN w/ 0.1% formic acid (85:15) Detection: UV 254 nm
 Column Oven

Constant temperature for solvent and column is required to perform

reproducible results.
 HPLC UV/Vis Detectors
 Important performance  Influence on...
characteristics

 Variable Wavelength and  Variable Wavelength and Diode

Diode Array Detector Array Detector

 Low noise, wander and  Detection limit, quantitation limit

drift  Confidence in quantitation at high
and low concentrations
 Wide linear range  Accuracy of peak areas/heights
 Precisons of peak areas/heights
 Very good wavel. accuracy
 Excellent wavel. precision  Diode Array Detector only

 Diode Array Detector only  Accuracy of spectra, peak

identification by spectra
 High spectral resolution
 Accuracy of spectra, peak
identification by spectra at low
 Excellent spectral sensitivity concentrations
Influence of Pathlength on Signal Sensitivity

mAU
80

70
10mm path length
60

50

40

30

20
6mm path length
10

3.8 4 4.2 4.4 4.6 min

Page 40
 Determining Peak Apex

1.0 1.8 2.6

Retention Time (min)
Fit highest data point and those on each side
to a quadratic equation, solve for highest point
 Determining Peak Apex

1.0 1.8 2.6

Retention Time (min)
Sensitivity
Data rate – Peak height

mAU
95 mAU
100 Data rate 80 Hz

Toluamide
80

Toluene
60

40

20

0
Data rate 10 Hz
0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 min

mAU
62 mAU
60

50
Peak height increases with 40
increasing data rate! 30

20

10

0
0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 min

Page 43
Sample Signal and Reference Optimization

signal
wavelength

reference
bandwidth
mAU

100 nm

30 nm reference
bandwidth wavelength
350 nm

Wavelength (nm)
 Total Signal with Diode Array Detection

reference
signal
wavelength bandwidth
100 nm

reference
wavelength
350 nm

Absorbance Sample wavelength + Absorbance averaged over Bandwidth Absorbance ref wavelength + Absorbance averaged over Bandwidth
= Total
# of wavelengths used # of wavelengths used Absorbance
 Isoabsorbance plot without reference

Isoabsorbance plot with reference

 Optimizing Slit

1 nm
8 nm

2 nm

16 nm

4 nm
Tip #8 - Routine Maintenance Procedure – DAD/MWD Uv Detectors

• Clean the leak sensors

• Check the waste tube
• Exchange the lamp (if necessary)
• Clean the flow cell (if necessary)
• Wavelength calibration
• Holmium Oxide Test
• Intensity test Available
• Cell test Diagnostic Tests
• Dark current test
• Filter test
- Covered by an annual Agilent LC PM contract -

Page 50
Replacing the DAD/MWD Lamps

Deuterium Lamp (1000 hours): 2140-0590

Deuterium Lamp (2000 hours): 2140-0820*
Step 1: Remove the lamp by unscrewing *includes RFID tag read by SL modules
both screws and unplugging it.
Step 2: Install the lamp into the housing
(auto-aligning) and tighten screws.
Step 3: Check wavelength calibration
Step 4: Check lamp intensity (for future
reference)
Tungsten lamp G1103-60001

Page 51
Tip # 9: Care of Detector Flow Cells
Avoid the use of alkaline solutions with pH > 9.5 which can attack
quartz and impair optical performance.

Prevent crystallization of buffers or salts which will lead to blockage

and damage.

Aqueous solvents can allow algae growth. Don’t leave 100% water
standing in the flow cell. When leaving LC idle, pump a mobile phase
with at least 5-10% of organic solvent.

Observe the pressure limits of flow cells. Be careful when using

detectors in series or fraction collectors.

Page 52
Dynamically Mixed vs. Premixed Mobile
Phases (prepared by one user)
6 dynamically mixed mobile phases
Norm.
30

25

20

15

10

1 2 3 4 min

6 premixed mobile phases

Norm.

40

35

30

25

20

15

10

1 2 3 4 min

ASTS March 14 2013

Non-ideal mixtures
Acetonitrile/Water 40 °C
Maximum viscosity of
0.8
acetonitrile/water
0.6
mixtures at approx. 10 %
Viscosity

0.4
acetonitrile.
0.2

0.0
0 20 40 60 80 100
% Acetonitrile

Methanol/Water 40 °C

1.2
Viscosity
0.8
Maximum viscosity of
methanol/water mixtures 0.4

at approx. 50 % 0.0
0 20 40 60 80 100
methanol. % Methanol

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