HPLC Column Care

HPLC Column Care
HPLC Column Care
HPLC Column Care Outline:
• Receipt of New Column
• Tubing and Fittings
• Mobile Phase Considerations
• Column Protection
• Guard Columns
• Column Cleaning
• Column Storage

Receipt of New Column
• Verify column identity
– Is it what you ordered?
– Are the dimensions correct?
– Correct phase?
• Inspect for physical damage
• Test column with standard test sample under standard
conditions to verify performance
• Every Phenomenex column is individually tested
Tubing and Fittings
• Chromatographic performance is dependent on entire
system, not just column
• Tubing and fittings contribute to system dead volume
• System dead volume results in:
– Band broadening
– Peak degradation
– Reduced efficiencies
– Reduced sensitivity

Tubing and Fittings
HPLC System Optimized for Maximum Performance
• Change in connecting tubing id
– 0.17 mm id to 0.12 mm id
• Decreased flow cell volume
– Standard cell – 13 µL, 10 mm path
– Semi-Micro cell – 5 µL, 6 mm path
Conditions
• Luna® 3µm C18(2), 10 x 2.0 mm
– (MercuryMS cartridge)
• 50:50 Acetonitrile/Water at 0.2 mL/min
• HP1100 w/binary pump
Tubing and Fittings
Before Optimization
• Peak width
– Peak 3: 0.072 min
– Peak 4: 0.085 min
– Peak 5: 0.103 min
• Resolution 3/4 = 2.06
• Resolution 4/5 = 1.89
After Optimization
• Peak width
– Peak 3: 0.057 min
– Peak 4: 0.076 min
– Peak 5: 0.099 min
• Resolution 3/4 = 2.73
• Resolution 4/5 = 2.35
Not Optimized
DAD1 B, Sig=254,8 Ref=off (F:\PROJECTS\0217_M~1\IN-PRO~1\INSTRU~1\CONFIG07.D)
mAU
350
Optimized
DAD1 B, Sig=254,4 Ref=off (F:\PROJECTS\0217_M~1\INSTRU~1\CHROMA~1\HPLCSY~1\TESTIN62.D)
mAU
350
300
0.467
300
250
200
150
0.697
0.972
1.275
250
200
150
0.399
0.650
0.958
1.308
100
100
50
0.280
50
0.204
0
0
0 0.25 0.5 0.75 1 1.25 1.5 1.75 2 2.25
min
0 0.25 0.5 0.75 1 1.25 1.5 1.75 2 2.25
min

Connecting Tubing
1. Pump to Injector
• Larger ID (0.010” = 0.254 mm) is OK to use (isocratic)
2. Injector to Column
• Minimize tubing ID
• Typically use 0.007” (0.178 mm) or 0.005” (0.127 mm) ID
3. Column to Detector
• Minimize tubing ID
• Larger ID negates separation achieved
• Typically use 0.007” or 0.005” ID
4. Detector to Waste
• OK to use larger ID (≥0.010”)
Fittings
• Not all column end fittings are equivalent
• Different types/shapes of ferrules used
• Different seating depths (0.080” to 0.130”)
• Seating depth is critical
– Tubing MUST be seated flush with column
– Improperly seated fitting results in increased dead
volume or leaking fittings
• Fingertight fittings (Polymeric)
– Easy to use
– One piece
– Conform to shape of column endfitting

Fittings
Polymeric
Fingertight
Male Nut
10-32 Threaded
Male Nut and
Ferrule
Female/Inverted
(internal) Column
Endfitting
Different Manufacturer’s Fittings

Connecting Fittings
• If length of tubing
beyond ferrule is too
long, then ferrule will not
seat properly and leaks
will occur
• If length of tubing beyond
ferrule is too short, then a
void will be present that
will result in band
broadening and tailing
Mobile Phase
• Flush HPLC pump and lines thoroughly to remove air
bubbles (open purge valve to speed process)
• Connect column to injector
– Check flow direction as indicated on column label
• Pump mobile phase at 0.1 mL/min, then increase to
normal flow rate
• Stop flow and connect column to detector inlet
• Resume flow and equilibrate column with mobile phase
for 10-30 column volumes
– Ion-pair containing mobile phase may require longer
to equilibrate

Mobile Phase
• Use HPLC-grade solvents
• Use highest purity chemicals and reagents
• Degas and filter all mobile phases prior to use
• Make certain that solvents are miscible
• Verify solubility of buffer salts in mixed aqueous/organic
mobile phases (especially with gradients run from high
aqueous to high organic)
• Check sample solubility in mobile phase
Mobile Phase
• Strongly recommend preparing aqueous buffers fresh
DAILY to minimize microbial growth, which can foul inline
filters and columns
Microbial growth on
packing material at
inlet of column

Other Considerations
• Maintain pH between 2.0 and 8.0 (older-type silica and
polar endcapped phases) or between 1.5 and 10.0
(newer-type silica)
• Low pH will strip (hydrolyze) the bonded phase from the
silica
• High pH will dissolve the silica
• If operating near pH extremes, use of a pre-saturator
column will minimize these effects
• Newer media available offers wider pH (1 – 12) stability
Other Considerations
• Backpressures must typically be kept

Column Protection
• Why Bother?
– Maximize lifetime of column
– Reduce system wear
– Save time and money in long run
– Achieve optimum results
• How?
– Mobile phase inlet filter
– In-line filter
– Guard column
– Degas mobile phase
– Filter all mobile phase components
– Filter all samples
Sample as Source of Contaminants
• Sample matrix is a primary source of contaminants on
HPLC/UHPLC columns resulting in:
• Increased back pressure
• Loss in chromatographic performance
– Peak shape problems (splitting, tailing)
– Decreased efficiency
– Loss of resolution

Sample Preparation
• Remove potential contaminants before injecting samples
into HPLC/UHPLC columns
• Filter samples (0.45 or 0.2 µm syringe filters)
• Sample cleanup – e.g. SPE, Protein Precipitation, etc.
Inlet frit – New column
Inlet frit - Inadequate sample prep
20000 x magnification Contaminants
20000 x magnification
Guard Columns
• Advantages
– Protect valuable analytical columns by removing
particulates and strongly retained sample
components
– Increase lifetime of analytical column
– Maintain high column efficiencies
– Cost-effective
• Disadvantages
– Extra tubing and fittings add dead volume to system
– Broader peaks
– Retention time shifts

Security Guard
• Universal Guard Cartridge System
• Direct connection reduces dead volume
• 100% inert and biocompatible flow path
– PEEK
– Titanium
• Patented design
• Simple to use
• Inexpensive
• Universal phases
Security Guard

Security Guard
Security Guard Ultra
• Protects against damaging chemical contaminants and
microparticulates
• Will NOT alter chromatography
• Easy to Use
• Compatible with virtually all UHPLC, core-shell and sub-2 µm
columns 2.0 to 4.6 mm ID
• Pressure rated to 20,000 psi (1,378 bar)

Increasing Column Lifetime
Column Care
Would You Rather?

Column Care
Column Cleaning
• Consult with column manufacturer for recommended
cleaning procedures and limitations
• Flush column with mobile phase minus buffer to remove
all buffer salts
• Flush with 10 column volumes of mutually miscible
solvent such as methanol or acetonitrile
• Flush with 20 column volumes of strong solvent (THF or
IPA) to remove strongly adsorbed sample components
• Reverse the process and equilibrate with mobile phase

Column Storage
• Column storage conditions affect column lifetime
• NEVER store columns (long-term) with buffers
• Flush with 10 column volumes of mobile phase without
buffer to remove buffers and salts
• Typical solvent storage conditions for silica-based
columns
• Reversed phase: 65/35 Acetonitrile/Water
• Normal phase: isopropanol or hexane
• Ion-Exchange: methanol
• SEC or Diol: 0.05% NaN 3 in Water or 10% Methanol
Column Care Summary
• Protecting Your Expensive Analytical Columns
– Extends column lifetimes
– Ensures optimal column performance
– Ensures quality analytical results
– Minimizes downtime
– Saves money
• Protecting Your HPLC System
– Minimizes system downtime
– Ensures quality results
– Saves money

“Chromatographic Hygiene”
• GUARD COLUMNS
– Particulate matter from piston seals and injection valve rotors can clog
columns if they are not removed
• 0.2 µm SYRINGE FILTER
– With sub-2 μm columns, every sample must be filtered through a 0.2
μm porosity filter
• MOBILE PHASE FILTERS
– Fresh buffers must be made daily and filtration through a 0.2 μm
porosity filter is required
• INLINE FILTERS:
– I strongly recommend using a 0.2 μm porosity in-line filter between the
autosampler and guard column
LC/GC Magazine
November 1, 2010
UHPLC Tips and Techniques
By John W. Dolan
Thank You!!