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

HPLC Column Care

• 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

Receipt of New Column

• 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

min0 0.25 0.5 0.75 1 1.25 1.5 1.75 2 2.25

mAU

0

50

100

150

200

250

300

350

DAD1 B, Sig=254,8 Ref=off (F:\PROJECTS\0217_M~1\IN-PRO~1\INSTRU~1\CONFIG07.D)

0.2

80

0.4

67

0.6

97

0.9

72

1.2

75

min0 0.25 0.5 0.75 1 1.25 1.5 1.75 2 2.25

mAU

0

50

100

150

200

250

300

350

DAD1 B, Sig=254,4 Ref=off (F:\PROJECTS\0217_M~1\INSTRU~1\CHROMA~1\HPLCSY~1\TESTIN62.D)

0.2

04

0.3

99

0.6

50

0.9

58

1.3

08

Not Optimized Optimized

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

Tubing and Fittings

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”)

Connecting Tubing

• 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

Female/Inverted (internal) Column

Endfitting

10-32 Threaded Male Nut and

Ferrule

Polymeric

FingertightMale Nut

Fittings

Different Manufacturer’s 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

Connecting Fittings

• 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

Mobile Phase

• Strongly recommend preparing aqueous buffers fresh

DAILY to minimize microbial growth, which can foul in-

line filters and columns

Microbial growth on

packing material at

inlet of column

• 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 <3500 psi

(conventional HPLC columns and systems)

• UHPLC columns have higher pressure limits (≥9000 psi)

• Flow rate must be consistent with backpressure

limitations for columns

• Avoid sudden pressure changes

• In event of high backpressure, reverse flush column

(check with column manufacturer)

Other Considerations

• 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

Column Protection

• 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 as Source of Contaminants

• 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.

Sample Preparation

Inlet frit – New column Inlet frit - Inadequate sample prep

Contaminants20000 x magnification 20000 x magnification

• 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

Guard Columns

• 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

• 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)

Security Guard Ultra

Increasing Column Lifetime

Column Care

Would You Rather?

Column Care

• 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 Cleaning

• 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% NaN3 in Water or 10% Methanol

Column Storage

• 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

Column Care Summary

• 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, 2010UHPLC Tips and Techniques

By John W. Dolan

“Chromatographic Hygiene”

Thank You!!