From goods-in inspection to the decision to retire a column, covering general questions on HPLC and UPLC columns, end fittings, guard columns, fitting screws and tubing. With 20 cross-sections, exploded views and perspective drawings, focusing on fitting specifications, understanding 1/16″ and 1/8″ tubing, where PEEK and stainless steel each apply, and locating leaks.
Between delivery and retirement, a column passes through four routine stages — goods-in inspection, installation, routine operation and shutdown storage — plus two non-routine states: leaks and performance problems. Most problems in use come not from the medium itself but from the connections outside the column: a mismatched fitting standard, the wrong insertion depth, the wrong tubing bore, a reused ferrule. This page sets these out in sequence and gives cross-sections and exploded views for the fittings and tubing.
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How to read it:read down and the problems group by responsibility; read across and the focus differs at each stage of the same column's life. Confirmation at goods-in is cheap while investigation during operation is expensive — confirming the fitting standard and flow arrow on receipt avoids most later rework on connections.
Extra-column factors account for a large shareA considerable proportion of peak shape and retention problems come from the connecting tubing and fittings rather than from a failed medium. Confirm extra-column factors first.
A ferrule cannot be undoneOnce tightened, a stainless steel ferrule deforms and bites into the tubing; moved to a fitting of different insertion depth, it usually will not seal again.
The size names are easily confused1/16″ and 1/8″ refer to the outside diameter and say nothing about the bore. The bore determines extra-column volume; the outside diameter determines which fitting to use.
Each material has its limitsPEEK allows tool-free work but has limited pressure and solvent resistance; stainless steel takes high pressure but is sensitive to halides and strong acids.
1 · Column body and end fitting anatomy
An analytical HPLC column consists of the column tube, an end fitting at each end, frits, seals and the packed bed. Understanding how these parts relate is the basis for locating a leak or a source of dead volume.
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Key point:the two ends of the column tube are not symmetrical. During packing the material is driven in from the inlet, so the bed near the outlet is less dense. The arrow on the column marks the direction in which the bed was consolidated; flowing with the arrow keeps the bed under compression, while reverse flow applies force in the direction that loosens it.
1.1 Function and failure mode of each part
Part
Function
Common failure
Externally observable sign
Column tube
A pressure vessel that constrains the bed
Internal corrosion, halide pitting
Metal ion interference on the baseline, worse peak tailing
An inlet frit that is
Stops particulates and distributes the flow
Blockage, local fouling
Column pressure rises progressively, with efficiency changing later
Outlet frit
Retains the packing
Damage, packing passing through
Rising detector noise, blockage downstream
End fitting
Provides the sealing face and the flow path transition
Scratched sealing face, damaged threads
Weeping at the fitting; still leaks after tightening
The order of parts at the end fitting determines whether the connection seals and whether dead volume is left. Below is the exploded arrangement of a common compression fitting.
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Key point:A compression fitting seals through the ferrule and cone together, while the tube is located by being seated against the bottom of the fitting. If the tube is not pushed fully home before tightening, the ferrule bites at the wrong position, and no amount of retightening afterwards will close that gap.
Once tightened, a ferrule deforms plastically onto the tubing. Moving the same tubing with its ferrule to a fitting of different insertion depth normally means a new ferrule and a fresh cut, not reusing what is there.
2 · Differences between HPLC and UPLC columns
The separation mechanism is the same; the differences lie in particle size, pressure rating and sensitivity to extra-column volume. Fitting a UPLC column to an HPLC system often means its efficiency advantage never appears, because system extra-column broadening is too large.
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Key point:The volume of a UPLC column is of the order of a tenth that of a conventional HPLC column. If the bore and length of the connecting tubing are carried over from an HPLC configuration, extra-column broadening takes up a large share of the peak width budget and the measured efficiency falls short of what the column can do.
Dimension
HPLC column
UPLC / UHPLC column
Operational consequence
Particle size
3–5 µm
1.5–2.0 µm
Halving the particle size raises column pressure roughly fourfold
Usual bore
3.0 / 4.6 mm
1.0 / 2.1 mm
The bore sets the linear velocity and the upper limit on injection volume
Pressure rating
Generally within 40 MPa
Can exceed 100 MPa
Fittings and tubing must be rated to match
Inlet frit pore size
About 2 µm
About 0.2–0.5 µm
The finer the pore, the more sensitive to particulates
Connecting tubing bore
0.17 mm (0.007″) is usual
0.10 mm (0.004″) or finer
Carrying over wider tubing causes marked broadening
Sample preparation
0.45 µm membrane
0.22 µm membrane
Inadequate filtration accelerates frit blockage
The particle size and pressure relationship above follows the usual consequence of Darcy's law (column pressure varies inversely with the square of particle size); the actual figures vary with column length, flow rate and mobile phase viscosity.
3 · Guard column systems
A guard column takes on particulates and strongly adsorbed components in a low-cost replaceable unit, slowing degradation of the analytical column's inlet frit and the front of the bed. The three common forms trade dead volume against replacement cost.
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Which to choose:for a relatively clean matrix where efficiency comes first, choose A; for large daily sample numbers with frequent replacement, choose B; where the method is still being adjusted and needs flexible assembly, choose C. All three add extra-column volume, so under UPLC conditions A or a low dead volume version of B is preferable.
Replacement CriteriaUse the rise in column pressure as the main indicator. Replacing at about 20% above the initial value allows intervention before efficiency falls.
Match the phase chemistryThe guard column should be C18 or the same class of bonded phase as the analytical column, so no different retention behaviour is introduced ahead of it.
Not a substitute for sample preparationA guard column takes on residual particulates. Filtration and centrifugation must still be done before injection.
Record when it is changedReplacing a guard column shifts retention slightly; recording when it was done helps distinguish method drift from a hardware change.
Part Two · Fittings, Threads and Tubing
What 1/16″ and 1/8″ actually mean
What are commonly called "1/16 tubing" and "1/8 tubing" in an LC system refer to the tubing'soutside diameter, which determines the fitting and ferrule to use; theInternal Diameteris stated separately and determines the internal volume and extra-column broadening. The two are independent, and several bores are available at the same outside diameter. Confusing them is a fairly common source of error in ordering and connecting.
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Key point:The outside diameter determines whether it fits; the bore determines how well it runs. Both must be given when ordering — for example "1/16″ OD × 0.17 mm ID PEEK tubing". Saying only "1/16 tubing" is not enough to specify what is needed.
4.1 Orders of magnitude for internal volume
The volume of tubing before and after the column adds directly to the peak width budget. Below are the volumes per 10 cm of length at common bores, for estimating the volume cost of a connection scheme.
Internal Diameter
Imperial
Volume per 10 cm
Typical use
Notes
0.064 mm
0.0025″
About 0.3 µL
UHPLC pre-column
Blocks easily; needs 0.22 µm filtration
0.10 mm
0.004″
About 0.8 µL
UPLC pre-column
The usual configuration for 2.1 mm bore columns
0.13 mm
0.005″
About 1.3 µL
UPLC / narrow-bore HPLC
Balances pressure drop against broadening
0.17 mm
0.007″
About 2.3 µL
Conventional HPLC pre-column
The usual configuration for 4.6 mm bore columns
0.25 mm
0.010″
About 4.9 µL
Column outlet to detector
A low-pressure section; the broadening effect must still be assessed
0.50 mm
0.020″
About 19.6 µL
Waste, pump to injection valve
Not for the critical flow path before or after the column
1.6 mm
1/16″ bore
About 201 µL
Preparative LC, solvent delivery
Usually with 1/8″ outside diameter tubing
Volumes are calculated as V = π(d/2)²L from the nominal bore, without allowing for the internal volume of the fittings. Measured values vary with tubing tolerance.
How to estimate: keeping the combined volume of tubing before and after the column within a suitable proportion of the column volume reduces broadening. For a 2.1 × 50 mm column (column volume about 173 µL at a porosity of 0.65), 30 cm of 0.17 mm bore tubing before the column alone is about 6.8 µL; switching to 0.10 mm bore brings it down to about 2.4 µL.
5 · Thread standards and fitting geometry
Thread standards for LC fittings are not universal. Even for the same 1/16″ tubing, the column end, injection valve end and detector end may use different threads and different cone depths. With the wrong standard, either the thread will not engage, or it engages but the sealing faces do not meet and the joint weeps.
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Suggested practice:keep a fitting list for each instrument, recording the thread standard and cone depth at the injection valve outlet, before the column, after the column and at the detector inlet. Check the list before changing column brand, then decide whether an adapter is needed.
5.1 How a ferrule seals
As it is tightened, a ferrule is squeezed by the cone, contracts radially and bites into the tubing wall to form a sealing band. This deformation is plastic and does not spring back when the fitting is removed.
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Order of operations:push the tubing to the bottom of the fitting and hold axial pressure, then screw the nut in finger-tight, and finally tighten by the specified number of turns or torque. Reverse the order and the ferrule bites with the tube unseated, creating the cavity shown in the upper half of the figure.
Ferrule material
Reusable
Indicative pressure rating
Suited to
Caution
Stainless steel
No (the position is fixed)
High
High-pressure pre-column, UHPLC
Changing fitting normally means a new ferrule and a fresh cut
One-piece PEEK
A limited number of times
Medium (within about 34 MPa)
Conventional HPLC, biological samples
Sealing force falls after repeated assembly
Two-piece PEEK
Yes
Medium
Where frequent assembly is needed
The orientation must not be reversed
Finger-tight, no tools
Yes
Medium to low
Method development, temporary connections
Over-tightening crushes the thread
The indicative pressure ratings are the usual published ranges; follow the technical data for the product in use.
6 · Insertion depth differences and dead volume
The distance from the sealing cone to the bottom of the flow path — the insertion depth, or stub length — differs between brands. Moving a length of tubing with a ferrule already swaged onto it to a fitting of a different standard gives one of two outcomes: the tube does not reach the bottom, or it goes in too far.
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How to tell:disconnect and look at the tube end. If the length protruding beyond the ferrule does not match the fitting's stated insertion depth, it is condition B or C. Before connecting across brands, use a universal adjustable fitting, or cut the tubing afresh and swage a new ferrule to the target fitting.
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Order of investigation:when tailing appears, first replace the column with a zero dead volume union and inject, to see the peak shape of the system alone. If the peak is normal with the union but tails with the column fitted, the problem is at the column or its fittings; if it still tails with the union, the problem is in the system flow path.
7 · Choosing between PEEK and stainless steel tubing
The two materials have different limits in pressure, solvent resistance, biocompatibility and ease of use, and are usually selected by flow path section and sample type rather than standardized across the whole system.
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Common configuration:stainless steel or reinforced PEEK (PEEKsil, fused-silica lined) for the high-pressure section before the column; PEEK from the column outlet to the detector; for biological samples and phosphorylated compounds, PEEK throughout or tubing with an inert surface treatment; with halide-containing mobile phases, avoid leaving them standing in stainless steel tubing.
PEEK swells in tetrahydrofuran, dichloromethane and dimethyl sulfoxide, showing up as a changed bore and weeping at the fittings, and degrades in concentrated nitric or sulfuric acid. Methods using these solvents call for stainless steel or reinforced tubing.
7.1 Effect of the cut end
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Key point:End quality problems often do not show at low pressure, appearing as intermittent weeping only as pressure rises or during a gradient, where they are easily mistaken for a fitting or column problem. Checking the end before changing the fitting saves wasted investigation.
Part Three · Goods-in, Installation, Operation and Storage
8 · Goods-in inspection
Confirmation at goods-in is cheap, while the cost of an omission is magnified later. There is a set of items to confirm before unpacking a new column and another afterwards. Checking the fitting standard and flow arrow at this point avoids rework at installation.
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Key point:End plugs coming loose is a fairly common problem in transit. With a plug missing, the storage solvent evaporates and the bed dries locally, so a void can form when liquid is passed through again. If a plug is found missing on arrival, or there are obvious signs of liquid having leaked from the tube, contact the supplier before running the column.
Check
Acceptable condition
Abnormal sign
Remedy
Outer packaging
No crushing, no liquid marks
Packaging deformed, liner damp
Photograph it and contact the supplier
End plugs
Both in place and tight
Loose or missing
Inform the supplier before running; do not simply pass liquid through
Column tube appearance
No dents or scratches
Tube deformed
Do not run; go through the returns process
Label information
Matches the order
Phase, particle size or dimensions do not match
Check against the order and hold off using it
Fitting standard
Matches the existing system
Different thread or insertion depth
Prepare the matching fitting or an adapter
QC report
Lot number matches the column
Lot number does not match, or pages missing
Request the report for that lot
First re-measurement
N and As close to the reported values
Clearly different
Investigate extra-column factors in the system first, then judge the column
A discrepancy on first re-measurement should not be attributed to the column straight away. Test conditions, system extra-column volume, detector time constant and data acquisition rate all affect the measured plate number. Use a known good column on the same system as a reference.
9 · Installation and connection
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How to leak-test:after connecting, start the flow at 0.2 mL/min and wipe around the fitting with lint-free paper, looking for a damp mark. Raise gradually to the working flow rate, repeating the check as the pressure rises. No leak at low flow but a leak at high flow usually points to the end quality or an under-tightened ferrule.
10 · Everyday operating limits
Column life is governed mainly by chemical conditions and particulate load. The four limits below apply to most bonded silica columns; follow the technical data for the product in use for exact figures.
Limit
Conventional bonded silica
Base-stable bonded phase
Polymer substrate
What happens outside the limit
pH range
2.0–8.0
1.5–12.0
1–14
Hydrolysis of the bonded phase at low pH; dissolution of the silica skeleton at high pH
Maximum temperature
About 60 °C
About 80 °C
About 80 °C
High temperature accelerates hydrolysis and markedly shortens life
Maximum pressure
As rated for the column
As rated for the column
Usually lower than silica columns
Bed consolidation or frit deformation
Fully aqueous conditions
C18 chains may collapse at high aqueous content
As left
Unaffected
Retention falls sharply and re-equilibration is needed to recover
pH and temperature act together more strongly than either alone. Operating near the pH limit at an elevated temperature shortens life by more than the sum of the two effects individually (inferred on this page; no unified quantitative model was found).
10.1 Using and switching buffers
Filter through 0.45 µm after preparation (0.22 µm for UPLC), to keep insoluble matter out of the system.
Salt concentration and organic proportion must be considered together for solubility. Phosphate solubility falls as the acetonitrile proportion rises, and it readily precipitates at the mixing point.
Before switching from a salt-containing mobile phase to a high organic content, flush for about 10 min with 5%–10% organic in water, then raise the organic proportion in steps.
Flush out the buffer salt before shutdown storage, to avoid crystallization blocking the frit or corroding stainless steel flow paths.
10.2 Sample preparation
Filtration grade0.45 µm for HPLC, 0.22 µm for UPLC. The membrane material must be compatible with the sample solvent.
Matching solvent strengthWhere the injection solvent is stronger than the starting mobile phase, early peaks broaden or split. Dissolve the sample in the starting mobile phase.
Matrix removalFor matrices such as serum and fermentation broth, use solid phase extraction or protein precipitation first; a guard column alone is not enough.
Injection volumeToo large a volume overloads the column head and broadens peaks. The acceptable injection volume on a narrow-bore column scales with the square of the bore.
11 · Storage and care
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Duration:for a shutdown of a few days, cap the column under the organic conditions of the last run; beyond a week, change to the storage solvent shown above. No periodic flushing is needed during long-term storage.
Phase type
Short term (days)
Long term (over a week)
Avoid
C18 / C8 / phenyl
The last mobile phase used (salt removed)
Neat acetonitrile or methanol
Prolonged storage in pure water
Hydrophilic C18 / polar-embedded
The last mobile phase used
Acetonitrile / water (65:35) or per the instructions
Strongly alkaline solutions
HILIC
The last mobile phase used
90% acetonitrile / water
High aqueous content
Normal-phase silica
Hexane / isopropanol
Hexane
Solvents containing water
Ion exchange
The last buffer used
Dilute salt solution with a preservative
Aqueous solutions without preservative
SEC / GPC
The last mobile phase used
As specified for the medium
Changing solvent systems without a transition
11.1 Regeneration and backflushing
Where the pressure has risen and does not recover after replacing the guard column, regeneration can be tried. Backflushing (reverse flow) can push out particulates retained by the inlet frit, but it applies force to the bed in the direction that loosens it.
Disconnect the column from the detector before backflushing and route the effluent to waste, so particulates do not reach the flow cell.
Use less than half the normal flow rate, for of the order of 10 min.
Re-equilibrate in the forward direction afterwards and re-measure plate number and asymmetry factor to confirm the bed is unaffected.
Some suppliers do not recommend backflushing their columns. Check the technical data for the product in use before proceeding.
Strong-solvent flush sequence (reversed-phase columns): water → methanol → isopropanol → dichloromethane → isopropanol → methanol → water, 10–20 column volumes each. Confirm tubing compatibility for the dichloromethane steps (PEEK tubing is not suitable).
Part Four · Leaks, Troubleshooting and Quick Reference
12 · Leaks
A leak gives fairly direct feedback but has a wide range of causes. The same damp fitting may come from an under-tightened ferrule, an angled end, a mismatched insertion depth or a scratched sealing face — or from an upstream component with the liquid running along the tubing to that point. Working through the points one by one is more effective than repeated tightening.
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Suggested order:wipe dry and record the starting state, then run at the working flow rate for 5–10 min and re-check each point. If point 5 is damp while point 4 is dry, the problem is at the analytical column inlet; if both 4 and 5 are damp, suspect a leak upstream running down the tubing.
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Key point:A leak of a purely organic mobile phase evaporates quickly and may leave no obvious trace, showing only as low pressure or an unstable baseline. Such cases need a pressure test and a flow rate check to identify.
Symptom
Possible cause
Order of action
Leaks when finger-tight, stops when tightened further
Under-tightened
Tighten by the specified number of turns; if it takes clearly more than usual force to stop, check the end
Weeps persistently even when fully tightened
Wrong standard / wrong insertion depth / scratched sealing face
Remove and check the thread and insertion depth; inspect the fitting cone
No leak at low flow, leaks as pressure rises
Angled end / under-tightened ferrule
Recut and deburr the tubing, fit a new ferrule and reassemble
intermittent weeping
Uneven end / material deformation from temperature variation
Check the end; watch for thermal deformation of PEEK parts inside the column oven
Thread strips after repeated tightening
Damage from over-tightening
Replace the fitting and ferrule; do not reuse them
Fittings dry but pressure low
Leak upstream / pump seal failure
Disconnect the column and check the flow rate, locating it section by section
13 · Pressure and peak shape troubleshooting
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Establishing the baseline:after the first equilibration of a new column, record the column pressure under standard conditions (fixed flow rate, temperature and mobile phase composition) as that column's baseline. Judgements thereafter are based on the change relative to the baseline rather than on the pressure reading itself — tubing configuration differs between systems by several MPa.
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How to use this:Look at the peak shape of the unretained component (the dead time marker) first. That peak undergoes no retention, so its broadening and tailing come mainly from extra-column volume and flow path geometry, which makes it useful for separating column from system.
14 · One-page reference
14.1 Fittings and tubing
Item
Point
What 1/16″ means
Tubing outside diameter 1.59 mm, unrelated to the bore; the bore must be given as well when ordering
What 1/8″ means
Tubing outside diameter 3.18 mm, mostly for preparative work, pump inlets and waste
Choosing the pre-column bore
0.17 mm for a 4.6 mm column; 0.10 mm or finer for a 2.1 mm column
Common threads
10-32 UNF (the analytical mainstream), 1/4-28 UNF (low pressure / preparative), 6-40 (miniature), M6×1 (some European systems)
Easily confused standards
10-32 UNF with M5×0.8; 1/4-28 UNF with M6×1
Reusing a ferrule
Once tightened, a stainless steel ferrule's position is fixed; changing to a fitting of a different standard means a new ferrule and a fresh cut
Assembly order
Seat the tubing → hold axial pressure → finger-tight → tighten by the specified number of turns
Halide ions and strong acids readily cause pitting; chelating analytes may interact with the metal surface
14.2 Symptoms and what to check first
Symptom
Check first
Then
Finally
Column pressure rising
Replace the guard cartridge
Remove the column and measure system pressure
Strong-solvent flush / backflush
Column pressure falling
Leaks at the fittings
Bubbles in the pump and the check valves
Bed collapse (with peak splitting)
Column pressure fluctuating
Whether it matches the pump stroke frequency
Mobile phase degassing
Column temperature stability
Peak tailing
Insertion depth at the pre-column fitting
Contamination at the column head
Mobile phase pH and silanol interaction
Peak fronting
Injection solvent strength
Injection volume
Column head overload
Peak splitting
Whether every peak splits
Partial blockage of the inlet frit
A void in the bed
General broadening
Peak shape of the dead time marker
Connecting tubing bore and length
Loss of column efficiency
Retention drifting
Column temperature stability
Consistency of mobile phase preparation
Whether equilibration is adequate
Weeping at a fitting
Thread standard and insertion depth
Quality of the tubing end
Damage to the sealing face
Rising baseline noise
Bubbles in the flow cell
Mobile phase impurities
A damaged outlet frit letting packing escape
14.3 Storage solvents
Phase type
Long-term storage solvent
Avoid
C18 / C8 / phenyl
Neat acetonitrile or methanol
Prolonged storage in pure water
HILIC
90% acetonitrile / water
High aqueous content
Normal-phase silica
Hexane
Solvents containing water
Ion exchange
Dilute salt solution with a preservative
Aqueous solutions without preservative
SEC / GPC
The storage solution specified for the medium
Changing solvent systems without a transition
14.4 Problem attribution matrix
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How to read it:The installation column has the most red cells, consistent with Fig. 1 — connections are where general problems concentrate. The goods-in column is mostly confirmation, which is cheap and pays well.
15 · Frequently asked questions
The questions below are drawn from common technical support enquiries, grouped into selection, use, lifetime and mobile phase. A shorter version is atFAQ。
Selection
C18A is the general-purpose grade, with higher retention and loading, suited to most reversed-phase applications; C18C has a wider usable pH range (about 1.5–12) and suits demanding mobile phases or small peptide separations; C18D is intended for hydrophilic compounds, retains stably under high aqueous conditions, and can be used directly with LC-MS without an ion-pairing reagent. Establish the sample polarity and mobile phase pH first, then choose the grade.
A guard column is advisable where: (1) the sample matrix is complex (serum, urine, food extracts); (2) more than 20 injections are run per day; (3) the sample may contain particulates. A guard column slows degradation of the analytical column's inlet frit and the front of the bed, and a cartridge costs far less than a new analytical column. How much life is gained varies with the matrix and the standard of sample preparation.
The base medium is the same; the particle size differs. Analytical columns commonly use 3 µm or 5 µm and preparative columns 10 µm or 20 µm. Larger particles give lower backpressure and suit high-flow preparative work; smaller particles give higher efficiency and suit fine analysis. Choose according to the objective — resolution first, or throughput first.
It can be fitted where the pressure allows, but the measured efficiency is usually below what the column can achieve. A UPLC column's volume is of the order of a tenth that of a conventional HPLC column, and the connecting tubing bore, flow cell volume and data acquisition rate of an HPLC system introduce relatively large extra-column broadening. To realize its efficiency, change to narrow-bore connecting tubing and raise the acquisition rate at the same time. SeeSection 2。
Use & Operation
Equilibrate with 10–20 column volumes of mobile phase before injecting. If the column is stored in methanol, transition to aqueous conditions via 50% methanol/water before pure water, rather than flushing directly at high aqueous content and risking C18 chain collapse (hydrophilic C18 and polar-embedded phases can go straight to pure water).
Yes. The arrow on the column marks the direction in which the bed was consolidated, and flow should follow it. Reverse flow applies force in the direction that loosens the bed, which can create voids and lower efficiency and broaden peaks, usually irreversibly. Confirm the arrow points towards the detector before installing.
Do not respond by applying more force. Check in this order: (1) does the thread standard match (stop if the first two threads feel tight on entry); (2) is the tubing seated against the bottom of the fitting; (3) has the ferrule been swaged onto a fitting of another standard; (4) is the tubing end square and free of burrs; (5) is the fitting's sealing cone scratched. Most persistent weeping comes from (2) and (3). SeeSection 6。
Yes. Phosphate, ammonium acetate and similar salts left in the column and tubing can crystallize and block the frit, or corrode stainless steel parts. The sequence is: (1) flush for about 10 min with 5%–10% organic in water; (2) raise the organic proportion to 50%; (3) change to neat methanol or acetonitrile for storage.
Performance & Lifetime
Under normal conditions an analytical column will take roughly 2,000–5,000 injections (inferred on this page; it varies considerably with matrix and conditions). The factors that matter most, in order, are: whether sample preparation is thorough, whether mobile phase pH and column temperature stay within range, whether a guard column is fitted, and whether the column is flushed correctly after each run. Tracking the rise in column pressure relative to baseline and the change in plate number is more reliable than counting injections.
Common causes: (1) particulates accumulating at the head; (2) buffer salt crystallizing; (3) protein or lipid deposited at the head; (4) bubbles in the mobile phase. In order: replace the guard cartridge; remove the column and confirm the system pressure is normal; flush forward with a strong solvent; if it still has not recovered, consider backflushing or replacing the inlet frit. The decision path is inSection 13。
Check in this order: (1) is the column temperature stable (retention changes by roughly 1%–2% per 1 °C); (2) is the mobile phase prepared consistently (organic proportion, pH, method of preparation); (3) is the column fully equilibrated (at least 10 column volumes); (4) is the flow rate accurate (pump calibration); (5) is the column pressure within its baseline range.
Look at the peak shape of the unretained component first. That peak undergoes no retention, so its broadening and tailing come mainly from extra-column volume and flow path geometry. If that peak is equally affected, the problem is usually outside the column; if only retained components are affected in a way related to compound properties, it is usually inside. You can also replace the column with a zero dead volume union and inject, to compare against the system alone.
Mobile phase and tubing
(1) Do not use non-volatile buffers such as phosphate or borate; (2) formic acid (0.1%), acetic acid (0.1%), ammonium formate and ammonium acetate are suitable; (3) acetonitrile (better ionization efficiency) or methanol is usual as the organic phase; (4) choose a column that needs no ion-pairing reagent (hydrophilic C18, HILIC); (5) a flow rate of 0.2–0.4 mL/min is usual, to reduce ion suppression.
Not recommended. Phosphate gives a low UV background but is incompatible with MS; ammonium acetate is MS-compatible but gives a slightly higher UV background. Where one method must serve both UV and MS, use ammonium acetate or ammonium formate with a column that needs no ion-pairing reagent. Do not mix batches of mobile phase, as differences in composition cause retention drift.
Select by flow path section and sample type. Use stainless steel or reinforced PEEK for the high-pressure section before the column; PEEK from the column outlet to the detector; for biological samples and phosphorylated compounds, PEEK throughout or tubing with an inert surface treatment. PEEK swells in tetrahydrofuran, dichloromethane and dimethyl sulfoxide and degrades in concentrated nitric or sulfuric acid; stainless steel is at risk of pitting with halides or strong acids. The comparison is inSection 7。
Both refer to the outside diameter, 1.59 mm and 3.18 mm respectively, which determines the fitting and ferrule to use. The bore is stated separately and determines the internal volume and extra-column broadening. Pre- and post-column connections in analytical LC mostly use 1/16″ outside diameter tubing, with the bore chosen to suit the column bore; 1/8″ outside diameter tubing is mostly for preparative LC, pump inlets and waste lines. Both must be given when ordering. SeeSection 4。
For anything not covered above, send the column grade, lot number, system configuration, mobile phase composition and the symptoms to our technical team, or submit them through theonline enquiry page.