Fronting peaks …

fronting peak

In HPLC, Fronting peaks are as common as tailing peaks.

Many analysts believe that sample overload causes peak tailing. They’re wrong.

Sample overload causes peak fronting.

If you have fronting peaks, don’t worry. Your column is fine. These are the reasons:

  • Sample overload
    • Reduce sample concentration.
    • Reduce sample volume.
  • Unresolved peak on the front
    • Change detection wavelength, or if you have one, use a diode-array detector.
    • Change mobile phase. Specifically, reduce the stronger solvent a bit.
  • Sample solvent incompatible with mobile phase
    • Dissolve sample in mobile phase.

Got it?



Peaks with tails …

tailing peak

Tailing peaks are a nuisance in HPLC. And they happen all the time.

The common misconception about peak tailing is that it is caused by sample overload. No, it’s not.

Most of the time, peak tailing is caused by a bad column.

Here’s why peaks tail, and what to do about it:

1) Active silanol sites on column – Use 0.1 % triethylamine in mobile phase. Or get a better column, with a higher carbon load.

2 ) Sample ionisation – Adjust pH to suppress ionisation.

3) K’ too large – Increase mobile phase strength.

4) Insufficient end-capping – Change column.

5) Hidden peak on tail – Change detection wavelength and/or change mobile phase strength.

If you have persistent peak tailing, you have a bad column. You have no choice but to change it.

Cheers … SKS




LC troubleshooting … baseline drift.


A drifting baseline is a common issue in HPLC. And easily resolved.

Typically, these are the causes:

Change in mobile phase composition. This is the most common cause of baseline drift. If you’re running a gradient, and you’re using a UV-vis detector, you can always expect a drift in the baseline, caused by refractive index effects. Avoid using a steep gradient. Change the mobile phase composition as gradually as possible.

Change in column temperature. Simple. Use a good column oven, even if you’re at room temperature. It’s always a good idea to invest in a reliable column oven.

Mobile phase changeover, from previous injection. You will need to wait for a while, to let the baseline stabilise.

Aging UV lamp. People forget to calibrate and change the UV-vis lamp.

Column bleed. Rarely, the bonded phase may bleed from the column. This is actually quite rare with modern-day bonded columns. If you have a column bleed, you can always tell by the continuously drifting baseline. It will never stabilise, no matter what you do. In this case, you will have to discard the column.

You can avoid column bleed in the first place, by buying a good brand and staying within the pH limit of the column.

Cheers … SKS.


LC fundas – Low backpressure.

As opposed to high back-pressure, it is relatively simple to figure out why the back-pressure on your HPLC system is below normal.

By “normal” backpressure, one means that you know what the usual backpressure for a given LC column under given operating conditions is.

Since we already know that the usual backpressure on a C18, 150mm, 4.6mm ID, 5µ column, for AcN:water:;70:30 at 1ml/min at 25 deg C is about 700 to 1000 psi, a backpressure lower than 600 psi requires your attention.

The first suspect is a trapped air-bubble inside the system, especially in the pump piston head. An air-bubble will cause the back-pressure to drop and fluctuate.

Disconnect the column and purge out your HPLC thoroughly. If the backpressure is still lower than normal, it means there’s a leak somewhere.

High-pressure sites, i.e., any fittings and seals before the column are most likely to leak. Check-valves, piston seals and injector fittings, in that order, are what you should be looking at.

When you locate the leaking fitting, do not overtighten it. You can cheat a bit, and use a small piece of teflon tape around the fitting. That should take care of it.

If the threads on the fitting are damaged, you have to replace it. Wherever possible, I use Fingertight fittings, and I’d suggest you try them out too.

Cheers … SKSrinivas.

LC fundas: Choked column frits.


A column frit is a small disk of sintered steel that is placed on each end of the HPLC column. Some frits are incorporated into the column end-fitting itself. And some frits are made of titanium or specialised alloys, instead of steel.

Frits have three things to do:

Protect the column packing from contamination.
Distribute the injected sample into a uniform band before the column head.
Keep in the column packing in.

Column frits are porous, and for obvious reasons, the pore size needs to be less than the particle size of the column packing. For an analytical 5μm column, the top frit may have a pore size anywhere from 0.2μm to 2μm. Bottom frits may have slightly larger pore sizes.

Which means of course, that frits make a significant contribution to the system backpressure. And it also means that frits can get choked and further increase the backpressure.

As we know, ignoring high backpressure is not a good idea.

Here’s how we deal with troublesome frits:

  1. Disconnect column from detector.
  2. Reverse the column, reconnect to injector.
  3. If it’s a C18 or C8 column, backflush column with AcN:water:70:30 @ 0.5ml/min for an hour. For any other bonded phase, check with the column data sheet for solvent compatibility. For a non-bonded normal phase Si column, I would back-flush with dry acetonitrile or iso-propyl alcohol.
  4. Keep an eye on the backpressure. Once it drops down to normal, continue backflushing for 15 minutes.

Backflushing works most of the time. If the backpressure hasn’t come down at all, then you have a really choked frit, and it’s time for drastic measures.

Remember, this is only a last resort.

  1. Clamp the LC column in a strong vise, if you have one. Or else, use two opposing spanners or wrenches.
  2. Carefully unscrew the end-fitting – of the top frit only.
  3. Leave the bottom frit strictly alone.
  4. If the top-frit is part of the end-fitting, that’s good. Or else, if the top-frit is a separate disk, then …
  5. Carefully slide the frit away from the column head. Don’t pull out the top-frit from the column head. Slide it out. Or, you will disturb the column packing.
  6. Place the frit in a 50 ml beaker, add enough acetonitrile to cover it, and sonicate the top-frit in an ultrasonic bath for 30 mins.
  7. Some chromatographers prefer a stronger sonication liquid like 50% nitric acid. Not advisable at all. Strong acids will physically corrode the frit.
  8. Sonication should clear the frit, and restore its normal perfomance.

Once again, you use this method as a last resort. If this doesn’t work as well, then you have a real problem. All you can do then, is to buy a new frit.

Or a buy a new LC column.  C’est la vie.

Cheers … SKS

LC fundas: Dealing with high backpressure.

High backpressure is a chronic issue in HPLC. But then, how high is high?

To give you an idea:

For a C18, 150mm, 4.5mm ID column packed with irregular 5μm particles, at a flow rate of 1ml/min, water: acetonitrile (30:70) at 25 degs C, the normal backpressure should not exceed 1000 psi (or about 700 bar).

Under these conditions, anything more than 1000 psi should make you worry a bit. Sure, modern HPLC pumps can take up 6000 psi backpressure or more, but that’s no reason to push your expensive pump to its limits.

High backpressure increases the wear on piston seals, check-valves and other components, and will eventually result in higher maintenance costs.

Backpressure can be affected by several factors, to wit:

Mobile phase composition: Water and isopropyl alcohol (IPA) are considerably more viscous than acetonitrile. Try to limit the content of the more viscous solvent to 30%.

Temperature: Higher column temperature = lower backpressure. In general, higher column temperature will also improve peak separation, so it’s not a bad idea to experiment a bit. But try and keep it below 60 deg C.

Buffers: Precipitated salts from buffers are the primary cause of choked frits and high backpressure. Don’t forget to flush! I’ll tell you how to deal with choked frits in the next post.

Column packing particle size: 3.5μm can provide better peak resolution, no doubt, but at the cost of higher backpressure.

A quick way to reduce elevated backpressure is to disconnect the column from the detector, reverse it and reconnect it to the injector, and backflush with AcN:water::70:30 (or the mobile phase you’re using), at 0.5 ml/min for half an hour. That should take care of the problem for the time being.

Whether it’s your heart or your HPLC pump, don’t ignore high pressure.

Cheers … SKS

It’s Bunsen Burner Day!

Public domain image

Robert Wilhelm Eberhard Bunsen, the brilliant photochemist who developed the laboratory burner that is named after him, was born on this day, March 31, in 1811 in Göttingen, Germany.

In collaboration with Peter Desaga, his assistant at the University of Heidelberg, Robert Bunsen made his eponymous burner in 1854 as an improvement over the inefficient lab burners of his day.

Using a mixture of air and a fuel gas like LPG. methane, propane or butane, the Bunsen burner can produce an intensely hot flame that is ideally suited for a variety of experiments that require that kind of heat.

Public domain image

Peter Desaga and his descendants held the sole marketing rights for the Bunsen burner for several years.

A highly respected scientist, beloved teacher and a thorough gentleman, Robert Bunsen never took out a patent for any of his inventions.

Even after his official retirement at the age of 78, Bunsen continued to work in geology and mineralogy, right until his death at 88.

And he was single (like me). Which is perhaps why he was one of the most productive German scientists of his time!