Now you should leave the equipment for a little time to equilibrate. The triumphant advance of HPLC began at the beginning of the s. The installation will also have an additional useful side effect: The equipment should be placed in such a way that it will be easily accessible from both sides, the front and the back. Names of HPLC stationary-phase materials.

The quest for high plate numbers and shorter analysis times has outshone Olympic disciplines. To avoid memory effects, improve your sample preparation and make sure your equipment is clean. In order to set up a list of libraries that you have access to, you must first login or sign up. Temperature is an important optimization parameter whose influence could increase in the near future. How could this happen, since the system worked perfectly well?

More Practical Problem Solving in HPLC: Stavros Kromidas: : Books

Leakage or flow inconsistency of the pump, e. These are restrictor capillaries, sometimes also simply soolving restrictors. The cause of this behavior is a denser packing. II-I a shows both good long time and short time constancy.

The only disadvantage of helium is its prwctical. These 3 locations in New South Wales: Since it can be very short. For the weakly acidic region you could also use a formate buffer. In each case the problem, the solution and the conclusions are presented over a maximum of 4 pages, and in addition the book contains manufacturers’ addresses, references, data tables and checklists.


Please enable cookies in your browser to get the full Trove experience. All interconnection pieces, ferrules and fittings should come from one manufacturer, because different brand names may have small differences in their dimensions, leading to a small dead volume and consequently to a deterioration of the anticipated separation see Tip No.

The Solution Table gives an overview of some tests.

Practical Problem Solving in HPLC – PDF Free Download

Check before the injection that acetonitrile does not cause a precipit. High Propaganda Liquid Chromatography. Check for precipitation when mixing mobile phase and dissolved sample. Pump with good short-term and long-term constancy; V c is in both cases excellent. The more homogeneous the surface of the phase phases of the newest generationthe less important is the strength of the buffer used.

The appendix includes some further information about HPLC.

stavros kromidas more practical problem solving in hplc

The sample is soluble in an acidic or alkaline solvent and most likely can be analyzed in it. The first sample After having prepared your equipment in the described way, you can attach the source of prepared mobile phase. Often it is easier to insist on help from your internal customer or to quiz your supplier for the most suitable column or solvnig run a detailed literature search.

My advice on the subject is to use a generally accepted maximum value for the dead volume for all isocratic HPLC equipment in the laboratory.

I totally agree with them.

Pump precision or pump constancy We differentiate prlblem long-term and short-term constancy a Long-term constancy One way to access this is the precision often incorrectly: I Rule of thumb for the equilibration time between gradient runs: Lists What are lists? However, bases are protonated in their ionic form, an interaction with the hydrophobic surface is unlikely, and they elute early in the chromatogram.


Practical Problem Solving in HPLC

The author provides guidance for selecting and evaluating methods, intstruments and columns practical help with everyday trouble-shooting advice for optimizing separations, always explaining the reason why. If the dead time and retention time increase, the cause is a malfunctioning pump or a leakage; in this case you do not have to prepare a new mobile phase! This procedure guarantees a reproducible preparation of your mobile phase and avoids problems associated with volume contraction.

There are a few rules. Non-ionic, lipophilic compounds elute later. Pproblem my experience after this comparative study I would also recommend t1ushing your column with an acidic eluent, e. Since the sample is present in this part of the instrumentation, its total volume should be minimized. A high proportion of the metal ions will be cleaned away.

stavros kromidas more practical problem solving in hplc


Install the necessary column. In order to test whether the whole equipment is functioning, inject a standard mixture, which is normally specified in the operating procedure. If the original heptane in our case was “wet”, say a water content of ppm, and the freshly prepared heptane is dry, with only 20 ppm water, this difference can have an enormous impact on the separation. The acid concentration can result in a different ionic strength! The resolution is influenced by the above three factors, which again means that a separation optimization can be reached exclusively through change of k, a and N. The situation is similar for HPLC stationary phases.

The author provides – guidance for selecting and evaluating methods, intstruments and columns, – practical help with everyday trouble-shooting, – advice for optimizing separations, always explaining the reason why. If all values are on a straight line, the interaction type for all investigated solutes with the stationary phase is the same. The acid concentration can result in a different ionic strength! Is there anything unusual about the pump noise? Part I Separation of ionic solutes: Your supplier is not too unhappy, but you would like to know the cause.

In the early fifties, gas chromatography lead the way in exploring the tremendous breadth of chromatography and thus the gas chromatograph has become the paradigm of a new era in analytical chemistry. The book also features a special chapter on the retention of slving components in RP-HPLC, references, data tables and check lists.

The time lapse up to the detection of the UV-active compound correlates to the delay. Always do the same things in the same way. It is simple to check for a memory effect: If you use a recorder or an integrator, you should set the paper speed at its maximum value for an kromodas measurement.


A critical limit is hours – in theory. In reversed-phase chromatography, these sudden changes in retention are only seen if the water pracical changes by a few per cent. Problems and their Solutions 19 20 21 22 Sample preparation – how critical are which mistakes? L1T, L1 column or L1 mobile phase.

The Solution We can categorically state that a constant temperature increases the robustness in HPLC and is an important aspect for precise results.

stavros kromidas more practical problem solving in hplc

The first has a peak width of 0. The strong base m-toluidine is dissociated and strongly retained by the stationary phase. Passivate the equipment with 6 N Oromidas 3: Iz Retention time before 1m: The Solution You obtain the solution from the chromatogram.

Practical Problem Solving in HPLC

Is the column still in the system or has it been removed? But now, let’s get going According to the operating procedure, inject samples for comparison, samples and standards in a sstavros sequence and evaluate the resulting chromatograms. Here we have t m “” 0. The Case You notice that compared to chromatograms recorded earlier, your peaks are smaller or simply appear to be smaller. You must expect major fluctuations in the resolution see Tip No.

Keep the computer running.

stavros kromidas more practical problem solving in hplc

The Solution Almost always, the reason is in a different delay volume of your equipment compared to the moee the method was developed with. The baseline was a straight line.


The possible causes are in principle the same for all HPLC mechanisms. Would you like to tell us about a lower price?

More Practical Problem Solving in HPLC

The operation of the equipment is best explained by a colleague, or when you participate in a seminar organized by the manufacturer. Dead time, very often in the lab.

He is author and editor of numerous books. probpem

stavros kromidas more practical problem solving in hplc

Is the baseline stable with no drift and are the peaks symmetrical? Using a flow of 1 mllmin.

More Practical Problem Solving in HPLC: Stavros Kromidas: : Books

It is in each case important to know, how these parameters are to be adjusted and which values the manufacturer recommends for his software. We assume that the column is not overloaded; only a possible peak broadening caused by the injection will be discussed here.

This fact allows the following quick decisions: Particle size a small number e. You work with the same column and observe different retention times in the afternoon or on the next day. What is HPLC anyway?