Noisy baseline

Compared with the flame ionisation detector, however, the ECD is more specialised and tends to be chosen for its selectivity which can simplify chromatograms. The ECD requires careful attention to obtain reliable results. Cleanliness is essential and the carrier gases must be very pure and dry. The two most likely impurities in these gases are water and oxygen which are sufficiently electronegative to produce a detector response and so give a noisy baseline. 

The symptoms of column contamination include irregular peak shape, loss of resolution, loss of retention, irregular or noisy baseline, and ghost peaks from semivolatile materials of a previous run or from sample decomposition. Some of these problems can be the result of a contaminated injector. 

A typical MALDI spectrum of a bacterial sample has a number of peaks that vary greatly in intensity superimposed on a relatively noisy baseline. This can be problematic for many peak detection routines. Therefore methods that eliminate the need for peak detection also eliminate problems associated with poor peak detection performance. Full-spectrum identification algorithms use the (usually smoothed) spectral data without first performing peak detection. 

Pressurization of the vials at both the inlet and the outlet ends of the CEC capillary column packed with particles to about 1.2 MPa is required to prevent formation of bubbles that lead to a noisy baseline. Typically, equal pressure of an inert gas such as nitrogen is applied to both vials to avoid flow that would otherwise occur resulting from the pressure difference. Hydraulic pressure applied only at the inlet end of the capillary column is occasionally used in pressure-assisted electrochromatography [38,39]. 

System suitability errors often include profiles that are not consistent with historical data such as (1) excessive peak tailing, poor resolution of critical components or noisy baseline (2) peak spikes due to micro bubbles or electric shock and (3) integration parameters such as percent main peak area out of range for the assay reference control sample. 

The most commonly reported symptom of a bad system is baseline drift the second is a noisy baseline. Baselines that drift up and down are almost always due to peaks coming off the column. Baselines that drift up continuously can be garbage, a bad detector lamp, or decomposition on the flow cell window. Noise can come from almost anywhere in the system. 

If the detector passes the static test, turn on the flow and watch the baseline. Noisy baseline at this point is probably coming from before the detector. Realize that a reciprocating pump is noisy and it now lacks pulse dampening from the column. While we re here, shoot a sample response should be instantaneous, straight up and down. I have not tried it, but you might be able to quantitate lamp strength by shooting a known standard over a time period and measure the deflection. 

Problem 3. Contaminant or air buildup in detector cell. 4. Plugged outlet line after detector. (High pressure cracks cell window, producing noisy baseline.) 3. Flush cell with methanol or other strong solvent. If necessary, clean cell with IN HN03 (never with HC1). 4. Unplug or replace line. Refer to detector manual to replace window. 

If the solvents are immiscible, the LC system will fail. If the pump will be delivering an eluent that is not soluble with the previous mobile phase or if the new mobile phase consists of two immiscible solvents, the net result is to have tiny slugs of different solvents traveling through the HPLC. Typical indications of this problem are (1) erratic flow rate, (2) noisy baseline, and/ or (3) baseline drift. To insure that these problems are not caused by a mismatch of solvents, refer to Table 6-4 for the miscibility numbers (M) and their use. The discussion on determining solvent miscibility using miscibility numbers is adapted from reference 20. 

The quality of solvents and inorganic salts is an important consideration. Soluble impurities can give noisy baselines and spurious peaks or can build up on the surface of the packing material, eventually changing chromatographic retentions. Furthermore, the eluate may need to be collected for further analysis (e.g. mass spectrometry) and all contamination must be avoided. In addition, particulate matter should be removed, otherwise pump filters, meshes, and tubing can become blocked. 

The high-resolution line-start technique can be used, but this is not amenable to scanning since the low concentrations necessarily employed generate a noisy baseline. An additional benefit of the new software is that it allows for particles whose density is lower than that of the surrounding liquid, thus making it suitable for emulsion sizing. 

Noisy baseline Short-term detector noise can be estimated by using an expanded scale and measuring the peak-to-peak signal fluctuation of the baseline. The noise of a modern UV detector should be close to the published specification or 1 x 10 5AU. Noisy baseline such as the one shown in Figure 10.7a is typically caused by low energy of an aging UV lamp which should be replaced. Detector noise can be caused by a large air bubble trapped in the flow cell or pressure fluctuations caused by a small leak in the flow cell. 

A noisy baseline of an RI detector can be caused by inadequate mobile phase degassing or temperature thermostatting. However, low light energy can also be caused by a contaminated detector flow cell or high UV absorbance of the mobile phase (Figure 10.7d). 

You get a noisy baseline, the early eluters have a lower response than normal, and the baseline may start to rise if the temperature is programmed. 

Expect a noisy baseline and fluctuating detection limits. There is nothing a priori wrong with uneven numbers. However, they are suspicious. Check the method validation closely. 

If your deuterium lamp is old, it can be checked by the reading of the hours of operation. A critical limit is 800-1000 hours - in theory. However, it may well be that you can work very well after more than 1500-1800 hours. An old lamp can be detected by from the noisy baseline and small peaks, which should not be confused with spikes. Spikes are sharp lines due to air bubbles. Modern detectors contain a counter for lamp operation hours and also indicate the actual lamp energy. It should therefore be quite straightforward to identify an old lamp as the problem. Remember the control charts (see Tip No. 21). 

Dirty mirrors contribute to a noisy baseline and an unfavorable signal-to-noise ration. For cleaning see Tip No. 21 or ask your manufacturer. 

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