Column blank

Blcrameters) by using a prepacked colunn as a restrictor down stream from the e tty column blank to be packed [159]. 

However, so that you don t confuse yourself, we recommend using the placement on the left. And be sure to leave unused grid columns blank. 

Connect one end of your column blank to the tubing from the injector outlet the other end is connected to the line leading to the detector flow cell. We have one more fluid line to connect to complete our fluidics. A piece of 0.02-in tubing can be fitted to the detector flow cell outlet port to carry waste to a container. In some systems, this line will be replaced with small-diameter Teflon tubing. 

With the column blank in place, we are ready to do a general system cleaning. The technique is called pacification. It involves washing the system with 20% nitric acid. It sounds harsh and is, but your system should be resistant to it. Check with your manual to ensure that all wetted surfaces are stainless steel, ruby, Teflon , and quartz. If you can t tell from the manual, call the manufacturer. Use pacification only with UV detectors, I can t guarantee other detectors will be resistant. Do not use pacification on mass spectrometers or their interfaces ... 

Before we proceed, make sure you have removed the column. Trying to do pacification with a column in the system will make a mess and, at the least, ruin the column. After removing the column and inserting the column blank, wash with water for 15 min at 2mL/min to remove any buffer. Follow this with 20% HN03 (6N nitric acid) for 15 min at 2mL/min, then wash out with water until neutral pH is reached. This last step takes a long time. 

Spare parts that are needed are compression fittings and ferrules, plunger and injector rotor seals, an extra plunger and seals, column filters, and injector needle port seals. If you do not use pacification, you might want to keep a set of check valves on hand. I always have one coil each of 0.01- and 0.02-in tubing in addition to my column blank. A replacement solvent inlet line with a porous stone is useful in case of corrosion. If you filter solvents, you need cellulose, nylon, and Teflon filters. You also need a back-up lamp for your detector. 

As we said in the column section, the first step is to remove the column and see if the problem goes away. Pressure problems can be determined by looking at the pump pressure. Other problems can be diagnosed from the detector s digital display and the recorder baseline. Of course, with the column gone, there is no path to the detector. Here is where the column blank shows... 

You now have the tools to find most of the problems in the system. Remember that 80% of the problems are column problems and about 60% of those come from dirty water. If the problem fails to leave with removing the column and putting in a column blank, then do reverse-order diagnosis. It will be amazing how much time you save. 

Pacification is a technique for removing organics and buffers from HPLC metal and Teflon surfaces and protecting them from salt corrosion with 6 N nitric acid (see Chapter 4). First, remove the HPLC column and replace it with a column bridge. Do not flush this wash into the mass spectrometer. Wash the system with water. Remove the column and replace it with a column blank. Flush with 6 N nitric acid for at least 30min, then overnight with water. Ensure the effluent pH is back to that of lab water. Replace the column and flush with mobile phase. This should be done at least once a month to clean check valves, line, and injectors. Under no circumstances should this wash be done with an HPLC column in place or into the mass spectrometer ... 

Column Blank—A length of tubing, fitted with compression fittings simulating column ends, used to replaced the column for system cleaning and diagnosis. 

Watch the recorder or computer baseline. When it is stable, slow the pump flow to 0.1 mL/min, remove the column blank, and connect the Cl8 column to the injector. Do not connect the column to the detector yet. Wash the column solvent into a beaker (start a slow flow ramp up from 0.1 to l.OmL/min) for six column volumes (12-18mL). Pressure should slowly increase to around 2000 psi at 1 mL/min due to column backpressure. (Lab note Always hook up a column with solvent running to prevent introducing air from the column head into the column.)... 

Very Important Lab Note Remove the column Replace the column with the column blank. Put end fittings on the column for storage. Put water in the reservoir and wash the system at 2mL/min with water. 

To prepare the silica gel (Anasorb, 130-140 mesh, Analabs, Inc.) for use, a portion is placed in an evaporating dish and baked overnight at 275° in a clean oven (15 g, for example, will fill five columns). Longer heating is permissible. When removed from the oven, the gel is immediately poured into a small bottle which is then tightly capped and allowed to cool to room temperature. Activated gel is not allowed to stand more than a day prior to use. Unused gel can be reheated. Hydrocarbon-free nitrogen is tested as part of column blank measurement. 

In practice, four unknown samples and a blank are analyzed as a group. The blank consists of 25-50 ml carbon tetrachloride which is handled as a sample. Small but persistent amounts of hydrocarbons are found for all column blanks and are subtracted from the unknown samples. Blank corrections are also applied to the UV, GC, and MS analyses. 

This column blank is not to be confused with sample blanks such as those determined for the 5-gal. jugs. This latter blank correction is applied to (1) extractable organics and nonvolatile hydrocarbon contents measured by the method and (2) UV and GC data. No correction from the sample blank is applied to the compound type analysis by mass spectrometer. Very little net error can be attributed to this omission of a blank correction. 

A mass spectrometer is used which is on-line with an IBM 1800 computer. This capability greatly simplifies the extensive calculations which are required. Prior to analysis of a sample(s), a silica gel column blank is analyzed to supply the computer with a spectrum which will subsequently be subtracted from each unknown sample. A blank corrects each set of four unknowns that were simultaneously run through the silica gel separation. In practice, column blanks are quite similar to one another, so a given blank can be used to correct other samples sets also. In each instance the entire fraction is charged to the spectrometer by introducing the sample with a gallium-sealed frit at room temperature and with the sample inlet system open to the vacuum pumps. After 3 min of pumping to remove most of the isooctane, the vacuum valve of the inlet system is closed, and the frit temperature is increased to 315 °G. This vaporizes the hydrocarbons from the frit, and the sample spectrum can be obtained. 

Table II. Recovery of abamectin from spiked water samples taken through the extraction protocol of Figure 6. Values for the samples are the mean and standard error for triplicates that fell within the working range of the standard curve (0.2 to 0.7 of the normalized response), which had an I50 of 8.1 ppb. Values preceded by the sign were below the limit of detection, i.e., they had a normalized response above the standard curve limit of 0.7. The column blank is the mean SE of the unspiked samples...

California Dept, of Food and Agriculture s HPLC detection limit for abamectin residues is 1 ppb, and contaminated strawberries are likely to have on the order of 2 to 5 ppb of residue. (Dr. Mark Lee, Calif. Dept, of Food Agriculture, personal communication to Dr. Karu). The first two spikes in Table III are thus comparable to residues that could be left on field samples. Given the efficiency of recovery of the spikes and the errors, it is reasonable to expect that the E1A method could detect 2 to 5 ppb above the column blank. ... 

On a sheet of newsprint make a Class Prediction Table" (see Figure 4-2). Leave the Results column blank. 

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