Frequently Asked Questions
Post-column equipment (general) PCX5200 PCX5100 or PCX3100 PCX5000 or PCX3000 Carbamate Pesticides Glyphosate Amino Acid Analysis Post-Column Reaction Equipment The post-column pressure is too high. What is wrong? There is a blockage (partial or total) in the system. Blockages are usually caused by precipitates forming in a reactor or a connecting tube. Precipitates seem to fall in three general categories:
Soluble in dilute acid: use 2-3 molar nitric acid Soluble in strong organic solvent: use acetone then hexane then acetone again Not soluble: replace the blocked component or use physical means
The general procedure is to remove the column and guard and replace them with a bypass tube. With the reagents turned off, flush the post-column reactors with water. Pump the cleaning agent with the HPLC pump for 20 minutes. Flush the system again with water until all the acid or solvent is purged from the pump. Re-install your column and guard. Other less common blockages: Pinched plastic tubing, usually at a fitting A heat-exchanger or connecting tube made of very small-bore tubing What caused the precipitate? Some common reasons precipitates form:
Minerals dissolved in the sample react with a reagent Wax or grease in the sample coming out of solution Chemical impurities in a reagent Breakdown of the column
How do I find the blockage? Do a systematic disconnection, while observing the post-column pressure. First turn off the reagent(s) and lower the reactor temperature to a safe level. Starting at the waste bucket, track the flow of the eluant backwards through the system. Disconnect each component one at a time. Do not disconnect the reagent parts of the system (restrictor, relief valve, anti-siphon valve, etc.) because that will give misleading information. Why is the baseline too noisy? Well, that is a complicated subject. Not only are there many sources of noise, there are many kinds of noise. By far, the most common source of noise is a mis-behaving pump, and a piston seal or a check-valve is a likely culprit. The next most common problem is chemical contamination of a reagent or eluant. Old reagents can cause noise. Extremely dirty samples will cause noise in later runs. Finally, the detector may have a problem, such as a dirty flow-cell, leaks, or needs a new lamp. Each of these causes has a characteristic signature in the baseline. I have turned off the Pickering unit, and the noise went away. That proves that the problem is in the post-column instrument. Right? Unfortunately, that has proved nothing, although it does eliminate a few possibilities. The noise comes from the combination of the reagent and the mobile phase; if there is a problem with either one, there will be noise. How do I make the Pickering unit turn off after the end of the run? The most commonly used way is to exploit the minimum-pressure requirement. Write a special shutdown method for your HPLC, and run it last in the sequence, or make it the time-out method. In this method, select the solvent you normally use to flush and store the column (methanol for carbamates, regenerant for amino acids and glyphosate, etc.). Set the initial flow to 0.02 mL/min for 5 minutes; this lets the column pressure fall below 500 psi so that the post-column system shuts itself off. Then ramp to the normal flow rate and hold for 10 minutes to clean the column and flush out the reactor. Ramp down to 0.02 mL/min and hold that flow until the next day. For the PCX5100, there is a remote off; a contact closure between the Ground and Remote Off terminals will turn it off. On the PCX5200, send *E0 to the serial port (1200 baud, 8 bits, no parity, 2 stop bits). I just changed the seal (or reagent) and now the reagent is not pumping and the pressure gauge reads zero. What is the problem? The problem in most cases is a bubble in the reagent pump; try priming the pump. Less commonly, a check-valve has failed, the piston is broken, or the pump was mis-assembled when serviced. There is not a blockage in the restrictor. How do I prime the pump? This is covered in detail in your User's Manual. These pumps will not work if there is air in them. You can try priming with degassed methanol. A better method is to vacuum prime it. Briefly, you shut off the reagent supply with a stopcock, draw a vacuum on the pump with the 20cc priming syringe, then open the stopcock while under vacuum. Do this twice; if the problem is air this will fix it. The reagent pressure is going up. What do I do? This is caused by fouling on the reagent filter or reagent restrictor. Replace the reagent filter frit. Try cleaning the restrictor by replacing the reagent with alcohol (to remove organics) or 3M nitric acid (to remove minerals). Flush thoroughly with water when finished cleaning. If cleaning fails, replace the restrictor. ^ Back to top PCX5200 specific questions Can I control the '5200 from my chromatography data system? Yes, but... The serial port allows commands to be sent to the PCX5200. The command set is documented in the User's Manual. None of the data systems now available has the programming to use this feature. If you want you may write your own program or macro to add remote control. Pickering has no plans to write any such software. You can also use terminal software to communicate with the PCX5200. Some software has macros and custom buttons that can be used for this purpose. ^ Back to top PCX5100 or PCX3100 specific questions The temperature of the reactor (or column heater) jumps up and down. Sometimes the temperature display shows "EE1" What is wrong? These symptoms mean that the electronics has a bad or even broken connection to the temperature sensor. Usually this results from a bad cable harness between the temperature control module and the main printed circuit board. The quick fix is to remove the cable harness (take notes!), re-crimp the slip-on terminals, and re-install it. The replacement part is 1500-0707 for the older boards (rev.B) or 1500-0711 for the newer (rev.D). Less common causes include: 1) the reactor or heater is unplugged, 2) the sensor has a broken wire, 3) a bad printed circuit board, 4) a bad temperature controller, 5) a bad plug on the back of the temperature controller. How do I get in to replace the restrictor, reactor, tubing, etc.? Usually it is not necessary to remove the front panel. Pull the system out where you can work on it. To make more space to work inside, you can remove the reagent pump. Also you can remove one of the support posts that blocks access to the plumbing. You may leave this post out for routine use, but please re-install it for shipping. Why did you make the PCX5100 so [censored] hard to work on? Mea culpa! We apologize. I think we did a much better design on the PCX5200 mainly because you kept griping. ^ Back to top PCX5000 or PCX3000 specific questions Do you still support these models? Yes we do. Some parts are no longer available, but there are upgrades available for most cases. Some of the service parts have been dropped from our published price lists, but the common ones are still kept in stock. We will advise you on specific cases. The bulkhead fitting for the reagent leaks and I can't stop it. This bulkhead fitting is actually a filter assembly. You need to replace the filter housing (3102-0261) and ferrule (3102-2108). You probably also need a new filter frit (3102-9080). How do I get a stuck piston out of the Minipump? This is usually caused by neglect of cleaning and lubricating the pump. You will have to hammer out the slider that holds the piston, polish the housing, and almost certainly replace the piston. Pickering can do this for you. ^ Back to top Carbamate Pesticides There is much information in chapter 3 of the Carbamate manual. Look there for detailed directions. My column resolved Aldicarb Sulfone and Aldicarb Sulfoxide when it was new, but now they are fused. Can I get the resolution back? Probably not. This is caused by degradation of the bonded phase caused in turn by repeated exposure to the chloroacetic acid preservative. Unfortunately, the type of silica that allows the separation also makes it vulnerable to this degradation. Normally, the column will last for 3 to 9 months of use. My baseline goes up and down, and it is full of noise. What is wrong? Most likely contamination of one of the mobile phases. Contamination can come from the source, be in your solvent reservoir, be in your HPLC pump, or any filter frit. Your column may have been contaminated by bad mobile phase or a dirty sample. How do I clean the column? Do this only when necessary. Make certain that the rest of the system is clean. Set your HPLC pump to 70:30 water:methanol. Make a solution of 67% water, 30% methanol, and 3% ChlorAC buffer. Make repeated 100 µL injections of this solution until the baseline comes down. How do I store the column? Flush it with methanol for 15 minutes. Remove the column, wash the end-fittings, and cap it. Why does my reactor get clogged? If you are analyzing water, it is usually due to minerals in the water precipitating under the alkaline conditions of the hydrolysis. Using Pickering CB130 Hydrolysis Reagent with C47 additive will reduce this problem. Minerals are soluble in 3M nitric acid. If you are analyzing agricultural products, it is typically fats or waxes causing the problem. Lipids are soluble in acetone or hydrocarbon solvents (such as hexane). Some peaks disappear or have low recovery, but others do not have a problem. This is caused by hydrolysis or oxidation of the sample. Remember to use ChlorAC preservative for water samples or you will lose oxamyl and carbaryl to hydrolysis. Hydrophobic carbamates such as methiocarb can stick to plastic sample vials. Sulfur containing carbamates such as aldicarb and methiocarb can be oxidized by chlorine or peroxides in the water or on your guard column. Glyphosate There is much information in chapter 3 of the Glyphosate manual. Look there for more detailed directions. There are no peaks! The most common problem is iron contamination of the guard or analytical column. Columns that have been in long storage often have this problem. Corrosion in the HPLC , or dirty samples are also common sources of iron. Use Glyphosate Restore to remove heavy metals and other stubborn contaminants from the column. Replace steel filter frits in the solvent reservoirs with non-metallic ones. Flush the HPLC (not the column!) with 3M nitric acid followed by a long flush with deionized water. Less commonly, there are problems with the reagents. Contamination of the plumbing in the first reagent can reduce all the hypochlorite so that you see no glyphosate, but plenty of AMPA. Too little or too much hypochlorite will do it. So will using the wrong OPA buffer. The glyphosate peak is split, or mis-shapen. This usually happens to standards made up in de-ionized water. Oddly enough, real drinking water rarely does this. Adjust the pH to about 3 with phosphoric acid. Another possible cause is slight iron contamination or the guard column. For injections greater than 50 µL, we recommend pH adjustment of the samples and standards with phosphoric acid to pH ~3. We have found it possible to inject 400 µL this way, perhaps more. ^ Back to top Amino Acid Analysis My peaks are only 20% of normal. What happened? Your Trione reagent is oxidized. To confirm this, look at Proline; it will be normal, but primary amino acids like Glycine will be small. After opening, the reagent must be kept under inert gas to prevent air oxidation. Is it safe to use out-of-date Trione? After the expiration date of the classic 1-part Trione, the risk of precipitate formation becomes greater with time. If you get hydrindantin precipitation in your instrument, it becomes nucleated, and will clog up easily in the future. This is very hard to clean out. Trione will still work after its expiration, but damages are not covered under warranty. You have been warned. How do I store the column? Store it in regenerant RG003 for Lithium columns or RG011 for Sodium columns. Do I need special storage for Pickering eluants or Trione? No. Store them un-opened at room temperature. An opened bottle of Trione should be stored under inert gas. Refrigeration is not necessary. If you refrigerate Trione below 5°C it separates into two phases; warm and mix to redissolve them. ^ Back to top |
