Translator for HPLC HINTS and TIPS for Chromatographers

Sunday, May 15, 2011

Power and Surge Protection for Computers & Analytical Instruments (e.g. Uninterruptible Power Supply AKA UPS)

(1) When selecting an Uninterruptible Power Supply (UPS) for your computers and analytical instruments, only purchase one which outputs a true sine wave voltage (just like real AC power). Most of the UPS systems sold at the local office and electronic supply stores output either square wave A/C or pseudo-sine wave (which is not the real thing). Some brands will tell you they offer true sine wave outputs right in the model number or package, but always double-check the specifications (e.g. APC's "Smart-UPS" offer sine wave outputs, but NOT the Smart-UPS SC models). Using a UPS that outputs something other than true sine wave power can damage the power supply of what it is attached to and this can lead to premature failure of system you are trying to protect.

(2) Calculate the maximum load of the system being protected in Volt Amps (Line Voltage x Amps = VA) and purchase a system that can provide power well above that load for the time period you want to protect (*I think 20 or 30 minutes of run time, at load, is a realistic guideline to go by). A 700 VA UPS will usually power most desktop computers and a monitor for that amount of time. Most brownouts last for just seconds, but that is enough time to shut everything down or loose communication. If a power outage is more than five minutes in length, then it may be a while before it is restored. In this case, be sure to safely shut everything down manually before you run out of battery reserve power. Once everything has been turned off, remember to turn OFF the UPS system to stop it from slowly draining the battery (most computers are still turned ON and using power when they are OFF). Doing so will preserve the remaining battery capacity. Just turn the UPS back 'ON'  when power has been restored. 

(3) Unless you want to spend really BIG dollars, just put a high quality UPS system on the computer. A high quality UPS system designed for a single high current draw instrument like a chromatograph may cost thousands of dollars. If the system must be maintained at all times, then a serious back up source of energy should be invested in. High current UPS systems are available from a number of manufacturers so it would be wise to investigate these for your application. For everything else, use a dedicated, high quality power conditioner and surge suppressor on the analytical instrument circuits ($85 and up each, not the $20 models sold at most box stores). The computer system is really what you want to protect first. If the computer gets knocked out you loose communication and control of the analytical system. This usually means it will continue to run on and on without stopping. If the computer is protected by a UPS and the power gets glitched (such as a voltage drop/brown-out) or goes out entirely, then the computer may sense this and abort the run or shut the system down (if it has not been shut down). This is the type of approach we have used in our labs for twenty years and it works great during brown outs and black outs. The computers stay on long enough to properly and safely shut down everything without loosing data (which is the key). The instruments are all connected to individual power conditioners/surge suppressors (Tripp lite brand units in our case) so are protected from brown outs and surges. To protect our equipment from large surges, our electrician installed a high amperage DELTA lightning arrestor (100,000 AMP, 3000 joules per pole, unlimited number of surge capacity) on the breaker panel and individual Delta surge capacitors on each critical 20A circuit that supplies power to laboratory equipment. These extras steps provide protection from direct lightning strikes and surges to the panel and equipment. 

(4) The UPS Batteries: The lead acid batteries in your UPS system have a finite life. Mark them with a date and keep records on battery changes. Depending on the quality, temperature and load over time, they may need to be replaced in as little as one year or perhaps last as long as four years. When the batteries loose their ability to hold a charge your on-battery run time will drop dramatically so replace them early rather than late. The battery pack can be (and should be) tested on a regular basis. To test the UPS refer to the Operator's Manual for the correct procedure [*the test often involves selecting a time when the computer is not running any critical applications or providing any networked services (such as right before you would normally shut it off for the day). Once it is safe to do so, go ahead and remove the UPS’s A/C plug from the wall. This will remove the UPS system’s source of input power just as if a real power failure had occurred. Your computer system should behave as if nothing has happened. Monitor how long the system stays on until the available UPS run time shows 15 to 20% remaining time left and you will know how long the system can handle a complete power outage. The system will need overnight to fully recharge after this test and you should repeat this test a few times each year to monitor the status of the system]. *Many of the modern UPS systems provide a software application that continuously monitors the UPS modules which are connected to your computer or entire network. This can provide a record of the systems used, battery change dates and current charge levels right from your desktop.

Sunday, May 1, 2011

Determination of HPLC Column Void Volume / Dead Volume, Dead Time (T zero):

Column Hold-up Volume, Column Dead Time or 'Column Void Volume' (the preferred name) are all different terms we apply to find the internal volume of a packed column  (divided by the flow rate and usually expressed in minutes for the Column Void Time). You must know what this value is BEFORE starting to run an HPLC method or perform liquid chromatography. The value for column void volume changes for different column dimensions and different column support types.

Are you peaks or samples eluting at or near the column void volume? If so, no chromatography has taken place and no HPLC method has been developed. People with no chromatography training or experience often make this mistake and create methods with little or no peak retention. Make sure you have sample retention for a long enough time period on the column. How do you know how long is long enough? Start by estimating the Column Void Volume (use our table or calculate it), and then, calculate the K prime value for your sample. The K prime for each peak should be at least 1.5 (>2.0 is the accepted standard) for the method to be useful and selective. *A more accurate answer will be found by measuring the void volume of your column (read on).

Knowing the Column Void Volume and the Flow Rate used allows you to calculate the Column Void Time (which is the most useful value). Determining  the column void time or T0 ("Tee Zero" as we call it), is necessary to find other important chromatography values such as: the Resolution, Separation Factor and Capacity Factor (K1) in a chromatography separation. Ideally, it is measured by injecting a sample which is unretained by the column & mobile phase (passes right through the column support with little to no interaction). It may also be easily estimated for most spherical, bare or coated silica supports if you know a few physical specifications of the column and media used. You should first estimate it, then measure it (the values should be close, +/- 15%). Note: A practical "tip". You can also estimate T0 by noting when the small injector valve pressure peak ('blip') appears on the baseline. It results from the pressure change which occurs from switching the injection valve from the "load" to "inject" positions.

Here is short list of typical HPLC column dimensions and their associated estimated void volumes for fully porous silica supports. At a flow rate of 1.000 ml/min these values would also be the same as the void time in minutes.

COLUMN DIMENSIONS (I.D. x Length (mm))                 VOID VOLUME (ml)

                         2.1 x  50                                                                  0.12
                         2.1 x 100                                                                 0.24
                         2.1 x 150                                                                 0.37
                         2.1 x 250                                                                 0.61
                         2.1 x 300                                                                 0.73

                         4.6 x  50                                                                  0.58
                         4.6 x 100                                                                 1.16
                         4.6 x 150                                                                 1.75
                         4.6 x 250                                                                 2.90
                         4.6 x 300                                                                 3.49

                       10.0 x 100                                                                 5.50
                       10.0 x 150                                                                 8.25
                       10.0 x 250                                                               13.75
                       10.0 x 300                                                               16.49

                       Column Void Volume Equation (ul) = (d^2 *Pi * L * Pore Volume) / 4 ;
                                  {Column Diameter & Length are in mm}.

[Note: All you need is the column's length and ID to estimate it. For most fully porous supports, use a 'Pore Volume' value of 0.70 in the above equation. This is the most commonly measures pore volume found for non-encapped, fully porous spherical bare silica support (please check with the manufacturer for the actual value of your support). Estimating the value will often get you close to the measured value, but due to the unique chemistries used to prepare supports, it is only an approximation].

Always measure the actual void volume of your specific HPLC column with a compound which is unretained by your column. For RP applications which utilize at least 20% organic, Uracil or Thiourea are often used, but some inorganic salts (e.g. sodium nitrite and sodium nitrate) have also been shown to work as well. When determining the "Column Void Volume", you are really measuring the void volume of the column plus any extra-column volume from the injection volume plus all lines connecting the injection to the column and the column to the flow cell. Note: This is very different from the "System Dwell Volume" which includes the volume from the pump (or gradient valve) to the column head.

A more detailed version of this table with other common HPLC Column Sizes and Tubing Volumes for capillary lines are available at the following links (Link #1) or (Link #2).