Column compartments

The viscometer assembly is placed in the constant temperature column compartment of the chromatograph between the column outlet and the refractometer. A combination of two Waters Associates M-45 hydraulic filters in series with a capillary tubing coil (length 10 ft., I.D. 0.01 in.) is used to dampen the line pressure fluctuations caused by the pump. With the above pressure damping modifications the overall system noise was reduced to less than 1 millibar at 1.0 ml/min flow rate in tetrahydrofuran (THF) for a set of six p-Styragel columns 10 ,... 

Column compartment The column oven should be capable of maintaining a temperature range of 5 °C above ambient to 60 °C. The oven should be within 3°C of the set temperature and the temperature precision should be <2.0%. 

Qualification Approaches If an analytical instrument is comprised of different functionally discrete modules, a modular approach to qualification testing that focuses on the specific operations of the individual module can be suitable for certain aspects of some operational testing (such as the flow rate precision and accuracy testing of a HPLC pump and the temperature accuracy column compartment). 

High-performance liquid chromatography (HPLC) is one of the premier analytical techniques widely used in analytical laboratories. Numerous analytical HPLC analyses have been developed for pharmaceutical, chemical, food, cosmetic, and environmental applications. The popularity of HPLC analysis can be attributed to its powerful combination of separation and quantitation capabilities. HPLC instrumentation has reached a state of maturity. The majority of vendors can provide very sophisticated and highly automated systems to meet users needs. To provide a high level of assurance that the data generated from the HPLC analysis are reliable, the performance of the HPLC system should be monitored at regular intervals. In this chapter some of the key performance attributes for a typical HPLC system (consisting of a quaternary pump, an autoinjector, a UV-Vis detector, and a temperature-controlled column compartment) are discussed [1-8]. 

Column compartment Temperature accuracy 2°C of the set temperature 6 months... 

The temperature accuracy of the column heater is evaluated by placing a calibrated thermometer in the column compartment to measure the actual compartment temperature. The thermometer readings are compared to the preset temperature at 40 and 60°C. Many LC equipment manufacturers have the requirement set at 2°C. The temperature stability of the column compartment can be assessed indirectly by comparing the retention time of certain peaks in the chromatogram over time. Acceptance criteria of the stability evaluation should be assessed on a case-by-case basis due to different laboratory conditions and environment. 

If a system comprises several modules, it is recommended that system tests be performed for parameters that are affected by multiple modules (holistic testing) rather than performing tests module by module (modular testing). Individual module tests should be performed if the parameter is affected by that module only (e.g., the wavelength accuracy of an HPLC variable-wavelength detector or the temperature accuracy of a column compartment). 

Row range (ml/min) Max. press. (p.s.i.) Injection Column compartment Standard column ... 

The HPLC system consisted of a Model 600E multisolvent delivery system equipped with a heated column compartment, a Model 484 variable-wavelength detector, and a Model 745B computing integrator, all from Waters Assoc., Inc., Milford, MA. The chromatograph was equipped with a Spherisorb ODS column (10 pm particle size, 25 cm x 4.6 mm ID.). 

The temperature in the column compartment can be very different depending on the oven type, therefore the temperature probe is attached as follows ... 

Work In HPLC Is generally carried out at a constant temperature usually lower than those typically used In GC. Hence, the automation of the thermal control of the column compartment is fairly infrequent because, in addition, the controlled variation of the temperature Is much less efficient than in GC. A straightforward self-contained electric thermostating system is more than adequate for most separations in liquid chromatography. 

It is important to provide your customer with reliable results that are fit for purpose. As forensic scientists, we look to do this by performing instrument qualifications (calibrations against verified reference materials) and method validations (using verified reference standards), and by ensuring that we apply rigorous system suitability (see Chapter 7) and quality procedures into our methodologies. In relation to HPLC, qualification relates to an assessment of the instrumentation (pump, injector, column compartment, detector) that is used to determine the measured result, whereas validation relates to the applications and methods that are used to generate the results. 

The instrumentation comprises the individual elements that make up the HPLC system, including the pump, injector, column compartment, and detector. Qualification is performed on any element that, if defective, will have a significant effect on the result. Each of these elements must be assessed against a predetermined specification before it can be deemed fit for purpose and used to measure real samples. The specifications used in the qualification of instrumentation are usually provided by the individual vendors of the equipment. Care should be taken because not all manufacturers specifications are the same. 

Temperature can be regulated with the use of a column compartment heater (typical temperature range of 25-80°C) or the air-conditioning can be switched on locally. An increase in temperature reduces the mobile phase viscosity and improves the mass transfer, and compounds elute at a faster rate. This can result in loss of resolution between compounds and can have a detrimental effect on the assay outcomes. A decrease in temperature will have the opposite effect, but can lead to problems nevertheless. It is usual to run HPLC systems at a temperature slightly above ambient (25 C) when a column compartment heater is used. 

SEC conditions instmment. Waters ALC/GPC -150C column, two PLgel mixed mobile phase, DMSO with 0.03 M NaNOj temperature, 80°C for injector and column compartments injection ... 

DMSO + 0.03 M sodium nitrate temperature of column compartment,... 

Up to sixteen separate samples can be loaded on to the carousel and their chromatographic characteristics (flow rate, sensitivity, scale factor, delay time before injection, run time and number of injections up to a maximum of 9), separately loaded into the microprocessor for automatic analyses. The instrument has three compartments which are separately thermostatted-pump, injector and column compartments. The upper temperature is 150°C. The temperature of the compartments are controlled by proportional heaters allowing slow heating and cooling rates to be adopted if required. This minimizes thermal shock to the gel columns and also overheating—two features which help to prolong the life of the columns. 

The column compartment is reasonably large and can hold up to ten 60-cm X 1-cm analytical columns as well as the differential refractometer—the only standard detector supplied. Other detectors can be fitted (IR, UV, viscometers and LALLS), but these are normally fitted externally and require heated extension tubes with additional plumbing to the equipment. The refractometer has a sensitivity of 1 x 10 RI units full-scale deflection, with an average noise level of 5 x 10 RI units. A quartz halogen bulb light source is used for increased sensitivity. Very good temperature control is required when using a RI detector. 

Figure 1 Chromatograph diagrams (L, lab P, process). LI, column compartment P2, column and valving compartment L2, autosampler P2, sample handling valve(s) L3, pneumatic connections (rear) P3, pneumatic connections (side) and regulator compartment L4, user keypad and electronics P4, electronics compartment L5, data and remote control connections (rear) P5, process computer and local or remote control connection (side) L6, splii/splitless injector with septum P6, process sample input to inject valve and return outlet L7, detector and vent P7, detectors (interior).

Some suppliers have integrated the installed analytical column in their hardware systems for identification purposes. This can be performed by a funk chip that is connectedto the column and can be recognized by the column compartment (Agilent) or by a clip that is connected to the column that is then fixed at the column compartment (Waters). 

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