Stationary phase resolution affected

The stationary phase is selected to provide the maximum selectivity. Where possible, the retention factor is adjusted (by varying the mobile phase composition, temperature, or pressure) to an optimum value that generally falls between 2 and 10. Resolution is adversely affected when k 2, while product dilution and separation time... 

Other specifications of the porous materials that affect the performance of HOPC include pore volume. A larger pore volume, or equivalently closer packing, of the porous materials increases the ratio of the volume of the stationary phase to the volume of the mobile phase. The difference causes a shift in the segregation boundary in the partitioning and a change in the resolution. 

The rate of flow of the carrier gas affects resolution. A simple analogy here will make the point. Wet laundry hung out on a clothesline to dry will dry faster if it is a windy day. The components of the mixture will blow through the column more quickly (regardless of the degree of interaction with the stationary phase) if the carrier gas flow rate is increased. Thus, a minimum flow rate is needed for maximum resolution. It is known, however, that at extremely slow flow rates resolution is dramatically reduced due to factors such as packing irregularities, particle size, column diameter, etc. 

Tesafova, E. and Bosdkova, Z., The factors affecting the enantiomeric resolution and racemization of oxazepam, lorazepam and promethazine on macrocyclic antibiotics-bonded chiral stationary phases, Chemia Analityczna, 48, 439, 2003. 

Mechanical and chemical stability of novel stationary phases are basic requirements concerning their application. A lack in stability generally causes a loss in resolution and thus reduces column efficiency. In addition, the reproducibility of retention times, being important for qualitative analysis, may be affected. Evaluation of the mechanical stability of polymeric stationary phases is usually accomplished by the determination of the pressure drop across the column, when employing solvents of different polarity within a wide range of flow rates. A stationary phase can be considered as mechanically stable if a linear relationship between applied flow rate and resulting back pressure is obtained. 

MDGC is useful for separating compounds of an essential oil using two columns in line with different polarities. Through column-switching techniques, selected impure compounds in the first column can be diverted to the second column to ensure their complete separation. If the second column is chiral, then enantiomers potentially can be separated. The selected chiral stationary phase affects the resolution and separation drastically [73]. 

Flow Rate Accuracy. One of the key performance requirements for the pump module is the ability to maintain accurate and consistent flow of the mobile phase. This is necessary to provide stable and repeatable interactions between the analytes and the stationary phase [8,9]. Poor flow rate accuracy will affect the retention time and resolution of the separation. The flow-rate accuracy of the pump can be evaluated simply by calculating the time required to collect a predetermined volume of mobile phase at different flow rate settings. For example, the flow-rate accuracy at 2 mL/min can be verified by using a calibrated stopwatch to measure the time it takes to collect 25 mL of effluent from the pump into a 25-mL volumetric flask. A calibrated flow meter can be used to determine the flow rate as well. The typical acceptance of the flow rate accuracy is listed in Table 11.1. A steady backpressure may be required, depending on the requirement of the system. 

Chiral resolution is a matter of great importance. For example, the food and beverage industry is increasingly concerned with enantiomeric separations, because they can affect flavor, fragrance, and nutrition and can be used to monitor fermentation or product adulteration. Separations are carried out mainly on chiral stationary phases (CSPs). 

Separation speed and ease of use seem to be the primary factors driving changes in HPLC instrumentation. Resolution efficiency and stationary phase stability, especially at high pH, are the primary factors affecting current changes in column technology. 

The quality of chromatographic separation depends on the composition of the stationary phase of the column and the instrument settings. The temperature regime of the oven the flow rate of the mobile phase gas through the column the temperature of the injection port—all of these factors influence compound retention time and peak resolution. The reduction in the chromatographic analysis time may adversely affect compound resolution. Because commercial laboratories always balance a need for a sufficient resolution with a need to perform analysis within the shortest possible period of time, the quality of chromatographic resolution is often traded for the speed of analysis. 

The chromatographic performance and separation mechanism seems to be sensitive not only to the type of stationary phase but also to the purity of the packing material, in particular to the metal content. At low pH, McKeown et al. observed differences in ion-exchange activity and EOF which affected the resolution and elution order between the columns [45]. The study concluded that better peak shapes could be achieved by employing silica packings with lowest metal content. However, the efficiency was significantly lower than in the case of the less pure phases. 

It has been known since the early days of liquid chromatography that the size of the particle used for the stationary phase affects the separation, or resolution, in a rather direct way the smaller the particle, the better the separation. 

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