Plate numbers HPLC standard

As explained elsewhere in this book, resolution in SEC can be expressed in terms of the peak standard deviation and the slope of the calibration curve. As in other HPLC modes, the efficiency of SEC columns can be improved by decreasing particle size. The relationship between column efficiency (or plate number N) and velocity can be expressed i dimensionless (reduced) parameters. The reduced plate height h is equal to the ratio of the height of a theoretical plate and the particle size as shown in Equation (1). The reduced velocity v is equal to the product of the linear velocity and particle size dp divided by the solute diffusion coefficient/), as shown in Equation (2). 

Effect of acetonitrile in HPLC When the recovery of small molecules analyzed in serum is calculated based on the peak height of aqueous standards also treated by acetonitrile similar to serum, a high recovery is obtained. However, when the calculation is based on aqueous standards diluted in the mobile phase (pump solvent) a low recovery is obtained. In analyses of drugs in serum, acetonitrile in the sample decreases the peak height and limits the amount of sample to be injected on the column due to the formation of a short gradient leading to a non-symmetrical peak shape with a decrease in the plate number. Alternative deproteinizing mixtures for acetonitrile in HPLC have been proposed. 

In practice, the design and development of sub-2 tm particles is a significant challenge. As particle size decreases, the optimum flow to reach maximum plate numbers increases and the use of smaller particles is then substantially limited by a rapid increase in pressure drop [49]. Standard HPLC instruments have a maximum operating pressure of about 400 bar (6000 psi), and these systems simply do not have the capability to take full advantage of sub-2 pm particles. 

The efficiency, or plate count of a column N is often calculated as 5.54 (tr/a)2, where tr is the retention time of a standard and a is the peak width in time units at half-height.1 2 5 This approach assumes that peaks are Gaussian a number of other methods of plate calculation are in common use. Values measured for column efficiency depend on the standard used for measurement, the method of calculation, and the sources of extra-column band broadening in the test instrument. Therefore, efficiency measurements are used principally to compare the performance of a column over time or to compare the performance of different columns mounted on the same HPLC system. 

Prior to performing a formal validation, the analytical chemist should have performed some prevalidation during method development. The expectation is that a well-developed HPLC method should subsequently be validated with no major surprises or failures. Prior to validation, specificity and some degree of robustness should be demonstrated. In addition, some form of system suitability criteria will have been established. System suitability evaluates the capability of an HPLC system to perform a specific procedure on a given day. It is a quality check to ensure that the system functions as expected and that the generated data will be reliable. Only if the system passes this test should the analyst proceed to perform the specific analysis. System suitability can be based on resolution of two specified components, relative standard deviation, tailing factor, limit of quantitation or detection, expected retention times, number of theoretical plates, or a reference check. 

The simplest form of an HPLC SST involves comparison of the chromatogram with a standard one, allowing comparison of the peak shape and the peak width baseline resolution. Additional parameters that can be experimentally calculated to provide quantitative SST report include the number of theoretical plates, separation factor, resolution, tailing or peak asymmetry factor, accuracy, and precision (RSD of six measurements). Resolution may also be combined with a selectivity test to check the resolution of the analytes from components present in the sample matrix. If matrix components interfere with a method, a matrix blank may be included in the SST. Peak shape and asymmetry, or tailing factor, can... 

The narrower the peaks will he, (i.e., the smaller wb ) the better the separation. The ability to achieve narrow peaks is what has made HPLC such a powerful technique. The peak width (the standard deviation) is an indication of peak sharpness and of the number of theoretical plates. Sharpness of peak reflects how good a column is. The sharper the peaks, the more sample components can be separated in a given time. 

Hexachlorobenzene exhibited an HPLC peak at a retention time of 13.36 min. The widlh of the peak at its base was 2.18 min. The standard deviations s for the two time measurements were 0.043 and 0.061 min. respectively. Calculate (a) the number of plates in the column and (b) the standard deviation for the computed result. 

For chromatographers who do not use a sufficient number of columns per year to economically justify the purchase of a HPLC column packing facility, or who cannot afford the time sometimes required to obtain consistent results, the purchase of prepacked columns is advised. Indeed, because some materials are difficult to pack, many manufacturers do not release the bulk material (see for example Tables 2 and 4). The quality and reliability of pre-packed columns has improved considerably over the last few years, and most manufacturers now provide a test chromatogram in which a standard procedure is used to calculate column performance often a written guarantee is supplied. The test report will specify the efficiency of the column expressed in terms of the number of theoretical plates per column or metre, and will also show the test conditions used to obtain the result (mobile phase composition, flow-rate, injection volume, detection wavelength and attenuation, temperature and test sample components). It will also indicate the back-pressure the column will typically exhibit, under the test conditions. This facility is extremely useful to the chromatographer who can, on receipt of the column, repeat the test procedure for the column. Any discrepancy in the result can be due to one or more of the reasons listed below. 

With respect to the TLC plates, there are two major advances. The first was the introduction to high performance TLC (HPLC) plates produced from silica gel of between 5 and 10 fim compared to the 12 to 25 p,m commonly used for standard TLC plates. This resulted in more rapid and efficient chromatographic separation and reduced bond broadening, thus producing higher sensitivity. Second were a large number of bond-phases, of the type originally produced for HPLC, such as C2, Cg, C12, C18, aminopropyl, cyanopropyl, diphenyl, and the so called chiral phase which are used in TLC. 

Henrich developed a comprehensive TLC method for identification of surfactants in formulations (4). She specified two reversed-phase and four normal phase systems, with detection by fluorescence quenching, pinacryptol yellow and rhodamine B, and iodine. Prior to visualization, one plate was scanned with a densitometer at 254 nm, and UV reflectance spectra were recorded for each spot detected. Tables were prepared showing the Rf values of 150 standard surfactants in each of the six systems, along with the reflectance spectra and response to the visualizers. This system allows for systematic identification of compounds of a number of surfactant types (LAS, alcohol sulfates and ether sulfates, alkane sulfonates, sufosuccinate esters, phosphate compounds, AE, APE, ethoxylated sorbi-tan esters, mono- and dialkanolamides, EO/PO copolymers, amine oxides, quaternary amines, amphoterics and miscellaneous compounds). Supplementary analysis by normal phase HPLC aided in exactly characterizing ethoxylated compounds. For confirmation, the separated spots may be scraped from one of the silica gel plates and the surfactant extracted from the silica with methanol and identified by IR spectroscopy. 

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