SMT Separation Journals: March 2018 – Volume 02
Hafeez Fatunmbi and Bukola Fatunmbi
Separation Methods Technologies, Incorporated (Newark, Delaware, USA)
Capillary columns made with less than 1 millimeter (mm) internal diameters (i.d.) offer significant theoretical advantages over conventional 4.6 mm i.d. columns. However, current instrumental constraints and manufacturing difficulties have limited their applications. SMT currently offers a comprehensive range of capillary HPLC columns with 0.1 through 0.5 mm i.d. that are very rugged and highly reproducible. These capillary columns offer unprecedented theoretical plate counts of well over a million with minimal loss of performance compared to the corresponding analytical columns and can be used with optimized conventional equipment.
Liquid chromatography/mass spectrometry, LC/MS, is a revolutionary tool in the chemical and life sciences. LC/MS is accelerating chemical research by providing robust separations and identification tool for chemists and biologists in diverse fields. LC/MS is best done with capillary HPLC. Capillary HPLC uses smaller column internal diameters than conventional HPLC. Smaller i.d. columns, for fixed amounts of injected material, produce taller peaks. Taller peaks provide better detection limits for mass spectrometry and other concentration sensitive detectors. For the same amount of material injected, the peak height is inversely proportional to the cross sectional area of the column. However, the use of smaller i.d. columns requires careful planning compared to normal 4.6 mm columns. Traditional problems associated with the inherent incompatibility of HPLC (high liquid pressure) and mass spectrometry (low vapor pressure) have been largely overcome. LC-MS has become a leading technique offering characterization of solute molecules. LC-MS interface techniques such as Atmospheric Pressure Chemical Ionization, Electrospray, Particle Beam or Thermospray can typically handle maximum flow rates of 200 μl/min. As microbore columns (2 mm i.d.) utilize such flows they are commonly used in LC-MS applications. A 50 x 2.1 mm i.d. column , for example, is used for fast speed analysis applications whilst a 250 x 2.1 mm i.d. column is often the preferred choice for more complex separations. Where sensitivity is an issue, as in the analysis of peptides and proteins, 1 mm i.d. columns have been columns of choice.
Capillary columns are ideal for very sample-limited applications because they provide enhanced sensitivity by reducing on-column sample dilution. The detector of choice is often mass spectrometer. Few range of LC-MS columns, in lengths from 10 to 250 mm with i.d. of less than 1.0 mm are currently available in the market. These columns have many potential LC-MS applications including drug metabolism structure elucidation and quantitative studies; protein and peptide identification and sequencing; combinatorial chemistry; and agrochemical identification and quantitative studies. Along this line of applications, rapid analysis columns have been designed to analyze large numbers of samples in as short a time as possible without major sacrifice of column resolution. Quality Control environments particularly benefit from the use of rapid analysis columns giving corresponding increases in productivity including analysis time reduction by over 90%; increased in productivity as high as 10 times; and solvent saving of up to 98% in a wide range of chemistries. Baseline resolutions in these applications have been maintained with use of 1.5 – 5μm particle size silica in variety of pore sizes.
Critical aspects of capillary HPLC include pump design and flow; sample injection volume and technique (focusing); detector internal volume and response; instrument internal volume and sample flow path design; column internal volume and uniform bed design. Sample volume affects measured band width, for example, and an optimum Instrument Bandwidth Performance has been recorded with a 500 nL partial loop (1,000 nL) when the flow rate of 10.0 µL/min was used on capillary columns [2-4].
Good starting flow rates are 50 µL/min for 1-mm columns, 10 µL/min for 0.5-mm columns, and 5 µL/min for 0.3-mm columns. In terms of resolution, there is less of a penalty for running too fast than too slow. For example, for 0.5-mm columns, it is better not to work below 10 µL/min and try working above like 12-15 µL/min in order to optimize the detection limits for the MS. The upper flow limit is generally determined by the associated back pressure. Above 140 bar (2,000 psi) backpressure connections may begin to leak.
In general, increasing the temperature of the column increases resolution. As a consequence, separations are often run at 30-50°C. Running the column in a thermostated column compartment also provides better reproducibility of retention times with consistent separations from day to day. Care must be taken not to exceed the maximum temperature specified for the use of the column. Silica reversed phase columns can have maximum temperatures of 90°C or more. However, operation of such columns at higher temperatures tend to increase the rate of hydrolysis of the siloxane bonds to the functional alkyl ligands.
Reproducibility is a critical issue when working with small sample sizes. SMT columns features include highest sensitivity for smallest sample sizes; high reproducibility; compatibility with all LC/MS interfaces; wide range of internal diameters of 0.1 through 0.5 mm; and bonded phases with variety of pore sizes made with extremely stable Total Coverage™ SAM technology for use with both small and large molecules.SMT capillary columns are now available in a wide variety of bonded phases, pore sizes (60Å, 100Å and 300Å), and column dimensions.
HPLC apparatus and measurement procedures
Chromatographic column, SMT-C3, Cat# PRO-3-100/10.3, (0.3 x 100 mm), was purchased from Separation Methods Technologies, Inc., Newark, Delaware, USA. Modified HP 1090 system (USA), equipped with auto injector, thermostatted column compartment, diode array detector was used. Chromatographic analysis was carried out at temperature of 30°C. The compounds were separated with a mobile phase consisting of acetonitrile/water in the proportion of (30/70, v/v) at a flow rate 0.1 mL/min with injection volume of 0.1 µL (see QC Test Mix Conditions)
QC Test Mix Conditions
Column Efficiency Test:
Column: SMT-C3, Pro-3-100/10.3, 0.3mm x 100mm, 3µm
Mobile Phase: Acetonitrile:water, 30:70 (v:v)
Flow rate: 0.1ml/min
Column Temperature: 30°C
Detector: UV, 254 nm
Injection volume: 0.1 µL
Sample: 1=Uracil, 2=Anthracene (concentration = 5 µg/mL)
Results and Discussion
The analyte peaks were well defined, resolved and free from tailing with tailing factor well below 2 for both peaks and the separation was complete within 10 minutes. Figure 1. Shows a comparison of a capillary column with a standard analytical column. System suitability tests, including column efficiency, selectivity factor and reproducibility, were performed on freshly prepared QC Test mixture on the capillary column and the system was found to be quite suitable Tables 1 and 2. It is interesting to note that SMT-C3 capillary column, when used at a flow rate as high as 100 µL/min (most current studies have been performed using recommended optimum flow rate of 10 µL/min) produces theoretical plate counts of well over two million with very high reproducibility (Table 2). The column offers a rather low backpressure that allows its usage with a 10 times increase in recommended optimum flow rate resulting in much higher resolution.
Figure 1: Typical Chromatogram of Test Solutes
Table 1: Column Performance: Standard Analytical vs Capillary Column
Table 2: Column Performance: Reproducibility of Capillary Column
SMT capillary column offers a rather low backpressure that allows its usage at higher than recommended optimum flow rate; the result is a much higher resolution and unprecedented high performance with theoretical plate counts of two million or more when used with optimized conventional equipment.
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