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1.9 µm HPLC Columns
Optimizing Performance –
Making Your Assay More Productive
– Faster Analyses – Improved Resolution – Enhanced Sensitivity – Increased Peak Capacity – Easier Method Development Analyze • Detect • Measure • Control™
1.9 µm Hypersil GOLD Columns
1.9 µm Hypersil GOLD Columns
Based on high purity silica technology, along with a proprietary bonding and end-capping procedure, Hypersil GOLD™ columns offer improved chromatography and a new solution to thechallenges facing your lab. The outstanding peak symmetry even for basic compounds deliveredby Hypersil GOLD has already improved productivity in laboratories all over the world.
The use of sub-2 µm particles is becoming increasingly popular for The 1.9 µm Hypersil GOLD columns build on the recent advances applications in either High Throughput Screening (HTS) assays or in in technology made with Hypersil GOLD media, and now offer a Ultra High Pressure Liquid Chromatography (U-HPLC). 1.9 µm practical solution to improving laboratory throughput with your Hypersil GOLD columns offer advantages over the more traditional existing HPLC equipment. While 1.9 µm Hypersil GOLD columns can systems containing 3 and 5 µm particles through: be used for U-HPLC applications, the narrow particle size distribution • Operating at higher flow rates without compromising
means that improved speed and resolution can still be attained while maintaining the pressure within the range of conventional • Shorter analysis times
HPLC systems.
• Improvements in resolving power and sensitivity
This technical guide will focus on using existing HPLC equipment • A choice of chemistries for different selectivity options
and how to optimize the system to get the best performance with1.9 µm particles and harvest their benefits.
Figure 1: Make your assay more productive –use 1.9 µm Hypersil GOLD columns to provide extra resolution, speed, sensitivityand selectivity.
C O N T E N T S
Section 1: Exploiting the Theory
Provides the theoretical considerations to the
use of sub-2 µm particles for speed, resolution
and efficiency.
Section 2: System Optimization
Provides instrument considerations to enhance
the performance of sub-2 µm particles.
Section 3: Tips for Method Transfer
Provides guidelines to help you switch your
current isocratic or gradient methods to
sub-2 µm particles.
Section 4: Column Reproducibility
Demonstrates the reliability of the 1.9 µm
Hypersil GOLD columns and provides
ordering information.
Section 1: Exploiting the Theory
The separating power of a chromatographic column can be described Linear Velocity and Flow Rate
by the height equivalent to a theoretical plate (H), which varies with The mobile phase linear velocity (u) is related to the mobile phase the linear velocity (u) of the mobile phase as it passes through the flow rate in the column (F) and the column cross sectional area by column, the particle size (dp), the diffusion coefficient in the mobile phase (Dm) and is given by Equation 1: where ε is the volume fraction of the column between the particles where A, B, and C are constants relating to particle morphology, and dc is the column internal diameter.
particle size and column packing.
Equation 4 tells us that in order to maintain a constant mobile The lower the plate height, the higher the separating power of phase linear velocity, the mobile phase flow rate needs to be lower the column, and there is an optimum linear velocity for which H is for a narrower column. Figure 3 illustrates the linear velocities and a minimum and the plate number, or efficiency, N, is a maximum.
flow rates for three common column internal diameters. For each of The plate height is related to the efficiency by Equation 2: these the optimum flow rate for columns packed with 1.9 µm particles is highlighted.
where L is the column length.
Figure 2: The Advantages of 1.9 µm Particles. The van Deemter plot highlightshow columns packed with smaller particles can operate over a wider range Figure 3: Optimal Flow Rates for Columns Packed with 1.9 µm Particles. of linear velocity and maintain higher efficiencies. This allows the use of As column ID decreases, the mobile phase flow rate must decrease in order higher flow rates, resulting in considerable improvements in speed, without to maintain a constant linear velocity.
loss in performance.
These equations also show that the efficiency is inversely proportional to the square of particle size. In short, the efficiency increases as particle size decreases. The van Deemter curve illustrates how the limitations in column efficiency at higher linear velocities can be overcome byemploying smaller particles. As the particle size is reduced, the optimum mobile phase velocity (u) is increased and the curvebecomes flatter. Therefore, columns packed with smaller particlescan be operated over a wider range of linear velocity while maintaining high efficiencies. This has the resultant effect ofenabling the use of higher flow rates to decrease analysis timewithout compromising performance.
1.9 µm Hypersil GOLD Columns
The aim of a chromatographic separation is to maximize resolution resolution, a 150 mm column packed with 5 µm particles would be while minimizing analysis time. Resolution (Rs) is proportional to the required. However, if the particle size is reduced to 1.9 µm then only square root of separation efficiency (N), as described by Equation 5 50 mm of column is needed to obtain the same 13,500 plates. For a (which expresses resolution as a function of capacity factor (k), constant flow rate, analysis time would be reduced approximately selectivity (α), and efficiency (N)). Efficiency is in turn inversely propor- 3-fold with this change in particle size and column length. tional to the square diameter of particle size (dp), as discussed above.
Equation 5 also points out another very effective way to increase Resolution and analysis time are determined by the ratio of the resolution of two chromatographic bands – increasing the column length to particle size. When particle size is reduced, column selectivity factor (α) by manipulating column chemistry or mobile length can also be reduced while keeping separation efficiency phase composition. Thermo offers 1.9 µm Hypersil GOLD columns constant (and therefore R in three chemistries. s if all other experimental conditions remain unchanged). Figure 4 illustrates this concept. For example, if • Hypersil GOLD gives outstanding peak shapes using generic
gradients with C18 selectivity. 13,500 plates (green line on graph) are needed to obtain the required • Hypersil GOLD aQ is a C18 polar endcapped stationary phase
which can be used for challenging reverse phase separationsemploying highly aqueous mobile phases. • Hypersil GOLD PFP is a perfluorinated phenyl stationary phase
which can offer alternative selectivity in reverse phase applications.
Figure 5 illustrates the difference in selectivity between the three phases.
Figure 4: Iso efficiency plots. High throughput, high efficiency separationscan be obtained with short columns packed with small particles.
Hypersil GOLD
Columns:
1.9 µm, 50 x 2.1 mm A: 25 mM NH4OAc pH 5 10 – 100% B in 3 min UV @ 254 nm (2 µL flow cell) Injection Volume: 0.5 µL Analytes: 1. 2,4-diaminotoluene2. 4,4-oxydianiline3. o-toluidine4. 2-methoxy-5-methylaniline5. 2,4,5-trimethylaniline6. 4,4-methylene-bis (2-chloroaniline)7. Impurity from analyte No. 6 Figure 5: Effect of column chemistry (C18 selectivity, C18 polar endcapped and pentafluorophenyl) on the separation of aromatic amines. Note the change in elution order for compounds 2 and 3 for Hypersil GOLD PFP.
Sensitivity or signal-to-noise ratio is related to concentration at Decreasing column length (Figure 6 step 1) results in a shorter peak apex, cmax, which depends on chromatographic parameters run time if all other experimental parameters are kept unchanged.
such as efficiency (N), injection volume (Vi), column length (L), Note that, when decreasing the column internal diameter (Figure 6 column internal diameter (dc) and capacity factor (k), as expressed step 2), it is necessary to adjust the mobile phase flow rate to main- tain a constant mobile phase linear velocity through the column.
By decreasing the particle size (Figure 6 step 3), efficiency is increased, which leads to more sensitivity. Using smaller particlesalso allows us to increase the mobile phase flow rate without losing This dependence points out some ways to increase the sensitivity: separation performance, which also increases the speed of analysis.
reduce the length and reduce the internal diameter of the columnand increase separation efficiency. This is illustrated in Figure 6 forthe separation of four steroids.
Hypersil GOLD (dimensions vary as shown)
2O/ACN (50:50) +0.1% Formic acid Injection Volume: 60 nL Step 2: Reducing column ID,
decreasing flow rate to maintain
Step 3: Reducing particle size
Step 1: Reducing column length
constant linear velocity
and increasing flow rate
Flow Rate: 1 mL/min Flow Rate: 1 mL/min Flow Rate: 0.2 mL/min Flow Rate: 0.55 mL/min Height = 2476
Height = 3381
Height = 13729
Height = 16728
Figure 6: Improving sensitivity by reducing column length (L), column ID (dc) and particle size (dp). This series of chromatograms demonstrates how a step-wisedecrease in column length, column ID and particle size can affect peak height, and therefore sensitivity. The parameters that have been changed in each stepare highlighted in red.
1.9 µm Hypersil GOLD Columns
The opportunity to increase sample throughput has generally been Numerous experimental parameters can be manipulated to improve compromised by a trade-off between column dimensions and opera- speed, efficiency or resolution of a chromatographic separation.
tional parameters associated with system capability. To demonstrate However, serious trade-offs existed to achieve both until the how the use of smaller particles in this environment gives an introduction of sub-2 µm particles, such as Hypersil GOLD 1.9 µm additional route to improve sample turnaround, a separation of columns. The exceptional range of optimal linear velocity that can seven phenone compounds performed on a 200 x 2.1 mm, 5 µm be achieved using sub-2 µm particles allows the use of high flow column was transferred to shorter columns packed with smaller rates to reduce analysis time while maintaining chromatographic particles to reduce analysis time. The flow rate and gradient time were efficiency. In addition, the smaller particles allow for the reduction adjusted to reflect the changes in column length and particle size.
in column length while maintaining efficiency, providing further The 1.9 µm particles facilitate a decrease in run time from 6 to 0.5 gains in speed of analysis at high flow rates without exceeding the minutes, while maintaining baseline resolution of the seven phenones, pressure limits of standard HPLC systems.
as seen in Figure 7. In the top chromatogram, the column temperaturehas also been increased to achieve additional speed.
Hypersil GOLD
247 nm (0.1s rise time) Injection Volume: 6. Heptanophenone7. Octanophenone 50 x 2.1 mm, 1.9 µm
Flow Rate: 1000 µL/mintg = 0.4 min; Temp.: 60 °C 50 x 2.1 mm, 1.9 µm
Flow Rate: 1000 µL/mintg = 0.4 min 100 x 2.1 mm, 1.9 µm
Flow Rate: 1000 µL/mintg = 0.7 min 200 x 2.1 mm, 1.9 µm
Flow Rate: 600 µL/mintg = 1.5 min 200 x 2.1 mm, 3 µm
Flow Rate: 400 µL/mintg = 2.3 min 200 x 2.1 mm, 5 µm
Flow Rate: 250 µL/mintg = 3.5 min Figure 7: Effect of column length, particle size and operating conditions on run time and peak width at baseline (W). Section 2: System Optimization
We have seen that when using columns packed with 1.9 µm particles, As a guide for good chromatography, the extra column band analyses can be performed with a high linear velocity through the broadening, originating from the injector, flow cell and tubing (see column without loss in performance. To obtain the best data using Equation 8), should not exceed 10% of the total band broadening.
fast chromatography, however, it is critical that the LC instrument The extra column effects are more significant for scaled down sepa- system is optimized to operate under these conditions. All system rations (column volume decreases) and for less retained peaks components for the assay should be considered. System volume which have a lower peak volume. It is therefore critical to minimize (connecting tubing ID and length, injection volume, flow cell volume extra column dispersion. Figure 8 highlights the improvements that in UV) must be minimized, detector time constant and sampling rate can be made in resolution, asymmetry and efficiency by reducing the need to be carefully selected, and when running fast gradients injection volume (left), the flow cell volume (center) and column to pump dwell volume needs to be minimal.
detector connecting tubing ID (right). cell + τ2F2 + c • lc • F Band broadening, which has a detrimental effect on the chromato- graphic performance, can occur in the column, in the autosampler, In addition to the volumetric effects, the time constant of the in the tubing connecting the column to injector and detector and in detector (τ, response rate) and the scan rate may also contribute to the detector flow cell. These band broadening effects which occur the broadening of the peak, and should be considered. in the fluid path of the HPLC instrument are volumetric effects; each Detector Time Constant
contributes a variance (σ) to the width of the chromatographic bandand are additive: The central term covering flow cell volume in Equation 8 also showsthat dispersion in the detector is dependent upon the detector time constant τ. Reducing the time constant will reduce the observed where the subscripts mean: col – inside column, ext – extra column.
19% increase in efficiency
163% increase
22% improvement in asymmetry
215% increase in efficiency
in efficiency
137% improvement in resolution
Hypersil GOLD
Column:
1.9 µm, 50 x 2.1 mm Mobile Phase: H2O/ACN (50:50) + 0.1% Formic acidFlow Rate: Analytes:1. Cortisone2. 11-α-hydroxyprogesterone3. 17-α-hydroxyprogesterone4. Progesterone Figure 8: The effects of minimizing volume dispersion within the system.
1.9 µm Hypersil GOLD Columns
Detector Sampling Rate
Dwell Volume
With 1.9 µm particles, operating parameters can be optimized to The HPLC pump dwell volume is extremely important when running give fast analysis. This results in narrow chromatographic peaks high speed applications using ballistic gradients, typical of high which may be of the order of 1-2 seconds in width. It is important to throughput separations on small particle packed columns. This is scan the detector (whether it is UV or MS) fast enough to achieve because the pump dwell volume affects the time it takes for the optimum peak definition, otherwise resolution, efficiency and gradient to reach the head of the column.
analytical accuracy will be compromised. This is illustrated in If we consider a method using a flow rate of 0.4 mL/min and Figure 9, which illustrates a loss of resolution and peak height a fast gradient of 1 minute, the theoretical gradient reaches the when only five scan points cover the peak.
column immediately (Figure 10). A pump with a 65 µL dwell volume(such as used in the Thermo's Accela HPLC) will get the gradientonto the column in 9.75 seconds. A traditional quaternary pump witha dwell volume of 800 µL will take 2 minutes to get the gradient tothe column. When running rapid gradients this is too slow as can beseen on the example chromatogram.
In Figure 10 A and B the same 2 minute gradient was run on a pump with a 800 µL dwell volume, and a pump with a 80 µL dwellvolume. The chromatograms are very different: for chromatogram A,it was necessary to introduce an isocratic hold at the end of the 2 minutes gradient to allow elution of the analytes. In these conditions the pump dwell volume can double the run time, and it also impacts on column re-equilibration at the end of the run.
Figure 9: Effects of detector sampling rate on peak shape. With the use of1.9 µm particles, it is important that the detector scan rate is fast enough tomaintain peak definition. In this figure, there is a dramatic difference in peakshape when only five data points (slower scan rate) are acquired comparedto 45 data points (faster scan rate). 1.9 µm, 50 x 2.1 mm 1. Sulphaguanidine 2O + 0.1% Formic acid B: ACN + 0.1% Formic acid 2. Sulphamerazine3. Sulphamonomethoxine 4. Sulphaquinoxaline Injection Volume: 0.5 µL • Theoretical gradient reaches the column at • 65 µL dwell volume at 0.4 mL/min, gradient
reaches the column at 9.75 seconds • 180 µL dwell volume at 0.4 mL/min, gradient
reaches the column at 27 seconds • 800 µL dwell volume at 0.4 mL/min, gradient
reaches the column at 120 seconds Figure 10: The effects of pump dwell volume on the separation. When running rapid gradients it is important to utilize a pump with a small dwell volume.
For the fastest results from small particle columns, you must In order to tune your assay to your HPLC system, remember that as ensure that your system can function reliably at higher operating the particle size is reduced, resistance to flow increases, causing pressure. Figure 11 shows that the flow rates for optimum efficiency pressure drop to increase within the system. This is approximated (taken from the van Deemter plot) can lie within the limits of by the Equation 9 below: conventional HPLC systems, even for 1.9 µm particles.
Pressure Drop (psi) 250 L η F / d2 d2 where L = Column Length (mm) η = Mobile Phase Viscosity (cP)F = Flow Rate (mL/min)dp = Particle Diameter (µm)dc = Column Internal Diameter (mm) This equation shows that the pressure drop across the column • The length of the column. Longer columns have higher
pressure drops.
• The ID of the column. Narrower columns have higher
pressure drops.
• The diameter of the particles packed within the column.
The smaller the particles the higher the pressure drop. This is a Figure 11: System pressure considerations with 1.9 µm particles. The shaded squared relationship and has a significant effect.
areas indicate typical system pressures at the optimum flow rates, measured • The flow rate. A higher flow rate will result in a higher
on a 20 mm length column.
pressure drop.
• The viscosity of the mobile phase. Higher viscosities will
result in higher pressure drops. Increased temperatures reducethe viscosity, enabling a higher flow rate to be used for an equivalent pressure drop.
1.9 µm Hypersil GOLD Columns
Section 3: Tips for Method Transfer
As well as particle size, column dimensions can be scaled down toachieve faster separations. Care must be taken when transferring amethod to shorter columns with smaller particles to ensure operatingflow rate and gradient profiles are scaled to keep the assay profileconsistent. An understanding of some practical calculation routineshelp to achieve the scaling.
Isocratic Method Transfer
1) Adjust flow rate, keeping linear velocity constant between the original and new method.
Column 1: 100 x 4.6 mm, 5 µm Column 2: 100 x 2.1 mm, 1.9 µm Flow Rate 1: 1.0 mL/min Flow Rate 2: 0.55 mL/min F1 – original flow rate (mL/min) F2 – new flow rate (mL/min) dc1 – original column internal diameter (mm) dc2 – new column internal diameter (mm) dp1 – original column particle size (mm) dp2 – new column particle size (mm) Entering the column dimensions for both systems and the original flow rate results in a calculated flow rate of 0.55 mL/min for system 2. Transferring this method to a columnpacked with 1.9 µm particles gives a 4.8x increase in sensitivity and 2x increase in speed.
Hypersil GOLD
Column: 100 x 4.6 mm, 5 µm
Column: 100 x 2.1 mm, 1.9 µm
+ 0.1% Formic acid Injection Volume: 2 µL Analytes:1. Cortisone2. 11-α-hydroxyprogesterone3. 17-α-hydroxyprogesterone4. Progesterone 4.8 x higher sensitivity; 2 x speed
Figure 12: Isocratic method transfer to narrower ID column packed with smaller particles.
2) Adjust injection volume.
Column 1: 100 x 4.6 mm Column 2: 50 x 2.1 mm Vi1 – original injection volume (µL) Vi2 – new injection volume (µL) dc1 – original column internal diameter (mm) dc2 – new column internal diameter (mm) L1 – original column length (mm) L2 – new column length (mm) Entering the column dimensions for both systems and the original injection volume results in an injection volume of 1 µL for system 2.
In practical terms it is often not possible to follow the equationexactly, due to sample constraints, but the chromatographer shouldbe aware that smaller columns packed with 1.9 µm particles willrequire a smaller injection volume.
Method Transfer for Increased Resolution
Method Transfer for Increased Speed
When transferring a method to a column packed with small particles If the analyte peaks are well separated and high throughput is the it is possible to optimize certain chromatographic parameters to most important consideration for a method, it is possible to increase further increase resolution for difficult separations in complex the chromatographic speed by further reducing the column length matrices. In the example below, using the same analytes, the column and increasing the flow rate. In Figure 14, the calculated method length was maintained and the flow rate and column ID decreased.
transfer flow rate comes out at 0.55 mL/min. This and the shorter This results in a 13% increase in resolution between peaks 1 and 2, column used reduces the retention time for peak 4 from 416 seconds as shown in Figure 13.
to 95 seconds. To obtain an even faster analysis, the separation hasbeen repeated on a shorter column with a higher flow rate, giving aretention time for peak 4 of 29 seconds. This is 14 times faster thanthe original method and underlines the power of small particles forfast analysis.
Hypersil GOLD
Mobile Phase:
+ 0.1% Formic acid Injection Volume: 2 µL Analytes:1. Cortisone2. 11-α-hydroxyprogesterone3. 17-α-hydroxyprogesterone4. Progesterone 13% Higher Resolution
Figure 13: Isocratic method transfer to improve resolution. Hypersil GOLD
Mobile Phase:
+ 0.1% Formic acid Injection Volume: 2 µL Analytes:1. Cortisone2. 11-α-hydroxyprogesterone3. 17-α-hydroxyprogesterone4. Progesterone 14 x Faster
Figure 14: Isocratic method transfer to improve speed. The column has been shortened further and the flow rate increased to 0.8 mL/min.
1.9 µm Hypersil GOLD Columns
Gradient Method Transfer
1) Adjust flow rate, keeping linear velocity constant between the original and new method.
(in accordance with the isocratic method transfer equations on page 10) 2) Adjust injection volume. (in accordance with the isocratic method transfer equations on page 10)
3) Adjust gradient, keeping initial and final composition unchanged.
Column 1: 150 x 4.6 mm Column 2: 50 x 2.1 mm Gradient Profile 1 (tg1): 5 to 50% in 15 mins Gradient Profile 2 (tg2): 5 to 50% in 5 mins Column Volume: 1.7 mL Column Volume: 0.11 mL tg1 – gradient time in original method (min) tg2 – gradient time in new method (min) V01 – original column volume (mL) V02 – new column volume (mL) F1 – original flow rate (mL/min) F2 – new flow rate (mL/min) Entering the column dimensions and flow rates for both systems and the original gradient gives a tg of 5 minutes for system 2.
Column Volume (V0)
V0 – column void volume (mL)L – column length (cm) r – column radius (cm) 0.68 is the approximate fraction of the column volume occupied by mobile phase (for porous particles) In the example chromatograms on the following page (Figure 15),a gradient method for separating sulphonamides has beentransferred from a standard 5 µm analytical column to a shortercolumn containing 1.9 µm particles. This has been performedstepwise to illustrate the effect of each parameter change. For each step the gradient and flow rate have been scaled in accordance with the previous calculations. The final chromatogram illustrates where the column has been furthershortened and the flow rate and gradient made even faster to increase throughput.
The table compares key parameters from the start and end methods and shows that even under ultra fast conditions (thefinal gradient is 15 times faster than the starting gradient),baseline separation is still achieved. The peak heights are similar even though the injection volume is 10% of the startinginjection volume accentuating the increased sensitivityachieved using 1.9 µm particles.
Hypersil GOLD
Column:
150 x 4.6 mm, 5 µm (V = 1.7 mL)
5 to 100% B in 15 minutes
1. Sulphaguanidine 2. Sulphamerazine 2O + 0.1% Formic acid 3. Sulphamonomethoxine B: ACN + 0.1% Formic acid 4. Sulphaquinoxaline Injection Volume: 5 µL
Flow Rate:
150 x 4.6 mm, 5 µm
30 x 2.1 mm, 1.9 µm
Column: 100 x 4.6 mm, 5 µm (V = 1.1 mL)
Gradient: 5 to 100% B in 10 minutes
Flow Rate: 1 mL/min
Column: 50 x 2.1 mm, 5 µm (V = 0.11 mL)
Gradient: 5 to 100% B in 5 minutes
Flow Rate: 0.2 mL/min
Column: 50 x 2.1 mm, 1.9 µm (V = 0.11 mL)
Gradient: 5 to 100% B in 2 minutes
Flow Rate: 0.55 mL/min
Injection Volume: 0.5 µL
Column: 30 x 2.1 mm, 1.9 µm (V = 0.07 mL)
Gradient: 5 to 100% B in 1 minutes
Flow Rate: 0.7 mL/min
Injection Volume: 0.5 µL
Cycle Time Reduced by 10 Fold
Figure 15: Stepwise transfer of a gradient method to 1.9 µm Hypersil GOLD. In each step, the gradient and flow rate are scaled using the calculations presented. In step 1, column length was reduced. Step 2 shows the effects of a reduction in column ID. In Step 3 particle size is reduced to 1.9 µm. In Step 4, the column length is decreased again, while the flow rate is increased.
1.9 µm Hypersil GOLD Columns
Section 4: Column Reproducibility
Hypersil GOLD columns are exceptionally reproducible for reliable columns, based on 1.9 µm particles, designed for improved chromatography, column after column. This allows the user to be chromatography. Hypersil GOLD columns are manufactured in ISO confident that assays developed with Hypersil GOLD columns will 9001:2000 accredited laboratories under strict protocols using a be robust and stable for the life of the assay, making them the ideal robust manufacturing procedure and extensive quality control testing.
choice for new method development.
To obtain the most accurate results when performing quantitative Built on 30 years of experience in product development and analysis it is important that key chromatographic parameters such manufacturing of HPLC media and columns, Thermo continues upon as retention time and peak area remain consistent. its success with the development of a new state-of-the-art family of Figure 16: 1.9 µm Hypersil GOLD Batch Reproducibility. The capacity factorwas measured for a series of peaks across 5 separate batches of 1.9 µm Figure 18: Batch Reproducibility with 1.9 µm Hypersil GOLD Columns. For all Hypersil GOLD. The reproducible values provide the chromatographer with batches, the tailing factor measured for a very basic analyte, is close to confidence that analyte peaks will elute at the same time, every time. unity, highlighting the excellent symmetrical peak shape which is characteristic of all Hypersil GOLD columns. Figure 17: 1.9 µm Hypersil GOLD Column Reproducibility. 1.9 µm HypersilGOLD columns show excellent reproducibility, column after column. In thisexample, the efficiency of 200 different columns is shown. Efficiency in Figure 19: System Back Pressure with 1.9 µm Hypersil GOLD. The system excess of 160,000 plates/m is consistently achieved, column after column.
back pressure has been measured over eight separate columns at two flowrates. The minimal variation in column back pressure can only be achievedusing consistently well-packed stationary phase with a reproducible narrowparticle size distribution. Peak Area Peak Area Peak Area
The analysis of a mixture of sulphonamides using 1.9 µm Hypersil GOLD column gives a linear response over a range of concentrations Level 1 (25 ng/µL) between 25 and 250 ng/mL (Figure 20).
Level 2 (50 ng/µL) The table shows excellent reproducibility for retention times Level 3 (250 ng/µL) (< 1% RSD) and peak area (< 2% RSD) over six injections. This highlights that accurate data can be obtained under fast chromato- graphic conditions, where peak widths might be as narrow as Level 1 (25 ng/µL) 1-2 seconds.
Level 2 (50 ng/µL) Level 3 (250 ng/µL) Hypersil GOLD, 1.9 µm, 50 x 2.1 mm
A: H2O + 0.1% Formic acid Level 1 (25 ng/µL) B: ACN + 0.1% Formic acid 1. Sulphaguanidine 2. Sulphamerazine Level 2 (50 ng/µL) 3. Sulphamonomethoxine 4. Sulphaquinoxaline Level 3 (250 ng/µL) Injection Volume: 0.5 µL Level 1 (25 ng/µL) Level 2 (50 ng/µL) Level 3 (250 ng/µL) Figure 20: Calibration Curves for Sulphonamides. 1.9 µm Hypersil GOLDcolumns produce a linear response over a range of concentrations.
Length (mm)
320 µm ID
Hypersil GOLD aQ
Hypersil GOLD PFP
Other custom column dimensions are available. Please call your local Customer Service for more information.

Source: http://www.hplc.at/Gold.pdf