Agilent Technologies Optimizing 1100 High Sample Throughput Technical Note

organic compounds per unit time are purposely produced in order to be screened against a variety of biological targets for drug detection.

Introduction

High sample throughput is particularly important in the pharmaceutical industry, for example, in combinatorial chemistry and metabolite studies. In combinatorial chemistry a large number of

An ideal instrument configuration for high sample throughput comprises:

• an autosampler such as a standard HPLC autosampler for 2-ml vials or a sampler with microtiter and deep-well plates,

• a high-pressure gradient pump for lowest delay volume,

• a UV detector such as a diode array detector or variable wavelength detector, and

• a mass selective detector for additional mass and structural information (optional).

Optimizing the Agilent 1100 Series System for High Sample Throughput

Technical Note

Agilent Technologies

Innovating the HP Way

The limiting factor for high throughput in such systems is the speed of the HPLC analysis. Standard HPLC cycle times from injection to injection for gradient analysis lie between 15 and 20 min using columns of 100 to 200 mm in length.

To significantly increase the sample throughput, cycle times must be shortened. This can be achieved using fast gradients with short columns and high flow rates. In the following example we demonstrate how to optimize the Agilent 1100 Series high-pressure gradient system to obtain rapid gradients and high sample throughput. Hints are given on the influence of chromatographic parameters on cycle times, and on how run times of less than two minutes can be expected to affect performance.

Equipment

All HPLC experiments were carried out on the Agilent 1100 Series high-pressure gradient system comprising:

• Agilent 1100 Series high-pressure pump for lowest delay volume. In this design each solvent is pumped by its own pump assembly, and mixing takes place on the high-pressure side. This means gradient changes reach the column much faster than in low-pressure gradient systems where mixing takes place on the low-pressure side.

• Agilent 1100 Series vacuum degasser for optimum baseline stability.

• Agilent 1100 Series autosampler for sampling from 2-ml standard vials.

• Optional Agilent 220 micro plate sampler for flexible sampling from deepwell and/or microtiter plates.

• Agilent 1100 Series thermostatted column compartment for highest stability from 10 °C below ambient up to 80 °C.

• Agilent 1100 Series diode array detector with standard flow cell (10-mm pathlength, 13-microliter volume).

• Optional Agilent 1100 Series variable wavelength detector.

• Optional Agilent 1100 Series LC/MSD module for mass and structural information.

• Agilent ChemStation with 3D HPLC single instrument software for instrument control, data handling and sample tracking.

Compounds and chromatographic conditions

For our experiments we selected the following compounds which differ considerably in polarity:

• caffeine

• primidone

• phenacetin

• mandelic acid benzylester

• biphenyl

The chromatographic conditions are listed next to the figures.

Optimization of chromatographic parameters

The following parameters have to be adapted to obtain short cycle times, sufficient resolution and best performance over a wide range of polarity:

• column length

• gradient

• flow rate

• delay volume

• data rate of detector

• column temperature

The aim was to achieve cycle times of about 2 min and baseline separation for all compounds.

Influence of column length on run time

For a standard column with a length of 100 mm and an id of

4.6 mm, run time cycles of about 15 min are good practice. In figure 1, the compounds mentioned in the previous paragraph were analyzed.

Cycle times of 14 min were obtained with excellent resolution for all compounds.

Shorter cycle times are obtained using a short column. In figure 2, the analysis of the same compounds is shown using a 50-mm colum. Cycle times are down to

2.8 min and baseline separation

for all compounds is given, despite decrease in resolution. Shortening the column length was the principal step in achieving reduced cycle times. The example on the next page demonstrates how to shorten the cycle even further.

Column 100 x 4.6 mm ODS

Hypersil, 5 µm

Flow rate 2 ml/min Mobile phase A = water, B = ace-

tonitrile (ACN)

Gradient 5 % B to 95 % B in

12 min to 5 % B in 13 min

Run time 13 min Post run 1 min Diode-array settings 210/8 nm,

ref. wavelength 360/100 nm

Injector volume 5 µl Column temperature 50 °C

Figure 1 Analysis of selected compounds using a 100-mm column

Figure 2 Analysis of selected compounds using a 50-mm column

Column 50 x 4.6 mm Zorbax

SB-C18, 3.5 µm

Flow rate 2 ml/min Mobile phase A = water, B = ace-

tonitrile (ACN)

Gradient 5 % B to 95 % B in

1 min 95 % up to 1.5 min to 5 % B in min

Run time 2 min Post run 0.8 min Diode-array settings 210/8 nm,

ref. wavelength 360/100 nm, response time 0.1 s

Injector volume 5 µl, autosampler in

bypass mode

Column temperature 50 °C

Absorbance [mAU]

400

350

300

250

200

150

100

-50

Absorbance [mAU]

1200

1000

800

600

400

200

1 Caffeine 4 Mandelic acid 2 Primidone benzylester 3 Phenacetin 5 Biphenyl

Time [min]

0.25

0.5

0.75

Time [min]

1.25

1.5

1.75

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