HPCE

HPCE Buffers

IEF Markers

Anion-HPCE-Kit

HPCE Buffers

• Introduction
• Quality Guarantee
• Product List

Introduction

The rapidly growing capabilities of HPCE have created a great demand for reagents of an appropriate quality.

With the introduction of a series of ready-to-use buffers covering the pH range from 2.5 to 11, Fluka now meets exactly the requirements of many analysts dealing with HPCE.

Quality Guarantee

We guarantee for each reagent:

• no insoluble impurities
  The HPCE buffers are filtered through a 0.2 mm filter membrane after production.
• minimal absorption over a wide wave length range
  see figure 1 below
• virtually no fluorescent impurities
  see figure 2 below
• tested for application
  see figure 3 below

figure 1: UV/VIS absorption of the buffer solution pH 2.5 for HPCE (Fluka 82581) in the wave-length range 200-800 nm.

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figure 2: Residual fluorescence of the buffer solution pH 2.5 for HPCE (Fluka 82581). The fluorescence is checked by exitation at 200, 230 and 260 nm. This illustrates the excellent suitability of this buffer for applications with fluorescence detection. Standard (S): 10^-7M ovalene, embedded in a polymethylmethacrylate (PMMA) matrix, excited at 342 nm.

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figure 3: Separation of the two peptides aprotinin (A) and soybean trypsin inhibitor (STI). Sample: containing 50 ng/ml of each peptide. Separation buffer: 20 nm sodium citrate pH 2.5 (Fluka 82581). Capillary: fused silica, 60 cm in length, 50 mm i.d. Injection: hydrostatic 3 sec, DH =9.8 cm. Field: 20 kV. Detection: 200 nm

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Product List: Buffer solutions for HPCE

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IEF Markers

• Application
• Product description
• Analysis conditions
• Product List
• Directions
• Reference

Application

IEF (Isoelectric Focusing) is a powerful analytical tool for the separation of ampholytes, mainly proteins. In order to ensure the high performance of analysis, standards of pI (pI markers) are needed. In addition to classical protein based standards, low molecular compounds were developed and successfully examined in capillary IEF and IEF-Gel electrophoresis. For capillary IEF, UV absorption is the most poular method in use. UV induced fluorescence emission is of interest if derivatizations of proteins with e.g. dansyl chloride, fluorescamine, o-phtaldialdehyde or coumarin moieties are used to increase sensitivity. Another advantage for IEF gel electrophoresis with Fluorescent IEF-Marker is the possibility to control the formation of gradient without further staining (using illumination UV).

Fluorescent IEF markers can also be detected by UV-absorption at 280 nm (20°C), allthough the signal is not as strong as with fluorescence detection. The absorption maxima of the individual markers are between 308 and 350 nm. For fluorescence detection an excitation wavelength of 310 nm (individual excitation maxima: 310 to 400 nm) is suggested; the emission maximum of the individual markers lies between 410 and 500 nm.

Product Description

Solids:

• 1 mg packages
• Fluorescenz: Em_max and Exc. (see Tabel 1)
• Storage at 4°C

Stock Solution:

• 200 ml packages
• Fluorescenz: Em_max, Exc. and conditions (see Tabel 1)
• Concentration: 1, 2, 3 mg /ml
• Solution: aqueous (see Tabel 1)
• Filtered through 0.45 mm membrane filter
• Store at 4°C; stable for at least 6 months

Analysis Conditions for CIEF using Pressure Mobilization

• Capillary: neutral capillary
• Anolyte: 91 mM phosphoric acide in gel buffer
• Catholyte: 20 mM sodium hydroxide in water
• Detection: 280 nm
• Temperature: 20 °C
• Injection: 20 psi, 1 min
• Polarity: inlet anode, outlet cathode
• Focussing voltage: 500 V/cm
• Focussing time: 2 min
• Mobilization: 0.5 psi, 500 V/cm anolyte -> catholyte (Mobilization should be stopped after the last marker is eluted to avoid the filling of the capillary with anolyte.)

	picture 1: Example for CE-IEF with fluorescent pI Markers pI Marker: Peak 1: 8.7 Peak 2: 7.6 Peak 3: 6.6 Peak 4: 6.2
	picture 2: The emission spectrum and emission maximum of the Fluorescent IEF-Marker pI 7.6
	picture 3: The UV spectrum of the Fluorescent IEF-Marker pI 7.6 (three different concentration)

Table 1. Conditions of Fluorescent IEF Markers stock solutions.
Additional information on Fluorescent IEF Markers in chapter Fluorescent Probes.

Directions for the use of Fluorescent IEF-Markers

The protocol for the use of Fluorescent IEF-Marker is following: Prepare the IEF-marker stock solution by dissolving the substance in the solvent according to Table 1. Sonicate for up to 10 min if necessary. Store at 4 °C (the solution is stable for approximately 6 months). For use in HPCE this stock solution should be diluted 1:100 in a suitable ampholyte solution (2% ampholyte 3-10; e.g. 10046). For uses in IEF-gels the stock solution can be loaded directly onto the gel (max. 1 ml per lane).

Reference

M. Horka, Th. Willimann, M. Blum, P. Nording, Z.Friedl, K. Slais, Capillary isoelectric focusing with UV-induced fluorescence detection, J. of Chromatography A, 916 65-71 (2001)

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Anion-HPCE-Kit Fluka 29244

• General Information
• Contents
• Application
• Working Instructions
• Peak Identification
• Quantification
• Literature
• Trouble Shooting Guide
• Electropherogramm

General Information

Capillary electrophoresis is an efficient analytical separation technique for analysis of minute amounts of sample and has several advantages, including fast separation and high resolution. The Fluka pH 7.7 buffer system is designed for the analysis of anions by indirect UV detection with reversed electro-osmotic flow. This buffer was validated for the analysis of eight common anions: fluoride, chloride, bromide, sulfate, nitrate, nitrite, thiosulfate and phosphate [1].

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Contents

• 100 ml Buffer solution pH 7.7 for HPCE (Fluka 82619)
  [Pyromellitic acid electrolyte buffer pH 7.7; composition: 2.25 mM pyromellitic acid, 6.50 mM sodium hydroxide, 0.75 mM hexamethonium hydroxide, 1.60 mM triethanolamine,
  pH: 7.7+/- 0.2 (250C); manufactured under clean room conditions; filtered through a 0.2 mm filter]
• 100 ml Water for HPCE (Fluka 95283)
  [manufactured under clean room conditions; filtered through a 0.2 mm filter]
• 10 ml Multielement Anion HPCE Standard Solution (Fluka 29235; 100 ppm)
  [composition: 100 mg/l fluoride, 100 mg/l bromide, 100 mg/l chloride, 100 mg/l phosphate, 100 mg/l sulfate, 100 mg/l nitrate; manufactured from highly pure salts and water; filtered through a 0.2 mm filter]
  
  When handling buffer solution pH 7.7 avoid contact with eyes or skin. Wear safety glasses. Store tightly closed.
  
  
• Needed but not supplied in this kit:
  0.1 M sodium hydroxide solution for HPCE (Fluka 72079) or an equivalent quality.

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Application

• The buffer solution pH 7.7 for HPCE is supplied ready to use. No dilutions or other treatments are required.
• The Multielement Anion Standard is supplied as a 100 ppm concentrate (0.1 g/l) of the six anions. Dilute the Multielement Anion Standard with Water for HPCE to your desired concentration (typically 1 ppm to 10 ppm). This working standard solution may be used as capillary test solution or for peak identification and quantification.
• Sample preparation:
  Samples are either used directly or diluted with water for HPCE (Fluka 95283) to a concentration of approximately 1 to 10 mg/l of the anion of interest. Detection limits are approximately at 0.5 mg/l of the anion of interest.

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Working Instructions

• Capillary treatment:
  For anion separation, use a fused silica capillary with an inner diameter of 50-75 mm, 50-60 cm total length (40-50 cm from inlet to detector). Rinse the capillary with 0.1 M sodium hydroxide solution for HPCE (Fluka 72079) for at least 10 min, then for 10 to 20 min with buffer solution pH 7.7. Do not use this capillary with other buffer systems, especially not with phosphate buffers (see also trouble shooting guide).
  
  
• Sample Injection:
  two modes of sample injection are frequently used in capillary electrophoresis: pressure injection or electrokinetic injection. For anion analysis commonly pressure injection is chosen; the injected sample has the same composition as the material in the sample vial. When using electrokinetic injection a preconcentration of faster migrating anions occurs: the composition of the injected sample has a higher concentration of faster migrating anions than the material in the sample vial.
  
  
• Electrophoresis conditions:
  

  Injection:	5 sec. at 0.5 PSI (corresponds to 30.2 mbar)
  Separation voltage:	- 30 kV ( [-] at the inlet side to [+] at the outlet side)
  Detection:	UV at 254 nm, inverted signal
  Run time:	5 to 15 min
  Expected current:	15 – 20 mA

  
  
• Capillary treatment between single runs:
  Between single runs a 3 min rinsing step with Buffer solution pH 7.7 for HPCE is sufficient.
  
  
• Capillary treatment at the end of the day:
  If the capillary will be used the next day, store the capillary in Buffer solution pH 7.7 for HPCE. If the capillary is not used for longer periods, rinse the capillary with 0.1 M sodium hydroxide solution for HPCE (Fluka 72079) for 5 min, then for 2 min with Water for HPCE (Fluka 95283). Blow the capillary dry for 2 min and store the capillary dry.

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Peak Identification

Single peaks are best identified by comparing the migration times with those of known anions. For final identification a standard anion should be added to the sample. The signal of the peak must be enhanced by the amount of the standard added (spiking).

Additional to the Multielement Anion HPCE Standard Solution (Fluka 29235) Fluka offers various anion standard solutions and multielement anion standard solutions for quantification or identification of detected anions. These standards are listed in the Fluka/Riedel catalogue as "Ion Chromatography Standard Solutions", "Multielement Atomic Spectroscopy Standard Solutions" and "Ionselective Electrode Standard Solutions".

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Quantification

For quantification of a specific anion in your sample prepare a calibration plot from a standard solution of the anion of interest:

Analyze serial dilutions of the anion standard solution and calculate the corrected peak areas (peak area / migration time). Plot the areas versus the concentrations of the diluted standard solution (Figure 1). From this calibration plot the concentration of your sample may be determined by comparing its corrected peak area with the calibration curve.

ppm corr. area 0 0 2 97 5 454 10 832 20 1720

Figure 1
Calibration plot of nitrate (at 0 ppm, 1 ppm, 5 ppm, 10 ppm, 20 ppm); without thiosulfate as reference anion.

For more accurate determinations, the use of a reference anion may be advantageous. This is done to omit run-to-run deviations caused by slightly variing injection amounts, buffer or run conditions. Chose a reference anion which is not contained in the samples analyzed. Add to all serial dilutions of the standard anion (e.g.: 1 ppm, 5 ppm, 10 ppm, 20 ppm nitrate) the choosen reference anion (e.g. 2 ppm thiosulfate). Add the same amount of reference anion also to your sample.

Calculate the quotients:

Plot the quotients Q_standard versus the concentrations of the diluted standard anions. From this calibration plot the concentration of an unknown sample may be determined by comparing its quotient Q_sample with the calibration curve. When run-to-run variations cause problems, this calibration curve will show a better correlation coefficient (R²).

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Literature

[1] Rhemrev-Boom, M. M. (1994): Determination of anions with capillary electrophoresis and indirect ultra violet detection. J. Chromatography 680 (2), pp. 675 – 684

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Trouble Shooting Guide

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Electropherogram

Separation of bromide (1), chloride (2), sulfate (3), nitrate (4), fluoride (5), and phosphate (6) each at a concentration of 6 mg/l (6 ppm).

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