Syringe Filter Efficiency and Effect of Filtration on HPLC Column Life

Compared to other manufacturers, Pall Acrodisc PSF syringe filters significantly prolonged HPLC column life.

Filtration as a preventative maintenance tool for HPLC analyses is well documented. It is commonly taken for granted that column life will be extended if samples are filtered prior to injection, but the extension of the column life has not been well quantified. It is the intent of this work to demonstrate that filter efficiency must be considered when choosing an HPLC sample-prep filter and that filtration will lengthen the life of a column.

In this paper, retention efficiency of three effectively equivalent 0.45 µm rated syringe filters was examined using 0.45 µm average diameter latex spheres. This work was conducted with latex spheres to offer the best possible reproducibility in both sample preparation and filter efficiency measurements.

In order to correlate the retention of spheres to the actual application, the quantitative effect of filtration on HPLC column life was investigated. This involved examining column life without filtration compared to column life when samples were filtered. It should be recognized that extending the column life is dependent on the particulate within the sample and actual column life extension may vary.

• Latex spheres and surfactant were purchased from Sigma. 0.45 µm average diameter latex spheres:
  L/N 31K1123, Cat #: LB-5, Triton X-100 L/N: 121K0090, Cat #: X-100
• 25 mm syringe filters with 0.45 µm pore size ratings were obtained from Pall, Company A, and Company B
• Pall Acrodisc PSF syringe filters with GHP membrane: P/N AP-4557, L/N: A224102521, A224227411, and A22422044
• Company A syringe filters with PVDF membrane: L/N: F2EN42569, F0BN28908, and F2NN83751
• Company B syringe filters with PVDF membrane: L/N: 11067, 11146, and 99297
  

• High Performance Liquid Chromatography (HPLC)
• Waters (Milford, MA, USA) 616 Pump
• Waters 600s Controller
• Waters 717plus Autosampler
• Ultraviolet/Visible Spectrophotometer (UV/VIS), HP 8452A Diode Array
  

• Luna 5 µm C18(2), columns were purchased from Phenomenex (Torrance, CA, USA): Size: 30 x 3.0 mm, P/N: 00A-4252-Y0, S/N: 160111-3, 159485-4, 159742-3, and 159485-3
  

The UV/VIS Spectrophotometer was used for measuring absorbance of latex-sphere solutions. The maximum absorbance of the latex-sphere solution was observed at 272 nm, which was used to correlate latex sphere concentrations with absorbance. The surfactant solution, 0.1% Triton X-100 that is free of latex spheres, was measured as the blank at 272 nm. A series of standard solutions of 0.0025%, 0.0050%, 0.0075%, and 0.01% 0.45 µm latex sphere concentrations were made and used for creating the calibration curve. The linear relationship between latex sphere concentrations and absorbance was established, which is in accordance with Beer’s law. A correlation coefficient of 0.9999 was obtained. The 0.01% 0.45 µm average diameter latex spheres solution was used for the retention efficiency study. The challenge solution was passed through each individual syringe filter and a 3 mL eluted aliquot was collected and analyzed at 272 nm. Three different filters from each of the three lots were tested (i.e., nine filters from each manufacturer were individually analyzed).

The HPLC was utilized for the column-plugging study. Column life was evaluated by comparing initial backpressure to backpressure after injections. A new LUNA* C18(2) 00A-4252-Y0 column (S/N: 160111-3) was installed. The outlet of the column was disconnected from the detector and allowed to run to drain. This modification allowed quicker injections for a more efficient determination of column backpressure. Acetonitrile:Water (35:65, percentage by volume) was used as mobile phase, with a flow rate of 1 mL/min. Column temperature was controlled at 25 °C. The system was set to automatically inject 50 µL each time. The column-plugging solution consisted of 0.05% (by weight) 0.45 µm average diameter latex spheres in 0.002% Triton X-100 solution. The first step was to inject this solution without any filtration to see how long the column would last without filtration. After the column was plugged (i.e., column backpressure reached 3500 psig, 241.3 bar, 507.6 kPa), a new LUNA C18(2) 00A-4252-Y0 column (S/N: 159485-4) was installed. This time the same latex-sphere solution was subjected to filtration using Company A filters. Thirty samples were generated with 30 Company A filters (ten from each of the three lots). The injections were carried out from sample vial one through vial 30 and then repeated in this sequence. The column backpressure was recorded with the number of injections. This procedure was repeated with new columns, S/N 159742-3 and 159485-3, for studies with Company B and Pall filters, respectively.

Table 1
0.45 µm Latex Sphere Retention Efficiency (in percentage) with 0.45 µm Pall GHP Filters

Table 2
0.45 µm Latex Sphere Retention Efficiency (in percentage) with 0.45 µm Company A Filters

Table 3
0.45 µm Latex Sphere Retention Efficiency (in percentage) with 0.45 µm Company B Filters

When the 0.05% latex-sphere solution was filtered through Pall Acrodisc PSF syringe filters with GHP membrane and injected to the HPLC system, the column backpressure did not increase after even 972 injections. The clear effluents from the filters suggested a more successful retention of latex spheres.

Figure 1
Effects of Filters on HPLC Column Life

We have shown that it is imperative that samples be filtered prior to their introduction into an HPLC system. Apparently, equivalent filters from various manufacturers with the same removal rating differ in capabilities. Using Pall Acrodisc PSF syringe filters with GHP membrane prolonged the column life 46 times with no increase in column backpressure.

Using Pall Acrodisc PSF syringe filters with GHP membrane prolonged the column life 46 times with no increase in column backpressure.

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