Essential Concepts of Mobile Phase Selection for Reversed Phase HPLC

 

V. Agrahari¹*, M. Bajpai², S. Nanda³

¹College of Pharmaceutical Sciences, RKGIT, Ghaziabad-201003, Uttar Pradesh, India

²Faculty of Pharmacy, Uttarakhand Technical University, Dehradun, India

³Department of Pharmaceutical Sciences, M.D. University, Rohtak-124001, Haryana, India

*Corresponding Author E-mail: v09world@gmail.com

 

In HPLC the mobile phase is pure or mixed solvents as well as solvents with solid modifiers. Chromatographers have a choice among hundreds of solvents for different application of HPLC. A particular selection is usually affected by solvent characteristics such as viscosity, refractive index, noncorrosiveness, toxicity, miscibility, transparency etc. Commercial availability in adequate purity at reasonable price is also important factor. The solvent strength or % organic solvent in mobile phase controls the retention time of the analyte. A useful rule of thumb in RPLC indicates that 10% decrease in the organic solvent in the mobile phase shows a 3-fold increase in k or t_R. Whenever acidic or basic samples are separated, it is strongly advisable to control mobile phase pH by adding a buffer. Several considerations should be kept in mind in selecting a particular buffer e.g. buffer capacity, solubility, interaction with sample or column, corrosion of HPLC system etc. A buffer concentration in the range of 10 to 50 mM is adequate for most reversed phase applications. During mobile phase preparation, premixing is done by measuring the volume of each solvent separately and combining them in the solvent reservoir. Buffered mobile phase pH must be adjusted before adding organic solvent. This approach leads to some uncertainty in the actual pH value of the final mobile phase because the addition of organic solvent can change the pH. But this problem is much less important than poor reproducibility of the mobile phase pH if it is measured after addition of the organic solvent. Aqueous mobile phases containing buffers must be filtered through a 0.45 or 0.2 μm membrane filter and degassed by vacuum filtration or sonication.

 

 

 

INTRODUCTION:

The mobile phase is the solvent that moves the analyte through the column. The analytes are present in a appropriate solvent to be injected into the chromatographic system after sample preparation. This solvent is not considered a new matrix, because it does not interfere with the analysis. The nature of the carrier (solvent) for the analytes can be chosen during the sample preparation to satisfy specific requirements such as volatility and miscibility with the mobile phase in HPLC.

 

In reverse phase columns, the mobile phase is more polar than the stationary phase. In general, polar mobile phase is preferred because the separations show better reproducibility.

 

The solubility of the analytes in the mobile phase also plays an important role in the column choice. For compounds soluble in water or partially aqueous solvents, the selection of a reverse phase HPLC is not a problem, but for compounds insoluble in polar solvents, a direct phase column is usually necessary. The evaluation of the polarity of the sample, that of the stationary phase, and that of the mobile phase are also important in the choice of the column. In HPLC, the mobile phase is pure or mixed solvents as well as solvents with solid modifiers[1]. The solubility in water of several solvents used in HPLC is given in Table 1. Besides polarity, some other parameters were developed for the characterization of the behaviour of a certain solvent towards a specific stationary phase. One such parameter is the elutropic strength ε⁰.

 

The equilibrium constant K_i of a specific compound "i" on a specific stationary phase and using a specific solvent is given by the expression:

log K_i = n log C_As + d (S⁰ - ε⁰ A_i)                           (1)

 

Where, n = A_i / A_m with A_i and A_m the areas occupied on the adsorbent by the solute and the solvent, respectively.

 

C_As is a parameter proportional with the adsorbent surface area,

d is characteristic for each solid phase,

S⁰ is a measure of adsorption energy of "i" onto a standard adsorbent surface,

ε⁰ is the elutropic strength.

 

As seen in expression (1), a number of factors are included, and its use for the calculation of K_i is limited. Some values for the elutropic strength ε⁰for solvents on alumina and C18 are given in Table 2.

 

In addition to pure solvents, it is common in HPLC to use solvent mixtures. The resulting elutropic strength is in between the ε⁰ of the two solvents.

 

In reverse phase chromatography it is common to chose a solvent with the polarity higher than that of the most polar analyte. For gradient HPLC the polarity of the solvent is slowly decreased to a solvent with polarity lower than that of the least polar component of the sample [1].

 

Table 1: Solubility in water of various solvents used in HPLC (Moldoveanu,2002)

Solvent	Solvent % in water	Solvent	Solvent % in water
Water	100	n-Butanol	0.43
Formamide	100	n-Propanol	100
Dimethylsulfoxide	100	Isopropanol	100
Dimethylformamide	100	Butyl acetate	7.81
Acetic acid	100	Methylene chloride	1.6
Acetonitrile	100	Diethyl ether	6.89
Ethanol	100	Benzene	0.18
Methanol	100	Carbon tetrachloride	0.08
Acetone	100	Cyclohexane	0.01
Ethyl acetate	8.7	Pentane	0.004
Chloroform	0.815	Hexane	0.001
Tetrahydrofuran	100	Heptane	0.0003

Table 2. Common HPLC solvents and their important properties. (Snyder et al., 1998)

Solvent	UV cut-off (nm)	Viscosity (cP)	Refractive index (200C)	Boiling Point (0C)	Miscibility number (M)	Elution Strength (E0)
						Alumina	C18
Acetone	330	0.36	1.36	56.3	15	0.56	8.8
Acetonitrile	190	0.38	1.34	81.6	11	0.65	3.1
Chloroform	245	0.57	1.44	61.15	19	0.4	…
Dimethyl formamide	268	0.92	1.43	153	12	…	7.6
Dimethyl sulfoxide	268	2.24	1.48	189	9	0.62	…
Hexane	195	0.31	1.38	68.7	29	0.01	…
Isopropyl alcohol	205	2.4	1.37	82.3	15	0.82	8.3
Methanol	205	0.55	1.33	64.7	12	0.95	1
Propyl alcohol	210	2.3	1.38	97.2	…	0.82
Tetrahydrofuran	212	0.55	1.4	66	17	0.45	3.7
Toluene	284	0.59	1.49	110.6	23	0.29	…
Trifluoroacetic acid	210	0.93	1.28	71.8	…	…	…
Water	190	1	1.33	100	…	…	…

 

Table 3. Absorbance at specified wavelength for a number of solvents and additives used in reversed-phase HPLC (Snyder et al., 1998)

	Absorbance at wavelength (nm) specified
	200	205	210	215	220	230	240	250
Solvents
Acetonitrile	0.05	0.03	0.02	0.01	0.01	<  0.01
Methanol	2.06	1	0.53	0.37	0.24	0.11	0.05	0.02
Isopropanol	1.8	0.68	0.34	0.24	0.19	0.08	0.04	0.03
Tetrahydrofuran	2.44	2.57	2.31	1.8	1.54	0.94	0.42	0.21
Acids and Bases
Acetic acid, 1%	2.61	2.63	2.61	2.43	2.17	0.87	0.14	0.01
Hydrochloric acid,                       6 mM (0.02%)	0.11	0.02	<0.01
Phosphoric acid, 0.1%	<0.01
Trifluoroacetic acid           0.1% in water                                                            0.1% in acetontrile	1.2                0.29	0.78                0.33	0.54                0.37	0.34                0.38	0.20                0.37	0.06                0.25	0.02                0.12	0.01                0.04
Ammonium phosphate, dibasic, 50mM	1.85	0.67	0.15	0.02	<  0.01
Triethylamine, 1%	2.33	2.42	2.5	2.45	2.37	1.96	0.5	0.12
Buffers and Salts
Ammonium acetate,10mM	1.88	0.94	0.53	0.29	0.15	0.02	0.01
Ammonium bicarbonate, 10mM	0.41	0.1	0.01	< 0.01
EDTA, Disodium, 1mM	0.11	0.07	0.06	0.04	0.03	0.03	0.02	0.02
Potassium phosphate Monobasic, 10mM      Dibasic, 10mM	0.03               0.53	< 0.01               0.16	0.05	0.01	< 0.01

GENERAL REQUIREMENTS:

A particular selection is usually affected by solvent characteristics such as viscosity, refractive index, non-corrosiveness, toxicity, miscibility, transparency etc. Commercial availability in adequate purity and at a reasonable price is also important factors [2,3,4]. Various characteristics of solvents used as HPLC mobile phases are discussed as below:

 

a)      Purity: All solvents should be of the highest purity and quality. HPLC grade solvents are prefiltered and purified to have minimal absorbance in the UV. So these contain no impurities to produce spurious peaks in a chromatogram baseline. Water used in buffer preparation is of highest purity. Deionized water often contains trace levels of organic compounds and so therefore is not recommended for HPLC use. Ultra pure HPLC water (18 MΩ resistivity) is generated by passing deionized water through an ion exchange bed. This mechanism is applied in modern water purification instruments to produce suitable quality of water in high volumes. Alternately, HPLC grade water can be purchased from solvent suppliers. 

 

b)      Viscosity: To maintain an acceptable pressure drop (< 2500 psi) with a reasonable flow rate through the column, the mobile phase viscosity should be as low as possible. Lower viscosity mobile phases produce narrower chromatographic peaks. For instance, acetonitrile is often the solvent of choice for reversed-phase HPLC because it has not only a lower viscosity than methanol or isopropanol but also has good retention characteristics. Viscosity values for several pure solvents are listed in Table 2.

 

c)      Refractive Index: When refractive index detector is being used, this is an important parameter for mobile phase selection. RI values of some pure solvents are listed in Table 2.

 

d)       Boiling point: The boiling point is important if sample recovery is concerned. A low boiling point solvent is easier to remove from the sample.

 

e)      Noncorrosiveness: The solvents must be noncorrosive to HPLC system components.

 

f)       Toxicity: Nowadays everyone is concerned with the health and safety risks associated with chemicals in the laboratory. Tetrahydrofuran is available as a unstabilized or stabilized mobile phase. The unstabilized version can be an explosion hazard if fully evaporated and decomposes readily when not stored under nitrogen. The stabilized version have a high UV absorbance at typical UV wavelengths used for detection.

 

g)      Miscibility: Not all HPLC solvents are miscible. Several problems may result if immiscible solvents are mixed such as unstable baseline, fluctuating pressures, high pressure etc. Miscibility data of some solvents are given in Table 2 [3]. All pairs whose M numbers differ by 15 units or less are miscible in all ratios at 15⁰C. A difference of 17 or more corresponds to immiscibility. For example, hexane has M=29 and acetonitrile has M=11, so the difference is 29-11=18 and these two solvents will not be miscible in all ratios. Isopropanol is miscible with most common HPLC solvents. So it can be used to flush the flow path of HPLC in case of uncertainty of last solvent system used.

 

h)      Transparency (UV-Cut off): The mobile phase (without sample) must transmit sufficiently at the wavelength used for detection. One study indicates that mobile phase absorbance should usually be less than 0.5 at detection wavelength. Table 3 summarizes absorbance vs. wavelength for a number of solvents and additives used in reversed-phase HPLC [3].

 

Another consideration is the typical mixture of organic solvent, water and additives that might be used with detection at 200 nm (A<0.5). Table 4 illustrate some examples. The solvent below or near its UV cut-off should not be used when utilizing a UV detector otherwise an unacceptable noise level will result. For instance, monitoring a compound at 220 nm, acetonitrile is selected over methanol because acetonitrile’s UV cut-off is lower than methanol resulting in better detection performance.

 

Table 4. Useful solvent mixtures with low background absorbances (< 0.5) at ≥ 200 nm (Snyder et al., 1998)

0-26% methanol-water

0-28% isopropanol

0-20% THF

0-100% acetonitrile-water

ACN-water with additives

 0.2% acetic acid

0.4% trifluoroacetic acid

 

SOLVENT STRENGTH AND SELECTIVITY:

Solvent strength is related to its polarity. It refers to the ability of a solvent to elute solutes from a column. Under Normal Phase Chromatography (NPC) solvent strengths are often characterized by Hildebrand’s elution strength scale (E⁰). Elution strength of some solvent is listed in Table2.3. In NPC, nonpolar hexane is a weak solvent whereas water is a strong solvent. In RPLC, stationary phase is hydrophobic hence water is a weak solvent and organic solvents are strong. Thus solvent strength increases as solvent polarity decreases in RPLC and Hildebrand scale becomes in reverse order. For example THF>ACN>MeOH>>Water.  The goal of solvent strength adjustment is to position all the bands within a k range of roughly 0.5 to 20 (0.5< k < 20). It has been observed that on lowering % of organic solvents (or solvent strength) in the mobile phase t_R, k, α and R_s increases. A useful rule of thumb in RPLC indicates that 10% decrease in the organic solvent in the mobile phase shows a 3-fold increase in k or t_R [2]. For a given solute, much lower % of THF is needed as compared to MeOH to attain the same k value. But use of THF is not popular due to its toxicity and peroxide formation. Instead of THF another ether, methyl t-butyl ether (MTBE) is used at low levels or with a co-solvent due to its low water-miscibility [5].

 

Mobile phase strength depends on both the choice of organic solvent and its concentration in the mobile phase (%B), where A is water. Fig.1 provides for the interconversion of reversed-phase mobile phases having the same strength. A vertical line connects %B values for mobile phase having the Iso-eluotropic (same elution power) property giving similar k values. For example, 40% Acetonitrile has the same strength as 30% THF or 50% Methanol.

 

BUFFERS:

A buffer solution is an aqueous solution consisting of a mixture of a weak acid and its conjugate base or a weak base and its conjugate acid. It resists changes in pH when small amount of acid or base are added to it. Buffer solutions are used as a means of keeping pH at a nearly constant value in a wide variety of chemical applications [6]. In liquid chromatography buffered mobile phase is used when the analyte contains acidic or basic moieties or when the column contains acidic or basic sites (such as ion exchange columns). Table 5 summarizes the common buffers used for reversed phase HPLC [7].

 

Table 5 Common buffers used for RP HPLC. (Rosario LoBrutto, 2007)

Buffers	pKa	Buffer range	UV cut-off (nm)
Trifluoroacetic acid (TFA)a	0.5	upto 1.5	210
Phosphate	2.1	1.1-3.1	< 200
	7.2	6.2-8.2
	12.3	11.3-13.3
Formic acida	3.8	2.8-4.8	210
Acetic acida	4.8	3.8-5.8	210
Citrate	3.1	2.1-4.1	230
	4.7	3.7-5.7
	5.4	4.4-6.4
Trisamino methane	8.1	7.1-9.1	205
Diethylamine	10.5	9.5-11.5	235
Triethylamine	10.8	9.8-11.8	200
Ammonium Hydroxidea	9.3	8.3-10.3	200
Ammonium bicarbonatea	6.4	5.4-7.4	200
	10.3	9.3-11.3
a Volatile buffers; can be used for LC-MS

 

Buffer capacity:

It is a measure of the strength of the buffer. It specifies the amount of hydronium ions or hydroxyl ions that are needed to change the buffer pH by a certain value. The larger the buffer capacity, the larger is the amount of acid or base that can be added to the solution without a change in pH. A buffer has always the best buffer capacity around its pKa. For example, acetic acid has a pKa of 4.8 (in water), therefore acetate buffers have the best buffer capacity at this pH. Phosphate has three pKa values (2.1, 7.2 and 12.3) and is most effective for pH ranges of 1-3, 6-8 and 11-13. It is not effective at pH 4 because it does not have any buffering capacity at that pH. The rule of thumb says that a buffer should never be used outside 1.0 pH units around its pKa, since it loses its buffering capacity outside this range [3].

 

When it is needed?:

In reversed-phase HPLC, mobile phase pH values are usually between 2 and 7.5. Buffers are needed when the analyte is ionisable under reversed phase conditions. The pH of the mobile phase will determine the degree of ionization of analyte, which in turn affects retention. For example neutral compounds are more hydrophobic in RPLC separations and thus more retained than polar, ionized components. When an analyte is ionized, it becomes less hydrophobic and, therefore its retention decreases.  Acids (HA) lose a proton and become ionized (on increasing pH) while bases (B) gain a proton and become ionized (on decreasing pH) Figure 2.

 

Fig 1: Relative Strength for Different Solvents. (Courtesy: Sigma-Aldrich, Technical Report) 

 

Figure:2 Effect of pH on Retention (Courtesy: Mac-Mod Analytical Inc.)

 

Table 6: Solubility of some phosphate buffers at room temperature and 5⁰C (Yuri Kazakevich et, al., 2007).

Buffer Salt	Buffer conc. (mM)	Initial pH value	Final pH value	Appearance of different ratio of ACN/ Buffer
				(70:30)		(80:20)		(85:15)
				RT	50C	RT	50C	RT	50C
Ammonium Phosphate monobasic	25	4.5	3	soluble	soluble	soluble	soluble	Precipitate	Precipitate
Di- ammonium hydrogen phosphate	25	8	7	soluble	soluble	Precipitate	Precipitate	Precipitate	Precipitate
Di-potassium hydrogen phosphate	10	9.1	7	soluble	soluble	soluble	soluble	Precipitate	Precipitate

The robustness of HPLC method is often dependent on mobile phase pH. Figure 3 shows a graph of retention time versus mobile phase pH for a basic, an acidic and a neutral compound. The retention of neutral compounds is little affected by mobile phase pH. However the retention of acidic compounds are sensitive to pH between 2 to 5 while basic compounds can change dramatically in the pH range of 5 to 10. For example, methylamphetamine (base) is fully protonated at pH < 3 and its retention is not affected by slight changes in mobile phase pH. Nevertheless, at pH 7 (near to its pKa) a change of 0.2 units in pH will shift retention by 1.2 minutes [8,9].

 

Peak shapes can also be affected by the degree of ionization of sample. Therefore it is often essential to buffer the mobile phase to control selectivity and to achieve reproducible separations with acceptable peak shape [6]. Considering the affects of pH on analyte retention, it is important in RPLC method development of ionic analytes, to select suitable  buffer to use, its concentration, solubility in the organic modifier and its affect on detection.  An improper choice of buffer can result in poor or irreproducible retention and tailing in reverse-phase separation of polar and ionizable compounds. Problems of partial ionization of the analyte and strong interaction between analytes and residual silanoles on the stationary phases can be overcome by proper mobile phase buffering (maintaining the pH within a narrow range) and choosing the right ionic species and its concentration (ionic strength) in the mobile phase.

 

Figure 3: Effect of mobile phase pH on basic, acidic and neutral compounds.   (Courtsey: Column troubleshooting, ACE Newsletter 2)

 

Buffer Concentration:

The solute retention in reversed phase HPLC is affected by buffer concentration. This situation occurs when ion exchange interactions take place between basic solutes and acidic silanols on the surface of silica stationary phase supports. The increasing buffer concentration sometimes lead to an improvement of the peak asymmetry (reduction of tailing) for protonated basic compounds. The concentration should also be low enough to avoid problems with precipitations in HPLC column. In case of phosphate buffers precaution to be taken to minimize the abrasive

 

effect on pump seals [10]. Although 50 mM buffers are specified in many older methods, the modern trend is to use low buffer strengths, typically in the range of 10-20        mM [2]. 

 

MOBILE PHASE PREPARATION:

Premixing:

Mobile phase premixing is done by measuring the volume of each solvent separately and combining them in the solvent reservoir. When organic solvents are mixed with water there is the negative volume of mixing. For e.g. to prepare 1 L of methanol/water (50:50), measure 500ml each of methanol and water separately in a measuring cylinder and combine them together. Due to the shrinkage of solvents upon mixing, it is not advisable to pour 500ml of methanol into a 1-L volumetric flask and fill it to volume with water, as more than 500ml of water is needed [2].

 

Buffer:

Samples containing acidic or basic analytes requires buffers in the mobile phase. The pH adjustments should be made in the aqueous buffer alone i.e before mixing it with any organic solvents. Precaution should be taken to prevent any possibility of buffer precipitation. Methanol is fairly miscible with most buffers. When phosphate buffer is blend with acetonitrile (ACN) the upper limit should be 60-80% ACN and concentration of the phosphate buffer should be less than 15mM to prevent precipitation. An acidic pH of 2.5 to 3 is a good starting point for most of the pharmaceutical applications. It is due to suppressed ionization of most acidic analytes (which provides a higher retention) and minimum interaction of basic analytes with surface silanols on silica based column (silanols are not ionized at that pH). Common acids used for mobile phase preparations are phosphoric acid, formic acid and acetic acid. The concentration of buffer additive must be kept constant throughout the entire gradient run. It is generally recommended to use the same concentration of TFA or other acid (UV absorbing) in both the aqueous and organic portions of the mobile phase in order to suppress any type of baseline shift during gradient run. Some considerations when using buffered mobile phase are as below:

·        Phosphate is more soluble in methanol/water than in ACN/water or THF/water.

·        Solubility order of buffer salts in organic/water mobile phases is as below: ammonium salts > potassium salts > sodium salts.

·        TFA and TEA degrade with time and their UV absorbance increases. Mobile phase containing these buffers should be made fresh.

·        Aqueous buffered mobile phase have the risk of bacterial growth, which will accumulate on column inlets and damage the chromatographic performance.

·        At pH more than 7, phosphate buffers accelerate the dissolution of silica and severely shorten the lifetime of silica-based HPLC columns.

·        Never let buffered mobile phases sit in the HPLC. Flush buffers from system with water (or 10% ACN in water) before switching to organic mobile phase.

·        Ammonium bicarbonate buffers usually are prone to pH changes (becomes more basic due to release of carbon dioxide) and usually stable for only 24 to 48 hours.

·        A ‘test tube test’ should be conducted to determine the buffer precipitation in the HPLC system. This test is performed by preparing buffered mobile phase in a 10 ml test tube and putting the test tube in the refrigerator, at room temperature and / or on water bath to determine if any precipitation occurs. Test tube-tests of some phosphate buffers are given in Table 6 [10, 11].

 

Filtration:

All aqueous mobile phases containing buffers must be filtered through a 0.45 or 0.2 μm membrane filter (typically 47-mm diameter). Cellulose acetate filters and PTFE or nylon filters are used for aqueous solvents and organic solvents respectively. HPLC grade solvents are already pre-filtered by the manufacturers. All-glass solvent filtration apparatus consisting funnel, clamp, filter holder and vacuum flask are used for filtration.

 

Degassing:

Mobile phase degassing is needed because gas bubbles in the pump head can cause malfunctions (air-lock) or flow errors (low flow). So it is important to prevent pump problems and inaccurate pump blending for gradient operation. Degassing by vacuum filtration or sonication is partially effective and adequate for isocratic operation. After this type of degassing, depending on humidity and atmospheric pressure, gasses like carbon dioxide almost immediately begin to re-introduce back into the mobile phase. Thus mobile phase will re-gas within a few hours and might cause blending error. The endothermic system (ACN/water) is difficult to degas while exothermic system (MeOH/water) is easy to degas. The most convenient way for solvent degassing is an in-line vacuum degasser. Helium sparging and pressurization is also very effective         means [2, 12]. 

 

CONCLUSION:

This article provides an overview of essential concepts in reversed phase HPLC mobile phase selection. The polarity of the sample, stationary phase, and mobile phase are also important in the choice of the column. Generally, in reversed phase HPLC polar mobile phase is preferred because the separations show better reproducibility. Different solvent characteristics such as purity, solubility, viscosity, refractive index, toxicity, miscibility, transparency etc discussed for particular selection of mobile phase. Ultra pure HPLC water (18 MΩ resistivity) is generated by passing deionized water through an ion exchange bed. Several problems may result if immiscible solvents are mixed such as unstable baseline, fluctuating pressures, high pressure etc. Absorbance at specified wavelength for a number of solvents and additives used in reversed-phase HPLC mentioned to find out transparency (UV cut-off). The solvent below or near its UV cut-off should not be used when utilizing a UV detector. The solvent strength increases as solvent polarity decreases in RPLC. Solvent strength nomograph showed the mobile phase having the Iso-eluotropic (same elution power) property giving similar k values. A useful rule of thumb in RPLC indicates that 10% decrease in the organic solvent in the mobile phase shows a 3-fold increase in k or t_R. In liquid chromatography buffered mobile phase is used when the analyte contains acidic or basic moieties. The robustness of HPLC method is often dependent on mobile phase pH and it depends upon the appropriate buffer selection. A buffer should never be used outside 1.0 pH units around its pKa, since it loses its buffering capacity outside this range. To minimize the abrasive effect on pump seals and to improve the peak symmetry buffer concentration should be used typically in the range of 10-20 mM. Various steps of mobile phase preparation also discussed including premixing, pH measurement, filtration and degassing. Mobile phase premixing is done by measuring the volume of each solvent separately and combining them in the solvent reservoir. The pH measurement should be made in the aqueous buffer alone i.e before mixing it with any organic solvents. Common acids used for mobile phase preparations are phosphoric acid, formic acid and acetic acid. It is generally recommended to use the same concentration of acid in both the aqueous and organic portions of the mobile phase in order to suppress any type of baseline shift during gradient run. All mobile phases must be filtered through a 0.45 or 0.2 μm membrane filter (typically 47-mm diameter) and degassed. Cellulose acetate filters and PTFE or nylon filters are used for aqueous and organic solvents respectively. Degassing is done by vacuum filtration or sonication.

        

REFERENCES:

1      Moldoveanu S and David V. Sample preparation in chromatography, Journal of Chromatography Library- volume 65 (2002) 182-190.

2        Dong M.W., Modern HPLC for Practicing Scientists, Wiley-Interscience publisher, New York (2006) 27-33,111-119.

3        Snyder L.R., Kirkland J.J. and Glajch J.L. Practical HPLC Method Development, 2^nd Edition, Wiley-Interscience publisher, New York (1998) 68-70,721-728.

4        Agilent Technologies, Student Manual - Practical High Performance Liquid Chromatography, Course Number H5930A, USA (2001) 25-28.

5        Ornaf R.M. and Dong M.W.  (2005), Key concepts of HPLC in Pharmaceutical Analysis In Ahuja S. and Dong M.W., Handbook of Pharmaceutical Analysis by HPLC, Elsevier Inc, 35-38.

6        Tindall G.W., Dolan J.W., Mobile Phase Buffers Part-II, LC.GC Europe, (2003) 2-4.

7        Rosario LoBrutto (2007), Reversed-Phasse HPLC. In Yuri Kazakevich and Rosario LoBrutto, HPLC for Pharmaceutical Scientists (pp158-160). John Wiley and Sons, Inc.

8        Column troubleshooting, ACE  Newsletter 2,Advanced Chromatography Technologies.

9        Mac-Mod Analytical Inc. Technical report, Number 02091 TR.

10     Yuri Kazakevich and Rosario LoBrutto, HPLC for Pharmaceutical Scientists, John Wiley and Sons, Inc (2007) 375-380.

11     Dong M.W. (2005), How to be more successful with HPLC analysis: Practical aspects in HPLC operation In S. Ahuja, M.W.Dong, Handbook of Pharmaceutical Analysis by HPLC, Elsevier Inc, 256-258.

12     McMaster, M.C. HPLC A Practical User’s Guide, VCH Publishers Inc, New York, (1994) 85.

13     Sigma-Aldrich, Technical Report, Approaches to Lessening the Impact of the Acetonitrile Shortage on Your Reversed-Phase Separations. See http:// www. Sigma aldrich.com/catalog/search.

 

 

Received on 21.02.2013       Modified on 03.03.2013

Accepted on 10.03.2013      © RJPT All right reserved