C190-E094 Tips for practical HPLC analysis —Separation Know-how— Shimadzu LC World Talk Special Issue Volume 2 ����� ������������� 2 ����� ������������� This is an compilation of articles related to tips for preparation of mobilephase and samples from introductory, laboratory and technical sections of past issues of the "LCtalk", Shimadzu's newsletter for HPLC users, Japanese version. Contents Page 1. Preparation of Mobile Phases 4 2. Differences between Acetonitrile and Methanol in Reversed Phase Chromatography 6 3. Preparing Mobile Phases - Solvent Mixing Ratio - 8 4. Preparing Buffer Solutions 9 5. pKa and Dissociation Equilibrium 10 6. Water Grade 11 7. Gradient Baseline for Acetonitrile Containing TFA 12 8. Ion-Pair Chromatography 14 - Choosing between Alkyl Sulfonate and Perchloric Acid - 9. Measuring Accurately with Electronic Balances 16 10. Causes of Quantitative Errors Originating in Sample Preparation 18 11. Peaks Caused by Dissolved Air in Sample Solvents 20 12. Influence of Sample Solvent on Peak Shape 22 13. Check Methods for Abnormal Increases in Solvent Delivery Pressure 23 14. Internal Standard Method 26 15. Formulas for Number of Theoretical Plates 27 3 ����� ������������� Preparation of Mobile Phases Aqueous solvents, organic solvents, and mixtures of these types of buffer solution that uses phosphoric acid, but that the counterions solvents are usually used as the mobile phase in high performance are unclear. If we assume that they are sodium ions, the next liquid chromatography (HPLC). Buffer solutions are often used as problem is that we do not know whether the "20 mM" refers to aqueous solutions. Specific preparation methods for some of the the concentration of the phosphoric acid or sodium phosphate. representative buffer solutions used in HPLC are given on page 9. If we think of this solution as "20 mM phosphoric acid (sodium) In general, however, there are many cases where the definition of buffer solution", we can consider "20 mM" to be the concentration buffer solutions is vague. There are also cases where, because of of phosphoric acid. If, however, we consider "20 mM" to be the differences between the instructions given in documentation and concentration of sodium, we can think of this solution as a "buffer the actual preparation methods used, disparities in mobile phases solution created by the pH adjustment of an aqueous solution occur that will affect the chromatograms and the analysis results. of 20 mM sodium dihydrogen phosphate". (The pH value of an There are many aspects of mobile phase preparation that can be aqueous solution of 20 mM sodium phosphate is roughly 5.0, so thought of as blind spots. This applies not just to buffer solutions in order to attain a pH value of 2.5, pH adjustment with some acid but also, for example, to solvent mixing methods. Here, using is required.) Depending on the acid used for pH adjustment, the phosphate buffer as an example, we will look at the effect that the ion-pair effect may occur, and there may be some influence on the mobile phase preparation method can have on analysis results. analysis results. We can see then that there are several possible interpretations for the term "buffer solution". 1) Preparation of Buffer Solutions Fig. 1 shows the effect on the analysis results of interpreting In general, how is something described as "20 mM phosphate the above example three different ways. The top line shows the buffer solution (pH 2.5)" actually prepared? We will look at several result obtained by interpreting "20 mM" as the concentration possible cases. First, let us assume that we are talking about a of phosphoric acid and using a solution prepared as "20 mM Peaks 1. Acetaminophen Peaks ������� �� 1: acetaminophen 2: dihydrocodeine 3: caffeine 2. Dihydrocodeine ���� � ���� Analysis Conditions �� Column: Shim-pack VP-ODS (150 mm × 4.6 mm I.D.) Mobile Phase: Buffer solution (pH 2.5) / Acetonitrile = 9/1 (v/v) Top (A): 20 mM phosphoric acid (sodium) 3. Caffeine Buffer solution (pH 2.5) Middle (B): 20 mM sodium dihydrogen phosphate � ��� Buffer solution (pH 2.5) (Phosphoric acid added for pH adjustment.) Bottom (C): 20 mM sodium dihydrogen phosphate ��� � � Buffer solution (pH 2.5) (Perchloric acid added for pH adjustment.) Flow Rate: 1 mL/min � Temperature: 40 °C Detection: 210 nm � � ��� Chemical Structures of Constituents Fig. 1 Influence of pH Adjustment Method Used for Buffer Solutions 4 ����� ������������� phosphoric acid (sodium) buffer solution (pH 2.5)" as the mobile (sodium) buffer solution (pH 2.5) to that of acetonitrile is 9:1; in phase. The middle and bottom lines show the results obtained by other words, amounts corresponding to this ratio are measured out interpreting "20 mM" as the concentration of sodium dihydrogen and mixed. On the other hand, if we consider this description to phosphate, and adjusting the pH value to 2.5 by respectively simply mean "10% acetonitrile", this implies that 20 mM phosphoric adding phosphoric acid and perchloric acid. As illustrated by acid (sodium) buffer solution (pH 2.5) is used, and is diluted with dihydrocodeine in this example, there are cases where the retention acetonitrile. Applying the latter interpretation changes the relative time and consequently the robustness of the analysis technique are volumes and consequently the amount of 20 mM phosphoric acid significantly affected. (sodium) buffer solution is larger. There is a tendency to think that Indicating the preparation method for buffer solutions so that the there is no significant difference between these two interpretations. solution can be accurately identified helps to prevent problems Fig. 2, however, shows how the mixing method used can have a resulting from differences in interpretation. significant effect on the analysis results (particularly retention times). In general, regarding the preparation of mobile phases for HPLC, it 2) Mixing Organic Solvents and Aqueous Solvents seems that the notation "A:B = 3:2 (V:V)", indicating that an amount Solutions obtained by mixing organic solvents and aqueous solvents of solution A corresponding to a relative volume of 3 and an amount are sometimes used as mobile phases. The way in which mixing is of solution B corresponding to a relative volume of 2 are separately performed can have a significant effect on the analysis results. As an measured out and mixed together, is commonly used. (In practice, example, let us consider a mixture that is 90% 20 mM phosphoric the total volume of the mixture will be less than a relative volume of acid (sodium) buffer solution (pH 2.5) and 10% acetonitrile. If we 5.) consider this description to indicate that the mixing ratio is 9:1, The problems mentioned above occur not only in the preparation this implies that the ratio of the volume of 20 mM phosphoric acid of mobile phases, but also in the preparation of sample solutions and other solutions. Also, different practices and conventions are used in different fields (e.g., pharmaceuticals, chemical industry), further adding to the potential causes of confusion. Official documents, such as the Japanese Pharmacopoeia, Standard Peaks Methods of Analysis for Hygienic Chemists, and Japanese Industrial 1: acetaminophen Standards (JIS) give general principles and definitions related to the 2: dihydrocodeine 3: caffeine preparation of solutions. It is advisable to refer to these documents and to strive on a daily basis to use notation that avoids confusion. Analysis Conditions Column: Shim-pack VP-ODS (150 mm × 4.6 mm I.D.) Mobile Phase: Top (A): 20 mM (phosphoric-acid) sodium buffer solution (pH 2.5) / Acetonitrile = 9/1 (v/v) Bottom (B): Acetonitrile diluted by a factor of 10 with 20 mM (phosphoric- acid) sodium buffer solution (pH 2.5) Flow Rate: 1 mL/min Temperature: 40 °C Detection: 210 nm Fig. 2 Influence of Mixing Method Used for Mobile Phase 5 ����� ������������� Differences between Acetonitrile and Methanol in Reversed Phase Chromatography 1. Acetonitrile Is More Expensive inconsistency. There is not much difference in the price and so, if The organic solvents acetonitrile and methanol are often used as the possible, we should use the HPLC-type. mobile phase in reversed-phase chromatography. Commercial prices of these solvents are relatively expensive, particularly Acetonitrile for 3. The Pressure with Acetonitrile Is Lower HPLC. Acetonitrile appears more often, however, in related literature The pressure applied to the column varies with the type of organic and conditions specified by HPLC manufacturers. Here, we will be solvent and the mixing ratio. Fig. 3 shows some examples illustrating the looking at the reasons for this. relationship between the mixing ratio and the delivery pressure for water/ acetonitrile and water/methanol mixtures. The pressure for methanol 2. HPLC-Type Acetonitrile Has Less Absorbance increases significantly with the proportion of water, whereas the increase Fig. 1 and 2 show absorption spectra for acetonitrile and methanol for acetonitrile is not so marked. Therefore, if acetonitrile is used, undue (commercial HPLC type and special grade). “HPLC type” does not pressure is not applied to the column for the same flow rate. indicate that the solvent has a high absolute purity. This type is created by removing impurities that have UV absorbance and the The two points given above explain why acetonitrile is used. Are there absorbance for specified wavelengths is suppressed so as to lie not, then, any benefits obtained by using methanol, other than the below certain levels. It can be seen that, out of these four reagents, lower price? Other aspects are compared below. HPLC-type acetonitrile has the lowest absorbance (particularly for short wavelengths). Using an organic solvent with lower absorbance 4. In General, Acetonitrile Has a Higher Elution Capacity as the mobile phase results in less noise in UV detection, and so If acetonitrile and methanol are mixed together with water in the same HPLC-type acetonitrile is suitable for high-sensitivity analysis in the proportion, in general, the elution capacity will be higher for acetonitrile.