L-Lysine on Γ-Alumina and Kaolinite

L-Lysine on γ-alumina and kaolinite Jan-Feb 2008

KEY FINDINGS o No lysine observed on γ-alumina samples treated (and then washed to remove pore water) with lysine solutions with an initial pH of 9.8.

o No lysine observed on γ-alumina samples treated (and then washed to remove pore water) with lysine solutions with an initial pH of 6.3 and concentrations ranging from 0.02 M – 0.001 M.

o Evidence of aliphatic carbon chain was found on the γ-alumina sample treated with 0.05 M lysine at pH 6.3. No evidence of amino or carboxylate groups was observed. This finding is consistent with incomplete exchange of pore water in the washing cycle for this sample which was treated with the most concentrated solution.

o Lysine was observed on kaolinite samples treated with 0.05 – 0.005 M solutions (for both of the pH 9.8 and the pH 6.3 sets). The kaolinite samples were very difficult to wash without large loss of solid and thus the observed lysine may be a result of residual solution.

o The observed results are consistent with the strong ability of lysine to complex with aluminum ions (particularly those with octahedral coordination) and to extract them from mineral substrates.

ABSTRACT The DRIFTS technique was successful in detecting lysine on the surface of γ- alumina and kaolinite, after treatment with solutions containing the amino acid (as described previously in a May 2007 report. These preliminary investigations used solutions with sufficient amino acid for multi-layer coverage of the mineral surface. Samples were decanted but not washed and thus and some dry down of the organic onto the surface occurred. The previous work was successful in showing that spectral windows were available, on γ-alumina and on kaolinite, using the DRIFTS technique (as developed at JLab Applied Science). The peaks observable in the spectral window include peaks for carboxylate and amine and the hydrocarbon chain. The spectra obtained for lysine was consistent with the work reported in the literature (for lysine adsorption onto TiO2) using in situ ATR –IR spectroscopy. In this study conditions are set for monolayer or less coverage and samples are washed, prior to drying so as to remove residual treating solution. Investigations are carried out with solutions of pH 6.3 and of 9.3.

INVESTIGATION

The DRIFTS technique was successful in detecting lysine on the surface of γ- alumina and kaolinite, after treatment with solutions containing the amino acid. This has been described in the May 2007 report “The use of DRIFTS to investigate the adsorption of amino acids onto alumina and kaolinite – preliminary investigations”. Samples were prepared such that the availability of amino acid in solution was sufficient for multi-layer coverage of the mineral surface and some dry down of the organic onto the surface occurred. The previous work showed that by using the DRIFTS technique (as developed at JLab Applied Science) spectral windows, in the 1800 -1300 cm-1 region and in the 3400 -2800 cm-1 region, were available for the study of amino acids on γ-alumina and on kaolinite. The peaks observable in the spectral window include peaks for carboxylate and amine and the hydrocarbon chain. The spectra obtained for lysine was consistent with the work reported in the literature (for lysine adsorption onto

i,ii,iii TiO2) using in situ ATR –IR spectroscopy .

Spectra obtained, in the initial study on kaolinite and on γ-alumina, are shown in Figure 1. -

12 O 2 5 6 7 O 9 0 8 H 11 d C 4 6 5 0 e N - 2 7 1 - 1 1 3 10 b 9 0 r 3 3 4 o 1

9 1 1 s ; d 8 9 a r 4

e 3 6 7 t 1 3 a M 6

6 w K 5 1 4 3 2 1 0 1800 1700 1600 1500 1400 1300 Wavenumbers (cm-1)

Figure 1 Difference spectra (both full scale) of Lysine adsorbed onto minerals (unwashed samples) (RED KW scale 0-12 KM) and (BLUE γ-alumina scale 0-3 KM).

In the previous study, L-lysine was observed on the surface of γ-alumina and on kaolinite, after treatment with 0.02 M lysine (at pH 9.8, the pH obtained by dissolving the lysine in 0.01 M HClO4, with no further manipulation). The samples were centrifuged to remove excess solution but not washed, thus it was possible that deposition from dry-down may have occurred. Further studies are now carried out with all samples undergoing a washing cycle (as for studies with salicylic acid and myristic acid).

A dilution series was prepared for each of γ-alumina and kaolinite with two sets of pH conditions used (4 sample sets in all); o In the region of the iso-electric point of lysine, pH 9.8, such that the dominant solution species is the uncharged but polarized Zwitter ion, and the surface charge on the minerals is negative. o A pH in the slightly acid to neutral region (pH 6.0-7.0) were the dominant solution + + + - species is L , (NH3 -(CH2)4-C(H)(NH3 )(COO ), exists in the region of pH 2.1 – 9.1 + (the pKa of the α-amino group is 9.1 and L ). At this pH the surface charge of γ- alumina is positive and that of the siloxane surface of kaolinite, negative.

MATERIALS

L-Lysine H2N-(CH2)4-C(H)(NH2)COOH o Molecular formula = C6H14N2O2; o GMW = 146.18 Minerals o γ-alumina, Science Minerals Corp, surface area (BET method) 51 m2/g o kaolinite, Ward’s Natural Sci., Edgar, Florida deposit, surface area (BET method) 27 m2/g

METHOD

A Solution of 0.1 M NaClO4 was prepared using Milli-Q water (14.04 g/L) for use as a high background strength medium for the lysine solutions. A stock solution of 0.05 M L- lysine was prepared (1.829 g in 250 ml of 0.1 M NaClO4). Sets of dilution series were prepared using the stock solution and 0.1 M NaClO4 for dilution. The pH of each solution sample was adjusted before the addition of mineral o sets Ai and Ki were adjusted to 9.8 ±0.1 (using NaOH)

o sets Aii and Kii were adjusted to 6.3 ±0.2 (using HClO4).

The solid to solution ratio for γ-alumina was 1g/20 ml to match that used in the previous study (with samples unwashed after treatment) – in practice, 0.75 in 15 ml was used to enable the use of the glass vial available. In the case of kaolinite, 1.5 g per 15 ml was used such that an approximately equivalent surface area to solution was used for each mineral.

The solutions were capped and placed on a shaker for 24 h. The solution was decanted from the solid (after the solid was allowed to settle). Each sample was then washed and decanted 3 X using Milli-Q water adjusted to either pH 9.8 or 6.3. Note that 0.1 M NaClO4 was not used for the washing, to minimize the dry down of NaClO4. Table 1: Summary of samples prepared Conc Code *molecules per nm2 M A = γ-alumina K = kaolinite i = pH 9.8 ii = pH 6.3 0.05 1 10 ± 1 A=12; K=11 0.02 2 5 ± 0.5 A=4.7; K=4.4 0.01 3 2 ± 0.5 A=2.4; K=2.2 0.005 4 1 ± 0.2 A=1.2; K=1.1 0.002 5 0.5 ± 0.1 A=0.47;K=0.44 0.001 6 0.2 ± 0.05 A=0.24; K=0.22 0.000 BLK 0.0

* Calculation of the number of molecules available per nm2

Molecules/nm2 = vol(L) x conc (M) x 6.00 x 1023 / (wt (g) x area (m2/g) x 1018)

DRIFTS proceedure

System purged with dry air Settings as in previous studies RESULTS

Table 2: Sample table

γ-alumina Final max lysine Sample Final max lysine Initial pH pH molecules observed Initial pH molecules observed 9.8 / nm2 pH 6.3 / nm2 A1i 9.5 10 no A1ii 8.2 10 yes A2i 9.3 5 no A2ii 7.9 5 no A3i 9.3 2 no A3ii 7.8 2 no A4i 9.0 1 no A4ii 7.7 1 no A5i 8.4 0.5 no A5i 7.2 0.5 no A5iR 8.7 0.5 no A5iiR 7.4 0.5 no A6i 8.2 0.2 no A6ii 7.0 0.2 no BLK Ai 7.9 0.0 no BLK Aii 7.2 0.0 no kaolinite Final kaolinite Final initial pH pH initial pH pH 9.8 6.3 K1i 9.7 10 yes K1ii 8.0 10 ? K2i 9.1 5 yes K2ii 7.8 5 Yes/ trace K3i 8.8 2 yes K3ii 7.3 2 yes K4i 8.4 1 yes K4ii 6.7 1 Yes /trace K5i 7.7 0.5 no K5ii 5.5 0.5 no K5iR 7.8 0.5 no K5iiR 6.4 0.5 no k6i 7.6 0.2 no k6ii 5.9 0.2 no BLK Ki 5.7 0.0 no BLK Kii 5.3 0.0 no

Observed changes in pH.

Solutions with an initial pH of 9.8 Kaolinite & γ-alumina o Biggest changes were observed with solutions with the least lysine, the greatest being the blank. o The observed changes were greater for kaolinite than for γ-alumina. The pH of the most concentrated solution was as a result of lysine dissolution in water – no extra OH- added.

Kaolinite At high pH mineral oxides deprotonate, thus in an unbuffered system the pH will fall. Aluminum cations may be dissolved from the surface (esp edges) See Chpt 5 .Chemistry of the solid water interface – Wener Stummiv 3+ - - -Al + 4OH ↔ Al(OH)4

4+ - Si +4OH ↔ Si(OH)4

The solution with the highest lysine concentration have the smallest pH change. This could be due to 1. Buffering by the lysine L ↔ L+ + OH- 2. Adsorption of lysine onto cations thus protecting them from OH- (competitive adsorption).

Γ-alumina samples

o All samples, showed an increase in pH, the blank being the least.

For the blank this is consistent with uptake of H+ by surface oxide groups.

The equilibrium L + H+ ↔ HL+ will also come into play when lysine is present.

Solutions with an initial pH of 6.3.

Γ-alumina samples o The range of final pH values (7.2 (blank) – 8.2 (most conc solution)) o Increase in pH of the blank is due to adsorption of protons onto the surface o Some contribution by the lysine in solution L + H+ ↔ HL+

The lysine extracts Al3+ from the mineral (forming a complex) Na ions would exchange into the lattice but to maintain charge balance, protons would move into the lattice as well, thus increasing pH.

3+ 3+ Al + 3L ↔ Al(L)3

Kaolinite samples.

o The pH of the blank solution and the most dilute lysine solution increased. This is consistent with the coordination of hydroxyl groups to surface cations, but it should be noted that a change from 6.3 to 5.9 is very small in terms of the change in hydrogen ion concentration. Calculation show a change of 7.5 x10 -7 moles per L in H+ which equates to 2 x 10-4 ions per nm2. Such a value is consistent with adsorption of OH- to under coordinated edge or corner sites. 3+ 2+ + Al + H2O → Al(OH) + H

o Samples treated with lysine conc > 0.005 M showed an increase in pH

The pH behavior can be explained by there being sufficient lysine to extract significant Al from the surface of the kaolinite (as was the case with alumina), such that H+ moves into the surface to counteract the charge imbalance from the Al3+ / Na+ exchange into the lattice. This effect competes for the adsorption of OH- onto the kaolinite surface.

3+ The process of removal of Al may also promote the release of Si as H4SiO4 (especially from the edge surface of the particles). (Se STUMM iv chpt 5)

4- + SiO4 + 4H ↔ H4SiO4

DRIFTS analysis

Regions examined o 2000 – 1300 cm-1 (amino and carboxyl groups)

o 3000 – 2800 cm-1 (aliphatic chain -(CH2)4- )

Γ-alumina samples; Initial pH 9.8 (Figures 2 and 3)

No lysine was detected on any of the samples o In 2000 – 1300 cm-1 region o In the 3000 – 2800 cm-1 region

12 11 10 9 8

7 KM 6 5 4 3 2 1 0 1800 1700 1600 1500 1400 1300 Wavenumbers (cm-1) Figure 2: Γ-alumina samples; Initial pH 9.8. Red curve is an unwashed sample 1-33 Lys (2005) all other curves are 2008 Ai series which have all been washed 0.05

0.00 KM

2980 2960 2940 2920 2900 2880 2860 2840 2820 2800 Wavenumbers (cm-1)

Figure 3: Comparison of set Ai samples after washing and drying with a sample (RED) equivalent to A2i (treated with 0.02 M lysine solution) which was dried without washing (3000 – 2800 cm-1 region for aliphatic chain).

Γ-alumina samples pH 6.3 (Figures 4,5,6)

No lysine was detected on any of the samples in 2000 – 1300 cm-1 region. In the 3000 – 2800 cm-1 region, signal for aliphatic carbon was observed on sample A1i, the sample treated with 0.05 M lysine. Since this was the sample treated with the most concentrated lysine solution it is possible that the washing was not sufficient to remove all of the residual lysine.

0.030 KM

0.000 3000 2950 2900 2850 2800 Wavenumbers (cm-1)

Figure 4 : Set Ai, γ-alumina samples pH 6.3 . Samples treated & washed; A1i - 0.05 M (black); A6i- 0.02 M (pink) 0.045 0.040 0.035 0.030 M

0.025K 0.020 0.015 0.010 0.005 0.000 3000 2950 2900 2850 2800 Wavenumbers (cm-1) Figure 5: Set Ai, γ-alumina samples pH 6.3 . A1ii cf A6ii

12 11 10 9

7 KM 6 5 4 3 2 1 0 1800 1700 1600 1500 1400 Wavenumbers (cm-1)

Figure 6: Set Ai, γ-alumina samples pH 6.3 . 1800 – 1300 cm-1 region; spectrum observed on an unwashed sample compared with the sample set treated at pH 6.3;

Discussion of the interaction of aluminum ions with lysine

Lysine forms a strong complex with aluminum ions which can result in

o Stopping the exchange of Al3+ into a resin (as does EDTA) o Lysine solutions extract Al3+ from exchange resins and from glass containers (resulting contamination of pharmaceutical products). Articlev

Denise Bohrer, Paulo Cícero do Nascimento, Patrícia Martins and Regina Binotto “Availability of aluminum from glass and an Al form ion exchanger in the presence of complexing agents and amino acids” Analytica Chimica Acta Vol 459 -2 May 2002 P267-276

Extract from paper

Al present in glass are supposed to replace the silicon in the silicate tetrahedra network. This is possible because of size similarities (rSi4+=0.50 Å, rAl3+=0.55 Å). Because of the difference in oxidation number, replacement of Si4+ by Al3+ raises the anion oxidation state by one unit and requires the presence of either a balancing cation (Na+) or a further isomorphous replacement of one cation by another of higher positive charge (Na+ by Ca2+). However, whereas silicon is always 4-coordinate toward oxygen, Al may be either 4 or 6-coordinate [44]. An AlO4 group would be the structural analog of an SiO4 group, but an AlO6 group would not. As Al is added as Al2O3 to the glass mass, the 6-coordinate Al must be converted into the 4-coordinate ion. It is only possible when Na2O is also present. It furnishes the polarizable O2− ions that allow the change in the coordination of the Al ions and the necessary Na+ in order to balance the charge [45]. When Al replaces Si in the glass network, it acts as a network former and is rigidly tied into the lattice. If, however, the AlO6 group is not able to be converted into the AlO4 group, Al remains 6- coordinate and acts as a network modifier, that has the role of balancing the charge of terminal oxygen ions (oxygen of one tetrahedron not linked to another). Network modifiers only balance the charges and this site can be occupied by any cation. Network modifiers can be replaced by other cations in an exchange procedure and this is the fundamental principle of the glass electrode [45 and 46]. Al is considered a network former and therefore not exchangeable, however based on anomalies in viscosity, expansion coefficient and electric properties of glass some authors consider that Al can exist in glass as 6-coordinate. Yoldas [47] considers that only above a critical concentration, Al ion exists as [AlO4]−, below this, it exists as [AlO6]−. Because the AlO6 octahederon is convered into a AlO4 tetrahedron in the glass mass only in the presence of Na2O (or other alkali metal oxide) and this oxide is randomly distributed in the glass structure [48], it is possible that most but not all of the Al ions are converted into the 4-coordinate tetrahedron. Only the existence of 6-coordinate residual ions, occupying network modifier sites, could justify how, amino acid solutions that do not interact with glass themselves, would be able to withdraw Al3+ from glass.

Conclusions from the investigation from the γ-alumina treated samples.

Some interaction of γ-alumina with hydroxide (note the change in pH of the blank from 9.8 to 7.9).

Al2O3(s) + 3H2O → 2Al(OH)3(s)

- - Al(OH)3(s) + OH + 2H2O → Al(OH)4(H2O)2

The presence of lysine countered this change in pH – with the most concentrated lysine solution restricting the change from 9.8 to 9.5. It is hypothesized that the lysine extracted Al3+ from the mineral surface, into solution. In the washing process any lysine coordinated with the surface was removed. In set Aii (pH 6.3) all solutions (including the blank) showed an increase in pH

o Blank - uptake of H+ onto surface sites – to take the alumina closer to its iso- electric point Kaolinite samples treated with lysine solutions

In the case of the kaolinite samples, the suspensions took over 1 day to settle. Traces of lysine was detected and samples treated with solutions treated with ≥ 1 molecule of lysine / nm2. This was true for both set of samples (with basic (Figure X1) and slightly acidic (Figure X2) initial pH.) pH 9.8 lysine solution (Figures 7, 8, 9, 10, 11, 12 )

2.8 2 H C

2.4 4 3

2.0 r e 1.8 t a M w

K s O O 1.4 2 d C O H a -

C n N o a 6 7 - e s a

e l l 3 5 1.0 0 h n 6 P k a i t b k 6 9 4 0 1 4 0.8 1 6 1 0.6 1 0.4 0.2 0.0 1800 1700 1600 1500 1400 Wavenumbers (cm-1)

Figure 7: Kaolinite samples treated with 0.02 M lysine solution at pH 9.8. The red curve is an unwashed sample and the green curve a washed sample.

1.0 0.9 0.8 0.7 0.6 M

K 0.5 0.4 0.3 0.2 0.1 0.0 1750 1700 1650 1600 1550 1500 1450 1400 Wavenumbers (cm-1)

Figure 8: Ki series initial pH 9.8: Red 0.05 M; Blue 0.02 M; Black 0.01; Aqua 0.005; Green 0.002; Pink 0.001 3.0 2.8 2.6 2.4 2.2

2.0 KM 1.8 1.6 1.4

1800 1750 1700 1650 1600 1550 1500 1450 1400 Wavenumbers (cm-1) Figure 9: Kaolinite samples K1i and K2i treated with lysine solution with an initial pH of 9.8. BLUE: blank (0-5 KM); RED K1i (0-3 KM) and GREEN K2i (0-2 KM): difference spectra of kaolinite exposed to 10 and 5 molecules / nm2 respectively. All spectra shown full scale

Note: in Figure 9: o 1636 cm-1 water o Broad pk centered on 1600 combination of o Antisymmetric carboxylate stretch o 1620 anti sym deformation of proximal and distal NH3+

0.032 0.030 0.028 0.026 0.024 0.022 0.020

0.018M

0.016K 0.014 0.012 0.010 0.008 0.006 0.004 0.002 0.000 3000 2950 2900 2850 2800 Wavenumbers (cm-1)

Figure 10: 3000 – 2700 cm-1 region – all spectra are on a common scale. Aliphatic carbon shows clearly on samples K1i & K2i but perhaps a hint on all samples. Red K1i; yellow K2i; blue K3i; black K4i; pink K5i; green K6i. Not clearly resolved over the noise in the signal for samples 4-6 0.030 0.025 0.020 0.015

0.010M K 0.005 -0.000 -0.005 -0.010 -0.015 3000 2950 2900 2850 2800 Wavenumbers (cm-1)

Figure 11: 3000 – 2700 cm-1 region - all shown full scale. Samples K1i (red); K2i (gold); K3i (blue)

0.19 0.18 0.17 0.16

M 0.15 K 0.14 0.13 0.12 0.11 0.10 3400 3300 3200 3100 3000 2900 2800 2700 Wavenumbers (cm-1)

Figure 12: Sample K1i (Blue); Blank (pink); Difference spectrum for K1i (Red )

PEAKS (figure 12 o 3264 ? NH3+ stretch o 3190 ? NH3+ stretch o 2947 CH stretching of methylene groups o 2868 CH stretching of methylene groups Spectra of kaolinite samples (Kii) treated with initial pH 6.3 (Figures 13, 14)

5.0 4.5 4.0 3.5 3.0 M K 2.5 2.0 1.5 1.0 0.5 0.0 1800 1750 1700 1650 1600 1550 1500 1450 1400 Wavenumbers (cm-1)

Figure 13: Kaolinite samples treated with lysine solution with an initial pH of 6.3. RED: blank (0-5 KM); Aqua and GREEN (0-3.5 KM); Grey and BLUE (0-1.25 KM): difference spectra of kaolinite exposed to from 10 to 1 molecules / nm2. All spectra shown full scale.

0.14 0.13 0.12 0.11 0.10 0.09 0.08 M

K 0.07 0.06 0.05 0.04 0.03 0.02 0.01 0.00 3300 3200 3100 3000 2900 2800 Wavenumbers (cm-1)

Figure 14: K1ii – Red difference spectrum cf blank (clue)

PEAKS o 3264 ? NH3+ stretch o 3190 ? NH3+ stretch o 2974 o 2947 CH stretching of methylene groups o 2870 CH stretching of methylene groups Conclusions

Investigation of γ-alumina treated with lysine showed no organic remained on the mineral surface after washing. This is not to say that no interaction occurred. Al ions on the surface of the alumina intact strongly with lysine and are extracted into solution. The washing step removes the soluble lysine –Al complex.

The kaolinite samples settled very slowly and it was difficult to decant solution from the solid. To confirm the presence of lysine on kaolinite samples treated with solutions >0.0005 M concentration in lysine, the experiments would have to be redone and the kaolinite vacuum filtered (or centrifuged) and then washed 3 times (with filtration each time). i Roddick-Lanzilotta, A. D. & McQuillan, A. J. (2000) Journal of Colloid and Interface Science, 227, 48-54. ii Bellamy L.J., The Infrared spectra of complex molecules. Third Edition, Wiley, New York. iii Brian Smith, B. (1999) Infrared Spectral Interpretation; a systematic approach. CRC Press LLC., Boca Raton, London, New York, Washington DC. iv Werner Stumm (1992) Chemistry of the Solid-Water Interface. Wiley-Interscience, New York, Chichester, Brisbane, Toronto, Singapore. v Denise Bohrer, Paulo Cícero do Nascimento, Patrícia Martins and Regina Binotto (2002) “Availability of aluminum from glass and an Al form ion exchanger in the presence of complexing agents and amino acids” Analytica Chimica Acta Vol 459 -2 May 2002 P267-276